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Posts Tagged ‘embryonic stem cells’


Embryo Stem Cells Out of Skin

Reporter: Irina Robu, PhD

Researchers at Hebrew University identified a set of genes able to transform murine skin cells into three cell types such as the embryo itself, the placenta and extraembryonic tissue i.e. umbilical cord which was published in the journal Cell Stem Cell.

Dr. Oren Ram, Institute of Life Science at Hebrew University, Prof. Tommy Kaplan, School of Computer Science and Engineering found a new combination of five genes that once inserted into skin cells, reprogram the cells into each of three early embryonic cell types. Researchers identified that the gene “Eomes” pushes the cell toward placental stem-cell identity and placental development, whereas the “Esrrb” gene arranges fetus stem cells development through the temporary procurement of an extraembryonic stem cell identity.The team used this to examine the molecular forces that oversee cell fate decisions for skin cell reprogramming and the natural process of embryonic development.

Even though this groundbreaking research could provide a path toward creating entire human embryos from human cell skin cells without need for sperm of organs, that is still a long way in the future. However, for now this work can have large implications for modeling embryonic disease and placental dysfunctions in addition to solving infertility problems by creating human embryos in a petri dish.

SOURCE
https://www.jpost.com/OMG/A-baby-from-skin-cells-Israeli-team-makes-embryo-stem-cells-out-of-skin-588531

 

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Embryonic Stem Cell differentiation

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1

Yan Tang1, Ya-Zhen Hong1, Hua-Jun Bai1, Qiang Wu1, Charlie Degui Chen2, Jing-Yu Lang1, Kenneth R. Boheler3 and Huang-Tian Yang1,4,*

STEM CELLS: 18 APR 2016     http://dx.doi.org:/10.1002/stem.2333

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8-/Y) model, we found that deletion ofphf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8+/Y) and ph8-/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown ofpmaip1 mimicked the phenotype of ph8-/Y by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8-/Y ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells2016

 

Significance Statement

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Thus, ESCs provide a unique model for the study of early embryonic development. We report here previously unrecognized effects of histone demethylase plant homeo domain finger protein 8 (PHF8) on mesodermal and early cardiac differentiation. This effect is resulted from the regulation of PHF8 on apoptosis through activating the transcription of pro-apoptotic gene pmaip1. These findings extend the knowledge in understanding of the epigenetic modification in apoptosis during ESC differentiation and of the link between apoptosis and cell lineage decision as well as cardiogenesis.

 

Embryonic stem cells (ESCs) have the unique ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Due to this plasticity, mechanisms controlling cell autonomous and regulatory events critical to in vivo mammalian development have benefitted from the in vitro study of differentiating ESCs [1, 2]. Early embryogenesis and cavity formation as well as early ESC differentiation, for example, are accompanied by a reduction in proliferation and increased apoptosis [3-5]. Withdrawal of leukemia inhibitory factor (LIF) from mouse embryonic stem cells (mESCs) cultivated in vitro causes approximately 20%-30% of the cells to die by spontaneous (constitutive) apoptosis [4, 5]. This occurs secondary to the induction of cleaved caspase 3 [3] and apoptosis-inducing factor (AIF)-complex proteins [6]. Blockade of spontaneous apoptosis in vitro by a p38 mitogen-activated protein kinase (MAPK) inhibitor alters the differentiation markers and increases the abundance of both antiapoptotic proteins (Bcl-2, Bcl-XL) and Ca2+-binding proteins [4, 7]. In addition, Ca2+ released from type 3 inositol 1, 4, 5-trisphosphate receptors (IP3R3) negatively regulates this apoptotic response, which in turn modulates the mesodermal lineage commitment of early differentiating mESCs [5]. These findings explain, in part, how apoptosis contributes to specific lineage commitment during early development. However, in contrast to the relatively advanced knowledge of signaling pathways [8], little is known about the contribution of epigenetic regulators, especially, histone lysine demethylases (KDMs), in the regulation of apoptosis during ESC differentiation and how the affected programmed cell death by KDMs contributes to the lineage commitment.

Epigenetic regulators and dynamic histone modifications by KDMs are emerging as important players in ESC fate decisions [9]. Histone modifications coordinate transient changes in gene transcription and help maintaining differential patterns of gene expression during differentiation [10-13]. The molecular and biological functions of many KDMs, however, remain enigmatic during ESC differentiation. PHF8, an X-linked gene encoding an evolutionarily conserved histone demethylase harboring an N-terminal plant homeo domain (PHD) and an active jumonji-C domain (JmjC), is able to catalyze demethylation from histones [14, 15]. It is actively recruited to and enriched in the promoters of transcriptionally active genes [14], and it functions to maintain the active state of rRNA through the removal of the repressive H3K9me2 methylation mark at the active rRNA promoters. Mutation of PHF8 is associated with X-linked mental retardation with cleft lip/cleft palate in human [16-18]. Knockdown of phf8 in mouse embryonic carcinoma P19 cells impairs neuronal differentiation [19] and leads to brain defects in zebrafish by directly regulating the expression of the homeo domain transcription factor MSX1/MSXB [20]. However, the precise function of PHF8 in the regulation of lineage differentiation derived from other germ layers remains to be identified.

Here, we report previously unrecognized effects of the PHF8 histone demethylase on germ layer commitment and differentiation of mESCs. The results are based on an assessment of early steps of differentiation to mesodermal lineages and cardiomyocytes using phf8 knockout (phf8-/Y) and wild-type (phf8+/Y) mESCs. The data show that PHF8 regulates gene transcription of a proapoptotic gene pmaip1 (also named Noxa) [21]. Activation or repression of pmaip1 controlled by PHF8 ultimately determines mESC lineage commitment through the regulation of caspase 3-independent apoptosis during mesodermal and cardiac differentiation. Our data reveal that PHF8-mediated the demethylation of histone proteins coordinates ESC lineage commitment through the regulation of apoptosis in early differentiating ESCs.

 

Deletion of phf8 Promotes Mesodermal and Cardiac Lineage Commitment

The PHF8 protein was detectable in undifferentiated ESCs, but its abundance significantly increased within one day of LIF withdrawal. Then it gradually decreased to a level at day 5 lower than that observed in the undifferentiated ESCs (Fig. 1A).    http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0001-t.gif

 

Figure 1. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and early cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Western blot analysis of PHF8 expression in undifferentiated and differentiating ESCs. n = 3. (B): quantitative RT-PCR (qRT-PCR) analysis of pluripotency markers nanog, rex1, sox2, and oct4. n = 8. (C): qRT-PCR analysis of gene expression of pluripotency marker oct4; early mesodermal markers brachyury (T), gsc, eomes, and mesp1; cardiovascular progenitor markers flk-1 and nrp1; and the cardiac transcription factors hand1, tbx5, and mef2c during ESC differentiation. n = 5. (D): qRT-PCR analysis of the early ectodermal markersnestin and fgf5 during ESC differentiation. n = 3. (E): qRT-PCR analysis of early endodermal markers afp, foxa2, sox17, and gata4 during ESC differentiation. n = 3. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.

To determine the significance of phf8 gene expression on ESC fate decision, we knocked out the X-chromosome-encoded phf8 gene in one allele of male SCR012 ESCs by deletion of exons 7 and 8 through Cre-mediated recombination (Supporting Information Fig. S1A). Gene inactivation was confirmed by the lack of Phf8 mRNA and PHF8 protein expression in these targeted ESCs (Supporting Information Fig. S1B). Transcripts for pluripotency marker genes nanog, rex1 (zfp42), sox2, and oct4 (pou5f1) were not significantly different between phf8+/Y and phf8-/Y ESCs (Fig. 1B). No significant difference was observed in cell morphology (Supporting Information Fig. S1C) of undifferentiated phf8+/Y and phf8-/Y ESCs or in alkaline phosphatase activity (Supporting Information Fig. S1D). Immunofluorescence staining confirmed that the expression of pluripotency marker SOX2 and SSEA-1 did not differ between the phf8+/Y and phf8-/Y ESCs (Supporting Information Fig. S1E). These results indicate that phf8 may be dispensable for normal growth and maintenance of mESCs.

We then analyzed the role of PHF8 in the mesodermal and cardiac lineage commitment. By microarray analysis of differentiating phf8+/Y and phf8-/Y cells from days 0, 1, to 3.5, we found a significant decrease in transcripts for pluripotency markers, accompanied by a significant increase in transcripts for ectoderm, mesoderm and endoderm, while in phf8-/Y cells some transcripts for mesodermal and cardiac lineage commitment were significantly enhanced compared with those in phf8+/Y cells (Supporting Information Fig. S2A). These differentiation-dependent changes in transcript abundance were confirmed by qRT-PCR for early mesodermal markers brachyury (T) [28], goosecoid (gsc),eomes[29], and mesp1[30], cardiovascular progenitor marker flk-1[31, 32] and neuropilin 1 (nrp1) [33]. Early cardiac transcription factors, including myocyte enhancer factor 2C (mef2c) [34], hand1[35], and tbx5[36, 37] were also up-regulated in phf8-/Y cells at differentiation day 5, while no difference in the expression levels of pluripotent markersoct4 (Fig. 1C), rex1, and nanog (Supporting Information Fig. S2B) were detected between phf8+/Y and phf8-/Y cells at the time points examined.

Because mESCs can differentiate into all three germ layers, we also examined whether phf8 affected ectodermal and endodermal differentiation. qRT-PCR analysis did not show any significantly difference in the expression of early ectodermal markers nestin and fgf5 between the phf8+/Y and phf8-/Y cells (Fig. 1D). Moreover, in the induced early ectodermal differentiation system [23], the expression of ectodermal markers nestin, fgf5, and pax6 were comparable between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3A). Besides, the expression of endodermal markers afp, foxa2, sox17, and gata4 were not significantly different between the phf8+/Y and phf8-/Y cells (Fig.1E). Consistently, the expression of endodermal markers foxa2, sox17, and gata4 were comparable during induced endodermal differentiation [24] between the phf8+/Y andphf8-/Y cells (Supporting Information Fig. S3B). Thus, phf8 appears not to affect early ectodermal and endodermal differentiation.

The increased mesodermal and cardiac marker expressions were associated with a significant increase in the total number of cardiac progenitors and cardiomyocytes in differentiating phf8-/Y cells. By flow cytometry analysis, marked increases in the population of FLK-1 positive (FLK-1+) cells were detected in phf8-/Y cells at differentiation day 3 and day 4 (Fig. 2A). Consistently, the percentage of contracting EBs was higher in phf8-/Y cells than in phf8+/Y cells (Fig. 2B). The transcripts for progenitor marker nrp1, early cardiac transcription factor tbx5, and cardiac specific genes tnnt2, myh6, myl2, and gja1 were higher in phf8-/Y EBs than those in phf8+/Y ones (Fig. 2C). The areas of immunostained EBs positive for the cardiac cytoskeletal and myofilamental proteins α-actinin and TNNT2 were also greater in phf8-/Y than in phf8+/Y EBs (Fig. 2D). Flow cytometry analysis of MYH6+ (Fig. 2E) and TNNT2+ (Fig. 2F) cells at differentiation day 9 further confirmed the increase of cardiomyocytes in phf8-/Y cells. Taken together, these data indicate that the phf8 deletion facilitates the differentiation of mesodermal and cardiac linage commitment.

Figure 2. phf8 deletion promotes cardiac differentiation of mouse embryonic stem cells (mESCs). (A): Left, representative flow cytometry plots showing FLK-1 expression at differentiation day 3 (n = 6), day 4 and day 5 (n = 3 each). Right, the quantification of flow cytometry data. (B): Differentiation profile of cardiomyocytes during embryoid bodies (EB) outgrowth. n = 6. (C): qRT-PCR analysis of ESCs for the expression of cardiac markers at differentiation day 14. n = 3. (D): Immunofluorescence analysis of TNNT2 and α-actinin in day 14 EBs. Scale bars = 400 μm. (E) Flow cytometry analysis of MYH6 positive cells and (F) TNNT2 positive cells in day 9 EBs. n = 3 each. Data are presented as mean ± SEM *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0002-t.gif

PHF8 Inactivation Increases Cell Viability but not Proliferation of the Differentiating ESCs

Differentiation of both phf8+/Y and phf8-/Y ESCs via EB formation produced normal round shaped EBs but, by day 3, phf8-/Y EBs were larger than those generated from phf8+/YESCs, and the size differences were visibly obvious at differentiation days 5 and 7 (Fig. 3A). Although no significant differences in cell viability could be demonstrated between undifferentiated phf8+/Y and phf8-/Y ESCs (Fig. 3B), the viability of phf8-/Y cells was significantly greater than that in phf8+/Y cells at differentiation days 3 to 7 (Fig. 3C). However, no significant change in BrdU staining was detected by flow cytometry between phf8+/Y and phf8-/Y ESCs at differentiation days 0, 3, or 5 (Fig. 3D). Moreover, no significant difference in the cell cycle distribution between the differentiating Phf8+/Y and Phf8-/Y ESCs was detected, although the percentage of cells in S phase gradually decreased while those in G1 phase increased upon differentiation (Fig. 3E). Knockout of phf8 thus increases cell numbers in the early differentiating ESCs through the improvement of cell viability without changes in cell proliferation or cell cycle progression.

Figure 3. phf8 deletion increases cell viability in differentiating mouse embryonic stem cells (mESCs) without affecting cell proliferation. (A): Left, phase-contrast images of embryoid bodies (EB) morphology during EB formation from ESCs. Scale bar = 200 μm. Right, the diameter of EB formed from ESCs. (B): Cell viability of undifferentiated and (C): differentiating ESCs analyzed by MTT assay for seven consecutive days. n = 3. (D): Flow cytometry analysis of BrdU positive proportion of undifferentiated (n = 4) and differentiating ESCs at day 3 and day 5.n = 5 each. (E): Flow cytometry analysis of cell cycle distribution by propidium iodide (PI) staining at differentiation day 3 (n = 6) and day 5 (n = 3). Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Y value.   http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0003-t.gif

PHF8 Regulates Apoptosis During the Early Stage of Cardiac Lineage Commitment

We then examined whether cell death might account for the differences in the cell viability observed between the differentiating phf8+/Y and phf8-/Y ESCs. In undifferentiated ESCs, no significant difference was demonstrated with Annexin V (an early apoptosis marker) staining, TUNEL assay, total DNA fragmentation or caspase 3 protein cleavage between phf8+/Y and phf8-/Y cells (Fig. 4A–4C, 4E). In contrast, Annexin V staining (Fig. 4A) and TUNEL assay (Fig. 4B) showed significant decreases in the number of apoptotic cells in phf8-/Y ESCs at differentiation days 3 and 5 compared with those in phf8+/Y cells. Genomic DNA fragmentation with a pattern typical for apoptosis was detected in phf8+/Y cells at differentiation days 3 and 5, but it was reduced in phf8-/Y cells at the same time points (Fig. 4C). Moreover, approximately 35% of Annexin V+ cells were present in FLK-1+/phf8+/Y cells at differentiation day 4, whereas only 9% of the cells were Annexin V+ in FLK-1+/phf8-/Y cells (Fig. 4D). The ratio of TUNEL+ to either NESTIN+ (ectoderm) or SOX17+ (endoderm) cells did not differ between the phf8+/Y and phf8-/Y cells (Supporting Information Fig. S3C, S3D). In addition, phf8+/Y ESCs at differentiation days 3 and 5 increased the caspase 3 cleavage (Fig. 4E, upper and lower left panels) and the ratio of cleaved caspase 3 to total caspase 3 protein (Fig. 4E, lower right panel). Unexpectedly, the ratio of cleaved caspase 3 to total caspase 3 in phf8-/Y ESCs did not significantly differ from that observed in phf8+/Y ones. Consistently, a significant enhancement of the downstream target PARP1 cleavage [38, 39] was observed at differentiation days 3 and 5, but it was comparable between the phf8+/Y andphf8-/Y cells (Fig. 4F). These data suggest that the cell death associated with phf8 function does not operate through the conventional caspase 3-mediated apoptosis.

Figure 4. Plant homeo domain finger protein 8 (PHF8) regulates apoptosis during the early mouse embryonic stem cells (mESC) differentiation. (A): Left, representative flow cytometry plots showing Annexin V (x-axis), and PI (y-axis) staining in ECSs at differentiation day 0 (n = 4), day 3 (n = 3) and day 5 (n = 7). Right, the quantification of flow cytometry data. (B): Flow cytometry detection of apoptotic responses of TUNEL positive cells at differentiation day 0 (n = 3), day 3 (n = 4), and day 5 (n = 4). (C): DNA laddering analysis at differentiation days 0, 3, and 5. n = 6 each. (D): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 3. (E): Western blot analysis of caspase 3 during the mESC differentiation. β-actin was used as a loading control. n = 4. (F): Western blot analysis of PARP1 expression during the differentiation. β-actin was used as a loading control. n = 4. Data are presented as mean ± SEM *, p < .05; ***, p < .001 compared with the corresponding phf8+/Yvalue; #, p < .05; ##, p < .01 compared with the corresponding d0 value.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0004-t.gif

pmaip1 is a Direct Target Gene of PHF8 in the Early Differentiating ESCs

To understand how PHF8 might regulate apoptosis during early ESC differentiation, we compared the profiles of apoptosis-related gene transcripts in undifferentiated and early differentiating phf8+/Y and phf8-/Y ESCs using gene expression microarrays. Among the apoptosis-related genes, the transcript to pmaip1, a proapoptotic Bcl-2 family member crucial in fine-turning the cell death decision [21, 40-42], was markedly increased during early differentiation of phf8+/Y cells but it was reduced in phf8-/Y cells at differentiation days 1 and 3.5 (Fig. 5A). These expression patterns were confirmed by qRT-PCR during cardiac differentiation (Fig. 5B), and the results were consistent with the apoptotic pattern observed during the early ESC differentiation (Fig. 4A–4C). In addition, qRT-PCR analysis showed that the expression of pmaip1 did not show any significant difference between the phf8+/Y and phf8-/Y cells during the induced ectodermal (Supporting Information Fig. S3E) or endodermal (Supporting Information Fig. S3F) differentiation.

Figure 5. pmaip1 is a direct target gene of plant homeo domain finger protein 8 (PHF8) in mouse embryonic stem cells (mESCs). (A): Microarray gene expression heat map depicting the expression of apoptosis-related genes at differentiation days 0, 1 and 3.5 in phf8+/Y and phf8-/Y ESCs. The expression values in log2 scale were calculated and presented on the heat map with red representing highly abundant transcripts and green representing poorly abundant transcripts. n = 3. (B): qRT-PCR analysis of pmaip1 during the ESC differentiation. (C): ChIP assay of PHF8 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation days 0 and 3. n = 4 each. (D): Western blot analysis of H3K9me2 and H3 in phf8+/Y and phf8-/Y ESCs during the differentiation. H3 was used as a loading control. n = 9. (E): H3K9me2 staining inphf8+/Y and phf8-/Y embryoid bodies (EBs) at differentiation day 1. Scale bars = 25 μm. (F): ChIP assay of H3K9me2 around the TSS of pmaip1 in phf8+/Y andphf8-/Y ESCs at differentiation day 3. n = 4. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y value or d0.  http://onlinelibrary.wiley.com/store/10.1002/stem.2333/asset/image_t/stem2333-fig-0005-t.gif

A direct link between the PHF8 and pmaip1 was then confirmed by ChIP analysis. We detected the endogenous binding of PHF8 at the transcription start site (TSS, from −45 bp to 104 bp) of pmaip1 in phf8+/Y ESCs and determined that binding was enhanced at differentiation day 3. The binding of PHF8 was not detectable above the IgG control levels in phf8-/Y cells (Fig. 5C). Global methylation (H3K9me2 normalized to H3) was unchanged at differentiation days 3 and 5, but it was significantly enhanced at differentiation day 1 in phf8-/Y cells (Fig. 5D). The augmentation of H3K9me2 methylation in phf8-/Y ESCs was then confirmed by immunostaining at differentiation day 1 (Fig.5E). An increase in the repressive mark of H3K9me2 was also observed at the TSS of pmaip1 in the early differentiating phf8-/Y ESCs (Fig. 5F), indicating that the PHF8 demethylase activity is actively involved in the regulation of pmaip1 gene.

Transient Knockdown of pmaip1 Decreases Apoptosis and Promotes Mesodermal and Cardiac Differentiation

To clarify the role of pmaip1 in mESC differentiation, we transfected specific siRNAs against pmaip1 (si-Pmaip1) into phf8+/Y ESCs followed by EB formation. The negative control siRNA (si-NC) did not alter pmaip1 transcript levels compared with untreated (NT) cells, while si-Pmaip1 significantly inhibited pmaip1 transcripts by 74%-76% at differentiation days 0 and 1 (Fig. 6A-a). si-Pmaip1 cells had fewer TUNEL+ cells compared with the NT and si-NC cells at differentiation day 3 in both phf8+/Y and phf8-/Y cells (Fig. 6A-b). We then examined whether the pmaip1 knockdown influences mesodermal and early cardiac differentiation. As shown in Figure 6B, the apoptosis of FLK-1+ cells was significantly decreased in si-Pmaip1 mESCs (Fig. 6B). The expression of T and gsc as well as nrp1 and flk-1 were increased in si-Pmaip1 cells compared with those in NT and si-NC cells at differentiation day 3. In addition, the expression of cardiac transcript factors mef2c and tbx5 was up-regulated at differentiation day 5, and myh6 andtnnt2 were up-regulated at differentiation day 9 (Fig. 6C). We also transfected si-Pmaip1 into phf8-/Y ESCs. The expression of pmaip1 was downregulated at differentiation day 0 and day 1 in phf8-/Y ESCs with si-Pmaip1 (Supporting Information as Fig. S4A-a), accompanied by a decrease in TUNEL+ cells compared with NT and si-NC (Fig. 6A-b), while Annexin V remained unchanged (Supporting Information Fig. S4A-b). The expression of nrp1 and flk1 did not significantly change in phf8-/Y ESCs with si-Pmaip1 at differentiation day 3, while mef2c was upregulated at differentiation day 5, and myh6 was upregulated at differentiation day 9 (Supporting Information as Fig. S4B). These results suggest that downregulation of pmaip1 in phf8-/Y ESCs may not lead to as robust of a phenotype as it did in phf8+/Y ESCs. This difference is likely due to the level ofpmaip1 during early differentiation of phf8-/Y ESCs was already decreased to a low level similar to that observed in the undifferentiated cells (Fig. 5B). Taken together, these data demonstrate that the decreased apoptosis via down-regulation of pmaip1 contributes, at least partially, to the phf8-/Y-facilitated mesodermal and cardiomyocyte commitment.

Figure 6. Plant homeo domain finger protein 8 (PHF8) regulates the mesodermal and cardiac differentiation through pmaip1. (A-a): qRT-PCR analysis of thepmaip1 expression in phf8+/Y ESCs after being transiently transfected with si-NC or si-Pmaip1. n = 4. (A-b): Apoptosis cells were quantified by flow cytometry analysis of TUNEL assay at differentiation day 3 in phf8+/Y and phf8-/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 3. (B): Cells double stained with FLK-1 and Annexin V were analyzed by flow cytometry at differentiation day 4. n = 4. (C): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8+/Y ESCs after transient transfection with si-NC or si-Pmaip1. n = 5. (D): Flow cytometry detection of TUNEL positive cells at differentiation day 3, Annexin V positive cells and double stained FLK-1 and Annexin V at differentiation day 4 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mouse embryonic stem cells (mESCs). n = 4. (E): qRT-PCR analysis of the expression of T, gsc, flk-1, nrp1, tbx5, mef2c, myh6, and tnnt2 in phf8-/Y, phf8-NC-/+, phf8-pmaip1-/+, and phf8-hPHF8-/+ mESCs. n = 3. Data are presented as mean ± SEM. *, p < .05; **, p < .01; ***, p < .001 compared with the corresponding phf8+/Y or phf8-/Y value.
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Overexpression of pmaip1 or hPHF8 in phf8-/Y ESCs Increases Apoptosis and Weakens Mesodermal and Cardiac Differentiation

To further determine whether PHF8 contributes to mesoderm and cardiac cell commitment through the regulation of apoptosis via targeting pmaip1, we rescued the expression of pmaip1 and phf8 in phf8-/Y ESCs by generating pmaip1-ovexpressing phf8-/Y mESCs (phf8-pmaip1-/+ mESCs) and hPHF8-overexpressing phf8-/Y mESCs (phf8-hPHF8-/+ mESCs). The qRT-PCR analysis confirmed that the expression of hPHF8 or pmaip1 was significantly upregulated in the respective overexpressing cell lines (Supporting Information Fig. S4C). The expression of pmaip1 in undifferentiated phf8-/Y mESCs was not affected by hPHF8 overexpression. However, Pmaip1 transcripts increased by differentiation day 3 in overexpressing cells (Supporting Information Fig. S4D), indicating that PHF8 does regulate the expression of pmaip1 during differentiation. Both TUNEL and Annexin V analysis revealed significant increases of apoptosis in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs compared with the phf8-/Y andphf8-NC-/+ mESCs at differentiation day 3 or day 4, accompanied by a higher apoptosis ratio in FLK-1+ cells (Fig. 6D). Moreover, the expression of T and gsc as well as nrp1and flk-1 were significantly decreased in phf8-pmaip1-/+ and phf8-hPHF8-/+ mESCs at differentiation day 3, followed by a down-regulation of mef2c and tbx5 at differentiation day 5, and myh6 and tnnt2 at differentiation day 9 (Fig. 6E). In addition, TUNEL analysis showed no changes in the apoptotic responses either through knockout or overexpression of phf8 compared with the corresponding wild-type cells or phf8+/Y cells during induced ectodermal differentiation (Supporting information Fig. S4E). These data are consistent with a regulatory role of phf8 on mesodermal and cardiac differentiation through targeting of pmaip1

Discussion

This is the first study to unravel a regulatory role of histone demethylase in the differentiation of ESCs through the control of apoptosis and subsequent effects on cell lineage commitment. The role of PHF8 in the regulation of ESC differentiation to the mesodermal lineage and cardiac differentiation is supported by selective changes in RNA markers for mesodermal lineages, and an increase in cardiomyocyte progenitors and cardiomyocytes (Figs. 1C, 2C). Moreover, deletion of phf8 specifically inhibits apoptosis of Flk-1+ mesodermal cells with a concomitant reduction in Annexin V+ staining (Fig. 4D) and cardiac differentiation (Fig. 2B–E), while the ratio of TUNEL+ to either NESTIN+(ectodermal cells) or SOX17+ (endodermal cells) cells does not differ between the phf8+/Y and phf8-/Y lines (Supporting Information Fig. S3C, S3D). Consistently, the proportion of early apoptotic cells (Annexin V+) in pmaip1-knockdown (Fig. 6B) is also decreased, while pmaip1-overexpression or hPHF8-overexpression in phf8-/Y cells increase the proportion of TUNEL+ and Annexin V+ cells simultaneously with a reduction in mesodermal and cardiac differentiation (Fig. 6D, 6E). These findings indicate that the PHF8 functions, at least partially, through regulation of apoptosis.

It is well known that the regulation of apoptosis is of critical importance for proper ESC differentiation and embryo development [8, 43]. ESC differentiation is regulated by apoptosis induced by MAPK activation [7] and IP3R3-regulated Ca2+ release [5]. Previously only histone 3 lysine 4 methyltransferase MLL2 had been shown to activate the antiapoptotic gene bcl2 to inhibit apoptosis during ESC differentiation [44]. The data presented, here, extends and reveals the importance of epigenetic controls in the activation of proapoptotic gene associated with ESC differentiation.

Mesodermal and cardiac differentiation have been shown to be regulated by the histone demethylase ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX)[13, 45] and jumonji domain–containing protein 3 (JMJD3) [12] through transcriptional activation of mesodermal and cardiac genes. These findings together with those presented in this paper support the critical role of histone demethylases in lineage commitment through regulatory mechanisms that control the expression of core lineage specific transcription factors and apoptotic genes. The decrease in apoptosis through deletion of phf8 can be attributed to the maintenance of repressive H3K9me2 mark on the TSS of pmaip1 after phf8 deletion, resulting in a ∼70% downregulation of pmaip1 at differentiation day 3 in the phf8-/Y cells (Fig. 5B). The pro-apoptotic gene pmaip1 is, therefore, epigenetically regulated by the histone demethylase, which subsequently affects the mesodermal and cardiac differentiation.

PMAIP1 is a Bcl2 homology domain 3 (BH3)-only protein that acts as an important mediator of apoptosis [46]. Its expression is regulated transcriptionally by various transcription factors and, when present, it acts to promote cell death in a variety of ways [21] including caspase 3 dependent [47] and independent apoptosis [48] and autophagy [40]. Here, we find that PHF8 and its regulation on the pmaip1 promote DNA fragmentation and cell death most likely through a caspase 3-independent pathway. This conclusion is based on the observation that neither the ratio of cleaved caspase 3 to total caspase 3 [49, 50] nor PARP1, a downstream target of caspase 3, is significantly affected. While this may be explained as the inhibitor of apoptosis proteins can counteract the function of caspase 3 [51, 52], the exact mechanisms we observed here need to be further explored.

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Conduction, graphene, elements and light

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New 2D material could upstage graphene   Mar 25, 2016

Can function as a conductor or semiconductor, is extremely stable, and uses light, inexpensive earth-abundant elements
http://www.kurzweilai.net/new-2d-material-could-upstage-graphene
The atoms in the new structure are arranged in a hexagonal pattern as in graphene, but that is where the similarity ends. The three elements forming the new material all have different sizes; the bonds connecting the atoms are also different. As a result, the sides of the hexagons formed by these atoms are unequal, unlike in graphene. (credit: Madhu Menon)

A new one-atom-thick flat material made up of silicon, boron, and nitrogen can function as a conductor or semiconductor (unlike graphene) and could upstage graphene and advance digital technology, say scientists at the University of Kentucky, Daimler in Germany, and the Institute for Electronic Structure and Laser (IESL) in Greece.

Reported in Physical Review B, Rapid Communications, the new Si2BN material was discovered in theory (not yet made in the lab). It uses light, inexpensive earth-abundant elements and is extremely stable, a property many other graphene alternatives lack, says University of Kentucky Center for Computational Sciences physicist Madhu Menon, PhD.

Limitations of other 2D semiconducting materials

A search for new 2D semiconducting materials has led researchers to a new class of three-layer materials called transition-metal dichalcogenides (TMDCs). TMDCs are mostly semiconductors and can be made into digital processors with greater efficiency than anything possible with silicon. However, these are much bulkier than graphene and made of materials that are not necessarily earth-abundant and inexpensive.

Other graphene-like materials have been proposed but lack the strengths of the new material. Silicene, for example, does not have a flat surface and eventually forms a 3D surface. Other materials are highly unstable, some only for a few hours at most.

The new Si2BN material is metallic, but by attaching other elements on top of the silicon atoms, its band gap can be changed (from conductor to semiconductor, for example) — a key advantage over graphene for electronics applications and solar-energy conversion.

The presence of silicon also suggests possible seamless integration with current silicon-based technology, allowing the industry to slowly move away from silicon, rather than precipitously, notes Menon.

https://youtu.be/lKc_PbTD5go

Abstract of Prediction of a new graphenelike Si2BN solid

While the possibility to create a single-atom-thick two-dimensional layer from any material remains, only a few such structures have been obtained other than graphene and a monolayer of boron nitride. Here, based upon ab initiotheoretical simulations, we propose a new stable graphenelike single-atomic-layer Si2BN structure that has all of its atoms with sp2 bonding with no out-of-plane buckling. The structure is found to be metallic with a finite density of states at the Fermi level. This structure can be rolled into nanotubes in a manner similar to graphene. Combining first- and second-row elements in the Periodic Table to form a one-atom-thick material that is also flat opens up the possibility for studying new physics beyond graphene. The presence of Si will make the surface more reactive and therefore a promising candidate for hydrogen storage.

 

Nano-enhanced textiles clean themselves with light

Catalytic uses for industrial-scale chemical processes in agrochemicals, pharmaceuticals, and natural products also seen
http://www.kurzweilai.net/nano-enhanced-textiles-clean-themselves-with-light
Close-up of nanostructures grown on cotton textiles. Image magnified 150,000 times. (credit: RMIT University)

Researchers at at RMIT University in Australia have developed a cheap, efficient way to grow special copper- and silver-based nanostructures on textiles that can degrade organic matter when exposed to light.

Don’t throw out your washing machine yet, but the work paves the way toward nano-enhanced textiles that can spontaneously clean themselves of stains and grime simply by being put under a light or worn out in the sun.

The nanostructures absorb visible light (via localized surface plasmon resonance — collective electron-charge oscillations in metallic nanoparticles that are excited by light), generating high-energy (“hot”) electrons that cause the nanostructures to act as catalysts for chemical reactions that degrade organic matter.

Steps involved in fabricating copper- and silver-based cotton fabrics: 1. Sensitize the fabric with tin. 2. Form palladium seeds that act as nucleation (clustering) sites. 3. Grow metallic copper and silver nanoparticles on the surface of the cotton fabric. (credit: Samuel R. Anderson et al./Advanced Materials Interfaces)

The challenge for researchers has been to bring the concept out of the lab by working out how to build these nanostructures on an industrial scale and permanently attach them to textiles. The RMIT team’s novel approach was to grow the nanostructures directly onto the textiles by dipping them into specific solutions, resulting in development of stable nanostructures within 30 minutes.

When exposed to light, it took less than six minutes for some of the nano-enhanced textiles to spontaneously clean themselves.

The research was described in the journal Advanced Materials Interfaces.

Scaling up to industrial levels

Rajesh Ramanathan, a RMIT postdoctoral fellow and co-senior author, said the process also had a variety of applications for catalysis-based industries such as agrochemicals, pharmaceuticals, and natural productsand could be easily scaled up to industrial levels. “The advantage of textiles is they already have a 3D structure, so they are great at absorbing light, which in turn speeds up the process of degrading organic matter,” he said.

Cotton textile fabric with copper-based nanostructures. The image is magnified 200 times. (credit: RMIT University)

“Our next step will be to test our nano-enhanced textiles with organic compounds that could be more relevant to consumers, to see how quickly they can handle common stains like tomato sauce or wine,” Ramanathan said.

“There’s more work to do to before we can start throwing out our washing machines, but this advance lays a strong foundation for the future development of fully self-cleaning textiles.”


Abstract of Robust Nanostructured Silver and Copper Fabrics with Localized Surface Plasmon Resonance Property for Effective Visible Light Induced Reductive Catalysis

Inspired by high porosity, absorbency, wettability, and hierarchical ordering on the micrometer and nanometer scale of cotton fabrics, a facile strategy is developed to coat visible light active metal nanostructures of copper and silver on cotton fabric substrates. The fabrication of nanostructured Ag and Cu onto interwoven threads of a cotton fabric by electroless deposition creates metal nanostructures that show a localized surface plasmon resonance (LSPR) effect. The micro/nanoscale hierarchical ordering of the cotton fabrics allows access to catalytically active sites to participate in heterogeneous catalysis with high efficiency. The ability of metals to absorb visible light through LSPR further enhances the catalytic reaction rates under photoexcitation conditions. Understanding the modes of electron transfer during visible light illumination in Ag@Cotton and Cu@Cotton through electrochemical measurements provides mechanistic evidence on the influence of light in promoting electron transfer during heterogeneous catalysis for the first time. The outcomes presented in this work will be helpful in designing new multifunctional fabrics with the ability to absorb visible light and thereby enhance light-activated catalytic processes.

 

New type of molecular tag makes MRI 10,000 times more sensitive

Could detect biochemical processes in opaque tissue without requiring PET radiation or CT x-rays
http://www.kurzweilai.net/new-type-of-molecular-tag-makes-mri-10000-times-more-sensitive

Duke scientists have discovered a new class of inexpensive, long-lived molecular tags that enhance MRI signals by 10,000 times. To activate the tags, the researchers mix them with a newly developed catalyst (center) and a special form of hydrogen (gray), converting them into long-lived magnetic resonance “lightbulbs” that might be used to track disease metabolism in real time. (credit: Thomas Theis, Duke University)

Duke University researchers have discovered a new form of MRI that’s 10,000 times more sensitive and could record actual biochemical reactions, such as those involved in cancer and heart disease, and in real time.

Let’s review how MRI (magnetic resonance imaging) works: MRI takes advantage of a property called spin, which makes the nuclei in hydrogen atoms act like tiny magnets. By generating a strong magnetic field (such as 3 Tesla) and a series of radio-frequency waves, MRI induces these hydrogen magnets in atoms to broadcast their locations. Since most of the hydrogen atoms in the body are bound up in water, the technique is used in clinical settings to create detailed images of soft tissues like organs (such as the brain), blood vessels, and tumors inside the body.


MRI’s ability to track chemical transformations in the body has been limited by the low sensitivity of the technique. That makes it impossible to detect small numbers of molecules (without using unattainably more massive magnetic fields).

So to take MRI a giant step further in sensitivity, the Duke researchers created a new class of molecular “tags” that can track disease metabolism in real time, and can last for more than an hour, using a technique called hyperpolarization.* These tags are biocompatible and inexpensive to produce, allowing for using existing MRI machines.

“This represents a completely new class of molecules that doesn’t look anything at all like what people thought could be made into MRI tags,” said Warren S. Warren, James B. Duke Professor and Chair of Physics at Duke, and senior author on the study. “We envision it could provide a whole new way to use MRI to learn about the biochemistry of disease.”

Sensitive tissue detection without radiation

The new molecular tags open up a new world for medicine and research by making it possible to detect what’s happening in optically opaque tissue instead of requiring expensive positron emission tomography (PET), which uses a radioactive tracer chemical to look at organs in the body and only works for (typically) about 20 minutes, or CT x-rays, according to the researchers.

This research was reported in the March 25 issue of Science Advances. It was supported by the National Science Foundation, the National Institutes of Health, the Department of Defense Congressionally Directed Medical Research Programs Breast Cancer grant, the Pratt School of Engineering Research Innovation Seed Fund, the Burroughs Wellcome Fellowship, and the Donors of the American Chemical Society Petroleum Research Fund.

* For the past decade, researchers have been developing methods to “hyperpolarize” biologically important molecules. “Hyperpolarization gives them 10,000 times more signal than they would normally have if they had just been magnetized in an ordinary magnetic field,” Warren said. But while promising, Warren says these hyperpolarization techniques face two fundamental problems: incredibly expensive equipment — around 3 million dollars for one machine — and most of these molecular “lightbulbs” burn out in a matter of seconds.

“It’s hard to take an image with an agent that is only visible for seconds, and there are a lot of biological processes you could never hope to see,” said Warren. “We wanted to try to figure out what molecules could give extremely long-lived signals so that you could look at slower processes.”

So the researchers synthesized a series of molecules containing diazarines — a chemical structure composed of two nitrogen atoms bound together in a ring. Diazirines were a promising target for screening because their geometry traps hyperpolarization in a “hidden state” where it cannot relax quickly. Using a simple and inexpensive approach to hyperpolarization called SABRE-SHEATH, in which the molecular tags are mixed with a spin-polarized form of hydrogen and a catalyst, the researchers were able to rapidly hyperpolarize one of the diazirine-containing molecules, greatly enhancing its magnetic resonance signals for over an hour.

The scientists believe their SABRE-SHEATH catalyst could be used to hyperpolarize a wide variety of chemical structures at a fraction of the cost of other methods.


Abstract of Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal 15N2-diazirine molecular tags

Abstract of Direct and cost-efficient hyperpolarization of long-lived nuclear spin states on universal 15N2-diazirine molecular tags

Conventional magnetic resonance (MR) faces serious sensitivity limitations, which can be overcome by hyperpolarization methods, but the most common method (dynamic nuclear polarization) is complex and expensive, and applications are limited by short spin lifetimes (typically seconds) of biologically relevant molecules. We use a recently developed method, SABRE-SHEATH, to directly hyperpolarize 15N2 magnetization and long-lived 15N2singlet spin order, with signal decay time constants of 5.8 and 23 min, respectively. We find >10,000-fold enhancements generating detectable nuclear MR signals that last for more than an hour. 15N2-diazirines represent a class of particularly promising and versatile molecular tags, and can be incorporated into a wide range of biomolecules without significantly altering molecular function.

references:

[Seems like they have a great idea, now all they need to do is confirm very specific uses or types of cancers/diseases or other processes they can track or target. Will be interesting to see if they can do more than just see things, maybe they can use this to target and destroy bad things in the body also. Keep up the good work….. this sounds like a game changer.]

 

Scientists time-reverse developed stem cells to make them ‘embryonic’ again

May help avoid ethically controversial use of human embryos for research and support other research goals
http://www.kurzweilai.net/scientists-time-reverse-developed-stem-cells-to-make-them-embryonic-again
Researchers have reversed “primed” (developed) “epiblast” stem cells (top) from early mouse embryos using the drug MM-401, causing the treated cells (bottom) to revert to the original form of the stem cells. (credit: University of Michigan)

University of Michigan Medical School researchers have discovered a way to convert mouse stem cells (taken from an embryo) that have  become “primed” (reached the stage where they can  differentiate, or develop into every specialized cell in the body) to a “naïve” (unspecialized) state by simply adding a drug.

This breakthrough has the potential to one day allow researchers to avoid the ethically controversial use of human embryos left over from infertility treatments. To achieve this breakthrough, the researchers treated the primedembryonic stem cells (“EpiSC”) with a drug called MM-401* (a leukemia drug) for a short period of time.

Embryonic stem cells are able to develop into any type of cell, except those of the placenta (credit: Mike Jones/CC)

…..

* The drug, MM-401, specifically targets epigenetic chemical markers on histones, the protein “spools” that DNA coils around to create structures called chromatin. These epigenetic changes signal the cell’s DNA-reading machinery and tell it where to start uncoiling the chromatin in order to read it.

A gene called Mll1 is responsible for the addition of these epigenetic changes, which are like small chemical tags called methyl groups. Mll1 plays a key role in the uncontrolled explosion of white blood cells in leukemia, which is why researchers developed the drug MM-401 to interfere with this process. But Mll1 also plays a role in cell development and the formation of blood cells and other cells in later-stage embryos.

Stem cells do not turn on the Mll1 gene until they are more developed. The MM-401 drug blocks Mll1’s normal activity in developing cells so the epigenetic chemical markers are missing. These cells are then unable to continue to develop into different types of specialized cells but are still able to revert to healthy naive pluripotent stem cells.


Abstract of MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency

The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.


Abstract of Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

 

 

How to kill bacteria in seconds using gold nanoparticles and light

March 24, 2016

 

zapping bacteria ft Could treat bacterial infections without using antibiotics, which could help reduce the risk of spreading antibiotics resistance

Researchers at the University of Houston have developed a new technique for killing bacteria in 5 to 25 seconds using highly porous gold nanodisks and light, according to a study published today in Optical Materials Express. The method could one day help hospitals treat some common infections without using antibiotics

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Understanding the Stem Cell Niche: A Webinar by The Scientist

Reporter: Stephen J. Williams, Ph.D.

 

The Scientist

nature stem cell

Schematic diagram showing some of the factors implicated in each process. Haematopoietic stem cells (HSCs) bound to the bone-marrow niche are mobilized in response to granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide, or after peripheral myeloablation following treatment with 5-fluorouracil (5-FU). After extravasation from the bone-marrow cords into the microvasculature, HSCs enter the circulation and are distributed to peripheral tissues such as the spleen or liver. HSCs locate close to endothelial cells in the splenic red pulp. They home to the bone-marrow cords through the circulation, a process that is controlled by a number of adhesion molecules such as very late antigen 4 (VLA4), VLA5, lymphocyte function-associated antigen 1 (LFA1) or selectins. After entering the bone marrow, HSCs specifically lodge in the niche, a process requiring membrane-bound stem-cell factor (SCF), CXC-chemokine ligand 12 (CXCL12), osteopontin (OPN), hyaluronic acid, and their corresponding receptors. CXCR4, CXC-chemokine receptor 4; E-selectin, endothelial-cell selectin; P-selectin, platelet selectin; PSGL1, P-selectin glycoprotein ligand 1.

 

Understanding the Stem Cell Niche

  This presentation will begin on Tuesday, December 01, 2015 at 02:30 PM Eastern Standard Time.
   

Free Webinar
Tuesday December 1, 2015
2:30 – 4:00 PM EST

Stem cells provide an attractive model to study human physiology and disease. However, technical challenges persist in the biological characterization and manipulation of stem cells in their native microenvironment. The Scientist brings together a panel of experts to discuss interactions between stem cells and external cues, and the role of the stem cell niche in development and disease. Topics to be covered include the molecular mechanisms of hematopoietic stem cell niche interactions and techniques for engineering 3-D stem-cell microenvironments. Following the presentations, attendees will have an opportunity to ask questions concerning their specific applications and receive answers in real-time.

Speakers:

Dr. Jon Hoggatt, Assistant Professor of Medicine, Cancer Center and Center for Transplantation Sciences, Harvard Medical School/Massachusetts General Hospital.

Dr. Todd McDevitt, Senior Investigator, Gladstone Institute of Cardiovascular Disease, Professor, Department of Bioengineering & Therapeutic Sciences, UCSF.

 

Understanding the Stem Cell Niche
Click Here To Watch The Video

To find out about our upcoming events follow us on Twitter @LabMgrEvents

 

Notes from Webinar:

Hematopoetic stem cells good model since now we have liquid biopsies (as a result field has skyrocketed).

Two processes involved with stem cells finding their niche

  1. Homing; CXCR4-SDK1 dependent process into the bone marrow.
  2. Mobilization: stem cells moving from bone into blood (found that GMCSF main factor responsible for this process)

Dr. Raymond Schofield was one of the first to propose the existence of this stem cell niche (each progenitor will produce a unique factor {possibly a therapeutic target} for example leptin+ receptor target perivascular cells so one target is good for only a small subset of stem cells)

Therefore it may be possible or advantageous to target the whole stem cell milieu. One such possible target they are investigating is CD26 (dipeptyl peptidase). The diabetes drug Januvia is an inhibitor of CD26.

It was also noticed if inhibit the GMCSF receptor complex can inhibit the whole stem cell niche.

Prostoglandins and stem cell niche

  • Indomethacin blocks the mobilization step
  • Prostaglandin E increases homing
  • GMCSF and malaxocam (COX2 inhibitor) flattens osteoblast cells and may be a mechanism how inhibition of prostaglandin synthesis blocks mobilization
  • Found that the PGE4 receptor is ultimately responsible for the NSAID effect

The niche after G-CSF

Dr. Hoggat found that macrophages are supplying the factors that support the niche. He will be presenting the findings at 2015 Hematology conference. (See information about his conference presentation here).

From the 57th Annual American Society of Hematology Meeting (2015) please see Dr. Hoggat’s moderated section Hematopoiesis and Stem Cells: Microenvironment, Cell Adhesion and Stromal Stem Cells: Hematopoietic Stem Cell Niche

 

Relevant articles from Dr. Hoggat

Anti-CD47 Therapy Is More Than a Dinner Bell October 19, 2015

Dr. Hoggatt looks at the therapeutic effects of blocking CD47 aside from alerting macrophages to devour tumor cells.

Hematopoietic Stem Cells Should Hold Their Breath August 12, 2015

Dr. Hoggatt and Hannah Rasmussen discuss new approaches to the use of hematopoietic stem cells considering observer effects that emerge due to our experimental systems for HSCs.

Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation. Hoggatt J, Singh P, Sampath J, Pelus LM. Blood. 2009 May 28;113(22):5444-55. doi: 10.1182/blood-2009-01-201335. Epub 2009 Mar 26.

 

Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness. Hoggatt J, Mohammad KS, Singh P, Pelus LM. Blood. 2013 Oct 24;122(17):2997-3000. doi: 10.1182/blood-2013-07-515288. Epub 2013 Sep 18.

 

Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment. Speth JM, Hoggatt J, Singh P, Pelus LM. Blood. 2014 Jan 9;123(2):203-7. doi: 10.1182/blood-2013-07-516336. Epub 2013 Oct 28.

 

Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development from hematopoietic progenitor cells. Singh P, Hoggatt J, Hu P, Speth JM, Fukuda S, Breyer RM, Pelus LM. Blood. 2012 Feb 16;119(7):1671-82. doi: 10.1182/blood-2011-03-342428. Epub 2011 Nov 22.

 

Recovery from hematopoietic injury by modulating prostaglandin E(2) signaling post-irradiation. Hoggatt J, Singh P, Stilger KN, Plett PA, Sampson CH, Chua HL, Orschell CM, Pelus LM. Blood Cells Mol Dis. 2013 Mar;50(3):147-53. doi: 10.1016/j.bcmd.2012.11.006. Epub 2012 Nov 30.

 

Pulse exposure of haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment and recovery. Pelus LM, Hoggatt J, Singh P. Cell Prolif. 2011 Apr;44 Suppl 1:22-9. doi: 10.1111/j.1365-2184.2010.00726.x.

 

Pleiotropic effects of prostaglandin E2 in hematopoiesis; prostaglandin E2 and other eicosanoids regulate hematopoietic stem and progenitor cell function. Pelus LM, Hoggatt J. Prostaglandins Other Lipid Mediat. 2011 Nov;96(1-4):3-9. doi: 10.1016/j.prostaglandins.2011.06.004. Epub 2011 Jun 21. Review.

 

Differential stem- and progenitor-cell trafficking by prostaglandin E2. Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. Nature. 2013 Mar 21;495(7441):365-9. doi: 10.1038/nature11929. Epub 2013 Mar 13.

 

Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking.Hoggatt J, Pelus LM. Leukemia. 2010 Dec;24(12):1993-2002. doi: 10.1038/leu.2010.216. Epub 2010 Sep 30. Review.

 

Hematopoietic stem cell mobilization with agents other than G-CSF. Hoggatt J, Pelus LM. Methods Mol Biol. 2012;904:49-67. doi: 10.1007/978-1-61779-943-3_4.

 

Mobilization of hematopoietic stem cells from the bone marrow niche to the blood compartment. Hoggatt J, Pelus LM. Stem Cell Res Ther. 2011 Mar 14;2(2):13. doi: 10.1186/scrt54. Review.

 

Engineering 3D Pluripotent Stem Cell Microenvironments by Todd McDevitt, Ph.D.

In recent years, it has finally been shown how to produce centrally derived (self assembling) organoids (microtissues).

 

How to specifically deliver specific morphogens in 3D organoids

 

  1. Microparticle (MP)-mediated delivery (can do in mouse and human): reduces the amount needed to be delivered

 

 

What are other effects of introduced MP in ES (embryonic stem cell) aggregates?

  1. a) physiocomechanical changes –mechanical effects of materials
  2. b) how changes in local presentation of factors affect bioavailbility and binding properties

 

 

 

 

 

 

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Mass-producing stem cells to satisfy the demands of regenerative medicine

Reported by: Irina Robu, PhD

Instead of culturing cell on round, flat Petri dishes, Steve Oh from A*STAR Bioprocessing Technology Institute he grew them in a tiny polystyrene beads known as microcarriers floating in a nutritional brew. The standard Petri dish fits fewer than 100,000 cells, a minuscule amount when stacked against the 2 billion muscle cells  that make up the heart or 100 billion red blood cells needed to fill a bag of blood. 

The average Petri dish fits fewer than 100,000 cells, a miniscule amount when stacked against the 2 billion muscle cells that make up the heart or the 100 billion red blood cells needed to fill a bag of blood. The approach Reuveny suggested potentially could produce cells in much vaster numbers to make them more practical for therapy.
 
Dr. Oh first tried the approach on human embryonic stem cells, because they have the potential to mature into any type of cell in the body and struggled to develop a coating that would make the stem cells stick to the microcarriers and formulate the right mixture of nutrients for cell to grow. After a year, one line of human embryonic stem cells survived past the 20 week mark of stability and found out that these cells were two to four times times more densely packed than grown in petri dishes.

However after six years of refining the processes, they were able to achieve three times higher cell densities than petri dishes approach by modifying the feeding strategy.  Their success started with cardiomyocytes wich are known as the fastest cell type to differentiate. The researchers developed a strategy to grow pure batches of cariomyocytes without adding growth factors but instead use small molecules to first inhibit and then activate a key cell differentiation pathway known as Wnt signaling. Then they apply the small molecule approach to grow and differentiate cardiomyocytes from embryonic stem cells directly on microcarriers. And according to Dr. Oh their method had beat the Petri dish methods on purity, yield, cost of cells and simplicity of process.

The main  goal of the research is to grow enough cells inexpensively in order to patch up one square-centimeter of damaged heart muscle following a heart attack.

Source

http://phys.org/news/2015-06-mass-producing-stem-cells-demands-regenerative.html

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Stem Cell Therapy for Coronary Artery Disease (CAD)

Author and Curator: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

There is great interest and future promise for stem cell therapy in ischemic heart disease.  This is another report for the active work in cardiology with stem cell therapy by MA Gaballa and associates at University of Arizona.

Stem Cell Therapy for Coronary Heart Disease

Julia N. E. Sunkomat and Mohamed A. Gaballa

The University ofArizona Sarver Heart Center, Section of Cardiology, Tucson, Ar
Cardiovascular Drug Reviews 2003: 21(4): 327–342

Keywords: Angiogenesis — Cardiac therapy — Coronary heart disease — Heart failure — Myoblasts — Myocardial ischemia — Myocardial regenera­tion — Stem cells

ABSTRACT

Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that

stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be ad­dressed. To name a few:

  1.  Which patient population should be considered for stem cell therapy?
  2.  What type of stem cell should be used?
  3.  What is the best route for cell de­livery?
  4.  What is the optimum number of cells that should be used to achieve functional effects?
  5.  Is stem cell therapy safer and more effective than conventional therapies?

The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of

  1. ischemia,
  2. species of donors and recipients,
  3. techniques of cell delivery,
  4. cell types,
  5. cell numbers and
  6. timing of the experiments

have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease.

  • Non-ischemic cardiomyopathy,
  • peripheral vascular disease, and
  • aging may be treated by stem cells.

Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with phar­macological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.

INTRODUCTION

Background

Congestive heart failure (CHF) is the leading discharge diagnosis in patients over the age of 65 with estimates of $24 billion spent on health care in the US (1,11). The number one cause of CHF is coronary artery diseases (CAD). Coronary care units, reperfusion therapy (lytic and percutaneous coronary intervention) and medical therapy with anti-pla­telet agents, statins, ACE-inhibitors and â-adrenoceptor antagonists all significantly reduce morbidity and mortality of CAD and CHF (9), but it is very difficult to regenerate new viable myocardium and new blood vessels.

Identification of circulating endothelial progenitor cells in peripheral blood that incor­porated into foci of neovascularization in hindlimb ischemia (4) and the successful engraftment of embryonic stem cells into myocardium of adult dystrophic mice (31) intro­duced a new therapeutic strategy to the field of cardiovascular diseases: tissue regeneration. This approach is supported by the discovery of primitive cells of extracardiac origin in cardiac tissues after sex-mismatched transplants suggesting that an endogenous repair mechanism may exist in the heart (35,45,54). The number of recruited cells varied significantly from 0 (19) to 18% (54), but the natural course of ischemic cardiomyopathy implies that cell recruitment for tissue repair in most cases is insufficient to prevent heart failure. Therefore, investigational efforts are geared towards

  • augmenting the number of multipotent stem cells and endothelial and myocardial progenitor cells at the site of ischemia to induce clinically significant angiogenesis and potentially myogenesis.

Stem and Progenitor Cells

Stem cells are defined by their ability to give rise to identical stem cells and progenitor cells that continue to differentiate into a specific tissue cell phenotype (23,33). The po­tential of mammalian stem cells varies with stage of development and age (Table 1).

In mammals, the fertilized oocyte and blastomere cells of embryos of the two to eight cell stage can generate a complete organism when implanted into the uterus; they are called totipotent stem cells. After the blastocyst stage, embryonic stem cells retain the ability to differentiate into all cell types, but

  • cannot generate a complete organism and thus are denoted pluripotent stem cells.

Other examples of pluripotent stem cells are embryo­nic germ cells that are derived from the gonadal ridge of aborted embryos and embryonic carcinoma cells that are found in gonadal tumors (teratocarcinomas) (23,33). Both these cell types can also differentiate into cells of all three germ layers, but are not as well inves­tigated as embryonic stem cells.

It is well established that embryonic stem cells can differentiate into cardiomyocytes (7,10,13,14,31,37,76), endothelial cells (55), and smooth muscle cells (5,22,78) in vitro, but it is unclear whether

  • pure populations of embryonic stem cell-derived cardiomyocytes can integrate and function appropriately in the heart after transplantation.
  • one study reported arrhythmogenic potential of embryonic stem cell-derived cardiomyocytes in vitro (80).

Adult somatic stem cells are cells that have already committed to one of the three germ layers: endoderm, ectoderm, or mesoderm (76). While embryonic stem cells are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is still unknown. The primary role of adult stem cells in a living organism is thought to be maintaining and repairing the tissue in which they reside. They are the source of more identical stem cells and cells with a progressively more distinct phenotype of specialized tissue cells (progenitor and precursor cells) (Fig. 1). Until recently adult stem cells were thought to be lineage-specific, meaning that they can only differentiate into the cell-type of their original tissue. This concept has now been challenged with the discovery of multipotent stem and progenitor cells (26, 50, 51).

The presence of multipotent stem and progenitor cells in adult mammals has vast im­plications on the availability of stem cells to research and clinical medicine. Recent publi­cations, however, have questioned whether the adaptation of a phenotype in those dogma-challenging studies is really a result of trans-differentiation or rather a result of cell and nuclear fusion (60,68,75,79). Spontaneous fusion between mammalian cells was first re­ported in 1961 (8), but how frequently fusion occurs and whether it occurs in vivo is not clear.

The bone marrow is a known source of stem cells. Hematopoietic stem cells are fre­quently used in the field of hematology. Surface receptors are used to differentiate hematopoietic stem and progenitor cells from mature cells. For example, virtually all

  • hematopoietic stem and progenitor cells express the CD34+ glycoprotein antigen on their cell membrane (73),

though a small proportion of primitive cells have been shown to be CD34 negative (58).

The function of the CD34+ receptor is not yet fully understood. It has been suggested that it may act as a regulator of hematopoietic cell adhesion in the bone marrow microenvironment. It also appears to be involved in the maintenance of the hematopoietic stem/progenitor cell phenotype and function (16,21). The frequency of immature CD34+ cells in peripheral circulation diminishes with age.

  • It is the highest (up to 11%) in utero (69) and decreases to 1% of nucleated cells in term cord blood (63).
  • This equals the per­centage of CD34+ cells in adult bone marrow.
  • The number of circulating stem cells in adult peripheral blood is even lower at 0.1% of nucleated cells.

Since hematopoietic stem cells have been identified as endothelial progenitor cells (29,30,32) their low density in adult bone marrow and blood could explain the inadequacy of endogenous recruitment of cells to injured organs such as an ischemic heart. The bone marrow is also home to another stem cell population the so-called mesenchymal stem cells. These may constitute a subset of the bone marrow stromal cells (2,43). Bone marrow stromal cells are a mixed cell popu­lation that generates

  1. bone,
  2. cartilage,
  3. fat,
  4. connective tissue, and
  5. reticular network that sup­ports cell formation (23).

Mesenchymal stem cells have been described as multipotent (51,52) and as a source of myocardial progenitor cells (41,59). They are, however, much less defined than the hematopoietic stem cells and a characteristic antigen constellation has not yet been identified (44).

Another example of an adult tissue containing stem cells is the skeletal muscle. The cells responsible for renewal and growth of the skeletal muscle are called satellite cells or myoblasts and are located between the sarcolemma and the basal lamina of the muscle fiber (5). Since skeletal muscle and cardiac muscle share similar characteristics such as they both are striated muscle cells, satellite cells are considered good candidates for the repair of damaged myocardium and have been extensively studied (20,25,38–40,48,56, 64–67). Myoblasts are particularly attractive, because they can be autotransplanted, so that issues of donor availability, ethics, tumorigenesis and immunological compatibility can be avoided. They also have been shown to have a high growth potential in vitro and a strong resistance to ischemia in vivo (20). On the down side

  • they may have more arrhythmogenic potential when transplanted into myocardium than bone marrow or peripheral blood de­rived stem cells and progenitor cells (40).

Isolation of Cells Prior to Transplantation

Hematopoietic stem and progenitor cells are commonly identified by the expression of a profile of surface receptors (cell antigens). For example, human hematopoietic stem cells are defined as CD34+/CD59+/Thy-1+/CD38low//c-kit/low/lin, while mouse hema-topoietic stem cells are defined as CD34low//Sca-1+/Thy-1+/low/CD38+/c-kit+/lin (23). Additional cell surface receptors have been identified as markers for subgroups of hema-topoietic stem cells with the ability to differentiate into non-hematopoetic tissues, such as endothelial cells (57,78). These can be specifically targeted by isolation methods that use the receptors for cell selection (positive selection with antibody coated magnetic beads or fluorescence-activated cell sorting, FACS). Other stem cell populations are identified by their behavior in cell culture (mesenchymal stem cells) or dye exclusion (SP cells). Finally, embryonic stem cells are isolated from the inner cell mass of the blastocyst and skeletal myoblasts are mechanically and enzymatically dissociated from an easily acces­sible skeletal muscle and expanded in cell culture.

FIG. 1. Maturation process of adult stem cells: with acquisition of a certain phenotype the cell gradually loses its self-renewal capability.  (unable to transfer)

METHODICAL APPROACHES 

j.1527-3466.2003.tb00125.x  fig stem cell

FIG. 2. Intramyocardial injection:

the cells are injected directly into the myocardium through the epicardium. Usually a thoracotomy or sternotomy is required. Transendocardial injection: access can be gained from the ar­terial vasculature. Cells are injected through the endocardium into the myocardium, ideally after identifying the ischemic myocardium by perfusion studies and/or electromechanical mapping. Intracoronary injection: the coronary artery is accessed from the arterial vasculature. Stem cells are injected into the lumen of the coronary artery. Proximal washout is prevented by inflation of a balloon. Cells are then distributed through the capillary system. They eventually cross the endothelium and migrate towards ischemic areas.

The intracoronary delivery of stem cells (Fig. 2) and distribution through the coronary system has also been explored (6,62,74). This approach was pioneered by Robinson et al. (56), who demonstrated successful engraftment within the coronary distribution after intracoronary delivery of genetically labeled skeletal myoblasts. The risk of intracoronary injection is comparable to that of a coronary angiogram and percutaneous transluminal coronary angioplasty (PTCA) (62), which are safe and clinically well established.

RESULTS IN ANIMAL STUDIES AND HUMAN TRIALS

Dif­ferentiation into cardiomyocytes was observed after transplantation of embryonic stem cells, mesenchymal stem cells, lin/c-kit+ and SP cells. The induction of angiogenesis was observed after transplantation of embryonic stem cells, mesenchymal stem cells, bone marrow-derived mononuclear cells, circulating endothelial progenitor cells, SP cells and lin/c-kit+ cells.

The use of embryonic stem cells in ischemia was examined in two studies (42,43). These studies demonstrated that mice embryonic stem cells transplanted into rat myo­cardium exhibited cardiomyocyte phenotype at 6 weeks after transplantation. In addition, generation of myocardium and angiogenesis were observed at 32 weeks after allogenic transplantation in rats. In these two studies no arrhythmias or cardiac tumors were reported.

Several studies have shown retardation of LV remodeling and improvement of cardiac function after administration of bone marrow-derived mononuclear cells. For example, decreases in infarct size, and increase in ejection fraction (EF), and left ventricular (LV) time rate change of pressure (dP/dtmax) were observed after direct injection of bone marrow-derived mononuclear cells 60 min after ischemia in swine (28). In humans, intra-coronary delivery and transendocardial injection of mononuclear cells leads to a decrease in LV dimensions and improvement of cardiac function and perfusion (49,62). A decrease in end systolic volume (ESV) and an increase in EF as well as regional wall motion were observed following intracoronary administration of CD34+/CD45+ human circulating en­dothelial cells (6). Injection of circulating human CD34+/CD117+ cells into infarcted rat myocardium induced neoangiogenesis and improved cardiac function (32). This study suggests that the improvement in LV remodeling after infarction appears to be in part me­diated by a decrease in apoptosis within the noninfarcted myocardium. Two other studies reported increased fractional shortening, improved regional wall motion and decreased left ventricular dimensions after transplantation of human CD34+ cells (29,30). Improved global left ventricular function and infarct perfusion was demonstrated after intramyo-cardial injection of autologous endothelial progenitor cells in humans (61).

DISCUSSION AND OUTLOOK

The idea of replacing damaged myocardium by healthy cardiac tissue is exciting and has received much attention in the medical field and the media. Therefore, it is important for the scientist to know what is established and what is based on premature conclusions. Currently, there are data from animal studies and human trials (Table 2). However, some of these data are not very concrete. For example,

  • many animal studies do not report the level of achieved neoangiogenesis and/or regeneration of myocardium.
  • In studies where the numbers of neovessels and new cardiomyocytes are specified, these numbers are often very low.

While these experiments confirm the concept that bone marrow and peripheral blood-derived stem and progenitor cells can differentiate into cardiomyocytes and endo­thelial cells when transplanted into ischemic myocardium, they also raise the question how effective this treatment is.

The results of the clinical trials that have been conducted are encouraging, but they need to be interpreted with caution. The common endpoints of these studies include left ventricular dimensions, perfusion, wall motion and hemodynamic function. While all studies report improvement after mononuclear cell, myoblast or endothelial progenitor cell transplantation, it is difficult to separate the effects of stem cell transplantation from the effects of the state-of-the art medical care that the patients typically received.

CONCLUSION

While the majority of studies demonstrate neoangiogenesis and some studies also show regeneration of myocardium after stem/progenitor cell transplantation, it remains unclear whether the currently achieved level of tissue regeneration is sufficient to affect clinical outcome. Long-term follow-up of patients that received stem/progenitor cells in clinical trials will provide important information on the potential risks of neoplasm and arrhythmias and, therefore, safety of this treatment. Ultimately, postmortem histological confirmation of scar tissue repair by transplanted cells and randomized placebo control trials with long-term follow-up are required to prove efficacy of this treatment.

REFERENCES (10)

1. American Heart Association Disease and Stroke Statistics-2003 Update, Dallas TX, American Heart Associ­ation; 2002 http://http://www.americanheart.org/downloadable/heart/10461207852142003HDSStatsBook.pdf

2. Arai A, Sheikh F, Agyeman K, et al. Lack of benefit from cytokine mobilized stem cell therapy for acute myocardial infarction in nonhuman primates. J Am Coll Cardiol 2003;41(Suppl 6A):371.

3. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.

4. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.

5. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki M. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159(1):123–134.

6. Assmus B, Schaechinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration en­hancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:r53–r61.

7. Bader A, Al-Dubai H, Weitzer G. Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions. Circ Res 2000;86(7):787–794.

8. Barski G, Sorieul S, Cornefert F. “Hybrid” type cells in combined cultures of two different mammalian cell strains. J Natl Cancer Inst 1961;26:1269–1291.

9. Boersma E, Mercado N, Poldermans D, Gardien M, Vos J, Simoons M. Acute myocardial infarction. Lancet 2003;361:847–58.

  1. 10.          Boheler K, Czyz J, Tweedie D, Yang H, Anisimov S, Wobus A. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002;91:189–201.

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Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This article is a presentation of recently published work on the basis for control of mesodermal and cardiovascular differentiation of embryonic stem cells, which has taken on increasing importance in the treatment of cardiovascular disease, with particular application to heart failure due to any cause, but with particular relevance to significant loss of myocardium, as may occur with acute myocardial infarction with more than 60% occlusion of the left anterior descending artery, near the osteum, with or without adjacent artery involvement, resulting in major loss of cardiac contractile force and insufficient ejection fraction. The article is of high interest and makes the following points:

  1. Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal
  2. Ablation of Jmjd3 in mouse embryonic stem cells compromised mesoderm and subsequent endothelial and cardiac differentiation 
  3. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin
  4. β-catenin s critical for Wnt signal–induced mesoderm differentiation. 

Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

K Ohtani, C Zhao, G Dobreva, Y Manavski, B Kluge, T Braun, MA Rieger, AM Zeiher and S Dimmeler

I The Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany (K.O., C.Z., Y.M., B.K., S.D.); Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany (G.D., T.B.); Department of Hematology/Oncology, Internal Medicine
II LOEWE Center for Cell and Gene Therapy, University of Frankfurt, Frankfurt, Germany (M.A.R.); and Department of Cardiology, Internal Medicine
III University of Frankfurt, Frankfurt, Germany (A.M.Z.).
This manuscript was sent to Benoit Bruneau, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Stefanie Dimmeler,  Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, University of Frankfurt, Frankfurt, Germany. E-mail dimmeler@em.uni-frankfurt.de
 Circ Res. 2013;113:856-862;  http://dx.doi.org/10.1161/CIRCRESAHA.113.302035   http://circres.ahajournals.org/content/113/7/856  

Abstract

Rationale: The developmental role of the H3K27 demethylases Jmjd3, especially its epigenetic regulation at target genes in response to upstream developmental signaling, is unclear.

Objective: To determine the role of Jmjd3 during mesoderm and cardiovascular lineage commitment.

Methods and Results: Ablation of Jmjd3 in mouse embryonic stem cells does not affect the maintenance of pluripotency and self-renewal but compromised mesoderm and subsequent endothelial and cardiac differentiation. Jmjd3 reduces H3K27me3 marks at the Brachyury promoter and facilitates the recruitment of β-catenin, which is critical for Wnt signal–induced mesoderm differentiation.

Conclusions: These data demonstrate that Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment. (Circ Res. 2013;113:856-862.)

  • Key Words: Brachyury protein ■ embryonic stem cells ■ epigenomics ■ Jmjd3 protein, mouse ■ mesodermn  –  Wnt signaling pathway

Introduction

Post-translational modifications of histone proteins represent essential epigenetic control mechanisms that can either allow or repress gene expression.1 Trimethylation of H3K27 is mediated by Polycomb group proteins and represses gene expression.2 The JmjC domain–containing proteins, UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) and Jmjd3 (jumonji domain–containing protein 3, Kdm6b), not only act as demethylases to remove the repressive H3K27me3 marks, but also exhibit additional demethylase-independent functions.3–6 Jmjd3 is induced and participates in Hox gene expression during development,7 neuronal differentiation,8,9 and inflammation,5,10–12 and recent data suggest that Jmjd3 inhibits reprogramming by inducing cellular senescence.13 Because previous studies suggest that H3K27me3 regulates endothelial gene expression in adult proangiogenic cells,14 we addressed the function of Jmjd3 in cardiovascular lineage differentiation of embryonic stem cells (ESCs).

Methods

A detailed description of the experimental procedure is provided in the Online Data Supplement.  The online-only Data Supplement is available with this article at   http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA. 113.302035/-/DC1.

Cell Culture

Plasmid Construction and Stable Transfection

The full-length Jmjd3, the mutants, and Brachyury were cloned into pEF1 vector (Invitrogen). The linearized plasmids were transfected in Jmjd3−/− ESCs using the Amaxa nucleofection system (Lonza).

Chromatin Immunoprecipitation

Nonstandard Abbreviations and Acronyms

EB                       embryoid body

ESC                    embryonic stem cell

WI                        wild type

Results

Jmjd3 knockout ESCs were generated by 2 rounds of gene targeting (Online Figure IA and IB). We obtained 7 Jmjd3−/− ESC clones, which lacked Jmjd3 mRNA and protein expression. All of the clones showed slightly increased global H3K27me3, but the expression of pluripotency genes, the morphology, the growth kinetic, and survival was indistinguishable from wild-type (WT) ESCs (Figure 1A–1C; Online Figure IC–IF). No significant changes of repressive H3K27me3 marks at the promoters of pluripotency genes were detected in Jmjd3−/− compared with WT ESCs (Online Figure IH). When

  • spontaneous differentiation was induced by leukemia inhibitory factor withdrawal,
  • Jmjd3 expression increased in WT ESCs (Figure 1D).
  • EBs derived from Jmjd3−/− ESCs were slightly smaller in size compared with WT EBs (Figure 1E).

mRNA expression profiling of Jmjd3−/− and WT ESC clones at day 4 after induction of differentiation showed

  • a distinct expression pattern of lineage-specific genes (Online Figure IIA).

Gene ontology functional analyses revealed a significant repression of genes that are involved in mesoderm development (Figure 1F; Online Figure IIB). Moreover,

  • repressed gene sets in Jmjd3−/− EBs were shown to be related to cardiac and vascular development,
  • consistent with impaired mesoderm differentiation (Figure 1F; Online Figure IIB).

Validation of the microarray results showed a similar reduction of pluripotency gene expression after leukemia inhibitory factor withdrawal in Jmjd3−/− compared with WT ESCs (Figure 1G). However, depletion of Jmjd3 substantially compromised the induction of mesodermal genes (Figure 1G). Especially, the pan-mesoderm marker, Brachyury, and the early mesoendoderm marker, Mixl1, were profoundly increased at day 4 of differentiation in WT ESCs, but not in Jmjd3−/− ESCs (Figure 1G). Moreover, the mesoendodermal marker, Eomes, and endodermal markers, such as Sox17 and FoxA2, were significantly suppressed, which is consistent with a very recent study showing that Jmjd3 is required for endoderm differentiation.19 Ectodermal markers were not significantly changed in Jmjd3−/− ESCs when using the spontaneous differentiation protocol (Figure 1G).

Because Jmjd3−/− ESCs showed a prominent inhibition of mesodermal markers after leukemia inhibitory factor withdrawal, we next questioned whether this phenotype can also be observed when directing differentiation of mesoderm using 2 different protocols. Consistent with our findings,

  • Jmjd3−/− ESCs showed a reduced expression of mesodermal marker genes when using the protocol for mesoderm differentiation described by Gadue et al20 (data not shown). Moreover,
  • mesoderm differentiation was significantly suppressed when Jmjd3−/− ESCs were cultured on OP9 stromal cells, which support mesodermal differentiation21 (Figure 2A).

Whereas WT ESCs showed the typical time-dependent increase in Brachyury+ cells, Jmjd3−/− ESCs generated significantly less Brachyury+ mesodermal cells (Figure 2B). Moreover, fluorescence activated cell sorting analysis revealed that fetal liver kinase (Flk)1+ vascular endothelial-cadherin−mesodermal cells were generated in WT ESCs but were reduced when Jmjd3−/− ESCs were used (Figure 2C). Interestingly, the formation of vascular endothelial-cadherin+ Flk+ cells was also significantly reduced by 96±1% and 88±3% in the 2 Jmjd3−/− ESC clones compared with WT ESCs (P<0.01), prompting us to explore the role of Jmjd3 in vascular differentiation further.
Endothelial differentiation was induced by a cytokine cocktail18 and was associated with a significant upregulation of Jmjd3 expression (Online Figure IIIA). Jmjd3−/− ESCs showed a marked reduction of endothelial differentiation as evidenced by

  • significantly reduced mRNA levels of the endothelial marker vascular endothelial-cadherin and endothelial-specific receptor tyrosine kinase Tie2 (Figure 3A).
  • The formation of endothelial marker expressing vascular structures after induction of endothelial differentiation was abolished in Jmjd3−/− ESCs (Figure 3B; Online Figure IIIB).
  • The impaired endothelial differentiation of Jmjd3−/− cells was partially rescued by the overexpression of Brachyury (Online Figure IIIC and IIID), suggesting that the inhibition of mesoderm formation, at least in part, contributes to the impaired endothelial commitment.

Because genes involved in heart development and morphogenesis were significantly downregulated in Jmjd3−/− ESCs on differentiation (Figure 1F; Online Figure II), we additionally determined the capacity of Jmjd3−/− ESCs to generate cardio-myocytes by inducing cardiac differentiation.17

  • Expression of cardiac progenitor cell markers, Mesp1 and Pdgfra, was inhibited in Jmjd3−/− ESCs compared with WT ESCs (Figure 3C).

Moreover, after plating on gelatin-coated dishes,

  • the Jmjd3−/− ESCs showed an impaired formation of EBs and
  • only 20% of EBs were contracting (Figure 3D).

Consistently, expression of the cardiac transcription factor Mef2c, the marker of working myocardium Nppa, and cardiac structural proteins TnT2 and α-myosin heavy chain were downregulated in Jmjd3−/− ESCs (Figure 3E and 3F; Online Figure IIIE).

  • Next, we addressed whether the impaired mesoderm differentiation observed in Jmjd3−/− ESCs might be mediated by an increase of repressive H3K27me3 marks at the promoters of developmental regulators. Of the various promoters studied, only Brachyury and Mixl1 showed a significant augmentation of H3K27me3 marks in Jmjd3−/− ESCs on differentiation (Figure 4A; Online Figure IVA). Consistently, the recruitment of RNA polymerase II to the transcription start sites of the promoters of Brachyury and Mixl1 was also significantly reduced (Online Figure IVC). In addition, Jmjd3 deficiency repressed polymerase II recruitment to the Flk1 and Mesp1 promoter but the inactivation of these promoters was not associated with changes in H3K27me3 marks (Figure IVA and IVC). These data were confirmed using protocols
  • that induce mesoderm differentiation by addition of Wnt3a (data not shown).20 Under these conditions,
  • Jmjd3−/− ESCs showed a 1.81±0.23-fold (P<0.05) enrichment of H3K27me3 marks at the Brachyury promoter compared with WT ESCs.

To determine whether the demethylase activity of Jmjd3 controls Brachyury expression by reducing repressive H3K27me3 marks during differentiation, we overexpressed full-length Jmjd3, the carboxyl-terminal part, including the JmjC-domain (cJmjd3: amino acids, 1141–1641), and a carboxyl-terminal mutant construct, which includes a point mutation (cJmjd3H1388A) to inactivate demethylase activity. Overexpression of full-length Jmjd3 and the carboxyl-terminal part of Jmjd3 in Jmjd3−/− ESCs partially rescued the expression of Brachyury on differentiation (Figure 4B and 4C). Howver, the inactive carboxyl-terminal part of Jmjd3 failed to rescue the impaired Brachyury expression in Jmjd3−/− ESCs (Figure 4C), suggesting that

  • the demethylase activity of Jmjd3 is required for the activation of the Brachyury promoter.

Because canonical Wnt signaling regulates the expression of Brachyury during development22,23 and Wnt/B-catenin–dependent genes were suppressed in Jmjd3−/− EBs compared with WT EBs (Online Figure V), we further explored whether Jmjd3 might interact with B-catenin signaling. Indeed,

  • B-catenin recruitment to the Brachyury promoter was significantly suppressed in Jmjd3−/− ESCs (Figure 4D) and
  • was rescued by Jmjd3 overexpression (Figure 4E).

Similar results were obtained when using the protocol for direct mesoderm differentiation described by Gadue et al20 (data not shown). To determine whether Jmjd3 might interact with B-catenin, we performed coimmunoprecipitation studies and showed that

  • Jmjd3 interacts with B-catenin in human embryonic kidney 293 cell and differentiated EBs (Figure 4F; Online Figure VI).

To assess a direct effect of Jmjd3 on B-catenin responsive promoter activity, we used a luciferase reporter assay. Coexpression of lymphoid enhancer binding factor 1 and the constitutive active form of B-catenin harboring a nuclear localization signal resulted in the activation of lymphoid enhancer binding factor 1 luciferase reporter activity in WT ESCs, but

  • this transcriptional activation was markedly impaired in Jmjd3−/− ESCs (Figure 4G).

Discussion

The data of the present study demonstrate that

  • deletion of Jmjd3 in ESCs does not affect self-renewal but
  • significantly impairs the formation of mesoderm on induction of differentiation.

The findings that Jmjd3 is not required for ESC maintenance are consistent with the dispensability of the Polycomb complex and the related demethylase UTX for self-renewal.1

  • The requirement of Jmjd3 for mesoderm differentiation was shown in spontaneous differentiation, as well as
  • when more specifically inducing mesoderm differentiation by the OP9 coculture system or under serum-free conditions followed by Wnt3a stimulation.
  • Jmjd3 deficiency profoundly suppressed the expression of Brachury, which is essential for mesoderm differentiation.

In the absence of Jmjd3,

  • repressive H3K27me3 marks at the Brachyury promoter are significantly increased, and
  • the recruitment of B-catenin, which is a prerequisite for Wnt-induced mesoderm differentiation, is impaired.
  • In addition, Jmjd3 is interacting with B-catenin and is contributing to B-catenin– dependent promoter activation.

This is consistent with the recent findings that cofactors can form a complex with B-catenin/ lymphoid enhancer binding factor 1

  • at Tcf/lymphoid enhancer binding factor 1 binding sites
  • at B-catenin–dependent promoters and
  • synergize with canonical Wnt signaling.24

Interestingly, a demethylase-independent regulation of B-catenin–dependent gene expression was recently described for UTX.25 However, our data provide evidence that

  • Brachyury expression in Jmjd3−/− ESCs is only rescued by catalytically active Jmjd3, which has maintained the demethylase activity.

On the basis of these findings, we propose a model in which Jmjd3 is recruited to the Brachury promoter to remove repressive H3K27me3 marks and on Wnt stimulation additionally promotes B-catenin–dependent promoter activation (Figure 4H). Such a model is similar to the recently described function of Jmjd3 in endoderm differentiation, whereby Jmjd3 associates with Tbx3 and is recruited to the poised promoter of Eomes, to mediate chromatin remodeling allowing subsequent induction of endoderm differentiation induced by activin A.19 The present study additionally demonstrates that

  • Jmjd3 contributes to endothelial and cardiac differentiation.
  • endothelial differentiation was profoundly impaired,

a finding that is consistent with previous findings in adult progenitor cells, showing a high H3K27me3 at endothelial genes.14 The relatively modest inhibition of cardiomyocyte differentiation in Jmjd3−/−  ESCs may be, in part, explained by a compensatory effect of UTX which was shown to regulate cardiac development.26 Together, our study provides first evidence for the regulation of B-catenin–dependent Wnt target genes by Jmjd3 during differentiation of ESCs.  However, the in vivo relevance of the findings is still unclear. The Jmjd3−/− mice that we have generated out of the ESCs, used in the present study, showed embryonic lethality before E6.5, suggesting a crucial role of Jmjd3 in early embryonic development.

Conclusions

Novelty and Significance

What Is Known?

•            Cell fate decisions require well-controlled changes in gene expression that are tightly controlled by epigenetic modulators.

•            The post-transcriptional modifications of histone proteins epigeneti-cally regulate gene expression.

•            Trimethylation of lysine 27 at histone K3 (H3K27me3) silences gene expression and can be reversed by the demethylase Jmjd3.

What New Information Does This Article Contribute?

•            The histone demethylase Jmjd3 is required for mesoderm differentiation and cardiovascular lineage commitment of mouse embryonic stem cells.

•            This effect is partially mediated by a silencing of the mesodermal regulator Brachyury.

•            Ablation of Jmjd3 further reduces β-catenin recruitment to the Brachyury promoter, which interferes with Wnt signaling that is required for proper mesoderm differentiation.

The differentiation of stem cells to specific lineages requires a well-defined modulation of gene expression programs, which is often controlled by epigenetic mechanisms. Although several epi-genetically active enzymes and complexes have been described, the function of the histone demethylase Jmjd3 for cardiovascu¬lar lineage commitment was unknown. Using mouse embryonic stem cells as a model, we now show that the demethylase Jmjd3 is required for mesoderm differentiation and for the differentia¬tion of embryonic stem cells to the vascular and cardiac lineage. We further identified the mechanism and showed that ablation of Jmjd3 resulted in a silencing of the Brachyury promoter that is associated with an increase in H3K27me3 marks. In addition, Jmjd3 was shown to facilitate the recruitment of β-catenin to the Brachyury promoter, which contributes to the Wnt-dependent ac-tivation of mesoderm differentiation. Together these data describe a novel epigenetic mechanism that controls cell fate decision.

Supplemental Methods

Generation of Jmjd3 knockout ES cell lines

Mouse genomic DNA encompassing the murine Jmjd3 gene region were isolated by PCR amplification and used to generate short (1.6kb) and long (6.2kb) arms of homology. The targeting vectors were constructed by inserting a loxP site together with an FRT flanked neomycin selection cassette within the intron 5 and a single distal loxP within the intron 3. This targeting strategy results in the deletion of 600bp coding sequences encoding for the ATG methionine codon and produces a frame shift of JmjC domain existing exon 19-21 required for Jmjd3 demethylase activity. The targeting vector was electroporated in 129Sv ES cells. G418 resistant ES cell clones were screened for homologous recombination by PCR analysis and targeting was verified by Southern blot analysis. Homozygous Jmjd3lox/lox ES cells were generated by electroporation of heterozygous Jmjd3lox/+ ES cells with the same targeting vector as above except that the neomycin resistance gene was replaced by puromycin gene using the Nucleofector (Lonza). Double-allele-recombined ES cells were selected for puromycin (1.3µg/mL, Invitrogen). Correct targeting of homozygous Jmjd3lox/lox ES clones were determined by PCR. To obtain Jmjd3-/- ES cells, Jmjd3lox/lox ES cells were electroporated with Cre-recombinase plasmid vector and loss of targeting cassettes was evaluated by loss of resistance of G418 and puromycin. Correct targeting of homozygous Jmjd3-/- ES cells was determined by PCR.

Reporter gene assays

3xLEF1 reporter plasmid, LEF1 expression construct and NLS-13-catenin were kind gifts from Rudolf Grosschedl. Mouse ES cells were seeded (5×104) on gelatin coated 24-well. After 24 hours of plating, 3xLEF1 reporter plasmid, LEF1, and NLS-13-catenin plasmids were transiently transfected with FugeneHD (Promega). 13-galactosidase plasmid was co-transfected for normalization of transfection efficiency. Each group was transfected in triplicates. 48 hours after transfection, cells were harvested. Cell lysis and luciferase assay were performed following the protocol of Luciferase Reporter Assay System (Promega). 13-galactosidase assays were performed using CPRG (Sigma) as substrate and the absorbance at 600nm was measured. Luciferase activity was normalized to 13-galactosidase activity.

Manuscript References

  1. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell 32, 491-502 (2008).
  2. Sargent, C.Y., Berguig, G.Y. & McDevitt, T.C. Cardiomyogenic differentiation of embryoid bodies is promoted by rotary orbital suspension culture. Tissue engineering. Part A 15, 331­342 (2009).
  3. Gadue, P., Huber, T.L., Paddison, P.J. & Keller, G.M. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A 103, 16806-16811 (2006).
  4. Ohtani, K. et al. Epigenetic regulation of endothelial lineage committed genes in pro-angiogenic hematopoietic and endothelial progenitor cells. Circ Res 109, 1219-1229 (2011).
  5. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. & McMahon, A.P. T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13, 3185-3190 (1999).

Selected Figures   (Figure may not be shown)

Figure 1. Aberrant differentiation of Jmjd3−/− embryonic stem cells (ESCs). A, Quantitative polymerase chain reaction analysis of Jmjd3 in wild-type (WT) and Jmjd3−/− ESCs. B, Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3−/− ESCs. Histone H3 is used as a loading control. Quantification is shown in the right (n=3–5). C, Top, Morphology of WT and Jmjd3−/− ESCs on feeder cells. Bottom, Alkaline phosphatase staining of undifferentiated WT and Jmjd3−/− ESCs. D, Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. α-Tubulin is used as a loading control. E, Bright field image of embryoid bodies at day 5. Scale bar, 200 μm. F, Gene ontology analysis for >2-fold repressed genes in Jmjd3−/− ESCs compared with WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and P values for the significance of enrichment are shown on the x-axis. G, Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3−/− ESCs after spontaneous differentiation by leukemia inhibitory factor withdrawal (n=4). Flk indicates fetal liver kinase.

Figure 2. Jmjd3−/− embryonic stem cells (ESCs) show an impaired ability to differentiate into mesoderm. A, Schematic illustration of the experimental protocol. Differentiation of ESCs (wild-type [WT] and 2 Jmjd3−/− ESCs clones) on OP9 feeder cells was analyzed. B, Left, Representative fluorescence activated cell sorting (FACS) plots showing Brachyury expression of ESC-derived cells. Right, Quantification of FACS analyses (n=3). C, Left, Representative FACS plots showing fetal liver kinase 1 (Flk1) and vascular endothelial-cadherin expression on ESC-derived cells. Right, Quantification of FACS analyses in Flk1+ cells (n=3).

Figure 3. Jmjd3 is required for embryonic stem cells (ESCs) differentiation to the endothelial and cardiac lineage. A, mRNA expression of endothelial markers at day 7 of endothelial differentiation (n=3). B, Platelet endothelial cell adhesion molecule (Pecam)-1 staining of wild-type (WT) and Jmjd3−/− ESCs at day 8 of endothelial differentiation. Phalloidin is used to stain F-actin. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 20 pm. C, Gene expression of cardiac progenitor markers at day 3 of cardiac differentiation. D, Number of beating embryoid bodies (EBs) at day 10 of cardiac differentiation (n=8). E, Gene expression of cardiac markers at day 7 of cardiac differentiation (n=6). F, α-Myosin heavy chain staining of WT and Jmjd3−/− ESCs at day 9 of cardiac differentiation. Nuclei are stained with Hoechst (blue). Scale bar, 20 pm. *P<0.05, **P<0.01, and ***P<0.001

Online Figure I. Generation and characterization of Jmjd3 ESCs  (A) Targeting strategy to generate Jmjd3 mutant ESCs by homologous recombination. Primers used for PCR are shown. (B) Genotyping of Jmjd3 ESCs by using 2 different primers. (C) Oct4 and Nanog staining in WT and Jmjd3−’− ESCs. Scale bar indicates 10µm. (D) Expression of Oct4 and Nanog in WT and Jmjd3 ESCs. Data are presented as fold changes compared with day 0 WT ESCs. N=6. (E) Tunel staining (green) of WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (F) Growth curves of WT and Jmjd3 ESCs. N=6-8. (G) H3K27me3 staining in WT and Jmjd3 ESCs. Nuclei are stained with Hoechst (blue). Scale bar indicates 20µm. (H) ChIP assay of undifferentiated WT and Jmjd3 ESCs for H3K27me3. ChIP enrichments are normalized to Histone H3 density and represented as fold change relative to WT. N=3. Data represent mean ± SEM

Online Figure II. Jmjd3 ESCs show an impaired mesoderm differentiation. (A) Microarray gene expression heat map depicting expression of representative pluripotency and lineage markers 4 days after differentiation in Jmjd3 ESCs versus WI ESCs. Coloring illustrates log2 fold changes between Jmjd3 ESCs and WI ESCs. Green and red colors represent down-regulation and up-regulation, respectively. (B) Gene ontology analysis for more than 2-fold altered genes in Jmjd3 ESCs compared to WI ESCs 4 days after differentiation. Red and green colors represent down-regulation and up-regulation, respectively.

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English: This diagram shows the chromosomes of...

This diagram shows the chromosomes of Drosophila melanogaster approximately to scale. Chromosome sizes were based on basepair lengths given on the NCBI map viewer, and A. B. Carvalho, 2002. Curr. Op. Genet. & Devel. 12:664-668. Centimorgan distances were derived from selected loci listed in the NCBI website. (credit  Wikipedia)

Introduction

Generally speaking sexually reproducing species are composed of individuals of two complementary mating types or sexes.  An essential aspect of the developmental history of each individual is thus sex determination and differentiation. There exist two sex determination mechanisms, somatic and germline, that based on the chromosomal mechanism in the Drosophila melanogaster.  In the somatic sex determination mechanism, each individual assesses the ratio of X-chromosomes to autosomal chromosome sets), the X:A ratio provides the primary sex-determining signal   (reviewed by Cline and Meyer, 1996).  When X:A=1, female differentiation ensues (Bridges, 1925), along with the male-mode of X-chromosome dosage compensation.  The X:A ratio is calculated within each cell of the developing embryo, 2 hrs after fertilization. The X:A ratio determines the sex in Drosophila (Bridges, 1916, 1921, 1925) in a somatic-cell-autonomous manner that occurs early in embryonic development (Baker and Belote, 1983; Baker, 1989). Females possess two X-chromosomes, and males possess one X-chromosome and one Y-chromosome.   The Y-chromosome is required only for spermatogenesis (Lindsley and Tokuyasu 1980; Bridges 1986), and will not be considered further.  The number of X-chromosomes is counted through a mechanism involving positive-acting X-chromosome-encoded transcription factors, termed X-numerator elements (Cline, 1988), negative-acting autosome-encoded transcription factors or denominators, and signal transduction factors provided maternally.  Among the X-numerators are sisterless-a, sisterless-b (sis-b), sisterless-c, and runt (Schurpbach, 1985; Cline, 1986, 1988; Steinmann-Zwicky et al., 1989; Parkhurst et al., 1990; Ericson and Cline, 1991, 1993; Estes, 1995; Hoshijima et al., 1995; reviewed by Cline, 1993).

The best candidate for a denominator gene is the deadpan (dpn) locus.  Both daughterless (da) and extramacrochaete (emc) fulfill the role of maternally contributed transduction loci (Cline, 1976; Cronmiller et al., 1988).  Both in vitro biochemical evidence and in vivo genetic evidence support the idea that transcription factors of the basic-helix-loop-helix (bHLH) family are able to form homo- and hetero-dimers; thus the X:A ratio counting mechanism seems to involve the relative affinities and chromosome-dependent stoiciometries of the bHLH proteins SIS-B, DA, EMC, and DPN.  When X:A=1, sufficient SIS-B protein is synthesized so that it can effectively compete with the EMC and DPN proteins for binding to DA protein.  DA:SIS:B heterodimers then bind to so-called establishment promoter (Pe) elements of the SXL gene and activates its transcription, resulting in an early burst of SXL protein that sets splicing and dosage compensation in to female-specific modes.  When X:A=0.5, too little SIS-B is produced, and DA protein remains sequestered with EMC and DPN.  The Sxl Pe remains inactive, and splicing and dosage compensation enters male-specific modes. In response to X:A ratio=1, an embryo specific promoter of the gene called Sex-lethal (Sxl) is activated (Keyes et al., 1932).

Sxl protein that acts as a master gene for the somatic germline sex determination, has three somatic functions. First, Sxl protein carries out autoregulation at the level of pre-mRNA splicing.  Second, Sxl controls female-specific differentiation at the level of pre-RNA splicing and polyadenylation at least two genes that code for transcription factors that effect terminal differentiation. Third, Sxl protein negatively regulates X-chromosome dosage compensation.  It does so in two ways, by alternative RNA splicing of a normally male-specific gene, and by translation-level regulation of many X-chromosomal transcripts during embryogenesis. In the male, with Sxl in the off state, male differentiation occurs because tra is in the off state and therefore the differentiation-effector transcription factors are produced in alternative male-specific modes.  Dosage compensation is active, and the male X-chromosome is decorated by a minimum of four proteins and two RNA molecules that form a complex along the entire chromosome (reviewed by Cline and Meyer, 1996).  Transcription of the male X-chromosome is elevated two-fold, and it produces the same amount of RNA per template as found in females.

Germline pathway for sex determination and dosage compensation is different than the somatic sex determination mechanism.  (Figure 1) Figure 1: Sex determination of D. melanogaster (1998)The vast majority of somatic sex determination loci have no function in germline cells.  For example, none of the X-chromosome numerators is required for proper oogenesis (Granadino et al., 1989, 1992; Steinmann-Zwicky 1991), despite the fact that proper oogenesis requires that X:A =1 in the germline (Schupbach, 1982, 1985) nor are tra, tra-2, and dsxF required for oogenesis.  Sxl and snf have germline functions but the former is not a binary switch gene between oogenesis and spermatogenesis (Despande et al., 1996; Bopp et al., 1993, 1995; Hager et al., 1997). Systematic screens for female-sterile mutations have identified a large number of genes required for normal oogenesis (e.g. Gans et al., 1975; Mohler, 1977; Perrimon et al., 1986; Schupbach and Wieschaus, 19889, 1991).  Female-sterility can arise in diverse ways, but one interesting class of mutations is germline-dependent and causes an “ovarian tumor” phenotype.  “Ovarian tumor” mutations cause under-developed ovaries, in which egg chambers and ovarioles are filled with an excess of undifferentiated germ cells that have adopted male-like characteristics that include a prominent spherical nucleus, assembly of mitocondria around the nucleus, and mis-expression of male-specific marker genes (Oliver et al., 1988, 1990, 1993; Steinmann-Zwicky, 1988, 1992; Bopp et al., 1993; Pauli et al., Wei et al., 1994).  Among the “ovarian tumor” class of genes are ovo, ovarian tumor (otu), fused, and two genes with somatic phenotypes, namely snf and Sxl. Strong mutations at the ovo and otu loci result in ovaries totally devoid of germ cells (King and Killey, 1982; Busson et al., 1983; Oliver et al., 1987; Mevel-Ninio et al., 1989; Rodesh et al., 1995), Weaker mutations at both loci result in viable germline cells that have abnormal male-like splicing at the Sxl gene (Oliver et al, 1993). The overall conclusion is that oogenesis requires a chromosomally female germline is wild type for ovo, otu, Sxl, and snf.  If one of these genes is defective, either the germline will die or male-like differentiation and tumor formation ensure.

However, there are soma-germline interactions for a normal sex determination. (Figure 2) Figure 2: Somatic-Germline Interactions. (1998)Unlike the somatic regulatory hierarchy, which genetic mosaic experiments clearly showed functions in cell-autonomous fashion, sexual differentiation of the germline requires inductive signaling from somatic cells.  This was shown by use of pole cell transplantation, the method of making mosaics in which germline cells surgically transferred from donor embryos  (Schubach. 1985; Steinmann-Zwicky et al., 1989).  These experiments show that proper germline differentiation requires a combination of germline-autonomous chromosomal cues and proper signaling from the soma.  Evidence with tra and dsx mutant somatic hosts indicates these soma-germline interactions have detectable effects by larval stages (Steinmann-Zwicky., 1996).

The ovo gene is genetically complex.  At least three transcripts are produced from the ovo region (Mevel-Ninio et al, 1991, 1995, 1996; Garfinkel et al., 1992, 1994).  Two of these are germline-specific and correspond to the ovo function, while the third corresponds to the somatic-epidermal, non-sex-specific shavenbaby (svb) function.  (For a schematic of the gene map please refer to Figure3) 

 The ovo function is transcribed from two closely spaced germline-specific promoters, ovo a and ovob, give rise to 5-kb mRNAs (Mevel-Ninio et al., 1991, 1995; Garfinkel et al., 1992, 1994).   First identified  promoter was ovob  Garfinkel et al., (1994)  and the leader exon it forms is called Exon 1b, 1028-codon-long open reading frame that contains four Cys2-His2 fingers at the carboxy terminus; protein MW of 110.6 kD.  A second germline promoter, ovoa, was identified by Mevel-Ninio et al (1995), 1400 codons long, and predicts a 150.8-kD protein.  This Exon 1a contains an in-frame AUG upstream of the translation start in Exon 2 utilized by the OvoB open reading frame.  The OvoB mRNA isoforms is predominant during adult life, with the OvoA isoforms only appearing during Stage 14 of oogenesis (Mevel-Ninio et al., 1991, 1996; Garfinkel., 1994).  The ovo zinc finger domain binds to its own germline promoter regions, to the otu promoter region (Garfinkel et al., 1997; Lee, 1998; Lee and Garfinkel 1998).  This is consistent with ovo playing an important role in a sex determination hierarchy operating in germline cells that involves these other genes. The svb function is transcribed from an incompletely characterized somatic promoter that forms a 7.1 kb poly(A)+ mRNA (Garfinkel et al., 1994).  This transcript accumulates 9-12-hr post-fertilization, in the somatic tissues that later in embryogenesis form the cuticular structures affected by svb mutations.  Wieschaus et al. (1984) observed that ventral denticle belts and dorsal hairs are defective in svb mutations; hence the name, and svb mutations are polyphasic larval lethals. Exons and exon segments that are found in all mRNA forms coded by the region correspond to genomic DNA where so-called svb-ovo- mutations map (Mevel-Ninio et al., 1989; Garfinkel 1992).  Finally, somatic-specific exons, exon segments, and transcriptional regions correspond to region mutable to the svb- ovo- phenotype.  Since al known mRNA forms utilize the same splice junctions to join Exon3 to Exon4, all protein forms coded by the locus are believed to contain the same four zinc fingers at the carboxy terminus.   A wide variety of evidence points to ovo playing a critical role in germline sex determination.  High-level of ovo transcription in germline cells, as detected with Xgal staining of ovo promoter-lacZ constructs requires that they have a female karyotype (Oliver et al., 1994).  Chromosomally male germline cells have low levels of ovo transcription even if the soma is transformed towards female through the use of hs-traF cDNA minigenes.  Likewise, chromosomally female germline cells have high levels of ovo transcription even if the soma is anatomically male through the action of tra loss-of-function mutations.  This argues that high-level of ovo transcription is a germline X: A ratio-autonomous property, and stands in contrast to related experiments with otu.  In the case of otu, there is evidence that chromosomally male germline cells, which normally have no need of otu+ function at all, require otu- for proliferation when they are in a female host (Nagoshi et al., 1995). The D. melanogaster ovo gene is required for cell viability and differentiation of female germ cells, apparently playing a role in germline sex determination.  While female X: A ratio in germline cells is required for high levels of ovo germline promoters.  Therefore we undertook to identify trans-acting regulatory regions of the X-chromosome, with a particular interest in identifying candidate germline X-chromosome numerator elements. In this study, I screened  X-chromosome using 45 deficiency strains, I found that these trans-regulating regions were grouped into 12 loci based on overlapping cytology.  Five regions were trans-regulating activators, and seven were trans-regulating repressors; extrapolating to the entire genome, this result predicts nearly 85 loci.  A subset of the dozen X-chromosomal regions correlated with previously identified E(ovoD) and Su(ovoD) loci (Pauli et al., 1995).  

Materials and Methods

 

Fly Strains and Growth Flies were maintained on standard yeast/cornmeal medium and kept at 25oC and 18oC unless otherwise indicated.  Mutants are described in Lindsley and Zimm (1992).  The ovo3U21 and ovo4B8 were obtained from Brian Oliver of NIH;  OvoD1rS1 FM3 is from the Garfinkel lab collection.  The remaining stocks were obtained from the Bloomington Stock Center (see Table 2.1 for the list of stocks that had been used and Figure 2.1 for their location on the X Chromosome). 

Outcrosses Outcrosses were designed to create transgenic flies so that screening of the X chromosome for trans-regulators of ovo in the germline can be done.   Virgin female flies were collected 14 hour long windows at 18oC or 8 hour long windows at 25oC, during which newly emerged males remained immature.  Collected females were kept 3-5 days to make sure they are virgin before outcrossing them.  Heterozygous virgin females (5-7), carrying deficiency X-chromosomes balanced over first chromosome balancers were mated with males homozygous for either of two P-element transformation constructs of a lacZ reporter gene fused to the ovo promoter.  Both events were inserted on third chromosome.  They were grown at 25oC unless otherwise noted. The control class of F1 progeny has a complete X-chromosome pair, whereas the experimental class has one complete and one deficient X chromosome in its genome.  The [ovo::lacZ constructs] were designed by Oliver et al., (1994).  In this study two of their strains, ovo4B8 (pCOW+1.9) and ovo3U21 (pCOW-2.1) respectively, were used to determine the ovo promoter activity.

Outcrosses to Remove Duplications Several X-chromosome deficiencies in the Bloomington collection are carried in males, with compensatory duplications of X material on an autosome.  These had to be crossed to eliminate the duplications (Fig 2.4).  This was done as follows:  FM3/FM7a virgin flies were mated to Df/Y; Dp males.  Among the F1 progeny, half of the Df/(FM3 or FM7a) daughters will carry the unwanted duplication, and half will be free of the duplication.  In some cases, presence of the duplication could be determined from the females’ phenotypes.  In other cases, up to twenty individuals virgin Df(FM3 or FM7) F1 progeny were backcrossed to FM7a/Y males to establish stocks.  In the F2, absence of the duplication could be established by examining sons; in all cases, the Df is male-lethal unless “rescued” by the duplication.  Also FM3 is itself male lethal.  Thus, single-female stocks that produce only FM7a sons had the desired genotypes and were kept for experiments.

X-Gal Staining In this assay ovaries from two-day-old adults were dissected in Drosophila Ringer’s solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10mM TrisHCl, pH 6.8).  Then, these tissues were transferred to a microtiter plate and fixed in 1% gluteraldehyde, 50mM Na-cacodylyte acid solution for 15 minutes. After rinsing the tissues, three times for 5 minutes each staining buffer (7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 1.0 mM MgCl2, 0.15 mM NaCl), they were transferred to incubation buffer (staining buffer, 5 mM Fe2 (CN)3, 5 mM Fe3 (CN)2, 0.2% X-Gal) for an hour at 37oC.  Next, tissues were washed three times 5 minutes each in washing buffer, which is a 1 mM EDTA, added PBS (130 mM NaCl, 7 mM Na2HPO4*2H2O, 3 mM NaH2PO4*2H2O, pH 7.0) solution.  Finally, the tissues were dehydrated in ethanol solutions of increasing concentrations (50%, 75%, 95%) and mounted on a slide in Permount.  Preparate concentrations were examined under a compound microscope to make correlations between staining and gene activity. Although it was easy to determine positive and negative controls, but this assay wasn’t sensitive enough to see subtle differences due to effects of deleted regions on ovo promoters driving LacZ.

Histochemical Assay of LacZ Activity This method allowed us to make quantitative measurements of lacZ activity due to ovo promoter function in animals heterozygous for X-chromosome deletions.  Emerging F1 flies were collected and aged for two days before dissecting ovaries under a dissecting microscope.  For each soluble assay, 10 flies were dissected.  This is repeated at least seven assays (N, sample number) completed per stock for each construct.  Ovaries from ten dissected outcrossed flies were out into eppendorf tubes containing 100ml of Assay Buffer (50 mM K-phosphate, 1 mM MgCl2 at pH 7.8) and homogenized about 20 strokes.  For each dissected pair of ovaries 100 ml  of assay buffer was used and the volume was completed to appropriate amount.  After centrifuging for one minute, 20 ml of the supernatant was transferred into 980 ml of assay buffer (Simon and Lis, 1987; Ashburner, 1989) to make 2mM chlorophenol red-beta-D-galactopyranoside (CPRG).  Absorbance at 574 nm was measured at half hour time intervals starting from zero to two hours hydrolysis of CPRG by chlorophenol (red CPRG).  CPR has a molar extinction coefficient of 75,000 M-1 cm-1 (Boehringer-Manheim data sheet) and this is a very easily detected product of b-galactoside enzyme activity. Range finding experiments showed that 2mM of CPRG gives linear data for 2-3 hours often, color changes could be seen with the unaided eye. Two controls are shown in Figure 2.8 that validates CPRG for this work.  Ovaries from a non-transformed strain (y w RD) were used to prepare soluble extracts.  A near zero-absorbance at 574 nm was observed that did not appreciably change over several hours.  In contrast, ovarian extracts from the ovo promoter-lacZ transformant strain ovo3U21 and ovo4B8 (Oliver et al, 1994) showed a steep linear increase in A 574 during the same period.  The slopes of these lines were proportional to the amount of ovo3U21 and ovo4B8 extract added.

Bradford (1976) Assay For Protein This protein determination method is based on the binding of Coomasie Brilliant Blue G-250 to the protein.  Preparation of protein reagent was done according to Bradford (1976).  After 100 mg of Coomasie Brilliant Blue G-250 was dissolved in 50 ml 95% ethanol, and then 100 ml 85% (w/v) phosphoric acid was added.  The resulting solution was diluted to a final volume of 1 liter [final concentrations in the reagent were 0.01% (w/v) Coomasie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid].  20ml of prepared soluble extract from the dissected tissues were used.  This volume is diluted to 0.1ml with ddH2O, then 5ml of protein reagent was added to the test tube and contents were mixed.  The absorbance at 595nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of the appropriate buffer and 5 ml of protein reagent.  A standard curve using known quantities of bovine serum albumin (BSA) was constructed.  Soluble extract absorbances were plotted on the standard curve and protein amount interpolated.

Statistical Analysis Average specific activity is calculated as nanomoles of substrate used per hour per nanogram protein expressed (nmole CPRG liberated /ng / hr).  Sample number (N) always exceeded seven.  Mean specific activity and standard error of the mean (SEM) were calculated for each experimental and control class.  The F test was used to determine whether variances were equal, and therefore,, which type of student’s t-test calculation was appropriate.  A significant difference between experimental and control values was identified by a P < 0.05 for the t-test score.

RESULTS

In this study and ovo mechanism study, the X-chromosome was screened, using 56 different deficiency strains    Table 1: List of Stocks for X-chromosome Screening (1998)Table 2: Stocks Made in This Study for X-Chromosome Screening Table 1: Stocks for Negative Autoregulation of ovo (1998)  to identify transregulation of ovo Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

The results are given in three sections: X chromosome deficiency screening, negative autoregulation of ovo exhibited by deficiencies removing ovo, and gene dose analysis using P element transformants carrying extra copies of ovo.

X Chromosome Screening The presence of polytene chromosomes in the salivary glands, which have distinctive, banding patterns allows the map positions of genes to be correlated with physical features of the chromosomes.  Breakpoint locations rearrangements, and the locations of cloned sequences can be easily established.  Each of the major chromosome arms is divided into 20 numbered segments, except chromosome 4, which is divided into 4 regions.  Each numbered region is then divided into six consecutive lettered regions, and each lettered region into numbered bands, for example 4E1. The precise relationship between physical length and the numbering scheme depends on local topography (Lefevre, 1976).  In the summary tables, each deficiency listed according to cytological positions. The map of the X chromosome, including the deficiencies used in this study is given in Materials and Methods (Fig 1). Figure 1: Sex determination of D. melanogaster (1998) In Drosophila melanogaster germ cells, ovo has a primary role in female sex specific cell viability, proliferation and differentiation.  Ovo responds to the number of X-chromosomes as assessed by high level expression (Oliver et al., 1994).  Thus, the ovo promoter may be dependent upon X germline numerator elements.  To identify possible trans-regulators of the ovo germline promoter (and, I hope, to identify germline numerators) I undertook deficiency screen for quantitative effects on ovo::lacZ reporter constructs.  Determination of trans-regulation effect by any of the deletion mutant, was based on two general rules.  If the excised part of the X chromosomes has any genes with the positive regulatory effects on ovo gene activity, then the levels of LacZ reporter gene function will be reduced in experimentals compared to control siblings.  If the experimental class results in the elevation of the LacZ activity by producing high levels of enzyme compared to controls, the elevated region having removed a repression locus. Significant effects were determined by statistical analysis, which using a student’s t-test P value is less than or equal to 0.05.  X-chromosome screening results are presented in Table 3.1 and 3.2.  The entire X-chromosome deficiency set was tested twice: once with a 3.3kb ovo promoter fragment driving LacZ (strain ovo3u21), and separately with a 3.1kb ovo promoter (ovo4B8).  Of  45 deficiencies that represent about 70% of the X-chromosome 17 deficiencies had significant effects in both ovo3U21 and ovo4B8 reporter activity, 1 deficiency had significant effects on only ovo3U21 and only 1 deficiency effect on ovo4B8.  Some of these deficiencies partly overlap, allowing the identification of 11 regions that apparently contain trans-acting modifiers of ovo promoter activity six are positive regulators and five are negative.

Region 1-4.  This region covers the eight overlapping deficiency lines, Df(1) BA1, Df(1)sc14, Df(1)64c18, Df(1)JC19, Df(1)dm75e19, Df(1)N8, Df(1)A113, DF(1)JC70.  For three of them, Df(1)A113, Df(1)JC70, and Df(1)BA1, the student’s t-test probabilities show a significant difference between control and experimental siblings.  The remaining strain has no significant trans-regulation effect on ovo gene activity.  Df(1)BA1 enhanced the ovo gene expression activity about 20% when either ovo3U21 or ovo4B8 is used.  It was suggested that a suppressor of ovoD (1F-2B+ locus) maps within 1E3-4 to 2B3-4 because of the dramatic gene dose effect of this region on the development of ovoD2/+ ovaries (Pauli et al, 1995).  In contrast, it was found that Df(1)A113 and Df(1)JC70 have repressing effects on ovo expression.  Df(1)A113 (3D6-E1; 4F5) removes several genes beside ovo, showed a very significant repression effect in outcrosses, about 82% and 47% (e/C), in ovo3U21 and ovo4B8 respectively.  That data obtained in Df/+ females has a particular quantitative significance, which implies that the missing loci have the complementary effect. It was shown that this region is contains a gene or genes resulting in genetic unbalance (Cline et al., 1987).  Also, Oliver et al., (1988) show that in deficiency lines, which they have used, strains removing both ovo and snf together are reducing viability of the progeny, that is, there is a synergistic interaction between ovo and snf.  

Region 5-8.  Twelve overlapping deletions have been tested in this region.  Two deletions Df(1)N73 (5C3-5;5E-8) and Df(1)Lz90b24 (8B-D) caused very significant repressing effects, implying the presence of trans-activating loci, one deletion Df(1)RA2 (7D10;8A4-5) resulted in heterozygous experimentals with significant elevation in LacZ compared to siblings, implying a trans-repressor locus.  It has been reposted that Df(1)RA2 strongly enhances ovoD  phenotypes due to the function of otu+ in germline sex determination (Pauli et al., 1993).  However, since out protein is cytoplasmic, it is unlikely that the Df(1)RA2 effect on ovo::lacZ promoter activity is due to changing dosage of otu.  It is also suggested that there is a synergistic interaction between ovo and lozenge, eye phenotype, which is deleted by Df(1)Lz90b24, and here the data showed a trans-activating effect due to this deletion.  The other deletions do not cause any significant effect on gene activity.

Region 9-10.  In this cytological position nine deficiency lines had been tested.  Since this region was very dense for putative trans-regulation repressors, it was group in a small region.  Among nine of the deficiencies were used six of them showed a repressor effect.  These effective regions were: Df91)vL15, Df(1)N110, Df(1)HC133, Df(1)vL11, Df(1)KA7, and Df(1)N71.  This region seems to have a very important effect on ovo, since in the 9Bto 10F interval there are various levels of repressor effect.  Two common overlapping regions were found; one was from 9C4 to 9D1-2, and the other was from 10A to10F6.  Other repressor effects from strongest to weakest was Df(1)vL11 (9C4;10A1-2), Df(1)HC133 (9B9-10;9E-F), Df(1)N110 (9B3-4;9D1-2), and Df(1)v-L15 (9B1-2;10A1-2), Df(1)KA7 (10A9;10F6-7) breakpoint was outside the first loci in the examined region.  Df(1)Ka7 and Df(1)vL15 show about 20% increase in the heterozygous siblings, the longest and the shortest breakpoints, respectively.  Three out of five repressing effect intervals, Df(1)v-L11 (9C4; 10A1-2), Df (1)HC133 (9B9-10; 9E-F), Df(1) N110 (9C4; 10A1-2) is the strongest of all in Df/+ and bearing the common region among the five strains, which is 9C4; 10A1-2.  

Region 11-13.     Eight deficiency lines were in this region, Df(1)JA26, Df(1)HF368, Df(1)N12, Df(1)C246, Df(1)g, Df(1) RK2, Df(1)RK4, and Df(1) sd 72b   .  It has been found that this region involves five overlapping deletions that gave rise to repressing effect on ovo gene expression.  According to common regions of the cytological positions, these overlapping deletions were grouped into three loci.  These three common regions, which are responsible from trans-regulation activity of ovo, reside on 11D0F; 12B-D, and 13F-B regions of the X-chromosome.  Df(1)N12 (11D12;11F1-2) and Df(1)C246 (11D-E; 12A1-2) were in the 11D-F loci, Df(1)g (12B;12E8) and Df(1)RK2 (12D2-E1; 13A2-5) were in the 12B0D region, and Df(1)sd72B (13F1-14B1) in the 13B-14B loci, all of which in this examined region showed a repressor activity. The strongest effect among the X-chromosome screening was located in 11D1-11F1-2 excised region of X-chromosome, this deletion corresponds to Df(1)N12 strain, which shows a significant effect as well as high gene activity repression, Around 140% to 240% E/C in Df/+ flies for both ovo::LacZ constructs.  In addition, it has been reported that reduced dose of the 11D-F region results in synergistic mutant phenotypes with a number of somatic sex determination genes (Belote et., 1985).  Furthermore, Flybase reports that this region seems to include locus involved in early sex determination examined by Scott and baker (1986). However, ambiguities in deficiency breakpoint assignments complicate interpretation.  For example, first loci, which includes Df(1)N12 and Df(1)C246 due to uncertainty at the distal end breakpoints of Df(1)C246 (12D-e; 12A1-2); the trans-acting repressor of ovo maybe located in 11E-F rather than 11D-F. Similarly, for the second loci in this region ambiguity at the distal breakpoint of Df(1)RK2 also cause a dilemma about the location of the trans-acting repressor, since the question was the common region between Df(1)g and Df(1)RK2 was whether in the 12D-E or in the 2E1-2E8 of X-chromosome. On the other hand, the last loci were determined by the only one deficiency strain.  In this case, the problem was whether determination of the loci was accurate enough, or whether another locus is involved in repressing of ovo reporter activity which Df(1)sd72b (13F114B1) may have a common region with.  This deficiency removes several lethal mutations, Myb, sd (scalloped), shi (shibiri), and exd (extradenticle).  Two genes previously cloned in the 13F cytological region are the Drosophila c-myb oncogene homolog (Katzen et al, 1985) and a G protein b-subunit (Yarfitz et al 1988).  It has been suggested that the sd+ gene might be associated with more than one product (perhaps a differential processing) or it might reflect differential tissue and/or temporal regulation (Campbell et al., 1991).

Region 14-20.   In this region eight deficiency strains, Df(1)4b18, Df(1)rD1, Df(1)B, Df(1)N19, Df(1)JA27, Df(1)HF396, DF(1)DCB1, and Df(1) A-209, were tested.  According to measured specific activities Df(1)4b18 (14B8; 14C1) and DF(1) B (15F9=16A6-7) showed significant activating effect on ovo promoter, activity of the former was weaker than that of latter.  Since there is no common region between these two putative trans-acting activators, interpretations of the results gave rise to two loci, 14B8-14C1 and 15F-16A1; 16A6-9. In addition, the Flybase report for Df(1) shows that 70 deletion that breaks within the second exon of the non A (no on or transient A) gene from Stanewsky et al (1993). As a result of X-chromosome screening, 45 deficiency strains were tested and found 17 regions were trans-regulating ovo promoter.  These regions were classified into 12 loci according to their overlapping common regions.  Among these, six, of which were showing trans-acting activator effect, and seven, of which were responsible for trans-acting repressor effect on ovo promoter.   Furthermore, one deficiency strain, Df(1)sc14, showed a significant trans-acting repressor effect in only ovo4B8 strain but not in ovo3U21 strain.  This maybe explained by position effect of P[ovo::LacZ] construct due to landing on P element transposase onto insertion site or by difference between the size of the ovo::LacZ constructs, e.g. ovo3U21 carries 200 bp longer than ovo4B8 at the N-terminal end that may cause a better translation product.  Consequently, among the X-chromosome screening data, it was found that two of the deficiency lines. Df(1)A113 and Df(1)JC70, which are removing ovo and snf along with the several genes due to deletions, and correspond to one loci acting as an repressor, were taking into more detailed investigations.  These results suggested a negative autoregulation mechanism in the ovo promoter.  Therefore, negative autoregulation of ovo was examined with three approaches: ovo point mutations, more defined deficiency strain, and downstream genes.

DISCUSSION

  The sex determination involves complex set of mechanisms.  The fly is chosen to be studied since Drosophila is inexpensive to rear, generates large numbers of progeny, and has nearly a century of accumulated data upon which to design experiments.  Mutational analysis of cell biological and developmental process is relatively simple, even if the resulting mutations are organism-lethal when homozygous.  This is decided advantage over mammalian genetics, in which lethal mutations often die in utero, which complicates the ability to examine and interpret mutant phenotypes. The Drosophila genome is one-twentieth the size of the mammalian genome, making insertional mutagenesis and positional cloning much less difficult.  Additionally, mammalian genetics lacks genetic tools such as balancers that make the maintenance of sterile and lethal-mutations nearly trouble free in Drosophila.  Nematodes have many of the same conveniences as Drosophila, with the added advantage of a highly stereotyped pattern of embryonic (and post-hatching) cell lineages.  The more-regulative character of Drosophila development induces complications lacking from worm genetics, with respect to cellular level analysis of mutant phenotypes.  Perhaps, the most compelling reason to take advantage of the specialized properties of Drosophila, is the extent to which prior studies have shown that genes, proteins, and developmental pathways and processes are conserved among metazoan groups.  We can, with high confidence, study sex determination in Drosophila with a reasonable confidence that what we learn can be extrapolated to other species, including man and his clinical diseases.

  The deletion mapping technique was used to identify the locations of genes that are required for ovo trans-regulation.  Each deficiency line removes several to many genes from the genome.  A sufficiently complete set of overlapping deletions can allow, potentially, every individual trans-acting gene to be localized. Seventeen deficiencies that have effects on the ovo germline promoters are shown in Table 4.1.  Twelve deficiencies showed repressor effects, and five deficiencies showed activator effects.  Deleted regions may affect any of several processes, such as numerator elements, cell viability and differentiation, dosage compensation, and response to inductive signals from soma.  Determination of which gene within a specific region is responsible for the effect on ovo requires more defined deletions or having null alleles for each gene. Estimation of the Number of Trans-Regulators.  Among the seventeen deficiencies in Table 4.1, overlapping common regions identify seven that function as trans-acting repressor loci, and five that function as trans-acting activator loci.  Thus, the entire euchromatic X-chromosome may have as many as ≈10 repressor genes and ≈7 activator genes for the ovo germline promoters.  If these results were extrapolated to the entire fly genome, ≈50 repressors and ≈35 activators of ovo transcription are predicted.  These are underestimates from the data, since any given deleted common region need not remove exactly one relevant gene. Is it reasonable for nearly 85 genes to be involved in regulating the ovo germline promoters?  Precedents from other developmental control systems suggest this is not an implausibly high number.

Regulation of the master sex determination gene Sxl is complex.  To establish somatic sex determination in the early embryo, nine genes are required to activate the Sxl early promoter.  These are sis-a, sis-b, sis-c, run, da, emc, gro, dpn, and her.  In biochemical terms, most are DNA-binding proteins.  In genetic terms, some are positive and are others are negative regulators.  Maintenance of Sxl expression involves positive autoregulation at the level of pre-mRNA alternative splicing.  At least five genes are known to play specific roles in this process: Sxl itself, snf, vir, her, and fl(2)d.  Function of Sxl in the germline is regulated in several ways.  Germline-specific transcriptional control of Sxl is still conjectural, but it is clear that the somatic functioning numerator elements play no role in the germline.  It is possible that ovo may play an important role in germline transcriptional control of Sxl (e.g., Lee. 1998); certainly it has an indirect role (e.g., Oliver et al., 1993).  Splicing-level autoregulation of Sxl is active in the female germline, and it involves the same genes that function in this process in somatic cells.  Once Sxl protein is produced in female germline cells, the otu protein plays an important role in this relocalization into the nucleus.  Thus, a minimum of sixteen genes is required for proper regulation of Sxl.

Establishment of the body plan in Drosophila is also under complex transcriptional control.  Maternally localized RNA and protein molecules establish the gross body axes: anterior-posterior and dorsal-ventral.  Hierarchically organized sets of zygotically activated genes are transcribed, and their protein products serve to refine the body axes into progressively finer-grained structures.  The metameric anterior-posterior body axis is specified by so-called gap genes, pair rule genes, and segment polarity genes, which create the segment-sized repeating units of the body.  Homeotic genes encoded by the Antennapedia Complex (ANT-C) and bithorax Complex (BX-C) then confer position-specific identities upon each segment. During the cellular blastoderm stage, gap genes and maternal coordinate genes regulated the activation of primary pair rule genes such as even-skipped (eve).  These are expressed in seven one-segment-wide stripes that alternate with on-segment-wide regions of non-expressing cells.  For example, the second stripe of eve expression is positively regulated by hunchback and bicoid, and negatively regulated by giant and Kruppel.  All four proteins directly bind to a 500-bp-long “eve-stripe 2 enhancer.”  Binding have giant and Kruppel is competitive with binding of hunchback  and bicoid, and vice versa.  Thus, spatially controlled concentrations of giant, Kruppel, bicoid, and hunchback proteins result in spatially restricted activation or repression of the eve stripe 2 enhancer.  The remaining six stripes of eve expression are similarly controlled by other DNA-binding proteins, which are acting another discrete stripe-specific enhancers. Ectopic expression of homeotic genes can have disastrous effects on development.  Thus, a special heterochromatin-like mechanism functions to ensure that ANT-C and BX-C genes are inactive in cells and tissues that do not require their expression.  Stable repression is mediated by the Polycomb class of proteins, which number over forty. Each of these examples illustrates that developmental control of individual gene transcription is mediated by both positive and negative effectors, and that sometimes the number of such upstream regulators numbers between one and several dozen.  Thus, our estimate of 85 regulators of the ovo germline promoters is not out of line with other developmentally regulated systems.

Evaluation of Candidate Loci Within Common Regions.   Based overlapping cytology, seventeen deficiencies that affected the ovo germline promoter fell into twelve common regions.  Each of these will be discussed in turn below. Of particular interest was the relationship each of our trans-acting may have with Su(ovoD) and E(ovoD) loci identified in a generic screen by Pauli et al. (1995).  In general, it is not straightforward to suggest identities between Su(ovoD) or E(ovoD) loci and our trans-acting repressor or activator loci because of the dissimilar means of assaying these gene-dose-sensitive interactions.  We use quantitative measures of LacZ reporter activity as a proxy for ovo transcription, while Pauli et al. (1995) use semi-quantitative measures of vitellogenesis.

Region 1 (polytene bands 1A1; 2A1-4):  The distal region of the X-chromosome showed a trans-regulating activator effect on the ovo promoters.  This region includes the acheate-scute complex (AS-C), home of the X-chromosome numerator element sis-b (Cline, 1988; Parkhurst and Ish-Horowicz, 1990), also known as scute-T4.  This numerator has no function in the female germline (Granadino et al., 1989).  Pauli et al., (1995), using other deficiency strains affecting this section of the X-chromosome, identified a strong Su(ovoD) locus in the polytene region 1E3-4; 2B3-4 that may correspond with our trans-activator.  Flybase indicates that this region contains over 100 genes, among them 23 unassigned open reading frames, 33 genes defined by apparent visible mutations, 53 lethal genes,, and two female sterile loci.

Region 2 (polytene bands 4C15-16; 4F15):  This region includes the ovo and snf loci, and was identified by Pauli et al., (1995) as a strong E(ovoD) due to the effects of these loci.  Further discussion is deferred to mechanism of ovo autoregulation, which deal with ovo negative regulation. Region 3 (polytene bands 5C3-5; 5E8):  This region has a trans-regulatory activation effect on the ovo germline promoters.  Deficiency for this region showed no interaction with ovoD in the vitellogenesis assay (Pauli et al., 1995).  Examination of Flybase records for this region reveals over twenty genes, and no strong candidates that may account for the interaction with the ovo promoters.

Region 4 (polytene bands 7D10; 8A4-5):  Results  showed that this region contains a transacting-repressor of ovo germline promoter activity.  This region reported by Pauli et al. (1995) to contain a strong E(ovoD) locus, which was identified as the ovarian tumor gene (Pauli et al., 1993, 1995).  It is virtually certain that the repressor-of-ovo is distinct from otu.  First, the otu protein is cytoplasmic and plays a role in egg chamber cytoskeletal function (Nagoshi et al., 1997).  Second, the ovo protein binds to the otu promoter in vitro (Garfinkel et al., 1997; Lee, 1998, Lee and Garfinkel 1998; Lu et al., 1998).  Third, under certain conditions, in vivo activity of the otu promoter is dependent upon ovo protein production (Hager and Cline, 1997; Lu et al., 1998).  Examination of Flybase reveals that this region contains fifty genes mutable to lethal, visible, or female-sterile phenotypes, but none appear to be a strong candidate for the repressor-of-ovo locus.

Region 5 (polytene bands 8B5-8; 8DE):  This region also has an apparent repressor of ovo germline promoter activity.  Deficiency for this region showed no interaction with ovoD mutations in the Pauli et al. (1995) vitellogenesis assay.  Examination of Flybase reveals that this region contains thirty genes mutable to lethal, visible, or female sterile phenotypes.  One gene stands out as a candidate for the repressor, namely, lozenge.  This is a complex locus that is mutable to female sterility (Green and Green, 1949, 1956), and it is named for a reduced-eye, smoothened-eye, mutant phenotypes.  Interestingly, certain ovo-mutant alleles are called “lozenge-like” in recognition of a similar eye defect (Oliver et al., 1987; Mevel-Ninio et al., 1989; Garfinkel et al., 1992).  The lz gene codes for a transcription factor (Dag et al., 1996). Region 6 (polytene bands 9C4; 9D1-2):  The cytological assignment of this region is based on the overlap of three deficiencies:  Df(1)N110, Df(1)H133, and Df(1)v L11.  Together, they mark a trans-acting repressor of ovo promoter activity.  According to  Pauli et al. (1995), only two of these three deficiencies behaved as if they exposed an E(ovoD) locus, while the third had no effect.  In combination with positive results from other deficiencies, Pauli et al. positioned the E(ovoD) locus at cytological region 9E-F.  Thus, it is again possible that the repressor-of-ovo we identified is distinct from a nearby E(ovoD) locus, and is among the half-dozen loci identified by Flybase as mapping into this interval.

Region 7 (polytene bands 10A6; 10F6-7):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoD alleles.  Examination of Flybase reveals that this region includes the somatic X-chromosome numerator element sis-a, which also has no function in germline development (Granadino et al., 1989, 1990, 1997).  Given the extent of this region, it is not  surprising that Flybase identifies 65 genes with diverse phenotypes and biochemical roles; however no strong candidate locus that may count for the repressor-of-ovo locus is apparent.

Region 8 (polytene bands11D1-2; 11F1-2):   This region contains perhaps the strongest trans-acting repressor of ovo promoter activity in the survey: deficiency heterozygous experimentals had 2-2.5 fold more lacZ specific activity in their ovaries that the balancer carrying controls.  According to Pauli et al (1995), one of the two deficiencies defining this common region showed a statistically weak enhancement of ovoDalleles, while the other had a significant Su(ovoD) phenotype.  Likewise, Belote et al. (1985) and Scott and Baker (1986) reported that the same deficiency later shown to have Su(ovoD) activity also interacted with loci in the somatic sex determination pathway.  It is an open question how these three results relate to one another.  Among sixteen genes that map into this region are two signal transduction loci: the Mek3 gene, a serine-threonine-specific protein kinase in the MAP kinase pathway, and a beta subunit of the heterotrimeric GTP-binding protein. A solitary female-sterile, fs(1) K4, also maps roughly into this region; it is germline-dependent, and yields fragile eggs, a phenotype occasionally seen in the eggs laid by ovoD3/+ females.

Region 9 (polytene bands 12D2-12E1; 12E8):  This region contains a trans-acting repressor of ovo promoter activity.  According to Pauli et al. (1995), neither deficiency defining this common region interacted with ovoDalleles.  This region contains the yolkless gene (DiMario et al., 1987), which has been cloned and codes for a member of 35 known genes, including a cluster of tRNA genes, the male-germline-specific Stellate genes, and several lethal and female-sterile genes.

Region 10 (polytene bands 13F1; 14B1):  This region contains a trans-acting repressor of ovo promoter activity.  Again, no significant interaction with ovoD allel4es was observed by Pauli et al. (1995).  Podry, Katzen and others have extensively mutagenized this region due to its containing shibiri (the Drosophila homolog of dynamin), c-myb, another Gb subunit, and the homeodomain protein extradenticle.  Their work revealed a total of twenty lethal genes, ten apparent visibles, and over a half-dozen unassigned open reading frames.

Region 11 (polytene bands 14B8; 14C1):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al., (1995), the defining deficiency had no significant interaction with ovoD alleles.  This region is surprisingly dense genetically, as it apparently contains over forty genes.  Several behavioral genes coding for neuronal functions map here, including nonA, paralytic, and easily shocked.  The nonA gene codes for an RNA-binding protein, and is mutable to a variety of phenotypes including recessive lethality, male-courtship-strong abnormalities, and defective vision.  The location of para (a sodium channel) is particularly intriguing since parats mutations fail to complement certain napts alleles, and nap genetically overlaps the dosage compensation function maleless.  Mutations in maleless are unique among the known dosage compensation loci in having a mutant phenotype in germline clones, and they are said to suppress the female-germline-lethality of ovo null mutations.  The easily shocked locus codes for ethanolmine kinase, and mutations at this locus also interact with mle.

Region 12 (polytene bands 15F9-16A1; 16A7):  This region contains a trans-acting activator of ovo promoter activity.  According to Pauli et al. (1995), the defining deficiency had no significant interaction with ovoDalleles.  Examination of Flybase reveals that this region contains at least a dozen female-sterile loci, a dozen lethal loci (including the Bar homeodomain protein gene). There is an ambiguity in compared mean of activities.  According to the negative autoregulation mechanism, there suppose to be a linear decrease pattern correlated to increase in copy of ovo.  However, the pattern of the gene dose was reaching plato, when three copies of ovo were present in the genome. Yet, this also shows that there is a protection mechanism that counts the number of ovo versus number of X chromosome exists.  Therefore, the sex determination mechanism turns off the extra ovo in the system immediately. 

Consequently, the system prohibits more wrong information to be processed according to its default setting where if the X:A ratio equals to one the outcome is going to be prepared as female, if not turn off the mechanism towards male-like, sterile mode, or death at the embryonic stage.  This discontinuity in the linear correlation may be due to position effect of P[w+ ovo+].  Future Directions and Concluding Remarks The results of this study suggest that the ovo germline promoters are regulated by a large set of upstream factors.  Nearly a dozen of these maps to the X-chromosome, some to region that are well characterized genetically.  Further deficiency mapping experiments, and assessment of the phenotypes of single-P insertion lines with female-sterile or perhaps lethal phenotypes, would be required to identify the relevant genes.  Some regions contain candidate loci that have been cloned (e.g. lozenge); in this example, either in vitro DNA-binding experiments using Lz protein and the ovo promoter region, or computational assessment of the likelihood that the ovo promoter contains binding sites for Lz can be done. Another potential upstream factor not assessed in these experiments is the ecdysone regulatory hierarchy.  The steroid ecdysone is the endocrine hormone that controls molting and metamorphosis in arthropods.  It is an allosteric effector for a heterodimeric receptor of the steroid-receptor superfamily.  The ovaries of adult females manufacture their own ecdysone, and the gene for the rate-limiting steroidogenic enzyme transcribed beginning in Stage 7-8 egg chambers.  This stage immediately precedes the onset of the highest level of ovo transcription (Mevel-Ninio et al., 1991; Garfinkel et al., 1994).  Mutations in the E74 and E75 genes, when made homozygous in germline clones, cause arrest of oogenesis at Stage 7-8, as if egg chambers are unable to respond to endogenous ecdysone and continue differentiation.  Both E74 and E75 code for transcription factors that are induced as immediate-early primary responses to added ecdysone both in-vivo and in tissue culture assays.  Thus, it is reasonable to suggest that one or both of these proteins will bind to the ovo germline promoter in an in vivo effect on expression of the ovo::lacZ reporter using the methods established in this dissertation.  

Acknowledgement:  This work had been comppleted in the laboratory of Dr. Mark Garfinkel at Illinois Institute of Technology.   Dr. Demet Sag initiated the project with her own  ideas, was fully supported by Turkish National Merit Fellowship, and  earned NATO Advanced Science institute  Grant on Genome Structure and Functional Genomics, Elba Island, Italy, accepted to work with Dr. Mevel Ninio, based on the proposal submitted by Demet Sag on Molecular Mechanism of  ovo, through EMBO long term scholarship in France.

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FIGURES and TABLES:

Figure 1: Sex determination of D. melanogaster (1998)

Figure 2: Somatic-Germline Interactions. (1998)

Figure 3: Molecular Structure of the ovo locus

Figure 4: In vivo Biochemical_genetic Assay for Regulators

Figure 5: ovo-LacZ Reporter Construction. (1998)

Figure 6 : Establishing Stocks From Duplication Carrying Lines.

Figure 7: Control Assay for b-galactosidase Assay. (1998).

Table 1: List of Stocks for X-chromosome Screening (1998)

Table 2: Stocks Made in This Study for X-Chromosome Screening

Table 3: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo3U21

Table 4: LacZ Specific Activities Obtained by Screening X-Chromosome with ovo4B8 (Results)

Table 5: Deficiency Lines Affecting the ovo Gene Activity (X-chromosome screening result)

 

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ovo Female Germline Specific Drosophila melanogaster Gene has two auto-regulation mechanism: negative and positive

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Author: Aashir Awan, PhD

The primary cilium is organelle that has garnered much attention in the field of cell biology during the last 15 years. It is a slender, solitary hair-like organelle that extends 5-10 uM from each mammalian cell (in the G0 cell cycle state) that is microtubule-based (9 outer doublets arranged in a circular fashion) and dependent on a process called Intraflagellar Transport (IFT). IFT is the bidirectional movement of motors (kinesin-2 in the anterograde and dynein-2 in the retrograde direction) responsible for the assembly and maintenance of the cilium (Pedersen et al., 2006).

Until this time, it had been labeled a ‘vestigial’ organelle not worthy of research. Yet, a breakthrough into the sensory role of the primary cilium came in 2000 based on Dr. Rosenbaum’s research on Chlamydomonas and the motile cilium or flagella. Along with Dr. George Whitman’s group, they were able to show the importance of Tg737 (IFT88) protein to the pathology of polycystic kidney disease in mouse (Pazour et al., 2000). Since then, research into the primary cilium has exploded and has been linked to diverse pathologies (collectively known as ciliopathies) such as

  • retinitis pigmentosa,
  • hydrocephaly,
  • situs inversus,
  • ovarian and pancreatic cancers among others (Nielsen et al., 2008; Edberg et al., 2012). Also, various
  • signal transduction pathways have been found to be coordinated by the primary cilia such as hedgehog, wnt, PDGF among others (Veland et al., 2008).

Thus, in 2006, the Christensen lab at the University of Copenhagen (Denmark) with the collaboration of Dr. Peter Satir’s group at Albert Einstein College of Medicine (Bronx, NY) began to investigate whether the human embryonic stem cells (hESCs) possess primary cilium and then to begin preliminary molecular dissections of the role that this organelle could play in the proliferation and differentiation profiles of these pluripotent cells. The Albert Einstein group, due to NIH restrictions, had to work with two federally-sanctioned cell lines. Working with the Laboratory of Reproductive Biology at RigsHospital, the Danish side had access to in-house derived stem cell lines from discarded blastocysts. The advantage for the Danish side was obvious since these newer cell lines hadn’t undergone as many passages as the NIH cell lines and were thus more robust. To begin preliminary characterizations of these lines, some basic hallmarks of hESCs (Bernhardt et al., 2012) had to be localized to the nucleus such as the transcription factor (TF) Oct4 (Fig. 1).

In addition, a single primary cilium can be seen denoted by the acetylated tubulin staining emanating from each cell in the micrographs. Also, the base of the cilium is marked by the presence of pericentrin and centrin which demarcate the centriole.

Fig1 Fig. 1 Primary cilia stained with anti-acetylated tubulin (tb, red) are indicated by arrows and undifferentiated stem cells are identified by nuclear colocalization of OCT-4 (green) and DAPI (dark blue) in the merged image (light blue). A primary cilium (tb, red, arrow) in undifferentiated hESCs emerges from one of the centrioles (asterisks) marked with anti-centrin (centrin, green). Inset shows anti-pericentrin localization to base of cilia (Pctn, green).

Together, the three labs were the first to discover primary cilia in stem cells while other groups have since then confirmed these findings (Kiprilov et  al. 2008; Han et al. 2008). Attention was then to characterize different signal transduction pathways in the stem cell cilium. Since the hedgehog pathway has been shown to be important for differentiation and proliferation (Cerdan and Bhatia, 2012), the groups characterized this signal pathway in these cells using immunofluorescence, electron microscopy and qPCR techniques. One particularly interesting experiment to show that the hedgehog pathway was functional in these cells was to add the hedgehog agonist, SAG (Smoothened agonist), and then to isolate the cells for immunofluorescence at different times.

Gradually, one can see the appearance of the smoothened protein into the cilium as indicated by increasing intensity of the immunofluorescence staining. Conversely, patched levels in the cilium, decreased. This is a hallmark of hedgehog activation (Fig. 2).
Fig. 2 copiaFig. 2 Immunofluorescence micrographs of hESC showing smoothened (green), acetylated tubulin (red) and DAPI (blue). The micrographs from left to right represents SAG treatments at t = 0, 1 and 4 hours.

However, an additional interesting observation was made concerning these stem cells. An important characteristic for stem cells is the presence of certain transcription factors which render these cells in the pluripotent or undifferentiated state. These include Oct4, Sox2, and Nanog whose localization had been observed in the nucleus as expected for other TFs.

However, the Danish groups curiously found a subpopulation of stem cells where these TFs were additionally localized to the primary cilium (Fig. 3). This had never been observed or investigated before.  Additionally, proper negative controls were  carried out to exclude this phenomenon from being an artifact (e.g. bleed through).
Fig. 3 copia Fig. 3 Stem cell markers (Sox2, Nanog, and Oct4) localizing to the nucleus and the primary cilia (arrows) of hESC line LRB003. This and the previous figure show shifted overlay images whereby the green and red channels have been slightly shifted so that the red channel doesn’t swamp out the intensity of the green channels.

Thus, it raises an intriguing possibility that perhaps the primary cilia plays a previously uncharacterized role in the differentiation/proliferation state of the hESCs via possible modifications of these TFs perhaps analogous to the processing of the Gli transcription factors (Hui and Angers, 2011). Another curious observation is that the subpopulation of cells whose primary cilia are positive for these TFs always occur in clusters which might hint at its mechanistic explanation.  In conclusion, since stem cells are now being more routinely used for regenerative medicine such as repair of severed spinal cord (Lu et al. 2012), it behooves us to better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state so that we can harness their full therapeutic potential.

REFERENCES

Awan A, Oliveri RS, Jensen PL, Christensen ST, Andersen CY. 2010 Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions. Methods Mol Biol. 584:195-210.

Bernhardt M, Galach M, Novak D, Utikal J. 2012 Mediators of induced pluripotency and their role in cancer cells – current scientific knowledge and future perspectives. Biotechnol J. 7:810-821.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Heart attack patients could one day have their heart repaired using their own skin cells. This research focused on the potential use of human pluripotent stem cells such as human embryonic stem cells for the treatment of post-myocardial infarction heart failure and on the utilization of genetically-engineered cell grafts for the treatment of cardiac arrhythmias by modifying the electrophysiological properties. Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. That is using a patient’s own cells would avoid the problem of patients’ immune systems rejecting the cells as ‘foreign’. It was aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue. Dermal fibroblasts from HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. That is the resulting stem cells were able to differentiate to become heart muscle cells (cardiomyocytes) just as effectively as those that had been developed from healthy, young volunteers who acted as controls for the study. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes. That is it was possible to make the cardiomyocytes develop into heart muscle tissue, which was joined together with existing cardiac tissue and within 48 hours the tissues were beating together. Human-induced pluripotent stem cells thus can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine. This technology needs to be refined before it can be used for the treatment of patients with heart failure, but these findings are encouraging and take us a step closer to the goal of identifying an effective means of repairing the heart and limiting the consequences of heart failure.

 

Articles may be reviewed:

 

Zwi-Dantsis L, Huber I, Habib M, Winterstern A, Gepstein A, Arbel G, Gepstein L. 2012. Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients. Eur Heart J. [Epub ahead of print] (http://www.ncbi.nlm.nih.gov/pubmed?term=Derivation%20and%20cardiomyocyte%20differentiation%20of%20induced%20pluripotent%20stem%20cells%20from%20heart%20failure%20patients)

 

Yankelson, L., Feld, Y., Bressler-Stramer, T., Itzhaki, I., Huber, I., Gepstein, A., Aronson, D., Marom, S., Gepstein, L. 2008. Cell therapy for modification of the myocardial electrophysiological substrate. Circulation 117, 720-731. (http://www.ncbi.nlm.nih.gov/pubmed/18212286)

 

Caspi, O., Huber, I., Kehat, I., Habib, M., Arbel, G., Gepstein, A., Yankelson, L., Aronson, D., Beyar, R., Gepstein, L. 2007. Transplantation of human embryonic stem cell-derived cardiomyocytes improves myocardial performance in infarcted rat hearts. J Am Coll Cardiol 50, 1884-1893. (http://www.ncbi.nlm.nih.gov/pubmed?term=Transplantation%20of%20human%20embryonic%20stem%20cell-derived%20cardiomyocytes%20improves%20myocardial%20performance%20in%20infarcted%20rat%20hearts)

Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M., Gepstein, A., Habib, M., Yankelson, L., Kehat, I., Gepstein, L. 2007. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J 21, 2551-2563. (http://www.ncbi.nlm.nih.gov/pubmed/17435178)

http://www.dailymail.co.uk/health/article-2148205/Skin-cells-heart-attack-victims-turned-healthy-heart-muscle-tissue-time.html

 

http://rappinst.com/Rappaport/Templates/ShowPage.asp?DBID=1&TMID=610&FID=77&PID=0&IID=241

 

http://www1.technion.ac.il/_local/includes/blocks/news-items/110814-liorprize11/news-item-en.htm

 

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