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In an effort to identify novel candidate genes involved in testis determination, we previously used suppression subtraction hybridisation PCR on male and female whole embryonic (12.0–12.5 days post coitum) mouse gonads. One gene to emerge from our screen was Redd1. In the current study, we demonstrate by whole-mount in situ hybridisation that Redd1 is differentially expressed in the developing mouse gonad at the time of sex determination, with higher expression in testis than ovary. Furthermore, Redd1 expression was first detected as Sry expression peaks, immediately prior to morphological sex determination, suggesting a potential role for Redd1 during testis development. To determine the functional importance of this gene during testis development, we generated Redd1-deficient mice. Morphologically, Redd1-deficient mice were indistinguishable from control littermates and showed normal fertility. Our results show that Redd1 alone is not required for testis development or fertility in mice. The lack of a male reproductive phenotype in Redd1 mice may be due to functional compensation by the related gene Redd2.

Sexual reproduction is essential for the propagation and the maintenance of fitness of our species, and is dependent on the correct development of the bipotential genital ridges into testes and ovaries. Although several transcription factors, secreted signaling molecules, and their receptors have been found to be important for testis determination and early gonad development, comparatively little research has been carried out into intracellular signal transduction pathways activated during these processes. Focal adhesion kinase (FAK) and protein tyrosine kinase 2 beta (PTK2B) form one group of cytosolic tyrosine kinases that are known to be important for processes such as cell proliferation, differentiation, and motility. Here, we describe the temporal and spatial expression patterns of Fak and Ptk2b mRNA and protein during sex determination and early gonadogenesis in mouse embryos. Ptk2b mRNA and PTK2B protein were expressed in testes from 11.5 days post coitum onward, predominantly in developing Sertoli cells, in a SOX9-dependent manner. Fak mRNA and FAK protein were expressed in gonads of both sexes at all stages examined. Our data suggest cell type- and stage-specific roles for PTK2B during early testis development. Developmental Dynamics 239:2735–2741, 2010. © 2010 Wiley-Liss, Inc.

GlutathioneS-transferases (GSTs) are an important family of multifunctional enzymes that play a role in the protection of tissues by the detoxification of hazardous and carcinogenic compounds. We found previously thatGstm6is upregulated in the somatic cells of male mouse fetal gonads relative to female gonads. In this study, we describe the spatial and temporal expression pattern ofGstm6during mouse development. We show thatGstm6is predominantly expressed in the reproductive system, at significantly higher levels in XY gonads compared with XX gonads from 11.5 dpc onwards, and remains expressed in the testes in adult mice. Its expression is associated with the Sertoli cell lineage, and is dependent on the expression of the male sex-determining geneSox9. Our data suggest thatGstm6plays a male-specific role in gonad development or function, possibly by modulating the exposure of somatic tissue and/or germ cells to endogenous or exogenous toxicants.

Reproductive capacity is fundamental to the survival of all species. Consequently, much research has been undertaken to better understand gametogenesis and the interplay between germ cells and the somatic cell lineages of the gonads. In this study, we have analyzed the embryonic expression pattern of the X-linked gene family Reproductive homeobox genes on the X chromosome (Rhox) in mice. Our data show that eight members of the Rhox gene family are developmentally regulated in sexually dimorphic and temporally dynamic patterns in the developing germ cells during early gonadogenesis. These changes coincide with critical stages of differentiation where the germ cells enter either mitotic arrest in the testis or meiotic arrest in the ovary. Finally, we show that Rhox8 (Tox) is the only member of the Rhox gene family that is expressed in the somatic compartment of the embryonic gonads. Our results indicate that the regulation of Rhox gene expression and its potential function during embryogenesis are quite distinct from those previously reported for Rhox gene regulation in postnatal gonads.

In an effort to understand the mechanisms that underpin gonadal differentiation at the time of sex determination, we identified a cDNA encoding a putative novel testis expressed scavenger receptor, Tesr. Based on its domain structure, we hypothesize that the function of Tesr is similar to that of other scavenger receptors that play roles in phagocytosis of apoptotic cells, cell-cell adhesion, and defense. Tesr mRNA was detected in fetal mouse gonads of both sexes at 11.5 days post coitum (dpc). From 12.0 dpc, Tesr expression rapidly decreased in the female and was maintained in the male. Expression was seen in embryonic mouse sites other than the testis, such as in brain, eye, head, heart, neural arch, and cartilage primordium. Tesr expression in the newborn testis was faint to undetectable, but it increased from 2 days postpartum (dpp) until 15 dpp and was found in a subset of interstitial cells and in germ and Sertoli cells. Tesr mRNA in the adult mouse testis was observed in Sertoli cells, spermatogonia, spermatocytes, round spermatids, and in a subset of interstitial cells. We conclude that Tesr is differentially expressed in the male vs. female embryonic gonad and is expressed in both the ovary and the testes postnatally after 2 dpp.

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail–chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-α are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2α, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.

Mammalian sex determination relies on the expression of SRY, which triggers a tightly regulated cascade of gene expression leading to male differentiation. Many elements of this pathway remain to be identified. Here, we characterise Annexin XI (Anxa11), a gene whose major site of embryonic expression was within the undifferentiated and differentiating testis. Lower level expression was also observed in both sexes in the Mullerian and Wolffian ducts, the somitic dermamyotome, and the dorsal intermediate zone of the neural tube. Anxa11 transcripts were detected in the indifferent gonad from 10.5 days post coitum (dpc), becoming male specific as development proceeded. Expression was within the testis cords, initially in germ cells, and then in both Sertoli and germ cells. Annexin XI protein was seen in the testis cords from 12.5 dpc, localising to the cytoplasm of the Sertoli cells. Expression of calcyclin (S100a6), shown previously to interact with annexin XI in vitro, was also observed in proliferating cells of the embryonic testis, supporting a possible in vivo interaction. Developmental Dynamics 234:432–437, 2005. © 2005 Wiley-Liss, Inc.

The hermes gene encodes an RNA-binding protein containing an RNA-recognition motif. Its expression has been described previously in Xenopus and in the developing heart of very young chicken embryos. We have analyzed the expression of cHermes in later heart development, where expression is maintained in the myocardium, and also in previously undescribed sites. cHermes expression first appears in the somites in the first terminally differentiated myocytes of both the epaxial and the hypaxial myotome. Expression is also seen in the primordium of the allantois and continues in the developing allantoic sac. cHermes expression in the pronephric and mesonephric kidneys coincides temporally and spatially with the appearance of the vascular components of the glomeruli. In addition, cHermes expression was seen in the mesoderm of the gut and in the notochord.

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.

We have identified a gene with gonad restricted expression throughout mouse development, which is orthologous to human EKI2 (ethanolamine kinase 2). Our studies showed that mouse Eki2 expression became upregulated in the male gonad during the period of sex determination. Expression was restricted to the Sertoli cells of the developing testis. Eki2 has sequence similarity to ethanolamine (73%) and choline kinases (54%).

The sex of most mammals is determined by the action of SRY. Its presence initiates testis formation resulting in male differentiation, its absence results in ovary formation and female differentiation. We have used suppression subtraction hybridisation between 12.0–12.5 days postcoitum (dpc) mouse testes and ovaries to identify genes that potentially lie within the Sry pathway. Normalised urogenital ridge libraries comprising 8,352 clones were differentially screened with subtracted probes. A total of 272 candidate cDNAs were tested for qualitative differential expression and localisation by whole mount in situ hybridisation; germ cell-dependent or -independent expression was further resolved using busulfan. Fifty-four genes were identified that showed higher expression in the testis than the ovary. One novel gene may be a candidate for interactions with WT1, based on its localisation to Sertoli cells and map position (16q24.3). genesis 37:84–90, 2003. © 2003 Wiley-Liss, Inc.

Vertebrate DM domain genes encode a novel group of proteins related to the Drosophila doublesex and Caenorhabditis elegans mab-3 transcription factors. It is shown here that the recently identified gene, DMRT3, has a restricted embryonic expression profile that is conserved in chicken and mouse embryos. DMRT3 is expressed primarily in the forebrain, neural tube and nasal placode of both species. In the chicken, DMRT3 is also expressed in newly forming tail somites at early developmental stages and, later, in the Mullerian ducts of the urogenital system.

We describe the characterisation of three gene trap integrations in embryonic stem cells in which the lacZ reporter gene is repressed by retinoic acid (RA) in vitro and is expressed in the developing heart in vivo. In one of these, the gene trap vector has integrated into a gene that is located on chromosome 17 and is homologous to the human transcription factor gene, TFEB. Embryonic and adult cardiac expression of both the fusion transcript and the endogenous gene was confirmed. However, we show that the integration has not resulted in a null allele, because wild type transcripts, possibly resulting from splicing around the vector, are observed in homozygous tissue. The other two cardiac-expressing gene trap integrations have occurred into exons on chromosomes 1 and 5 and have used cryptic donor sites within the vector to generate functional fusion transcripts. One of these exon integrations results in a lethal neonatal phenotype. Dev. Dyn. 1998;212:267–276. © 1998 Wiley-Liss, Inc.

In this report we describe the construction of anti-5-iodo-4-hydroxy-3-nitrophen-acetyl (NIP) mouse/human immunoglobulin (Ig)G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described: Gln-268 is substituted by His in γ4 Q268H, Ser-331 is substituted by Pro in γ4 S331P, and in γ4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement-mediated cell lysis (CML) and phagocytosis by FcγRII- and FcγRIII-bearing polymorphonuclear leukocytes (PMN) were measured. In CML, γ4 Q268H was inactive, but both γ4 S331P and γ4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants γ4 S331P and γ4 Q268H/S331P were both active only when complement was introduced. γ4 Q268H was not active in phagocytosis under any conditions. We conclude that His-268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by FcγRII and/or FcγRIII. Pro-331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect FcγRII/FcγRIII-mediated phagocytosis. In this manner the mutants γ4 S331P and γ4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.

To increase the number of markers on distal mouse chromosome 4, knowledge of the synteny homology between this region and human chromosome 1p (HSA1p) was used to identify candidate homologous mouse genes. Ten probes corresponding to loci on human chromosome 1p were tested to reveal polymorphisms between C57BL/6 and DBA/2 mice, the progenitors of the 26 BXD recombinant inbred strains. These strains were typed with six of these probes and typed for inheritance of mouse-specific microsatellite markers. Five mouse homologues of human genes were assigned to distal mouse chromosome 4 in the following order (including MIT microsatellites): (cen)-D4Mit71-Lap18-D4Mit69-C1qB-Plod/T nfr2-Cd30-(tel). Furthermore, an additional HSA1p marker, Cd53, mapped close to Amy1 on mouse chromosome 3, and a sequence related to Lap18 mapped to mouse chromosome 17, near D17Mit3. This comparative approach suggests that the human counterparts of these genes may have a similar order on human chromosome 1p and also indicates that Lap18, C1qB, and Nhe1 are candidates for a recently described diabetes susceptibility gene on mouse chromosome 4.

Insulin-dependent diabetes mellitus is a multigenic autoimmune disease, for which one of the best animal models is the nonobese diabetic (NOD) mouse strain. In both humans and NOD mice, major histocompatibility complex genes are implicated as risk factors in the disease process. Other susceptibility genes are also involved, and a number have been mapped in the mouse to specific chromosomal locations. To identify further susceptibility genes, diabetic backcross mice, produced after crossing NOD/Lt to the nondiabetic strains SJL and C57BL/6 (B6), were examined for markers not previously associated with disease susceptibility. Linkage was found to loci on chromosomes 4 and 14. Of the candidate loci on chromosome 4, the gene encoding the Na+/H+ exchanger-1, Nhe-1, was the most likely, since the NOD allele was different from that of both nondiabetic strains. NOD lymphocytes were found to have a higher level of Na+/H+ exchange activity than lymphocytes from either B6 or SJL mice. Since the chromosome 4 susceptibility gene is recessive, the B6 allele should prevent diabetes. This prediction was tested in fourth-generation backcross mice, selected for retention of the B6 allele at Nhe-1. Mice homozygous for Nhe-1 developed diabetes after cyclophosphamide treatment, but heterozygotes were largely protected from disease. These results implicate the Na+/H+ exchanger (antiporter) in the development of type 1 diabetes and may provide a screening test for at-risk individuals as well as offering prospects for disease prevention.

Sex determination in vertebrates is controlled by a variety of mechanisms. We compared the expression of SF1, DAX1, DMRT1, SOX9 and AMH during gonadogenesis in the mouse, chicken and alligator embryo. In contrast to the expression profile of Sf1 in mouse embryos, chicken and alligator embryos show higher levels of Sf1 expression in the developing ovaries compared to testes. This may reflect the higher level of sex hormone synthesis in the ovary compared to the testis in chickens and alligators. The DAX1 gene has a similar expression profile in all three vertebrate species but appears to have different gene structure. As in mouse, DMRT1 was expressed at very high levels in the chicken and alligator male gonad. The male-specific up-regulation of SOX9 expression appears to be a common feature in all three vertebrates. In the chicken and alligator AMH is expressed prior to SOX9, suggesting that in these species SOX9 cannot initiate AMH expression as it does in mammals. SOX9 acts at multiple points in the vertebrate testis pathway but it appears that only some of these functions have been conserved through evolution.

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

During mouse embryogenesis, the fate of the bipotential gonads is sealed around 10.5 dayspost coitum(dpc) when the Y-linked geneSryspecifies the differentiation of testes in males, whereas in females, absence ofSryresults in ovary formation. Apart from the pivotal action ofSry, many other genes are known to be involved in sex determination and subsequent differentiation. Much is still unknown regarding the regulatory hierarchy governing these events and many more sex differentiation genes are yet to be discovered. In this study, we investigated the expression ofTmem184a,a novel gene encoding a protein of unknown function, but with predicted kinase activity, during mouse embryogenesis. We show thatTmem184ais expressed at high levels in the developing testis from 11.5 dpc, a time of active proliferation and differentiation.Tmem184aexpression is further shown to be expressed exclusively within the Sertoli cells of the developing testis cords, suggesting that it may mediate sex-specific signaling events during Sertoli cell differentiation.

The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sex-determining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY, SOX9, SF-1, DHH and FGF9. Genes not expressed included WT1, DAX1 and the ovary-specific genes FOXL2 and WNT4. Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.

Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by sectionin situhybridisation, in some instances followed by immunofluorescent detection of protein products.Collagen type-I,inhibinβA,caldesmon 1andtropomyosin 1were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

In a screen for transcripts differentially expressed during gonadal development in mouse embryos, we identified the novel armadillo-related gene, Alex2. The armadillo (arm) family of proteins share a 42 amino acid tandem repeat motif called the arm domain, through which they interact with different binding partners. These intracellular proteins are implicated in a variety of developmental processes, including cell proliferation, migration, maintenance of tissue integrity, and tumorigenesis. Alex2 is a member of a novel subgroup within the arm family, encoding a protein with a single arm domain and a putative transmembrane or signal sequence. Alex2 has a developmentally regulated expression profile during embryogenesis in the mouse. In the urogenital system, it is strongly expressed in the developing testis but is down-regulated during ovarian development. Alex2 expression is localized within the interstitial cell lineage of the developing testis, which gives rise to peritubular myoid, endothelial, and fetal Leydig cells. Alex2 is also expressed in the developing forebrain and somites and in dorsal root ganglia. In testicular cell lines, Alex2 fusion proteins localize to membrane structures within the cell. The expression profile of Alex2 suggests that it plays a role in the development of several tissues during embryogenesis, notably testicular differentiation. In the developing testis, its expression profile suggests that Alex2 has a role in specification or development of the interstitial cell lineage.

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.

Vertebrate DM domain genes encode a novel group of proteins related to the Drosophila doublesex and Caenorhabditis elegans mab-3 transcription factors. It is shown here that the recently identified gene, DMRT3, has a restricted embryonic expression profile that is conserved in chicken and mouse embryos. DMRT3 is expressed primarily in the forebrain, neural tube and nasal placode of both species. In the chicken, DMRT3 is also expressed in newly forming tail somites at early developmental stages and, later, in the Mullerian ducts of the urogenital system.

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.

Mutations in the transcription factor SOX9 give rise to campomelic dysplasia, a syndrome characterized by skeletal abnormalities and XY sex reversal. Sox9 is expressed at sites of chondrogenesis and in the developing testis, and, thus, it plays a role in two overtly different pathways of differentiation. Previous studies have identified the gene for type II collagen, Col2a1, as a target of Sox9 in mouse chondrocytes and implicated Col9a3 as a Sox9 target in testis. Using differential expression analysis combined with reverse transcription-polymerase chain reaction and whole-mount in situ hybridization, we have identified nonchondrocytic collagen transcript isoforms that are expressed in the early male mouse gonad. Male-specific, gonadal expression of nonchondrocytic Col2a1 was first seen at 11.5 days postcoitum (dpc) and was undetectable by 13.5 dpc. This was accompanied by increasing expression of nonchondrocytic Col9a1, Col9a2, and Col9a3, first detected at 11.5 dpc. Expression was analyzed in testes that had been depleted of germ cells by the cytotoxic drug busulfan. These studies showed Col9a3 and Col2a1 to be expressed in Sertoli cells within the developing testis cords. Nonchondrocytic type II collagen contains a cysteine-rich domain that has been shown to bind members of the transforming growth factor b superfamily of signaling molecules. Thus, this interaction may play a role in the morphogenesis and differentiation of the testis. developmental biology, early development, embryo, Sertoli cells, testis

Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4.

Vertebrates exhibit a surprising array of sex-determining mechanisms, including X- and Y-chromosome heterogametes in male mammals, Z- and W-chromosome hetero-gametes in female birds, and a temperature-dependent mechanism in many reptiles1. The Y-chromosome-linked SRY gene initiates male development in mammals2,3, but other vertebrates lack SRY and the genes controlling sex determination are largely unknown. Here we show that a gene implicated in human testis differentiation, DMRT1, has a gonad-specific and sexually dimorphic expression profile during embryogenesis in mammals, birds and a reptile (Alligator mississippiensis). Given the different sex-determining switches in these three groups, this gene must represent an ancient, conserved component of the ver-tebrate sex-determining pathway.

The Na{sup +}/H{sup +} exchanger-1 molecule (NHE1) regulates intracellular pH, cell volume, and cell growth. NHE1 is a phosphoprotein of approximately M{sub r} 110000 with 10 or 12 transmembrane domains. NHE1 is ubiquitously expressed. Three other family members have been identified which show close similarity to NHE1 but are significantly more restricted in their expression: all are found in the gastrointestinal tract, while NHE2 and NHE3 are also expressed in the kidney. 14 refs., 2 figs.

The non-obese diabetic (NOD) mouse is a polygenic model of insulin-dependent diabetes. To better understand the biology, immunology and genetics of this mouse strain NOD mice were typed for RFLP at 29 loci and microsatellite variation at a further nine loci. Mice were also typed for the expression of lymphocyte differentiation antigens. Comparisons were made with two non-diabetic strains, C57BL/6 and SJL, for these markers. A number of restriction fragment length polymorphisms (RFLP) were defined: seven between C57BL/6 and NOD; five between SJL and NOD; and four in which both C57BL/6 and SJL differed from NOD. Previously reported microsatellite results were extended by defining the SJL alleles at these loci, finding five loci at which NOD differed from B6 alone, and four loci at which NOD differed from both B6 and SJL. In addition, the NOD Igh allotype and alleles for a number of lymphocyte differentiation antigens were defined for the first time. The expression of uncommon alleles of these antigens suggests candidates whose role in the diabetogenic process can be tested.

We have devised a PCR-based sexing method that is quick, simple, and highly reproducible. DNA is first extracted from embryonic mouse yolk sac via a 15 min, two-step incubation procedure utilizing PCR-compatible proteinase K buffer. Without any further manipulation the lysate is subjected to 30 cycles of PCR, optimized to run in less than 1 hr. The reaction includes multiplexed primer pairs for Sry and Myog (myogenin) that generate a male specific band of 441 bp and an internal control band of 245 bp, respectively. This robust method is used routinely in our laboratory and gives rapid genotyping results with 98% reliability and 100% accuracy.

We present a detailed study of the expression pattern of WD repeat and SOCS box-containing 2 (Wsb2) in mouse embryonic and adult gonads. Wsb2 was previously identified in a differential screen aimed at identifying the genes involved in male- and female-specific gonadal development. Wsb2 expression was analysed during mouse gonadogenesis by real-time PCR, whole-mount and section in situ hybridisation and immunofluorescence. Wsb2 mRNA expression was initially detected in gonads of both sexes from 11.5 days post coitum (dpc) until 12.0 dpc. By 12.5 dpc and thereafter, Wsb2 expression rapidly decreased in the female, while persisting in the male gonads. In foetal, newborn and juvenile testes, Wsb2 mRNA and protein were readily detected in the seminiferous cords within both Sertoli and germ cells. Wsb2 mRNA was present in spermatogonia, spermatocytes and in Sertoli cells of the adult mouse testis. The differential expression of Wsb2 in male versus female embryonic gonads suggests some male-specific role in gonad development, and its expression in the first wave of spermatogenesis indicates a role in germ cells. Real-time analysis of adult mouse testis tubules cultured in the presence of the Hedgehog signalling inhibitor, cyclopamine, showed a downregulation of Wsb2 mRNA after treatment which suggests that Wsb2 may be a target of Hedgehog signalling.