Brief background:

Germline stem cells (GSCs) are essential for fertility and fecundity so the molecular characterization of factors involved in initiation, maintenance and differentiation is an important goal not only for in Drosophila but also in stem cell research.  In addition to genetic studies genomics studies carry a weight to explain the function of these factors, genes and structures. For example ovo, female germline specific gene, is an complex transcription gene producing three proteins from a one transcript in Drosophila yet in human ovo is expressed from three different chromosomes to produce three proteins with various roles.  There can be a relevance as a biomarker for fertility and ovarian cancer in human. Genomics increase our knowledge of these pathways in cancer development. Thus, in first section I will present the classical genetic work.  Then,  in the second section, I  will include modern genomics studies like http://www.biomedcentral.com/1471-213X/12/4  in both model organisms and human to correlate ovo and its importance in female cancers.

1.1  Germline Stem Cells and Sex determination

In chromosomally based sex determination, the two sexes differ in karyotype, and the task embryos face is to “count chromosomes” and to set and maintain sex-dimorphic regulatory mechanisms to the appropriate state.  Often associated with sex determination is the problem of adjusting transcription rates of the sex chromosomes so that individuals with dissimilar chromosome and gene doses become equalized for gene product dose, a process called “dosage compensation”.  One example of chromosomal based sex determination is Drosophila melanogaster, the common laboratory fruit fly.  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.   As it turns out the only obligatory function of the Y-chromosome in Drosophila is to provide genes required for the completion of spermatogenesis; the Y-chromosome has no primary role in sex determination (Lindsley and Tokuyasu 1980; Bridges 1986).

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.

Germline Pathway for Sex Determination and Dosage Compensation

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 dsx^F 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.

 

The ovo locus

A wide variety of evidence points to this gene playing a critical role in germline sex determination.

Mutations:  ovo mutations are female-sterile, with no discernible effect in male germline or in somatic tissues.  The latter conclusion is based on clonally analysis, which showed that the ovo mutant phenotype is germline-dependent (Perrimon and Gans 1983; Perrimon, 1984).  Homozygous null ovo mutations are female-sterile because germline cell death begins during gastrulation (Oliver et al., 1987; but see Rodesh et al., 1995 and Staab and Steinman-Zwicky et al., 1995), resulting in females whose ovaries lack germ cells altogether.  A second type of ovo mutation results in viable germ cells that adopt a morphology resembling male germ cells (Oliver et al., 1990).  A third type of ovo mutation results in defective oogenesis, but has no apparent germline sex transformation (Busson et al., 1983; Oliver et al, 1987).

 

Expression:  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-tra^F 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).

Genetic complexity of ovo: 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 Figure 1.3. Molecular Structure of the ovo locus

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.

The ovo function is transcribed from two closely spaced germline-specific promoters, and gives rise to 5-kb mRNAs (Mevel-Ninio et al., 1991, 1995; Garfinkel et al., 1992, 1994).  The promoter identified by Garfinkel et al., (1994) codes for an mRNA with a 1028-codon-long open reading frame that contains four Cys2-His2 fingers at the carboxy terminus; the predicted protein has a molecular weight of 110.6 kD.  This promoter now called ovob, and the leader exon it forms is called Exon 1b.  The open reading frame is called OvoB.  OvoB mRNA appears in germline cells during embryogenesis and is present the throughout the life cycle.  It is relatively love abundance in germaria and early egg chambers, but accumulates dramatically beginning in Stage 8 of oogenesis.  Substantial quantities are deposited into the egg as a maternal RNA.  A second germline promoter, ovoa, was identified by Mevel-Ninio et al (1995).  This Exon 1a contains an in-frame AUG upstream of the translation start in Exon 2 utilized by the OvoB open reading frame.  As a result, the OvoA open reading frame is 1400 codons long, and predicts a 150.8-kD protein.  Both proteins are collinear, with the OvoA protein possessing an N-terminal extension relative to OvoB.  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).

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.

That the svb-ovo protein isoforms code for putative transcription factors is supported by the primary sequence of the predicted products, and by in vitro biochemical data showing the zinc finger domain binds to DNA with sequence specificity.  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 Goals of This Study

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 that study, it had been found that certain regions affecting negative auto-regulation of ovo. Then it had been asked how having disturb expression of ovo by deletions within the gene, upstream or downstream of the gene, and increase of number of gene as well as origin of the copy could affect the germline sex determination mechanism.

Interestingly, deficiencies that removed ovo were scored as trans-acting repressors.  This implied that one of the functions of ovo+ is to down-regulate its own expression, which we called negative autoregulation.  Several point mutations in ovo had the same effect; P [ovo+] transgenes were predicted to have the opposite effect, but this was not observed.  Other gene regions containing candidate downstream targets of ovo, such as Sxl, had no effect on the ovo promoter, as expected.

 

 

Materials and Methods

 

2.1 Fly Strains and Growth

Flies were maintained on standard yeast/cornmeal medium and kept at 25^oC and 18^oC unless otherwise indicated.  Mutants are described in Lindsley and Zimm (1992).  The ovo^3U21 and ovo^4B8 were obtained from Brian Oliver of NIH; the snf–^e8H:snf^e8H, snf¹⁶²¹ FM7c, Sxl^7B0/FM7c from Helen K. Salz of Case Western Reserve University; ALK coded deficiency stocks from Alisa Katzen of the University of Illinois at Chicago; w¹¹¹⁸ Sco/Cyo, w¹¹¹⁸ TM3, Sb/TM6 Ubx from Chris Schonbaum of the University of Chicago; y w ovo^D1rS1/ y+ Y FM6; TM3, Sb/TM6, Ubx from Rod Nagoshi of University of Iowa.  Ovo^D1rS1 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).

2.2 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 (Fig 2.2).  Virgin female flies were collected 14 hour long windows at 18^oC or 8 hour long windows at 25^oC, 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 25^oC 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.

2.2.2. 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.

2.2.3. Outcrosses for Negative Autoregulation.  In these experiments to prove the negative autoregulation of ovo three types of mutation groups were examined (Table 2.3).  The first group was ovo locus point mutations that were tested to determined whether or not any region of the locus was involving in negative autoregulation.  Four strains of mutant ovo, ovo^D1rS1/FM3, y w ovo^D1rS1 v24/FM6/y+ Y, which are spontaneous mutations, ovo^D1rG2/FM7c, ovo^D1rG3/FM7c, which are gamma irradiations from spontaneous mutations, and one svb mutant (svb- ovo +) svb ^YP17B/FM7c, null of svb, were used.  (Table 2.3).  In the second group deficiency lines due to deletions Df (1) JC70 and Df(1)RC-40, which are null for ovo and snf, and DF(1)A113, which takes out several genes including ovo, were used.  The third group strains were chosen to test downstream genes, such as Sxl and snf, via point null point mutations of these genes.  Thus, snf^e8H/FM7a, snf¹⁶²¹/FM7c, and w cm Sxl ^7B0) / FM7cwere examined.  All of the tree groups were outcrossed as it is described in basic experimental design for regular stocks outcrosses (Figure 2.2).  Except for snf^e8H which is kept as a homozygous (snf^e8H / snf ^e8H) in the stock one extra outcross is carried to produce snf^e8H / FM7a stock for my study purposes.  Thus to create balancer carrying stock virgin females of homozygous snf^e8H had mated with FM7a males, as a result snf^e8H / FM7a stock established.

2.2.4. Outcrosses for ovo Gene Dosage Analysis.    The X:A ratio primary sex determination signal is required in both soma and germline sex determinations.  In germline sex determination ovo receives the X:A signal and responses to the number of X chromosome in the genome for proper female germline sex determination and differentiation.  Moreover, ovo plays a role in transcription by producing zinc finger protein.  Then, the question is how increasing copy number of ovo either in X chromosome or in the autosome would interfere with the ovo’s function and negative regulation character.

Therefore sets of outcrosses were prepared to test these effects by use of Tf(1)OW-10kb-17B (Tf(1) in short) and Tf(A)OW-10kb-13B (Tf(A) in short) stocks (Garfinkel et al., 1992).  Both contain P element mediated transformed of 10kb ovo- with white minigenes as a cell marker, but the difference is the location of the insertion.

 

ovo Dose Effects in X-Chromosome.  Since there is the negative regulation of ovo in the genome of ovo (Sag-Ozkol et al., 1997), what is the effect of having extra dose of ovo in the genome has decided to be examined Oliver et al., 1994.  Also, considering the fact that ovo is counting the number of X chromosomes, presumably, gene functioning as numerator elements, and itself, increasing the number of ovo more than two copies in the genome would yield similar negative autoregulation effect, too.

Therefore, appropriate outcrosses were designed to ascertain the regulation of ovo.  First, two copies versus three copies of ovo carrying lines were established (Fig 2.5) in two generation outcrosses.  In G0, generation zero, homozygous Tf(1)OW10.0-17B virgin females were mated to FM7a males to get heterozygous females bearing Tf(1)OW10.0-17B Oliver et al, 1994 balanced with FM7a in the F1.  These F1 females then outcrosses to males homozygous for ovo::LacZ reporter constructs, ovo3U21 and ovo4B8.  Control group, carrying two copies of ovo, (FM7a w; ovolacZ /+) gene expression activity was compared to of experimental group, carrying three copies of ovo, (Tf(1) / w; ovo:lacZ /+).

The Tf(1)OW10.0-17B second set of outcrosses is designed to examine the loss of ovo with complete ovo insertion (Fig 2.6).  Therefore, first Tf(1)/w; P[ovo::LacZ];+ (from first test, above) and FM7a/w; P[ovo::LacZ] (F1 of G0) stocks were created.  Then outcross between these stocks were made to generate Tf(1)/FM7a; P[ovo::LacZ]/P[ovo::LacZ] (f1 of G1) line.  Finally, selected F1 to G1 virgin females were mated with ovo^D1rS1 males to establish one copy versus two copies of ovo in the F1 of G2.  Then these flies were tested for their gene activity.  In this case, control group has one copy number of ovo, and experimental group has two copies of ovo (figure 2.6).  In third type of outcrosses genes, snf and Sxl, place downstream of ovo in the germline sex determination hierarchy were evaluated (Fig 2.6).  The same methods of basic regular outcross design (Fig 2.3) were applied.

ovo Dose Effect in Autosome.  The effect of increased ovo+ copy number was also analyzed using an autosomal source ovo+ gene insertion.  P element transformed line, Tf(A)OW10.0-17B (Garfinkel et al., 1992).  At the start of this work the insertion was localized.  Therefore, two rounds of outcrosses were designed to identify the chromosome carrying insertion whether in second or fourth chromosome (Fig2.7).  Standard second chromosome and third chromosome balancer stocks were used; X-chromosome location had been ruled out by early segregation data (e.g. Garfinkel, 1991, unpublished).  In these crosses, an assigned location to chromosome four can be inferred.  Chromosomes were chosen appropriate to insertion place for selection of control and experimental class (Fig 2.7).  After collecting yellow eyed balancer marker, like curly, showing phenotypic class (y w-; Bal.  Tf(1);lacZ-) of female flies, they had mated to males homozygous for ovo::LacZ insertion.  These flies were analyzed for their effect of gene activity.

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 37^oC.  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 previous study (X-chromosome Screening), about 70% of the euchromatic X-chromosome was screened, using 56 different deficiency strains, to identify transregulation of ovo. 

 

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.

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: 1. ovo point mutations,  more defined deficiency strain, and 2. downstream genes, 3. gene dose and origin.

Negative Autoregulation

Table: Stocks for Negative Autoregulation of ovo (1998),

1. ovo point mutations

Deficiency screen identified the ovo region itself as a having negative effects on ovo promoter activity.  Ovo showed significant depressant effect, negative autoregulation, according to preliminary data results (Sag-Ozkol, et al. 1997), along with, in our lab it was shown that ovo protein binds ovo promoter in vitro (Garfinkel and Lee, 1997).  Furthermore, other data are also mimic the ovo autoregulation includes females containing two copies of the ovo^D1 transgene, or those containing one recessive allele at the ovo locus, were as sterile as ovo ^D1 females, which (Mevel-Ninio et al., 1994).  In addition, it is suggested that two transcripts of ovo, a, and b, are regulating itself for their expression at different times of the development (Mevel-Ninio et., 1996).  These data imply that transcription factor function of ovo is also tittering its gene activity in the germline sex determination of D. melanogaster.  Therefore, ovo locus was tested for the negative regulation as well as the presence of genes where trans-acting repressor effect which are found to be downstream of ovo to ascertain that ovo is overting itself and genes reside downstream of ovo to ascertain that ovo is overting itself and genes reside downstream of ovo.

 

Deficiencies in the ovo locus.  In addition to the previously described experiments with Df(1)JC70, and Df(1)A113 a third deficiency, Df(1)RC40, was also used [Tables 3.3. and 3.4].  The new deficiency is smaller, as it has breakpoints at ovo and snf, thus, it was used to better localize the negative autoregulation Df(1)RC40 effect.  Ovo deficient lines due to deletions that remove ovo along with the other sets of genes have been examined.  According to the gene of interest in this study, Df(1)JC70, and Df(1)RC40 (4D-F) remove both ovo and snf, but Df(1)A113 only removes ovo, but these deletions also take out several other genes in that deleted region.  All three of deficiency lines in the heterozygous Df / + (experimental) are significantly different from the controls (+/Balancer), that is, negative autoregulation of ovo was supported.Table: Stocks for Negative Autoregulation of ovo (1998)

 

ovo region point mutations.  In these preliminary experiments ovo ^D1rS1 had been used.  This strain has an 5.8 kb insertion at +4.2 kb of ovo region and produces svb+ ovo- putative null mutant strain, (LOF) mutation, that homozygous mutants cannot produce germ cells and gives sterile females.  ovo^D1rS1/FM3 strain is outcrossed and tissues from F1 progeny were examined with b-gal assay (Fig. 3.1).  This graph shows the results of ovo mutant dose on ovo::LacZ reporter activity.  In both reporter constructs enzyme activity of controls showed about two-times higher than that of experimentals.  Differences between LacZ activities of the constructs may depend on either position effect of the P[ovo::LacZ] insertion onto chromosome or better translation product due to 200 bp longer N-terminal of ovo3U21 construct.Table: Stocks for Negative Autoregulation of ovo (1998)

 

2. Point Mutations for genes Downstream of ovo.  Candidate downstream genes were tested via point mutations,snf^eH8, snf¹⁶²¹, and w cm Sxl ^7B0.

It was important to test snf point mutations since Df(1)JC70 and Df(1)RC40 takes out ovo and svb as well as several additional genes including snf.  Two, gain of function snf point mutations were used: snf¹⁶²¹, is an arginine-histidine missense mutation at codon 49 in RNA reading motif (R49H in RRM1), and snfe8H, is an threonine-proline missense mutation at codon 97 in RNA reading frame motif (T97P) (Salz and Flicker, 1996).  As is seen in Tables 3.3 and 3.4, these two mutations have surprisingly different effects, one of which gave evidence of being a trans-acting repressor, contrast to predictions.  snf^e8H shows 20% and 30% decrease in gene activity, with ovo3U21 and ovo4B8, respectively.  However, snf¹⁶²¹ had no significant effect on ovo::LacZ activity with either construct.  These results do not correlate simply with Salz and Flicker, 1994.  Flicker and Salz 1996 that snf ^e8H is a weaker allele than snf¹⁶²¹ (example: snf^e8H can be kept as a homozygous stock, but snf¹⁶²¹ must be kept heterozygous over a balancer chromosome. Table: Stocks for Negative Autoregulation of ovo (1998)

Sxl^7B0  ,which is a molecular-null for all of Sxl (Salz et al., 1987) had no transregulation effect on ovo expression when assayed with either construct strains.  This is consisted with other genetic data (Oliver et al., 1993) that showed ovo to be upstream of Sxl in a germline regulatory hierarchy.

 

3. Gene dose and origin: Effects of Increasing ovo+ Gene Dose

 

Negative autoregulation of ovo as seen with deficiency and point mutations that reduce ovo+ function, predicts that extra doses of ovo+ (introduced by P[w+ ovo+] ) would have an overall repressing effect on ovo repressor activity.  Crosses and soluble extract assays to determine this were performed, and described below.Table: Stocks for Negative Autoregulation of ovo (1998),

 

 insertion of ovo+ Gene onto X-Chromosome with Tf(1)OW-10.0-17B.  Additional ovo into the genome is decreasing the viability of organism as it is seen in this study, therefore keeping the stock for these strains were not easy at 25^oC.  Also, as it is seen in summary tables (Table 4 and Table 5) of gene dose assay increasing the number of ovo is causing high ovo protein production in the experimental strains, hence a relatively large amount of enzyme is processed per time.  At the same token, decreasing number of ovo in the genome has caused an increase in the gene activity in the experimentals.

In this study, it was found that wild type ovo expression is counting the number of ovo in the germline and if the number is low or higher than two repressing its gene expression.  In Table 3.5 and 3.5, transformed ovo insertion showed negative autoregulation in all the outcrosses.  First positive internal control outcrosses were made and they showed about the same level of enzyme activity in both transformed insertion lines, Tf(1), 0.043± 0.006, and Tf(A), 0.038± 0.003.

Two types of outcrosses were designed to identify relation between number of ovo copies and ovo promoter expression.  In first set, two copies of ovo was compared to thee copies of ovo and found that there is about 24% and 20% (E/C) increase in the significant enzyme activity.  In the second set, genotypically recessive ovo mutation carrying males were crossed to heterozygous female genotype transformed ovo insertion with balancer.  Collected transgenic female progeny was tested for the ovo gene dose effect.  One dose of ovo in ovo^D1rS1 /FM3, controls, causes about two fold low enzyme activity than that of two doses of ovo in the experimental genotype Tf(1)/ovo^D1rS1.  Furthermore comparison of E/C ratios between 1 vs.2, and 3 vs. 2 doses of ovo showed bout 2.2 fold, (270%/124% of E/C) in ovo3U21, and 1.5, fold, (176% /120% E/C) in ovo4B8, increase in repressing activity of ovo.  In the ovo4B8 construct controls of the one dose versus two dose heterozygous females have high standard error of mean that may effect the level of repression.  The lower dose becomes the higher the negative autoregulation activity results.

 

 Parental Origin Effect of Tf(1) OW-13B.

 

  In addition, whether it is from maternal or paternal origin of ovo insertion has many affect on gene activity of ovo was tested (Table 3.5 and Table 3.6),

 

since ovo gene expression is female germline sex specific in germline sex determination of Drosophila melanogaster. Although in both outcrosses there is repressing effect, the paternal origin of ovo insertion of ovo cause 1.2 fold higher repression gene activity. 

 

Insertion of ovo Gene onto Autosomal Chromosome with Tf(A)OW10.0-13B.  In this study, the effect of ovo insertion onto autosome was also measured to ascertain the information gathered from X-chromosome insertion of ovo.  The results of these experiments showed that elevation of ovo copies in the genome is also tittering itself and causing high ovo protein is produced in the experimental lines.  Therefore, it is also concluded from the insertion data that ovo expression is independent from the location of insertion but dependent on number of ovo in the genome.  Autosomal insertion also showed similar repressing effect, 54%, and 26%, as insertion of ovo in X-chromosome in heterozygous deficiency female progenies.

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.  This discontinuity in the linear correlation may be due to position effect of P[w+ ovo+]. 

DISCUSSION

The goal of this study was to find transcriptional trans-regulation of the ovo germline promoters and to identify candidate germline X-chromosome numerator elements.  We surveyed approximately 70% of the X-chromosome using 45 deficiency strains, assessed the negative autoregulation of ovo with ten deficiency or recessive-mutant strains; and performed a complementary gene dose analysis of ovo+ with two P element insertion strains.  In all cases our assay for ovo germline promoter activity relied on measuring ovo::lacZ reported expression in dissected ovaries.

ovo Negative Autoregulation

Table 3.3. Negative Autoregulation of ovo Results Obtained w/ovo3U21

Table: Negative Autoregulation of ovo Results Obtained (result) with/ovo4B8

Table: Gene Dose Assay with ovo3U21 Construct (1998) (result)

Table: Gene Dose Assay with ovo4B8 Construct (1998). (result)

 

One of the key findings of the X-chromosome screen was the observation that deficiencies that removed the chromosomal interval harboring ovo+ results in a marked increase in the b-galactosidase specific activity of experimental females carrying the ovo: lacZ reporter constructs.  This suggested the possibility that one of the functions of the ovo+ gene is to down-regulate its own expression.  The ovo genetic function is derived from two germline-specific promoters that generate 5-kb mRNAs translatable into a nested pair of proteins, the 1028-aa-long OvoB isoforms and the 1400-aa-long OvoA isoforms (Garfinkel et al., 1994; Mevel-Ninio et al., 1995, 1996).  Both the OvoA and OvoB proteins contain four zinc finger motifs at the carboxy terminus (Garfinkel et al., 1994; Mevel-Ninio et al., 1991, 1995, 1996).  Their amino-terminal domains contain homopolymeric runs of alanine, histidine, aspargine, typical of many transcription factors.  Acidic patches of the sort that the play a role in protein-protein interactions and transcriptional activation are present; OvoB has three acidic regions (Garfinkel et al., 1994) and OvoA has four (Mevel-Ninio et al., 1995).  Bacterially expressed Ovo zinc finger domain is capable of binding a variety of germline-specific promoters in vitro, including the ovo germline promoter region (Garfinkel et al., 1997; Lee, 1998; Lee and Garfinkel, 1998; Lu et al., 1998).  In vivo experiments suggest that the OvoA protein isoform may down-regulate the promoter that generates OvoB protein during the late stages of oogenesis (Mevel-Ninio et al., 1996), and that mutations causing heterochronic production of OvoA-like proteins may result in a heterochronic reduction in OvoB protein synthesis (Mevel-Ninio et al., 1996).  Furthermore, increasing ovo+ gene dose seems to activate ovo::lacZ reporter genes in testes (Oliver et al., 194; Lu et al., 1998).  Thus, both in vitro biochemical data and in vivo genetic data support the idea that ovo regulates its own expression.

To corroborate our findings obtained with deficiencies, we repeated the outcrosses using ovo::lacZ reporter constructs and strains carrying each of several point mutations affecting the svb-ovo gene region.  Every mutation we tested-two gamma-ray-induced svb-ovo- mutations, one spontaneously arisen svb+ovo- mutation in two different chromosomal backgrounds, and one EMS-induced svb-ovo+ mutation-recapitulated the effects seen with Df(1)A113, DF(1)RC-40, and Df(1)JC70.  The results with the gamma-ray-induced svb-ovo- mutations and the svb+ovo- mutation were expected, since all three represent putative null alleles for the germline-specific ovo function.  Unexpected was the finding that the EMS-induced svb- ovo+ mutation also affected the ovo::LacZ reporter constructs.  This was surprising, because svb mutations have no phenotype in germline clones, and thus they were believed to have no effect on oogenesis.

If deficiencies and point mutations that eliminate ovo function cause a depression of ovo::lacZ reporter activity, one would predict that P element transgenes carrying extra copies of ovo+ would have the opposite effect, namely the reduction of ovo::lacZ reporter activity.  Using of different P element insertions of a single w+ minigenes / ovo+ construct (Garfinkel et al, 1992), we performed crosses that generated progeny flies carrying one copy of the ovo::lacZ reporter in combination with one or two ovo+ copies, and in combination with two or three copies.  We anticipated that the three-dose progeny would have less b-galactosidase activity than the two-dose sibling progeny, and that other two-dose progeny would have less than their one-dose siblings.  This was not the case.  One possibility is that the two independent insertions of the ovo+ transgene are subject to different chromosomal position effects and that neither produces precisely one “unit” of ovo function.  Another possible cause of non-linear gene-dose-response is the summation of both positive and negative autoregulatory effects.

Effects of Downstream Genes on Expression of ovo::lacZ Reporters

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

 

The previously proposed germline sex determination hierarchy (Pauli et al., 1993; Oliver et al., 1993) laced Sxl and snf downstream of both ovo and otu.  This was based on weakening of the Sxl autoregulatory loop in female germline cells carrying mutations in ovo or otu, and the restoration of proper Sxl autoregulation by dominant Sxl ^M#1 mutation.  In contrast to its role in somatic sex determination, Sxl does not act as a binary switch gene for sexual identity in the germline, rather it is thought to control a variety of female-specific germline differentiation and cell proliferation activities (Bopp et al., 1993, 1995; Despande et al., 1996; Hager and Cline a997; Horabin et al., 1995, 1997).  Placement of Sxl downstream of ovo was corroborated by our results that showed no significant effect of cytologically visible deficiencies that remove the Sxl region on the expression of the ovo::lacZ transgenes.  Likewise, a molecular-null for Sxl also had no effect.

The snf gene, despite its original detection as a female-sterile mutation that had dominant-lethal and dominant-sterile synergistic interactions with Sxl (Steinmann-Zwicky and Nothiger, 1985; Steinmann-Zwicky 1988; Oliver et al., 1988, 1990), is not strictly speaking a “sex determination” gene.  While the mutant phenotype of certain missense snf alleles is a breakdown in the efficiency of Sxl autoregulation (e.g., Salz, 1992), both the null phenotype (lethality to both male and female embryos) and the protein contains two tandem copies of the so-called RRM (RNA recognition motif), and is the functional equivalent of mammalian U1A snRNP.  The two characterized snf point mutations are both amino acid substitutions: snf¹⁶²¹replaces arginne-49 with histidine in the amino-terminal RRM, and snf^e8H replaces threonine-97 with proline (Flickinger and Salz, 1994).  The snf1621 mutation is predicted from structural studies of the mammalian U1A protein (Nagai et al., 1990; Jessen et al., Howe et al., 1994; Oubridge et al., 1994) to destabilize the RRM and to weaken its binding to U1A snRNA.  The snf^e8H mutation, in contrast, maps to a loop structure that is less highly conserved.  Consistent with the predicted structural properties of the mutant alleles, snf¹⁶²¹ is the stronger of the two mutations in the Sxl lethal/synergy assay (Oliver et al., 1988, 1990; Salz, 1992; Salz and Flickinger, 1996).  Paradoxically, the snf¹⁶²¹ mutation had no effect on the ovo::lacZ reporter constructs while the snf^e8H mutation did.  One possible explanation is that the two snf mutations differentially affect the protein’s ability to stimulate splicing of generic intervening sequences, the unregulated type such as appears in the ovo pre-mRNA fragment fused to the LacZ reporter gene.

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:  The experiments were completed in the laboratory of Dr. Mark D. Garfinkel Department of Biological Chemical and Physical Sciences of  Illinois Institute of Technology at Chicago.  Dr. Demet Sag was supported by the Turkish National Merit Fellowship.

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Figures and Tables:

Figure: Sex determination of D. melanogaster (1998)

Figure: Somatic-Germline Interactions. (1998)

Figure: Molecular Structure of the ovo locus

Figure: In vivo Biochemical_genetic Assay for Regulators

Figure: ovo-LacZ Reporter Construction. (1998)

Figure: Establishing Stocks From Duplication Carrying Lines.

Figure: Two versus three copies of ovo in the genome.

Figure: Locating the Autosomal Insertion of ovo. (1998)

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

Table: Stocks for Negative Autoregulation of ovo (1998)

Results:

 

Table1: Negative Autoregulation of ovo Results Obtained with ovo3U21

Table 2: Negative Autoregulation of ovo Results Obtained with 4B28 (result)

Table 3: Gene Dose Assay with ovo3U21 Construct (1998) (results)

Table 4: Gene Dose Assay with ovo4B8 Construct (1998). (results)