Pre-Sertoli Specific Gene Expression Profiling Reveals Differential Expression of Ppt1 and Brd3 Genes Within the Mouse Genital Ridge at the Time of Sex Determination1

Abstract In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.


INTRODUCTION
The SRY gene was identified more than 14 yr ago and is considered the dominant positive genetic factor respon- sible for initiating testis development in eutherian mammals [1][2][3]. It is generally accepted that Sry triggers the differentiation of Sertoli cells from a cell population that would otherwise become follicle cells. Surprisingly, the molecular mechanisms by which SRY accomplishes this remain enigmatic. In the mouse, Sry expression within the genital ridge is first detected at 10.5 days postcoitum (dpc); expression peaks at 11.5 dpc and ends at 12.5 dpc [4,5]. Lineage tracing experiments demonstrate that Sry is expressed within pre-Sertoli cells [6,7]. Pre-Sertoli cells induce cell proliferation, vascularization, and testis cord formation within the developing testis [8,9] as well as the migration of myoid, endothelial, and interstitial precursor cells into the gonads from the underlying mesonephros [10,11]. Sertoli cells provide morphogenic signaling to other cell types, inducing steroidogenic precursor cells to differentiate into Leydig cells, causing germ cells to enter mitotic arrest, and resulting in the elimination of paramesonephric duct cells via apoptotis.
Most of the genes currently known to play a role in the process of sex determination and differentiation have been identified from human and mouse models. Studying human patients showing sex reversal or dysgenesis of the gonads has revealed roles for genes such as Sry itself, and Sox9, Dmrt1, Dax1, Wt1 [1,[12][13][14][15]. Phenotypic anomalies in sex determination observed in knockout mouse models have further implicated genes such as M33, Lhx9, and Sf1 [16][17][18]. One gene, Dmrt1, was identified via a combination of homology with Drosophila and Caenorhabditis elegans genes [19] as well as human dysgenesis of the gonads [20]. More recently, to identify additional candidate genes as well as signaling pathways implicated in these processes, differential expression-based screening approaches were undertaken by several labs [21][22][23][24][25]. Although procedures varied somewhat, all of the screens reported to date have in common the tissue level comparison of developing testis with developing ovaries at a given developmental date.
To characterize in more detail the expression profile of pre-Sertoli cells at the beginning of sex differentiation as well as to identify new candidate genes involved in sex determination and differentiation, we used a transgenic mouse model expressing green fluorescent protein (GFP) under the control of 4.5 (kilobases) kb of the pig SRY 5Ј flanking region (pSRYp-GFP) [7] to isolate and purify pre-Sertoli cells via fluorescence. RNA from 12.0 to 12.5 dpc pre-Sertoli cells was subtracted against nonfluorescent cells from the male genital ridge using suppression subtraction hybridization (SSH). Gene sequencing followed by wholemount in situ hybridization (WISH) on selected clones al-821 PRE-SERTOLI CELL GENE EXPRESSION PROFILING lowed us to identify new candidate genes involved in mammalian sex determination and differentiation and to confirm the utility of using transgenically labeled cell populations for SSH applications in development.

Transgenic Mice, Cell Collection, and RNA Isolation
The cloning and characterization of 4.5 kb of pig SRY 5Ј flanking sequences and the generation of pSRYp-GFP transgenic mice have been described previously [7,26]. Mice were housed and handled according to guidelines established by the Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science) and according to the standards of the institution, and protocols were approved by the institutional ethics committee on use of animals in research. For staging of embryonic development, noon on the day of vaginal plug detection was designated as Day 0.5 postcoitum (0.5 dpc). To isolate male genital ridge cells from 12.0-12.5 dpc embryos, timed matings were performed. Genital ridges were dissected, male ridges were identified via fluorescence, and mesonephroi removed. The male genital ridges were then pooled and digested for 30 min at 37ЊC in M2 media (Sigma, St. Louis, MO) supplemented with collagenase (50U ml Ϫ1 ) (Gibco, Canadian Life Technologies, Burlington, ON, Canada) and dispase (2.4U ml Ϫ1 ) (Gibco). Fluorescent cells (pre-Sertoli) and nonfluorescent cells (remainder of genital ridge) were separated using fluorescent-activated cell sorting (FACS) isolation on a FACSTAR-Plus machine (Becton Dickinson, Mountain View, CA) equipped with an EGFP filter. In total 21 400 fluorescent cells and 22 000 nonfluorescent cells were recovered. Total RNA was extracted using RNeasy total RNA extraction kit (Qiagen, Mississauga, ON, Canada). The concentration of total RNA was quantified by measurement of optical density at 260 nm and was deemed sufficient to continue with the procedure (data not shown).

Suppression Subtractive Hybridization (SSH)
The SSH procedure was performed essentially as previously described [27], with changes appropriate to the mouse model system as described herein. SSH was used to compare gene expression between fluorescent and nonfluorescent cells of 12.0-12.5 dpc pSRYp-GFP transgenic mouse testis. To generate sufficient amounts of double-stranded cDNA for SSH, fluorescent and nonfluorescent cDNAs were amplified separately using the SMART polymerase chain reaction (PCR) cDNA synthesis kit according to the manufacturers' instructions (BD Biosciences Clontech, Mississauga, ON, Canada). To generate the first strand cDNA, 0.3 g of total RNA from fluorescent and nonfluorescent cells was reverse-transcribed with an oligo-dT30 primer (CDS: 5Ј-AAGCAGTGGTAACAACGCAGAG-TACT (30) (A/C/G/T)(A/G/C)-3Ј) using PowerScript reverse transcriptase (BD Biosciences Clontech). Second cDNA strands were produced with the SMART II 5Ј-anchored oligo, followed by PCR amplification for 21 cycles using Advantage 2 DNA polymerase (BD Biosciences Clontech). The final PCR products consisted of pools of cDNA fragments in the range of 300 base pairs (bp) to 4 kb. The fluorescent cell cDNAs (F) were subtracted against the nonfluorescent cell cDNAs (NF) using PCR-Select cDNA subtraction technology according to the provided protocol (user manual: PT1117-1; BD Biosciences Clontech), to give the (F-NF) subtraction cDNA pool.

Cloning of Subtracted cDNAs
Sequences from the (F-NF) genital ridge cell subtracted cDNA pool were cloned into the pT-Adv plasmid (BD Biosciences Clontech), to construct the fluorescent minus nonfluorescent (F-NF) subtracted cDNA li-brary. Ligated DNA was used to transform competent TOP10FЈ Escherichia coli, which was plated onto X-Gal/LB agar containing kanamycin (40 g/ml). Individual colonies were transferred into 96-well plates, grown in LB freezing media (8.8% glycerol, 55 mM K 2 HPO 4 , 1 mM MgSO 4 , 15 mM NH 4 (SO 4 ), 26 mM KH 2 PO 4 ) for 14 h at 37ЊC and then frozen at Ϫ70ЊC.

Differential Hybridization Screening
Differential hybridization screening was performed following previously described methodologies [27]. The (F-NF) genital ridge cell cDNA library was used to established macroarrays for differential screening. The insert of each cDNA clone was amplified in 96-well plates by PCR (25 cycles) using the PCR-nested primers 1 and 2R as supplied by the manufacturer (Clontech) and AmpliTaq DNA polymerase (Roche Molecular Systems Inc, Laval, PQ, Canada). To establish the cDNA macroarrays, an aliquot of each amplification product was denatured in 0.3 M NaOH with 5% bromophemol blue, and 10 l was vacuum transferred onto a nylon membrane (Hybond-N ϩ , Amersham Pharmacia Biotech, Baie d'Urfé, PQ, Canada) using a 96-well dot-blot apparatus. Membranes were then exposed to 150 mJ ultraviolet light to perform DNA cross-linking (Gs Gene Linker, Bio-Rad, Mississauga, ON, Canada). Control cDNAs (Gapdh, GFP) were transferred onto the microarrays. For each 96-well plate, three identical cDNA microarray replicate membranes were generated.
The (F-NF), (F), and (NF) cDNA pools were used to generate complex hybridization probes for differential screening of microarrays of the (F-NF) genital ridge cell cDNA library. The cDNA pools were first amplified by performing the secondary nested PCR, and products were purified using Qiaquick PCR purification kit (Qiagen). To prevent nonspecific interaction of the probes to cDNAs on microarrays during hybridization, the adaptors were removed by three successive digestions with AfaI, SmaI, and EagI. The cDNA mixtures were again purified (Qiaquick PCR purification kit; Qiagen) and 75-100 ng was labeled with 32 P-dCTP via random priming (Megaprime DNA Labeling System; Amersham Pharmacia Biotech). The radioactive probes were purified (QIAquick Nucleotide Removal kit; Qiagen) and quantified using a beta-counter.
The cDNA microarray membranes were incubated for 4 h at 71ЊC in 20 ml of prehybridization solution (600 mM NaCl, 120 mM Tris, pH 7.4, 4 mM EDTA, 0.1% Na 4 P 2 O 7 , 0.2% SDS, and 500g/ml heparin), to which heat denatured (5 min, 100ЊC) oligos were added to prevent nonspecific binding. These oligos included 1 mM each of PCR-Nested 1, PCR-Nested 2R, PCR-Nested 1 INV (5Ј-ACCTGCCCGGGCGGCCGCTCGA-3Ј), PCR-Nested 2R-INV (5Ј-ACCTCGGCCGCGACCACGCT-3Ј), as well as 2 mM poly dAdT (Amersham Pharmacia Biotech). Equal counts-per-minute amounts of each of the three heat-denatured cDNA probes were added to separate 15-ml aliquots of hybridization solution and used to individually hybridize the replicated (F-NF) cell microarray membranes. The hybridization solution was identical to the prehybridization solution with the addition of 15 g salmon sperm DNA and 10% dextran sulfate. The membranes were hybridized for 16 h at 71ЊC, then were washed for 15 min in 2ϫ saline sodium citrate (SSC) and 0.1% SDS followed by two 1-h washes in 0.1ϫ SSC and 0.1% SDS at 71ЊC. Membranes were exposed to a phosphor screen for 4 h and the images were digitized (Storm 840; Amersham Pharmacia Biotech). The differentially hybridizing cDNA clones were further characterized by DNA sequencing and WISH.

DNA Sequencing and Sequence Analysis
The cDNA clones identified as differentially expressed by the (F-NF) cell cDNA probe were amplified by PCR for 15 cycles with the PCRnested 1 and PCR-nested 2 oligos from the PCR product generated initially for the microarray analysis. The PCR product was purified (QIAquick; Qiagen) and verified by agarose gel analysis for the presence of a single cDNA band before proceeding with sequencing. Sequencing reactions were performed on cDNA clones via the dideoxy sequencing method (Big dye terminator 3.0; ABI prism; Applied Biosystems, PE, Branchburg, NJ) using the oligo PCR-Nested 1 (1 mM). Sequencing reactions were analyzed on an ABI Prism 310 sequencer (Applied Biosystems). Nucleic acid sequences were analyzed by BLAST (Basic Local Alignment Search Tool) against GenBank data banks (NR, EST, and mouse genome). A cDNA sequence was considered homologous to a GenBank sequence when at least 100 bp matched with an E probability value of less than e Ϫ10 .

Whole-Mount In Situ Hybridizations
The cDNA clones of the known genes that were differentially expressed in the SSH experiment were further characterized via WISH. To 822 BOYER ET AL. provide a standardized method for generating linear template DNA, clones were amplified by PCR using SP6-PCR-Nested1 (5Ј-TAATACGACT-CACTATAGGGAGCGTGGTCGCGGCCGAGGT-3Ј) and T7-PCR-Nest-ed2 (5Ј-ATTTAGGTGACACTATAGAATCGAGCGGCCGCCCGGGCA GGT-3Ј). The PCR products were used as templates to generate digoxigenin-labeled antisense riboprobes for WISH as previously described [28]. The PCR products were transcribed using Sp6 or T7 RNA polymerase as appropriate. Expression was analyzed in 11.5, 12.5, 13.5, and 14.5 dpc gonads, using two pairs of embryos of each sex per gene.

Depletion of Germ Cells by Busulfan
Two cDNA sequences were selected for an expression analysis in embryos lacking germ cells. These embryos were generated by injecting pregnant females with 40 mg/kg busulfan at 9.5 dpc and then harvesting embryos at 12.5-13.5 dpc [29]. As a control for this experiment, expression of the Chk1 gene, known to be expressed in germ cells but not in pre-Sertoli cells, was verified in germ cell-depleted embryos.

Isolation of Pre-Sertoli Cells
The pSRYp-GFP-positive embryos from timed matings were dissected at 12.0-12.5 dpc and fluorescent (pre-Sertoli) cells and nonfluorescent cells from the genital ridge were efficiently separated via FACS purification, with an efficiency of 98%. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on RNA from fluorescent cells to reveal the presence of endogenous Sry transcripts and the absence of Oct4 transcripts (data not shown), consistent with the fluorescent cells being of pre-Sertoli cell lineage and not of germ cell lineage as previously shown [7].

Production of Subtracted Libraries and Assessment of Normalization and Subtraction Efficiencies
To verify the efficiency of the SSH procedure, several control experiments were performed. To ensure that normalization of cDNAs had occurred, the quantity of the housekeeping gene Gapdh was assessed in subtracted (F-NF) and unsubtracted (F) cDNA pools. As expected, Gapdh abundance was decreased following subtraction (Fig. 1A). To assess the efficiency of enrichment of differentially expressed genes, the abundance of transcripts for GFP and Sox9 (pre-Sertoli cell expressed gene) was examined. Both GFP and Sox9 transcripts were enriched in the pre-Sertoli cell subtracted (F-NF) cDNA pools (Fig. 1, B and C). Interestingly, GFP but not Sox9 transcripts were still visible in PCR cycles taken from the unsubtracted cDNA pool. This can be explained by the PCR cycling conditions chosen and possibly also reflects relative differences in concentrations of message at the start of the procedure.
Once the subtraction efficiency was shown to be satis-823 PRE-SERTOLI CELL GENE EXPRESSION PROFILING factory, a differential hybridization screening procedure was performed on 658 bacterial colonies randomly selected from the (F-NF) subtracted cDNA library. The (F-NF) subtracted cDNA pool as well as (F) and (NF) nonsubtracted cDNA pools were used to generate three different hybridization probes for the differential screening of the selected clones spotted on three identical sets of microarrays. Representative differential screening results are illustrated in Figure 2. The differential screening procedure identified 154 cDNA clones as true positives, i.e., showing enhanced expression via the (F-NF) probe compared with the (F) probe. One hundred ten of these clones were analyzed by sequencing and 90 generated adequate sequencing results. which was identified 15 times. Among the known expressed genes identified was Sf1, a gene expressed in the genital ridge and implicated in sex determination/differentiation. In addition, Vanin-1, Vcp1, Sparc, and Aldh3a1 were identified, representing genes that were previously identified in screens of fetal testes versus ovaries [21,22,25].

Analysis of mRNA Expression
To verify that genes identified by SSH are differentially expressed between pre-Sertoli cells and the Leydig/endothelial and germ cell compartments and to test whether these genes are also differentially expressed between male and female embryos, WISH was performed on male and female embryos. Initial screening was carried out using 12.5-dpc embryos of both sexes for the 32 known genes. Most of these genes (23) were expressed in both sexes; 5 genes were overexpressed in testis; 1 gene was overex-pressed in ovary; and no expression was detected for 3 genes (Table 1). Representative examples of WISH results are shown in Figure 3 for the genes Catna, Sparc, Fdxr, Hmgcs2, and Rgs2. For the clones showing sex-specific or overexpression patterns in gonads of one sex, additional in situ experiments were performed at 11.5, 12.5, 13.5, and 14.5 dpc. We present here the expression patterns for Bromodomain-containing 3 (Brd3) (Fig. 4, A-D) and Palmitoyl-protein thioesterase 1 (Ppt1) (Fig. 4, E-H). At the detection level of the WISH procedure, Brd3 is ubiquitously expressed but is more strongly expressed in the testis compared with the ovary (see Fig. 4B) and to other tissues (data not shown). In contrast, Ppt1 was only detected within the developing testis. To further confirm that these genes are expressed in the pre-Sertoli cell population and not within germ cells, WISH was performed on embryos lacking germ cells after treating pregnant females Brd3 gene expression at 11.5, 12.5, 13.5, and 14.5 dpc, respectively. At 11.5 dpc, a comparable diffuse expression pattern was seen within both testes and ovaries but not within the mesonephros. By 12.5 dpc, strong expression was seen within the testicular cords, and this pattern continued until at least 14.5 dpc. Faint expression was still evident within the ovaries at 13.5 and 14.5 dpc. E-H) Ppt1 gene expression at 11.5, 12.5, 13.5, and 14.5 dpc, respectively. At 11.5 dpc, faint expression was detected within the genital ridges of both sexes. By 12.5 dpc, strong expression of Ppt1 was seen within the testicular cords of the male genital ridge; this pattern continued until at least 14.5 dpc. Expression of Ppt1 was no longer evident within the ovary by 14.5 dpc. Testes (t) are presented on the left and ovaries (o) are presented on the right. Magnification ϫ32.
with busulfan (Fig. 5). Expression of Brd3 and Ppt1 was not diminished within busulfan-treated compared with nontreated testes.

DISCUSSION
In mammals, Sertoli cells are thought to be the first cell lines of the genital ridge to commit to the male differentiation pathway and additionally to recruit other cell types to the male pathway via paracrine and contact signaling. Characterization of the specific subset of genes differentially expressed in pre-Sertoli cells compared with the other cell types present within the genital ridge at the moment of sex determination should provide further understanding of the molecular processes involved in sex determination and sex differentiation. To further study these processes, a mouse transgenic line expressing GFP under the control of 4.5 kb of pig Sry promoter [7] was used to isolate pre-Sertoli cells at 12.0-12.5 dpc. These cells were purified by FACS, and a SSH screen was performed to identify genes that were differentially expressed in fluorescent cells (F) compared with nonfluorescent cells (NF).
Screening the (F-NF) subtracted cDNA library by membrane hybridization allowed the identification of 32 known gene candidates, the analysis of which is presented herein. These genes included Sf1, known for its role in sex determination/differentiation, as well as Vanin-1, Sparc, Vcp1, and Aldh3a1, four genes previously identified in published screens for genes overexpressed in developing testis compared with ovary [21,22,25]. Furthermore, we confirmed by RT-PCR that Sry is expressed in fluorescent cells and that Sox9 is overexpressed within the (F-NF) subtracted cDNA pool compared with the (F) nonsubtracted cDNA pool. The identification of these genes provides an important confirmation of the biological validity of our current model. Other genes known to be implicated in sex deter-mination, such as Wt1, Dax1, Lhx9, or Gata4 were not identified in our current hybridization screen, and although Sry and Sox9 were subsequently identified by RT-PCR, they were not identified within the hybridization screen. This is possibly due to the relatively small sample size of clones screened as well as the potentially large complexity and low redundancy of the cDNA population of the pre-Sertoli cell. These observations are consistent with the findings of other reported genital ridge differential screens [21,22,25] but are in contrast with expression profiling experiments reported for other tissues such as granulosa cells of the developing bovine follicle [27].
The known genes identified by SSH were further characterized via WISH to determine differential expression patterns within the developing testis and also between developing testis and ovary. A high proportion of the genes tested by WISH (21/32) were expressed in both developing testis and ovary. This is not surprising due to the common origin of Sertoli and granulosa cells [6] and the fact that our subtraction was not between testicular and ovarian cells. This included genes known to be implicated with Sertoli cells, such as Cftr, implicated in nutrition of spermatozoids [30] and Gatm involved in creatine biosynthesis. In addition, genes involved with energy metabolism were identified, including Ptpn1, which acts as a negative regulator of insulin signaling. Recently, it was demonstrated that insulin receptor family members are needed for testes determination [31]. The fact that, for three genes, there was no WISH signal detected could be explained by the moderate sensitivity of the WISH method or could simply be due to technical failure.
For the six genes showing a differential expression between testes and ovaries, Sf1, Vanin-1, and Ppt1 were only expressed in pre-Sertoli cells, Brd3 and Sel1L were more strongly expressed in pre-Sertoli cells compared with in-826 BOYER ET AL.
FIG. 5. Effect of busulfan treatment on WISH expression of Brd3 and Ppt1 genes (from pre-Sertoli cell expression profiling screen) and of Chk1 gene (a germ cell marker) within mouse fetal testes between 12.5 and 13.5 dpc. Left testis is from a normal embryo; right testis is from a busulfan-exposed embryo. For the Brd3 and Ppt1 genes, expression was equivalent in the control and the busulfan-treated testes, suggesting that expression is within the pre-Sertoli cell compartment and not within the germ cell compartment. Expression of the Chk1 gene, known to be expressed within germ cells but not within pre-Sertoli cells, is reduced (but not absent) in the busulfan-treated testes compared with control (untreated) testes. Magnification ϫ32. terstitial cells, while Catna1 and Sparc were expressed equally in pre-Sertoli cells and interstitial cells. Two genes, Fdxr and Hmgcs2, were expressed in both testicular and ovarian genital ridges. One gene, Rgs2, was found to be overexpressed in ovaries compared with testes at 12.5 dpc but on closer examination was observed to be downregulated in ovaries at 13.5 dpc, underscoring the fact that sex differentiation occurs later in ovaries than in testis.
Ppt1 and Brd3 were selected for more detailed characterization of genital ridge expression. Ppt1 encodes a soluble lysosomal enzyme that hydrolyzes the thioester bond that attaches long-chain fatty acids, mostly palmitate, to the cysteine residue of S-acylated proteins [32,33]. Palmitoylation has been shown to be important in different cellular processes, including neural cell differentiation (review: [34]) and possibly in insulin-regulated cellular functions [35]. Recently, nonlysosomal roles for Ppt1 were described in synaptosomes [36] and during apoptosis [37]. It was previously reported that Ppt1 is ubiquitously expressed in adult tissue, with increased expression within the testis [32,38], while in Drosophila, Ppt1 shows testicular expression levels five time that of other tissues [39]. Here, we report that, within the mouse genital ridge, Ppt1 was only expressed in pre-Sertoli cells of the developing testis via SSH and WISH but was not seen in developing ovary via WISH. These observations suggest that Ppt1 may be involved in mammalian sex determination and differentiation.
Brd3 is a potential transcription factor containing two bromodomains. Members of this family include Brd2, a gene that is strongly expressed in ovary and testis (both Sertoli and germ cells) [40] and BrdT [41], a gene only expressed in the germ cells of the testis. Significantly, this family of genes is related to the female sterile homeotic protein gene in Drosophila, a gene required maternally for proper expression of other homeotic genes, such as Ubx, which is involved in pattern formation. Ubiquitous expression of UBX protein within the mesoderm in Drosophila results in the formation of ectopic gonad tissue anterior to the normal location of the gonads [42]. We now provide evidence that Brd3 may play a role in testis formation in mammals. Identification of Brd3 expression within the male genital ridge, with increased expression within pre-Sertoli cells compared with other cell types, provides evidence that Brd3 may play a role in testis formation in mammals and may help to define the developmental tissue code required for testis formation. Further studies are now required to either confirm or refute these ideas.
In summary, we have combined fluorescent marking of cell populations in transgenic models with differential expression screening to study gene expression in pre-Sertoli cells of the mammalian genital ridge. Using these methods, we have identified new candidate genes, including Ppt1 and Brd3, the genital ridge expression patterns of which suggest they may be involved in the processes of sex determination and differentiation.