Eva M. Eicher

Migration of mesonephric cells into the mammalian gonad depends on Sry.

In mammals, the primary step in male sex determination is the initiation of testis development which depends on the expression of the Y-linked testis determining gene, Sry. The mechanisms by which Sry controls this process are unknown. Studies showed that cell migration from the adjacent mesonephros only occurs into XY gonads; however, it was not known whether this effect depended on Sry, another Y-linked gene, or the presence of one versus two X chromosomes. Here we provide genetic proof that Sry is the only Y-linked gene necessary for cell migration into the gonad. Cell migration from the mesonephros into the differentiating gonad is consistently associated with Stys presence and with testis cord formation, suggesting that cell migration plays a critical role in the initiation of testis cord development. The induction of cell migration represents the earliest signaling pathway yet assigned to Sry.

Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two mimic mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.

A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis.

In mouse and man, deletions of specific regions of the Y chromosome have been linked to early failure of spermatogenesis and consequent sterility; the Y chromosomal gene(s) with this essential early role in spermatogenesis have not been identified. The partial deletion of the mouse Y short arm (the Sxrb deletion) that occurred when Tp(Y)1CtSxr-b (hereafter Sxrb) arose from Tp(Y)1CTSxr-b (hereafter Sxra) defines Spy, a Y chromosomal factor essential for normal spermatogonial proliferation. Molecular analysis has identified six genes that lie within the deletion: Ube1y1 (refs. 4,5), Smcy, Uty, Usp9y (also known as Dffry), Eif2s3y (also known as Eif-2γy) and Dby10; all have closely similar X-encoded homologs. Of the Y-encoded genes, Ube1y1 and Dby have been considered strong candidates for mouse Spy function, whereas Smcy has been effectively ruled out as a candidate. There is no Ube1y1 homolog in man, and DBY, either alone or in conjunction with USP9Y, is the favored candidate for an early spermatogenic role. Here we show that introduction of Ube1y1 and Dby as transgenes into Sxrb-deletion mice fails to overcome the spermatogenic block. However, the introduction of Eif2s3y restores normal spermatogonial proliferation and progression through meiotic prophase. Therefore, Eif2s3y, which encodes a subunit of the eukaryotic translation initiation factor Eif2, is Spy.

Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells.

The nuclear receptor transcription factor Dax1 is hypothesized to play a role in testicular development, although the mechanism of its action is unknown. Here, we present evidence that Dax1 plays an early essential role in fetal testis development. We hypothesize that upregulation of Sox9 expression in precursor somatic cells, a process required for their differentiation as Sertoli cells, depends on the coordinated expression of Dax1, Sry and another gene, Tda1. Our conclusion and model are based on the following experimental findings: (1) presence of a mutant Dax1 allele (Dax1-) results in complete gonadal sex reversal in C57BL/6JEi (B6) XY mice, whereas testes develop in DBA/2J (D2) and (B6×D2)F1 XY mice; (2) B6-DAX1 sex reversal is inherited as a complex trait that includes the chromosome 4 gene Tda1; (3) B6 Dax1-/Y fetal gonads initiate development as ovaries, even though Sry expression is activated at the correct time and at appropriate levels; (4) upregulation of Sox9 does not occur in B6 Dax1-/Y fetal gonads in spite of apparently normal Sry expression; and (5) overexpression of Sry in B6 Dax1-/Y fetal gonads upregulates Sox9 and corrects testis development.

Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Ovotestis development in B6-XY(POS) mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY(POS) ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.

Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice.

Previous reports suggested that humans and mice differ in their sensitivity to the genetic dosage of transcription factors that play a role in early testicular development. This difference implies that testis determination might be somewhat different in these two species. We report that the Fog2 and Gata4 transcription factors are haploinsufficient for testis determination in mice. Whether gonadal sex reversal occurs depends on genetic background (i.e., modifier genes). For example, C57BL/6J (B6) XY mice develop testes if they are heterozygous for a mutant Fog2 (Fog2−) or Gata4 (Gata4ki) allele. However, if the B6 Y chromosome (YB6) is replaced by the AKR Y chromosome (YAKR), B6 Fog2−/+ XYAKR mice develop ovaries, and B6 Gata4ki/+ XYAKR mice develop ovaries and ovotestes (gonads containing both ovarian and testicular tissue). Furthermore, DBA/2J (D2) Fog2−/+ XYAKR mice and (B6 × D2)F1 hybrid Gata4ki/+ XYAKR mice develop testes. Sry is expressed in the mutant XY gonads, indicating that the lack of Sry expression is not the cause of ovarian tissue development in B6 Fog2−/+ or Gata4ki/+ XYAKR mice. However, up-regulation of Sox9 expression, which is critical for normal testicular development, does not occur in mutant XY gonads that develop as ovaries. We conclude that under certain genetic conditions, Sox9 up-regulation depends on the proper dosage of Fog2 and Gata4. We propose that in humans the FOG2 and/or GATA4 genes might be haploinsufficient for normal testis determination and thus could be the cause of some previously unassigned cases of XY gonadal sex reversal.

INHERITANCE OF T-ASSOCIATED SEX REVERSAL IN MICE

We previously identified a primary sex-determining locus, Tas, on mouse Chr 17 that causes ovarian tissue development in C57BL/6J Thp/+ and TOrl/+ individuals if the AKR/JY chromosome is present. We hypothesized that Tas is located within the region of Chr 17 deleted by Thp and TOrl and that C57BL/6J carries a diagnostic Tas allele, based on the observation that ovarian tissue develops in XY mice when Thp is on a C57BL/6J inbred strain background, whereas normal testicular development occurs when Thp is on a C3H/HeSnJ inbred strain background. To test this hypothesis, we mated (C57BL/6J x C3H/HeSnJ)F1 females to C57BL/6J Thp/+ hermaphrodites. As expected, half of the XY Thp/+ offspring developed ovarian and testicular tissue while half developed exclusively testicular tissue. Unexpectedly, the inheritance of selected Chr 17 molecular loci was independent of gonadal development, as half of the male and hermaphroditic offspring inherited C3H/HeSnJ-derived Chr 17 loci and half inherited C57BL/6J-derived Chr 17 loci. We conclude that for ovarian tissue to develop in an XY Thp/+ or XY TOrl/+ individual (1) Tas must be present in a hemizygous state, which is accomplished by heterozygosity for the Thp or TOrl deletions; (2) the AKR/J-derived Y chromosome must be present; and (3) an additional locus involved in primary sex determination must be present in a homozygous C57BL/6J state. This newly identified gene may be one of the previously defined loci, tda-1 or tda-2.

Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.

Mapping of the murine Ly-15(LFA-1) locus to chromosome 7

A number of loci coding for murine Ly antigens have already been described, and many of these have been mapped to different chromosomes in the mouse genome (reviewed by Sutton et al., in press). We now report that the Ly-15 (LFA-1) locus is located on chromosome (Chr) SWXL 4, 15 C57L/J + 7, near the M o d 2 (mitochondrial malic enzyme-2) and 7, 12, 14, 16, 17 SWR/J Hbb (~-hemoglobin) loci. NX129 1, 5 1291J + The Ly-15 locus defines a polymorphism of LFA-1 7, 10, 12 NZB/BINJ molecules detectable with the murine Ly-15.2 monocloLXPL 1, 4 C57L/J + nal antibody (Hogarth et al. 1985). LFA-1 molecules are 6 PL/J of interest because they, together with the T-cell receptor, A K X L 5, 6, 8, 9, 14, 16, 24, 25, 29, 38 AKR/J the Ly-2/3 antigen, and other molecules, are implicitly 7, 12, 13, 17, 19, 21, 37 C57L/J qinvolved in T-cell function (Springer et al. 1982). LFA-1 cxs C, D, E, H, I, K, L, M STS/A + and Ly-15.2 monoclonal antibodies identify a cell-surface A, B, F, G, J, N BALB/cBy glycoprotein consisting of two noncovalently associated CXB -D, H, I, J C57BL/By + chains with molecular masses of 180 kd (t0 and 95 kd (13) E, G, K BALB/cBy -that are present on all lymphocytes and granulocytes, but are absent from other tissues. The Ly-15/LFA-1 molecule belongs to a family of similar molecules present in man and mouse, including Mac-1 and gp150/95, which have different a chains but identical [3 chains (Sanchez-Madrid et al. 1983). The Ly-15.2 phenotype of strains used in this study is shown in Tables 1 and 2. Segregation of the Ly-15 b allele was performed using monoclonal antibody to the Ly-15 .2 specificity encoded by the Ly-15 b allele (Potter et al. 1981). The antibody was purified by protein A affinity chromatography, radiolabeled with 125I and then used in Mod-2 b Hbb d Ly-15 a 45 a direct radioimmunoassay (Hogarth et al. 1984). We Mod_2 a Hbb s Ly_15 b 45 have used the following assay because the purification Mod-2 b Hbb s Ly-15 b 2 and radiolabeling ofmonoclonal antibody followed by its Mod-2 ~ Hbb d Ly-15 a 2 use in a direct binding assay is both a sensitive and conMod_2b Hbbd Ly_15b 6 venient technique for the examination of large numbers of Mod.2 a Hbb s Ly.15 a 7 mice in different laboratories. In this assay, 5 x 104 ceils Mod_2b HbbS Ly.15a 0 in 50 ~tl of phosphate-buffered saline supplement with Mod-U nbb d Ly-15 b 0 0.5% bovine serum albumin (PBS/BSA) were incubated Total 108 for 30 rain with an equal volume of 125I-anti-Ly-15.2 at 50 ng/ml, 25 ng/ml, and 12.5 ng/ml in PVC microliter plates coated with BSA. Unbound antibody was removed Table 1. Ly-15.2 typing of RI strains

Absence of mitochondrial malic enzyme in mice carrying two complementing lethal albino alleles.

Mitochondrial malic enzyme (MOD-2) was found to be missing in partially complementing genotypes between lethal deletion alleles at the albino locus in Chromosome 7 of the mouse. Since such partial complementers survive to adulthood, the absence of normal mitochondrial malic enzyme is compatible with life; however, the sterility of both females and males may be correlated with this enzyme deficiency. Of the six radiation-induced lethal albino mutations, five includedeletions for the Mod-2 locus mapping 1 centimorgan distally from c. Our data indicate that the maximum genetic distance occupied by any of these deletions is 6 centimorgans.