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. 2011 Jan 5;84(5):894–899. doi: 10.1095/biolreprod.110.088997

Redundant and Differential Roles of Transcription Factors Gli1 and Gli2 in the Development of Mouse Fetal Leydig Cells1

Ivraym Barsoum 3, Humphrey HC Yao 4,5,2
PMCID: PMC4574634  PMID: 21209421

Abstract

Appearance of mouse fetal Leydig cells requires activation of the Hedgehog pathway. Upon binding to the membrane-bound receptor patched, Hedgehog ligands induce intracellular responses via a combined effect of Gli transcription factors. Szczepny et al. (Biol Reprod 2009; 80:258–263) found that Gli1, one of the three Gli transcription factors, is present in the fetal testis and that its expression is suppressed by the Hedgehog inhibitor cyclopamine. In this study, we investigated the involvement of the Gli1 and Gli2 factors in mouse fetal Leydig cell differentiation. The Gli1 and Gli2 transcription factors showed an overlapping expression pattern in the testis interstitium at the time when fetal Leydig cells appear. Despite their similar expression, Gli1 and Gli2 patterns were differentially regulated. Initial Gli1 and Gli2 expression depends upon an active Hedgehog pathway; however, maintenance of only Gli1, but not Gli2, expression requires activation of the pathway. Inactivation of either the Gli1 or Gli2 gene did not affect fetal Leydig cell development and testis morphology, suggesting a functional redundancy. When the transcriptional activity of both GLI1 and GLI2 was suppressed by a selective inhibitor, GANT61, in cultured fetal testes before the appearance of fetal Leydig cells, Gli1 and Gli2 expression and steroidogenic marker activity were completely abolished. However at later stages when Leydig cells were already present, GANT61 treatment inhibited Gli1 expression but had no effects on Gli2 expression and fetal Leydig cell appearance. Our results reveal overlapping and redundant Gli1 and Gli2 roles in fetal Leydig cell differentiation and a novel regulation of Gli2 expression in the fetal testis.

Keywords: developmental biology, Gli, hedgehog, Leydig cells, steroidogenesis, testis

The transcription factors Gli1 and Gli2 are redundant initiators of fetal Leydig cell differentiation, while Gli2 maintains fetal Leydig cells in a Hedgehog-independent manner.

INTRODUCTION

In mammals, the Hedgehog (Hh) pathway is known to regulate patterning of fetal organs, and its aberrant activation has been linked to the development of cancers in adult life [1]. Three mammalian Hh genes have been identified: desert hedgehog (DHH), Indian hedgehog (IHH), and Sonic hedgehog (SHH) [2]. Intracellular signaling pathway induced by SHH is the most well-characterized among the three mammalian HH genes [1]. Once SHH is processed and secreted, it binds to its receptor, patched (PTCH), in a concentration-dependent manner [3]. In mammals, PTCH, a 12-span transmembrane receptor, is encoded by two different genes, PTCH1 and PTCH2 [4]. PTCH1 has been shown to be the dominant receptor [4]. In the absence of SHH, the PTCH1 receptor is expressed at a basal level, which is sufficient to inhibit the activity of the seven-transmembrane-span SMO protein [5]. The binding of SHH to PTCH1 results in the loss of the ability of PTCH1 to inhibit SMO. The consequent activation of SMO transduces the Hh signal to the cytoplasm, leading to the activation/inhibition of the Gli family of transcription factors [6]. Both GLI1 and GLI2 act primarily as transcriptional activators, whereas GLI3 is a transcriptional repressor [7]. Evidence suggests that GLI2, but not GLI1, is cleaved to generate repressor forms of the protein, dependent upon the tissue context [8]. The immediate molecular action in response to Hh activation is the up-regulation of Gli1 and Ptch1 [2]. Thus, Gli1 or Ptch1 up-regulation is used as an indicator of an active Hh pathway.

The Hh pathway has been implicated in the development and differentiation of the fetal testis [9] and postnatal and adult ovary [10, 11]. In the fetal testis, Sertoli cell-derived DHH is responsible for differentiation of the fetal Leydig cells, the primary steroidogenic cell type in the developing testis [12]. At embryonic Day 11.5 (E11.5) in mouse embryos, Sertoli cells start to synthesize DHH, which triggers the differentiation of fetal Leydig cells in the testis interstitium [13, 14]. When Dhh was inactivated, numbers of fetal Leydig cells were significantly reduced, leading to lowered androgen production and consequent underdevelopment of androgen-dependent male accessory organs [14, 15]. On the other hand, when E11.5 testes were cultured for 48 h in the presence of cyclopamine, a general inhibitor of SMO [16], the fetal Leydig cell population was completely abolished [14, 17]. These observations together suggest the possibility of compensation by other Hh ligands in addition to Dhh in the fetal testis. Indeed, the ability of Hh ligands to compensate for one another has been reported in other tissues such as the prostate [18], where loss of Shh is accompanied by an up-regulation of Ihh expression.

In addition to the redundant action of Hh ligands, a second tier of compensation has been reported at the level of the Gli transcriptional factors. It was found that replacement of the Gli2 with the Gli1 gene was able to restore the function of the Hh signaling pathway in the Gli2 knockout mice [19]. Additionally, Gli1 knockout mice were viable, whereas Gli1 knockout mice heterozygous for the Gli2 knockout allele (Gli1ZFD/ZFD;Gli2ZFD/+) died perinatally due to severe developmental defects, indicating gene dosage-dependent compensation of Gli2 in the absence of Gli1 [20]. Taking into account the complexity of the mammalian Hh pathway and its different levels of compensation and redundancy, we investigated the roles of Gli1 and Gli2 in mediating Hh-induced differentiation of fetal Leydig cells in the mouse embryos. We did so by first identifying the Gli genes targeted by SMO and then we sought to determine their functional roles in the establishment of fetal Leydig cells, using ex vivo testis culture and knockout models.

MATERIALS AND METHODS

Animals

Mouse colonies were maintained following the University of Illinois Institutional Animal Care and Use Committee regulations and in accordance with the Animal Welfare Act and Public Health Service Policy. Both Gli1-LacZ and Gli2-LacZ mouse lines were generously provided by Dr. Alex Joyner (Sloan-Kettering Institute, New York City, NY) and were maintained in a mixed genetic background [19, 21, 22]. Mice heterozygous for the LacZ allele were used as reporter lines, and mice homozygous for the LacZ allele were used for knockout analysis.

X-Gal Staining

Gonads with the mesonephroi attached were isolated from embryos and placed in X-gal substrate (0.25–1 mg/ml in final concentration of 2 mM MgCl2 plus 0.02% NP-40 in PBS plus 3.5 mM K ferrocyanide plus 3.5 mM K ferricyanide). Once color development had occurred, samples were fixed in 4% paraformaldehyde for 30 min, dehydrated through a sucrose gradient, and embedded in a sucrose-OCT embedding compound (Bayer) medium at a ratio of 1:3. Samples were cryosectioned at 8–10 μm on treated glass slides. FastRed nuclear counterstain (Vector Laboratories, CA) was applied to the slides for 30 sec to 1 min. The slides were rinsed in double-distilled water and then dehydrated through an ethanol-xylene gradient and mounted.

Ex Vivo Organ Culture

E11.5 and E12.5 testis and mesonephros complexes from CD1 or LacZ reporter embryos were isolated. The complexes were cultured in 35-mm cell culture dishes (Greiner Bio-One, Germany) floating in drops of Dulbecco modified Eagle medium with high glucose, supplemented with 10% fetal calf serum (Hyclone, UT) and 50 μg/ml ampicillin, at 37°C with 5% CO2/95% air flow. Cyclopamine (25 μM; Sigma Aldrich, MO), GANT61 (15 μM; Alexis Biochemicals, CA), or an equal volume of vehicle, dimethyl sulfoxide (DMSO), was added to the culture medium [16, 23]. The concentrations of cyclopamine and GANT61 represent the minimal concentrations that result in effects on the Hh pathway in the fetal testes in our studies. All culture experiments were repeated three times, and in each experiment, at least three gonads were exposed to the same treatment.

Whole-Mount In Situ Hybridization

Tissues were collected and fixed overnight in 4% paraformaldehyde in PBS at 4°C and dehydrated through a methanol gradient in PTW (0.1% Tween 20 in diethyl pyrocarbonate-PBS). Samples were rehydrated though a methanol gradient and digested in 10 μg/ml proteinase K solution at 37°C for 5–7 min, followed by immediate fixation in 4% paraformaldehyde-0.1% glutaraldehyde for 20 min at room temperature. Samples were washed three times for 5 min each in PTW, rinsed in PTW-hybridization buffer (1:1), and washed once in hybridization buffer at room temperature. Samples were incubated in the hybridization buffer consisting of 5× SSC (1× SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), pH 5, 50% formamide, 0.1% CHAPS, 0.1% Tween 20, 1 mg/ml yeast tRNA, 50 ng/ml heparin, and 5 mM EDTA (pH 8) for 2 h at 65°C. Digoxigenin-labeled RNA probe was added to the solution, and samples were incubated in an oven at 65°C overnight (12–16 h). On the following day, samples were washed with prewarmed hybridization buffer and then washed with MABTL (5% maleic acid buffer [MAB], 0.1% Tween 20, and 0.05% Levamisol) at room temperature. Samples were then incubated at room temperature in 20% heat-inactivated sheep serum in MABTL blocking (20 mg/ml, blocking powder; Boehringer) for 2–4 h prior to the addition of alkaline phosphatase-conjugated anti-digoxigenin antibody (1:1000 dilution). Samples were incubated on a shaker at 4°C overnight. After three 1-h washes with MABTL, samples were incubated in 20 μl/ml alkaline phosphatase substrate (NBT/BCIP; Roche Diagnostics) in alkaline phosphatase buffer for color development. The reaction was stopped at the appropriate color intensity by washing the samples in PTW, and this was followed by fixation in 4% paraformaldehyde for 20 min at room temperature.

Immunohistochemistry

Fixed samples were rehydrated from methanol back to PBS. Samples were washed with a sucrose gradient in PBS (10%, 15%, and 20% in a 20% sucrose-OCT medium at 1:1 ratio) and embedded in a 20% sucrose-OCT medium (at 1:3 ratio). The blocks were cryosectioned into 8- to 10-μm sections, which were mounted on glass slides. Sections were blocked for 1 h at room temperature in blocking solution (5% donkey serum and 0.1% Triton X-100 in PBS). Sections were then incubated overnight at 4°C in different concentrations of the following primary antibodies: anti-CYP17 (1:100 dilution; from Dr. Buck Hales, University of Illinois, Chicago), anti-laminin (1:200 dilution; Sigma), anti-anti-Müllerian hormone (anti-AMH; 1:100 dilution; Santa Cruz Biotechnology), and anti-TRA98 (1:1000 dilution; from Dr. Tanaka, Osaka University, Japan). After overnight incubation, slides were washed three times for 10 min each in washing solution (0.5% donkey serum and 0.1% Triton X-100 in PBS) and treated with either fluorescein isothiocyanate or rhodamine-conjugated secondary antibody at 1:500 concentration for 1 h at room temperature. Then, slides were washed three times for 10 min each in PBS, dried, and mounted with Vectashield mounting medium with 4′,6′-diamidino-2-phenylindole nuclear counterstain (Vector Labs).

RESULTS

Gli1 and Gli2 Expression in the Fetal Gonads

We first characterized the Gli1 and Gli2 expression patterns in gonads obtained from Gli1-LacZ and Gli2-LacZ embryos, where the LacZ reporter gene is inserted into the endogenous Gli1 or Gli2 locus [19, 21]. At E11.5, no Gli1-LacZ or Gli2-LacZ gene was detected in the male or female gonads (Fig. 1, A, D, G, and J). At E12.5 and E13.5, when steroidogenic fetal Leydig cells start to appear, both Gli1-LacZ and Gli2-LacZ expression became positive in the fetal testis (Fig. 1, B, C, H, and I). In contrast, the fetal ovary remained devoid of Gli1-LacZ and Gli2-LacZ expression (Fig. 1, E, F, K, and L). In the testis, both Gli1-LacZ and Gli2-LacZ expression was confined to the interstitium outside of the testis cords (Fig. 1, M and N), overlapping with that of fetal Leydig cell marker P450 side chain cleavage (Scc) mRNA (Fig. 1). We also obtained identical Gli1 and Gli2 expression patterns by using mRNA in situ hybridization (data not shown), confirming the fact that the LacZ activity faithfully reflects endogenous Gli1 and Gli2 expression. These results indicate that both the Gli1 and Gli2 genes are expressed in the fetal Leydig cells. In the mesonephros, Gli1-LacZ and Gli2-LacZ staining started to appear in the anterior tip of the mesonephros in both sexes at E11.5 and spread posteriorly afterward.

FIG. 1.

FIG. 1.

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Patterns of Gli1-LacZ (A–F and M) and Gli2-LacZ (G–L and N) expression are shown in fetal mouse gonads at E11.5, E12.5, and E13.5. Gonads and mesonephroi from male or female embryos carrying the LacZ reporter gene were stained with X-gal substrate (blue color). M and N) Gli1-LacZ and Gli2-LacZ E13.5 gonads (respectively) were X-gal stained, sectioned, and counterstained with nuclear FastRed. O) E14.5 testis was stained for Scc expression by whole-mount in situ hybridization and then cryosectioned. A) g, gonad; m, mesonephros; (O) tc, testis cord. A–L) Bar = 500 μm; (M–O) bar = 200 μm.

Effects of the SMO Inhibitor on Gli1 and Gli2 Expression and Appearance of Fetal Leydig Cells

To examine which Gli factors are regulated by Hh signaling in fetal testes, we cultured Gli1-LacZ or Gli2-LacZ fetal testes with cyclopamine, which inhibits the SMO activity [16]. When the organ culture started at E11.5 (Fig. 2, Treatment 1) before the time of Gli1-LacZ and Gli2-LacZ expression (Fig. 1) and fetal Leydig cell appearance, cyclopamine treatment abolished Gli1-LacZ, Gli2-LacZ, and Leydig cell marker Scc expression compared to those in the DMSO-treated control (Fig. 2, A–F, Treatment 1). On the other hand, when cyclopamine treatment was given to E12.5 testis, where Gli1-LacZ and Gli2-LacZ and fetal Leydig cells were already present, cyclopamine only suppressed Gli1-LacZ but not Gli2-LacZ and Scc expression (Fig. 2, G–L, Treatment 2). These results indicate that initial induction of both Gli1 and Gli2 and fetal Leydig cell differentiation requires an active Hh signaling via SMO. However, only maintenance of Gli1 activity, but not Gli2 and fetal Leydig cell differentiation, requires an active Hh pathway via SMO.

FIG. 2.

FIG. 2.

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Effects of the Hh inhibitor cyclopamine on Gli1-LacZ (A, B, G, and H), Gli2-LacZ (C, D, I, and J), and Scc (E, F, K, and L) expression in gonad explant culture are shown. E11.5 (Treatment 1) or E12.5 (Treatment 2) testes were cultured for 72 h in the presence of either DMSO or cyclopamine. After culture, samples were processed for LacZ staining or whole-mount in situ hybridization for Scc expression as a marker for steroidogenic cells. All culture experiments were repeated three times, and in each experiment, at least three gonads were exposed to the same treatment. A) m, mesonephros; t, testis. Bar = 500 μm.

Effects of Loss of Gli1 or Gli2 on Fetal Testes

We next investigated Gli1 and Gli2 functions in the establishment of fetal Leydig cells by examining testes from embryos carrying either the Gli1 or the Gli2 knockout allele. The Gli1 knockout animals in our colony developed normally and were fertile when they reached adulthood, consistent with the original observation [24]. At the time of birth, the reproductive organs in Gli1 and Gli2 knockout newborns were indistinguishable from their respective wild-type littermates (Fig. 3, A–D [only Gli2 animals are shown]). In the Gli1 and Gli2 knockout animals, formation of the testis cords, marked by laminin staining (Fig. 3, E–G), development of germ cells (Fig. 3, E–G, TRA98 staining), differentiation of Sertoli cells (Fig. 3, H–J, anti-Müllerian hormone or AMH staining), and differentiation of fetal Leydig cells (Fig. 3, H–J and K–M, 3-beta hydroxyl steroid dehydrogenase [3βHSD] and CYP17 staining, respectively) were not different from those of the wild-type animals at birth. Gli2 knockout animals died before birth, and therefore their reproductive status in adulthood remains unknown. Loss of either Gli1 or Gli2 expression did not affect testicular morphogenesis, establishment of fetal Leydig cells, and male sexual differentiation, suggesting that the Gli1 and Gli2 genes may function redundantly.

FIG. 3.

FIG. 3.

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Development of reproductive tracts and testes are shown in the wild-type and the Gli knockout newborn mice. A–D) Reproductive tracts were isolated from wild-type or Gli2 knockout embryos and processed for whole-mount imaging. B and D) White arrows and green arrows indicate epididymis and seminal vesicle, respectively. E–M) Testis sections were immunostained for the germ cell marker TRA98 (red in E–G) and laminin (green in E–G), the Sertoli cell marker AMH (red in H–J) and 3βHSD (green in H–J), and CYP17 (green in K–M). A) a, adrenal; k, kidney; t, testis. A–D) Bar = 500 μm; (E–M) bar = 100 μm.

Effects of the GLI1/GLI2 Inhibitor on Fetal Leydig Cell Differentiation

We intended to generate Gli1/Gli2 double-knockout embryos; however, none of our breeding yielded viable double-knockout embryos at E11.5. We therefore utilized a chemical inhibitor, GANT61, which was shown to suppress GLI1/GLI2-mediated transcription [23, 25]. GANT61 treatment inhibited Gli1-LacZ, Gli2-LacZ, and Leydig cell marker Scc and Hsd3b expression in fetal testes when the culture started at E11.5 or before the onset of Gli1-LacZ, Gli2-Lac, and Leydig cell differentiation (Fig. 4, A–H, Treatment 1 is identical to that in Fig. 2). Sertoli cell differentiation (Fig. 4, G and H, AMH staining) was not affected by GANT61 treatment, indicating that the observed effects did not result from compromised Sertoli cell differentiation. When GANT61 treatment started at E12.5 (Fig. 2, Treatment 2) after the appearance of Gli1-LacZ, Gli2-LacZ, and fetal Leydig cell markers (Scc and Hsd3b [only Scc is shown]) expression, the treatment inhibited only Gli1-LacZ expression (Fig. 4, I and J) but not those of Gli2-LacZ (Fig. 4, K and L) and Scc (Fig. 4, M and N). These results together demonstrate that Gli1 and Gli2 transcription activity is necessary for induction of fetal Leydig cell differentiation. However, once fetal Leydig cells appear, their maintenance does not require activation of the Hh pathway via GLI1 or GLI2. In addition, Gli1 and Gli2 expression is under differential control by their own actions. Gli1 expression requires the presence of Gli transcription activity. For Gli2, on the other hand, once its expression is initiated, maintenance of its expression does not require the transcriptional activity of Gli factors.

FIG. 4.

FIG. 4.

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Effects of GANT61 on Gli1-LacZ (A, B, I, and J), Gli2-LacZ (C, D, K, and L), and Scc (E, F, M, and N) expression in gonad explant culture are shown. E11.5 (Treatment 1 as shown in Fig. 2) or E12.5 (Treatment 2) testes were cultured for 72 h in the presence of either DMSO or GANT61. After culture, samples were processed for LacZ staining, whole-mount in situ hybridization for Scc, or immunohistochemistry for AMH (red) and 3βHSD (green in panels G and H). All culture experiments were repeated three times, and in each experiment, at least three gonads were exposed to the same treatment. A–F and I–N) Bar = 500 μm; (G and H) bar = 200 μm.

DISCUSSION

Steroidogenic cells are present in many organs including adrenal, ovary, testis, placenta, and brain. Gonads, in particular, synthesize sex steroids that are required for reproductive functions and fertility [26]. Steroidogenic cell function in adult gonads is regulated mainly by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary [27, 28]; however, steroidogenic fetal Leydig cells in fetal testes do not require LH or FSH [29, 30]. Mouse fetal Leydig cells depend upon Hh signaling to acquire their steroidogenic identity [14, 15, 31, 32]. Androgen and insulin-like growth factor 3 (INSL3) produced by fetal Leydig cells are required for masculinization, testicular descent, and male reproductive tract development [13]. In this study, we examined the potential involvement of Gli1 and Gli2 transcription factors in the initial differentiation of fetal Leydig cells. Gli1 and Gli2 expression in fetal gonads is testis-specific and exclusively present in the testicular interstitium, overlapping with the Hh receptor Ptch1 expression [33] and that of the steroidogenic marker Scc. Testis-specific Gli1 and Gli2 expression indicate these two transcription factors may act downstream of the Hh pathway and play roles in fetal Leydig cell differentiation.

Initial Gli1 and Gli2 expression is sensitive to the common Hh signaling modulator, SMO, as is evident by the loss of their expression in the presence of the SMO inhibitor cyclopamine. The inhibitory effects of cyclopamine on fetal Leydig cell development and testicular structure of mouse embryo have been reported previously [14, 17]. Szczepny et al. [34] documented a similar inhibitory effect of cyclopamine on Gli1 mRNA expression in testis at 11.5 days postcoitum. However, the spatial and temporal expression of the Gli1 and Gli2 genes and their involvement in fetal Leydig cell development were not known. Our findings place both the Gli1 and Gli2 genes downstream of SMO in the Hh pathway in the fetal testes. We discovered a differential SMO activity is required to control Gli1 and Gli2 expression. Initial Gli1 and Gli2 expression is dependent on SMO and Hh signaling. On the other hand, maintenance of Gli1, but not Gli2, expression requires a constant activation of SMO in the fetal testis. Research of other organ systems such as limbs has shown, contrary to our finding, that initial Gli2 expression is usually independent of Hh signaling [19, 21, 35], whereas Gli1 expression is dependent upon the activation of SMO [2, 19, 21, 35–39]. Our findings reveal a novel regulation of Gli2 expression in the fetal testis.

Loss of either Gli1 or Gli2 expression did not affect fetal Leydig cell differentiation and male sexual differentiation regulated by androgens, suggesting a potential compensation between these two factors. Compensation between the Gli1 and Gli2 genes is present in other tissues in the mouse [18, 19, 21], as well as in cultured cell lines [40, 41]. For example, in the Gli2 knockout mouse, introduction of the Gli1 gene into the Gli2 knockout allele restored the Hh signaling [19]. In addition, infection of Gli2−/− mouse embryonic fibroblasts with adenovirus encoding a mouse GLI1-GFP fusion protein was able to partially restore the Hh signaling [21]. In the development of lung, nervous system, and limbs, Gli2 knockout embryos had abnormal phenotypes that were aggravated by introduction of the Gli1 knockout allele [20]. Also, in regard to optic development, Gli1 and Gli2 knockout embryos had normal optic cup length, while the Gli1/Gli2 double-knockout embryos showed significantly elongated optic cup [42]. We intended to generate the Gli1/Gli2 double-knockout embryos [20] but failed to produce any viable double-knockout embryos at or after E11.5. A possible explanation is that the mixed genetic background of the Gli1 and Gli2 mouse strains contributes to an early onset of embryonic lethality. To circumvent this problem, we used testis organ culture with GANT61 inhibitor to suppress GLI1/GLI2-moderated transcription and studied its effect on fetal Leydig cell development.

GANT61 treatment inhibited the Hh pathway and Scc and Hsd3b expression in fetal testis. To exclude the possibility that these defects were the results of nonspecific action of GANT61 in general testis development, we investigated the differentiation of Sertoli cells, the cell type responsible for testis morphogenesis, and found no apparent defects in Sertoli cells. This finding indicates that the loss of Hh signaling and fetal Leydig cells in GANT61-treated testis was not secondary to loss of functional Sertoli cells. It has been shown that Amh mRNA expression in Sertoli cells was up-regulated in response to cyclopamine treatment [34]. In our study, we used the presence of the AMH protein as an indicator of Sertoli cell development and testis cord formation. The overall effects of GANT61 were similar to those of cyclopamine, which inhibited SMO and the appearance of fetal Leydig cells. Therefore, we concluded that both GLI1 and GLI2 are downstream components of the Hh pathway that regulate the initial steroidogenic enzyme expression in fetal Leydig cells.

Similar to the effects of cyclopamine, the initial Gli1 and Gli2 expression was sensitive to GANT61 treatment, while maintenance of their expression at later stages showed a different pattern of regulation. Both initiation and maintenance of Gli1 expression in the fetal testis requires the transcriptional activation of GLI factors. Gli2 expression, once initiated, becomes insensitive to GANT61, indicating that its maintenance is independent of transcriptional activation of the GLI factors. The Hh-independent Gli2 maintenance, as shown by GANT61 and cyclopamine experiments, may partly explain the inability of cyclopamine to inhibit fetal Leydig cell differentiation once they appear in the fetal testis [14]. In summary, we demonstrate that both GLI1 and GLI2 are involved in appearance of fetal Leydig cells induced by the Hh pathway. GLI1 and GLI2 appear to work in a redundant fashion to ensure the onset of steroidogenesis and appearance of fetal Leydig cells in the testes. However, once Gli2 expression and differentiation of fetal Leydig cells are initiated, the maintenance of these two events no longer requires an active Hh pathway.

Acknowledgments

We thank Drs. Buck Hales and Ken-Ichirou Morohashi for supplying antibodies. We also thank Dr. A. Joyner for the generous supply of Gli1-LacZ and Gli2-LacZ mouse lines.

Footnotes

1

Supported by U.S. National Institutes of Heath grants NIH-HD-46861 and HD-059961 to H.H.C.Y., March of Dimes Birth Defects Foundation to H.H.C.Y., and by a UIUC College of Veterinary Medicine Billie Field Graduate Fellowship to I.B.B. Also supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

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