Nuclear receptors Sf1 and Dax1 function cooperatively to mediate somatic cell differentiation during testis development

Sf1 and Dax1 are nuclear receptors found predominantly in the endocrine tissues that control reproduction: the ventromedial hypothalamus, pituitary gonadotropes, adrenal gland and gonad(Ikeda et al., 1996). Sf1 is considered to be a master regulator of the reproductive system because it regulates the expression of a large array of genes required for gland development and hormone synthesis (Parker et al., 2002). Sf1 protein acts by binding to a cognate DNA-response element (Morohashi et al.,1992; Rice et al.,1991) in the promoter regions of these genes. Unlike most nuclear receptors, Sf1 binds DNA as a monomer(Wilson et al., 1993). Sf1 interacts with a number of transcriptional co-activators(Val et al., 2003), and phosphatidyl inositol lipids may serve as ligands to regulate transcriptional activity (Krylova et al.,2005).

Dax1 is atypical among nuclear receptors because it lacks the highly conserved zinc-finger DNA-binding domain(Zanaria et al., 1994). Instead, the amino terminus of Dax1 contains a repeated peptide sequence comprising variations of a hydrophobic LXXLL-like motif(Zhang et al., 2000) that mediates protein-protein interactions(Suzuki et al., 2003). Dax1 may bind to certain DNA hairpin structures and to RNA(Lalli et al., 2000), but its major transcriptional regulatory action involves direct interactions with other nuclear receptors, including Sf1(Holter et al., 2002; Ito et al., 1997; Zhang et al., 2000). The carboxy terminus of Dax1 contains a transcriptional repressor domain(Ito et al., 1997; Zazopoulos et al., 1997) that interacts with several different co-repressors(Altincicek et al., 2000; Crawford et al., 1998). One model of Sf1 and Dax1 action proposes that the N terminus of Dax1 interacts with Sf1, and recruits repressors to the Sf1 transcription complex, thereby inhibiting the expression of Sf1-regulated genes such as Star, Dax1, Lhb,3βHSD (Hsd3b – Mouse Genome Informatics), Cyp19, Cyp11a1 and Amh(Lalli et al., 1998; Nachtigal et al., 1998; Salvi et al., 2002; Suzuki et al., 2003; Tabarin et al., 2000; Wang et al., 2001; Zazopoulos et al., 1997).

Sf1 is expressed in the urogenital ridge at E9.5(Ikeda et al., 1994; Ikeda et al., 2001). In the male gonad, Sf1-positive cells can be found in a population of coelomic epithelial cells that give rise to both Sertoli and interstitial cell precursors (Schmahl et al.,2000). In the Sertoli population, one target of Sf1 is anti-Müllerian hormone (Amh/Mis)(Hatano et al., 1994; Shen et al., 1994). Later in testis development, Sf1 expression intensifies in the interstitial Leydig population where it regulates the expression of multiple steroidogenic enzyme genes necessary for testosterone production(Hatano et al., 1994; Ikeda et al., 1996; Morohashi et al., 1993). Dax1 expression peaks in the male gonad at E11.5 and then remains relatively low until E17.5 when it increases significantly(Ikeda et al., 2001). At E11.5, Sf1 and Dax1 proteins overlap in the XY bipotential gonad, and by E12.5, they are co-localized mainly within the testis cords(Ikeda et al., 2001). The spatial and temporal overlap of Sf1 and Dax1 expression during the crucial time of gonadal differentiation raises the possibility of a functional interaction, but this has not been explored directly in vivo.

Loss-of-function mutations in Sf1 and Dax1 suggest important roles in gonadal development. Homozygous deletion of Sf1 in mice prevents adrenal gland and gonadal development, reflecting increased programmed cell death in the cell populations that normally give rise to these tissues (Luo et al., 1994). In the male, the failure of testis differentiation results in absence of anti-Müllerian hormone production and, consequently, there is persistence of Müllerian structures at birth. Prior to regression of the gonad rudiment in the Sf1 homozygous knockout, primordial germ cells are detectable in the gonadal ridge (Luo et al., 1994). Thus, the primary role of Sf1 in gonad development involves the somatic cell lineages.

Dax1-null hemizygous male mice have testicular dysgenesis and delayed regression of the fetal X-zone in the adrenal gland(Yu et al., 1998b). Although Dax1 had been suggested as a possible ovarian determining gene,ovaries develop in females with deletion of Dax1 on both X chromosomes. Adult female mice lacking Dax1 exhibit multi-oocyte follicles but they are fertile. The loss of Dax1 function in the testis has been shown to impair testis cord formation. This is caused in part by reduced numbers of peritubular myoid (PTM) cells(Meeks et al., 2003a), which normally surround the testis cords and, together with Sertoli cells, form the basement membrane of the developing seminiferous tubules. In the adult, the efferent duct epithelium is hyperplastic and there is obstruction of seminiferous tubules (Jeffs et al.,2001). Sertoli cells appear to be incompletely differentiated and germ cells progressively degenerate. As a consequence, male Dax1 null mice are infertile. Leydig cell development is also altered in the absence of Dax1. Fetal Leydig cells are restricted to the coelomic side of the interstitial compartment rather than extending across the full diameter of the gonad (Meeks et al., 2003a). In the adult, Leydig cells are hyperplastic and aromatase expression is elevated, leading to increased intratesticular estradiol production(Wang et al., 2001). On a genetic background with a weakened Sry allele (Mus domesticus poschiavinus), the phenotype associated with Dax1 deletion changes from dysgenetic testes to complete sex reversal(Meeks et al., 2003b),indicating that Dax1 functions in parallel with, or downstream of, Sry in the sex determination cascade to mediate normal testis development. Thus, Dax1 plays a more crucial role in testis differentiation than it does in ovary development.

Dax1-deficient mice exhibit overactivity of some Sf1-regulated genes, consistent with the idea that Dax1 antagonizes Sf1 function. For example, Cyp19, an Sf1-regulated steroidogenic enzyme gene, is overexpressed in the testis of adult male Dax1-knockout mice(Wang et al., 2001). Cyp21, a key enzyme for mineralocorticoid and glucocorticoid synthesis, is overexpressed in the adrenal gland of Dax1-deficient mice (Babu et al., 2002). These findings are consistent with a model in which Dax1 represses Sf1-mediated transcription.

The phenotypes seen in Sf1 and Dax1 knockout mice are largely predictive of the clinical manifestations in humans with mutations in SF1 or DAX1 (Achermann et al., 2001b). SF1 mutations cause adrenal insufficiency and XY gonadal dysgenesis. These features occur even with heterozygous mutations and vary across a wide phenotypic spectrum(Jameson, 2004). Thus, in humans, the function of SF1 is strikingly dose dependent(Achermann et al., 2002). Human DAX1 mutations cause X-linked adrenal insufficiency, hypogonadotropic hypogonadism and gonadal dysgenesis(Bardoni et al., 1994; Muscatelli et al., 1994; Zanaria et al., 1994). Some mutations with partial loss of DAX1 activity are associated with delayed onset and milder clinical features(Ozisik et al., 2003b; Salvi et al., 2002; Tabarin et al., 2000),suggesting that DAX1 action is also dose dependent(Achermann et al., 2001a; Reutens et al., 1999).

The phenotypic similarities associated with Sf1 and Dax1loss-of-function mutations are somewhat at odds with their proposed antagonistic actions at the transcriptional level. To further explore their functional relationship in vivo, we examined testis development in the context of combined loss of function of Sf1 and Dax1. We hypothesized that Sf1 heterozygosity would reduce the expression of some Sf1 target genes, such as steroidogenic enzymes genes(Lala et al., 1992; Morohashi et al., 1992). Moreover, because Dax1 acts as a repressor of Sf1-mediated transcription(Ito et al., 1997; Lalli et al., 1997), we predicted that Dax1 deficiency might partially or completely compensate for Sf1 heterozygosity in the Sf1/Dax1 double mutant. In contrast to a model in which Dax1 acts as a universal antagonist of Sf1 action, combined loss of Sf1 and Dax1 further impaired Sertoli cell differentiation and fetal Leydig cell development. In the embryonic gonad, we found that Sf1 and Dax1 act coordinately to enhance the expression of the Sertoli-derived factors Dhh and Amh. Dhh is a paracrine signaling factor that regulates fetal Leydig cell development(Yao et al., 2002). Amh is a key regulator of Müllerian regression and testis differentiation(Behringer et al., 1990; Behringer et al., 1994; Ross et al., 2003; Vigier et al., 1985). Thus early male gonadal development depends on the cooperative function of Sf1 and Dax1.

All procedures were approved by the Northwestern University Animal Care and Use Committee, Master Protocol #2004-0494. Dax1^+/–females were mated to Sf1^+/– males to attain animals of all four genotypes. For analysis of embryos, females were placed with individually housed males at 17.00 h. Vaginal plugs were identified at 08.00 h the following morning. Twelve o'clock noon corresponded with embryonic day 0.5. Visceral yolk sac taken from each embryo was used to obtain genomic DNA. Sf1, Dax1 and Sry genotyping was performed for all embryos as previously described (Babu et al.,2002; Yu et al.,1998b). Only males within each of the four genotype groups were studied.

In situ hybridization was performed by a standard protocol(Wilkinson, 1998). Plasmid constructs for synthesis of probes to detect Amh, Sox9; Cyp11a1,Cyp17; and Dhh were generously provided by B. Capel (Duke University), P. Koopman (University of Queensland), S. Tevosian (Dartmouth College) and A. McMahon (Harvard University), respectively. Whole-mount tissue was viewed with a Leica MZLFIII (Leica, Heerbrugg, Switzerland) dissecting microscope and images were taken with a Color MagnaFire (Optronics, Goleta,CA) digital camera. Littermates were used as wild-type controls.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) of fragmented DNA in apoptotic cells on tissue sections of embryonic gonads was performed using the Fluorescein-FragEL kit (Calbiochem, San Diego,CA) according to the manufacturer's instructions. De-paraffinized sections were treated with Proteinase K. Incubation with terminal deoxynucleotidyl transferase catalyzed the addition of fluorescein-labeled deoxynucleotides to exposed 3′-OH ends of DNA fragments. Nuclei were counterstained with Propidium Iodide mounting medium (Vector Laboratories, Burlingame, CA). Fluorescence microscopy images were captured on a Zeiss Axioskop (Zeiss,Thornwood, NY) with a Color MagnaFire (Optronics, Goleta, CA) digital camera.

Paraffin wax-embedded tissue was sectioned at 5 μm using a Jung RM 2025(Leica, Nussloch, Germany) microtome. Sections were deparaffinized by serial washes in xylenes and ethanol, followed by antigen retrieval in sodium citrate buffer (pH 6.0) at high temperature. 3β-hydroxysteroid dehydrogenase antibody (1:2500) was provided by Ian Mason (Edinburgh, Scotland). Secondary antibodies were applied for two hours at room temperature (Goat Cy3-anti-rabbit from Jackson Immunoresearch, West Grove, PA, DAB Vector,Burlingame, CA). Sections were washed and mounted with DAPI Hard Set (Vector,Burlingame, CA). Hematoxylin (Fisher Scientific, Fairlawn, NJ) and Eosin(Surgipath, Richmond, IL) (H&E) staining was performed following de-paraffinization of tissue and xylene-to-ethanol washes. Sections were viewed with a Zeiss Axioskop. Pictures were taken with a Color MagnaFire(Optronics, Goleta, CA) digital camera.

In wild-type males, fetal Leydig cells can be identified by in situ hybridization for the steroidogenic enzymes genes Cyp17 and Cyp11a1 at E13.5 (Fig. 1A-H). The Leydig cell population was diminished in Sf1^+/– mice, particularly in the periphery of the gonad. In the Dax1^-/Y testis, Leydig cells were restricted to the coelomic surface on the dorsal side of the gonad. Both markers were expressed on the ventral side, indicating a spatial requirement for Dax1 in the dorsolateral aspect of gonad near the mesonephros(S.Y.P., unpublished). In the Sf1/Dax1 double mutants, the Cyp17 and Cyp11a1 markers were not detected. Thus, in contrast to the hypothesis that elimination of Dax1 might compensate for loss of Sf1 activity, these results indicate a combinatorial role for Sf1 and Dax1 in fetal Leydig cell development. These effects of Sf1 haploinsufficiency are consistent with a gonad-specific knockout of Sf1, which showed failure to express the steroidogenic pathway genes Cyp11a1 and Star expression at E14.5 and 16.5, respectively (Jeyasuria et al., 2004).

Desert hedgehog (Dhh) is produced by the Sertoli cells(Bitgood et al., 1996). Its cognate receptor patched 1 (Ptc1) is located on the Leydig cell membrane and initiates an intracellular cascade to induce Cyp11a1 expression in an Sf1-dependent manner (Yao et al.,2002). We hypothesized that loss of Sf1 and Dax1might alter Dhh expression, providing a basis for impaired Leydig cell development. Dhh was measured by whole-mount in situ hybridization starting at E11.5. In wild-type mice, Dhh was expressed uniformly in the male gonad (Fig. 2A-D). In heterozygous Sf1 mutants, Dhh was expressed in the central region of the gonad but it was diminished in the anterior and posterior poles. In the Dax1 mutant, Dhhexpression was lower than in wild type, but it was present throughout the entire length of the gonad. In Sf1/Dax1 double mutants, Dhh expression was undetectable at E11.5. TUNEL staining was performed to assess whether the reduced expression of Dhh was associated with programmed cell death, as observed in Sf1 homozygous knockouts (Luo et al., 1994). However, there was minimal apoptosis in wild type, single, or double mutants(Fig. 2I-L). Sf1homozygous tissue (see insert) served as positive control. By E12.5, Dhh was localized to the embryonic testis cords and its expression was comparable in all four genotypes (Fig. 2E-H). The reduced Dhh expression associated with loss of Sf1 and Dax1 is therefore transient, suggesting coordinated regulation by Sf1 and Dax1 without affecting cell survival.

Amh activation is directly regulated by Sf1(Hatano et al., 1994; Shen et al., 1994). Other transcription factors that act with Sf1 on the Amh promoter include Wt1, Sox9, Sox8 and Gata4 (De Santa Barbara et al., 1998; Nachtigal et al., 1998; Schepers et al., 2003; Viger et al., 1998). Dax1,however, represses Sf1-mediated activation of Amh when tested in vitro (Nachtigal et al.,1998). Amh transcripts were first detected at E11.5 in the wild-type male gonad (Fig. 3A). The Sf1 heterozygous gonad did not express Amh (Fig. 3B), whereas sparse staining was seen in the anterior region of the Dax1 null gonad at E11.5 (Fig. 3C). The double mutant also showed minimal Amh expression(Fig. 3D). By E12.5, Amh levels returned to normal in the single Sf1 mutant(Fig. 3E,F). However, Amh expression remained low in the Dax1 mutant and in the Sf1/Dax1 double mutant (Fig. 3G,H), compatible with distinct roles for Sf1 and Dax1 in Amh expression.

Sox9 expression was examined at E11.5 to assess whether reduced expression of Dhh and Amh is selective or whether it reflects a more general delay in Sertoli cell differentiation. In the wild type, Sox9 was strongly expressed at E11.5(Fig. 4A). There was some reduction of Sox9 expression in the Sf1 heterozygote (B) and in the Sf1/Dax1 double mutant gonad (D), but less reduction in the Dax1 null (C). At 12.5 dpc, Sox9 expression was similar in Sertoli cells of all genotypes (Fig. 4E-H). Thus, Sox9 expression is partially impaired in single or double mutants. However, the reduction of Sox9 is not as pronounced as Dhh or Amh.

Testis cord morphogenesis is a characteristic feature of male gonad development (Tilmann and Capel,2002). In the wild-type male at E12.5, testis cord morphogenesis is apparent by cross-sectional histology. Sertoli cells surrounding primordial germ cells are enclosed by flattened, peritubular myoid cells within primitive tubules (Fig. 5A). The appearance of testis cords is not disrupted in the Sf1 heterozygous gonad (Fig. 5B). However, in the Dax1 null gonad, the number of testis cords is reduced and many are incompletely enclosed, reflecting impaired peritubular myoid differentiation (Fig. 5C)(Meeks et al., 2003a). A similar phenotype is observed in the Sf1/Dax1 double mutant gonad(Fig. 5D), indicating that the Dax1 mutant phenotype is not rectified by allelic loss of Sf1.

Although temporal regulation of Leydig cell differentiation is delayed in the double mutant, steroidogenic enzyme gene expression begins to recover as early as E14.5 (Fig. 6A-H). Thus, Leydig cell recovery allows the progression of steroidogenesis during embryonic development. This finding is consistent with the recovery of Dhh by E12.5 following a period of delay. Immunostaining for 3β-hydroxysteroid dehydrogenase (3βHSD) was performed two weeks after birth, prior to the proliferation of adult Leydig cells. Similar amounts of 3βHSD were found in Leydig cells of all four genotypes(Fig. 6I-L).

In this report, we examined the consequences of combined loss of function of nuclear receptors Sf1 and Dax1 in the context of male gonad development. Previous studies indicate that Sf1 acts in a dose-dependent manner (Babu et al.,2002; Bland et al.,2004; Bland et al.,2000; Ozisik et al.,2002), and that Dax1 binds to Sf1 to inhibit its transcriptional activity (Ito et al., 1997). We initially hypothesized that loss of Dax1 might complement Sf1 haploinsufficiency and partially rescue features associated with reduced Sf1 action. Unexpectedly, however, combined loss of Sf1 and Dax1 generated a more severe phenotype than that seen with the individual mutations, suggesting that these factors function in partially independent and complementary pathways. The most striking feature of the combined Sf1/Dax1 mutant is a delay in the development of fetal Leydig cells and their expression of steroidogenic enzyme genes. These features are heralded by delayed Sertoli cell expression of Dhh and Amh. Dhh is known to act in a paracrine manner to induce steroidogenic enzyme gene expression in fetal Leydig cells via its cognate receptor Ptc1 (Yao et al.,2002). In addition to its primary role in the regression of the Müllerian duct, Amh is thought to promote testis cord formation(Behringer et al., 1990),perhaps by inducing mesonephric cell migration(Ross et al., 2003), and to antagonize adult Leydig cell function(Behringer et al., 1994; Racine et al., 1998). Hence,the compound loss of Sf1 and Dax1 was associated with a transient delay in Sertoli cell function, as Dhh and Amhexpression recover, followed by fetal Leydig cell differentiation.

In this study, the dose-dependent effects of Sf1 seen previously in the adrenal gland (Babu et al.,2002; Bland et al.,2004; Bland et al.,2000) were also found in the developing testis. Heterozygous Sf1 mice have delayed adrenal development and reduced adrenal size but adrenal function is ultimately normal, indicating that compensatory mechanisms activate Sf1 target genes and stimulate adrenal growth. In the developing testis, heterozygous Sf1 mice have reduced expression of Leydig and Sertoli marker genes but these cell types recover, indicating a transient delay when Sf1 expression is reduced. The effects of Sf1 deficiency in the early stage of gonad development are more pronounced in the periphery of the gonad than in the central region. Immunohistochemical analyses have shown that Sf1 is expressed throughout the developing bipotential gonad at 11.5 dpc(Ikeda et al., 2001). In the coelomic epithelial layer of the male gonad, a population of Sf1-positive cells are proliferative and contribute to the sexually dimorphic growth seen in the testis (Schmahl et al.,2000). In addition, Sf1 acts on numerous target genes(Val et al., 2003) that are expressed during gonadal development. Thus, the delay in Sertoli and Leydig cell differentiation observed in the Sf1 heterozygous male gonad might reflect reduced numbers of progenitor cells, impaired differentiation,reduced transactivation of marker genes, or several of these mechanisms. A gonad-specific knockout of Sf1 similarly failed to express Cyp11a1 and Star at E14.5 and 16.5, which resulted in hypoplastic testes and cryptorchidism(Jeyasuria et al., 2004). Furthermore, the number of proliferating cells observed at E12.5 in the XY gonad-specific Sf1 knockout was significantly lower than in wild type. It is notable that humans with heterozygous SF1 mutations exhibit a spectrum of adrenal insufficiency and gonadal dysgenesis, including XY sex reversal (Jameson,2004; Ozisik et al.,2003a). Thus, the dose-dependent effects of SF1 are even more pronounced in humans than in murine models.

The delay in Sertoli and Leydig cell function associated with Sf1haploinsufficiency provides an opportunity to assess functional interactions with Dax1. The consequences of Dax1 deficiency on testis development have been documented previously(Meeks et al., 2003a). Although the loss of either Sf1 or Dax1 function alters early testis development, their effects are distinct. Absence of Dax1predominantly alters the differentiation of peritubular myoid cells, and the patterning of fetal Leydig cells along the ventromedial to dorsolateral axis. Sf1 regulates the temporal differentiation of Sertoli and Leydig cells, and there is a predominant spatial dependence exhibited in the anterior and posterior poles of the gonads. Sry expression begins in the central region of the gonad and extends anteriorly, followed by completion at the posterior end (Bullejos and Koopman,2005). It is possible that factors downstream of Sryfollow this spatiotemporal pattern.

Because the Dax1 promoter contains Sf1-regulatory elements(Burris et al., 1995; Hoyle et al., 2002; Kawabe et al., 1999; Yu et al., 1998a), the defects observed in the Sf1 heterozygote may be explained in part by reduced Dax1 expression. Semi-quantitative RT-PCR analysis of urogenital ridge tissue confirmed there was a decreased number of Dax1transcripts in Sf1 heterozygous gonads at E11.5 by about 50% (data not shown), consistent with previous reports(Hoyle, 2002). Nevertheless,the phenotypic features of the Sf1 heterozygous gonad are more pronounced than those seen in Dax1 null mutant, indicating distinct functions.

Although in vitro studies of Dax1 inhibition of Sf1 transactivation provide a relatively straightforward model for how these factors interact, the distinct effects of individual Sf1 and Dax1 mutations presage the consequences of the combined Sf1/Dax1 mutation. Indeed,we found that testis development was affected to a greater degree in the combined Sf1/Dax1 double mutant. In particular, the fetal Leydig cell markers Cyp17 and Cyp11a1 were absent at E13.5. The fetal Leydig cells ultimately recover in the Sf1/Dax1 mutant and their population is normal by 2 weeks after birth, prior to the proliferation of adult Leydig cells. Sertoli cells appear to have selective roles for Sf1 and Dax1. Amh expression was reduced in the Sf1heterozygote, consistent with the presence of Sf1-regulatory elements in this gene (Hatano et al., 1994; Shen et al., 1994). Unexpectedly, Dax1 null gonads have reduced Amh production in vivo, although Dax1 has been shown to mediate repression at the transcriptional level in vitro (Nachtigal et al., 1998). By E12.5, both of the single mutants recover Amh expression, but recovery is more delayed in the double mutant. Of note, adult Leydig cells are present in Sf1/Dax1 double mutant mice and steroidogenesis is normal in the adult testis (data not shown). In spite of delayed steroidogenic enzyme gene expression, testis descent and secondary male reproductive features were unaffected in single and double mutants, and there was no persistence of Müllerian ducts (data not shown).

The phenotype of the combined Sf1/Dax1 mutation is reminiscent of the Dhh knockout, which also exhibits a delay in fetal Leydig cell development and function (Yao et al.,2002). Another feature of the Dhh knockout male gonad is a defect in peritubular myoid cell development(Clark et al., 2000; Pierucci-Alves et al., 2001),a feature also seen in the Dax1 null mutant(Meeks et al., 2003a). Dhh is expressed by Sertoli cells and acts via the Ptc1 receptor on fetal Leydig cells. At E11.5, Dhh expression was greatly reduced in Sf1/Dax1 double mutant gonads but was partially recovered by E12.5. Hence, a temporal delay in Dhh expression in Sf1/Dax1mutants from E11.5 to E12.5 precedes the delay in fetal Leydig cell differentiation from E13.5 to E14.5. In addition to Sf1 and Dax1,Pdgfra also acts upstream of Dhh. Homozygous knockouts of Pdgfra show delayed Dhh expression from 11.5 to 12.5, with a concomitant decrease in Cyp11a1(Brennan et al., 2003).

The delay in Dhh and Amh expression suggests that Sf1 and Dax1 converge on Sertoli cells to modulate key molecular pathways in male differentiation. However, Sox9 expression is relatively preserved, suggesting differential effects on specific genes. The crucial genetic interaction of Sf1 and Dax1 appears to occur early in the bipotential stage at 11.5 dpc or before. The precise mechanism for this interaction is currently unknown. It remains plausible that Dax1 exerts a repressive function for a subset of Sf1-regulated genes that somehow regulate the timing of Sertoli cell differentiation, or Dhhand Amh expression. Given the multiple functions of Sf1 in male gonad development (coelomic epithelial proliferation, Amh production, and steroidogenesis), Dax1 repression of Sf1 target genes might be highly variable and depend on both the amount of Sf1 protein present and the number of Sf1 DNA-binding elements on the target gene promoter and (Hanley et al.,2001). At present, the regulatory elements of the Dhhpromoter have not been characterized but may contain regulatory elements that allow cooperative rather than antagonistic actions of Sf1 and Dax1. Alternatively, Sf1 and Dax1 may act indirectly to delay Dhh expression by altering cell lineage restriction prior to Dhh expression. This could include paracrine effects on Sertoli cells that express Dhh. The observation that single mutations of Sf1 and Dax1 influence the spatial expression of genes in the developing gonad is consistent with effects on positional cues or cell-cell interactions.

In a previous study, we analyzed the effect of allelic loss of Sf1on a Dax1 null background with respect to adrenal development and function (Babu et al., 2002). Double mutation of Sf1 and Dax1 restored adrenal weight and corticosterone production. Allelic loss of Sf1 corrected the overexpression of Cyp21 and the Acthr in the Dax1null adrenal gland. These findings in the adrenal gland are reminiscent of the selective repression of Cyp19 in adult Leydig cells of the testis(Wang et al., 2001). Taken together, these studies of Sf1 and Dax1 interactions in vivo suggest antagonistic interactions for some target genes, such as Cyp21,Acthr and Cyp19. However, target genes, such as Dhh and Amh, require cooperative functions of Sf1 and Dax1. It is also likely that these nuclear factors function independently, as evidenced by the distinct features found in single gene mutant phenotypes of the adrenal gland and gonad. These discrete actions of Sf1 and Dax1 are also consistent with the clinical consequences of human SF1 and DAX1 mutations, each of which impair adrenal and testis development but exhibit distinct histological characteristics.

The authors would like to thank B. Capel, P. Koopman, A. McMahon, I. Mason and S. Tevosian for generously providing reagents. We acknowledge Baxter Jeffs and David Bavers for initiating the work on this project in adult mice. We thank E. J. Lee, Jeanne O'Brien, Silvia Novelli, and Parham Yashar for technical expertise and helpful discussions. All procedures were approved by the Northwestern University Animal Care and Use Committee, Master Protocol#027-07-193. This work was supported by NIH grants U01 HD043425-01 and R01 HD044801. S.Y.P. is a Fellow of The Dolores Zohrab Liebmann Fund.