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Adrenal, testis, and ovary are steroidogenic organs derived from a common primordium that consists of steroidogenic factor 1 (SF1)-positive precursor cells. SF1 not only defines the steroidogenic lineages in these organs but also controls their differentiation. Recent evidence implicates the Hedgehog (Hh) signaling pathway as a downstream regulator of SF1 in the appearance of steroidogenic cells in these organs. The Hh signaling pathway serves as a common crosstalk component, yet has evolved diverse functions in the expansion and differentiation of the steroidogenic cells in a tissue-specific manner. The purpose of this review is to compare and contrast the different roles of Hh signaling in these three organs during development.
Adrenals and gonads (ovaries and testes) are the major sources of steroids essential for energy and salt homeostasis, immune functions, and reproduction. These organs share not only the ability to produce steroids, but also a common primordium when they first arise in embryos. In the mouse embryo around 9.5 days postcoitum (dpc), the adrenogonadal primordium forms as a result of proliferation of the coelomic epithelium that cover the urogenital ridge (Luo et al., 1994; Keegan and Hammer, 2002). Cells in the adrenogonadal primordium become distinguishable from other somatic cells as a result of the expression of the orphan nuclear receptor steroidogenic factor1 [Sf1, also known as Nr5a1, Ad4BP, or Ftzf1 (OMIM 184757)]. SF1 marks the steroidogenic precursors, which later become adrenal cortex and somatic cells in the future gonads (Fig. 1). SF1 is essential for the formation of the adrenals and gonads and development of pituitary gonadotrophs and ventromedial hypothalamic neurons (Rice et al., 1991; Luo et al., 1994; Ikeda et al., 1995; Shinoda et al., 1995). Between 10–11 dpc, via an unknown mechanism, the SF1-positive cells in the adrenogonadal primordium separate into two populations, which eventually differentiate into the adrenal primordium and gonadal primordium, respectively (Fig. 1).
In the developing adrenal primordium, SF1-expressing cells serve as the foundation for the fetal adrenocortex (Luo et al., 1994). Lineage tracing experiments suggest that the SF1-expressing fetal cortical cells also contribute to the adult adrenocortex (Zubair et al., 2008). The adrenal primordium is later encapsulated by the mesenchymal capsular cells, which is thought to provide stem/progenitor cells in adult or regenerating adrenal (Kim and Hammer, 2007; Kim et al., 2009). In the absence of Sf1, the adrenal primordium forms initially but then degenerates via programmed cell death, leading to adrenal agenesis at birth (Luo et al., 1994; Sadovsky et al., 1995).
Once separated from the adrenal primordium, the gonadal primordium undergoes dimorphic differentiation based on the genetic make-up of the embryos. In the XY embryo, a subset of the SF1-positive cells in the gonadal primordium starts to express the testis-determining gene Sry (Sex-determining region of the Y chromosome) and becomes Sertoli cells. SF1 participates in the establishment of Sertoli cells by regulating the expression of Sry, Sox9, and anti-Mullerian hormone (Amh) (Shen et al., 1994; Giuili et al., 1997; Sekido and Lovell-Badge, 2009). Sertoli cells then produce paracrine factors that induce differentiation of SF1-positive/SRY-negative Leydig cells outside the testis cords (Fig. 1). In the Leydig cells, SF1 controls the transcription of insulin-like growth factor 3 (Insl3) as well as various enzymes involved in steroidogenesis (Morohashi and Omura, 1996; Zimmermann et al., 1998; Cui et al., 2004; Park et al., 2007). In the male, testicular decent requires Leydig cell-derived Insl3, expression of which ould be regulated by androgen (Nef and Parada, 1999; Brokken et al., 2009; Tremblay et al., 2009).
In the XX mouse embryo that lacks the Sry gene, transcription and translation of SF1 decrease dramatically at 13.5 dpc, soon after the male and female gonads become morphologically different. SF1 level remains low in somatic cells of the fetal ovary but its expression rises again at the onset of folliculogenesis around the time of birth (Ikeda et al., 1994). SF1 is critical for granulosa cell development as granulosa cell-specific SF1 knockout females lack ovarian expression of SF1 target genes, resulting in decreased follicle numbers and infertility (Pelusi et al., 2008).
The presence of steroidogenic cells and their shared origin in adrenals and gonads raise the possibility that a common regulatory mechanism is present for the establishment of these steroidogenic cell lineages. In this review, we focus on the involvement of the Hh signaling, a conserved pathway in organogenesis among many species, in adrenal and gonadal development. We discuss recent findings on how this signaling pathway controls the growth and differentiation of the adrenocortex, testis, and ovary via a tissue-specific crosstalk among the SF1-positive cells.
The Hh ligands are secreted proteins involved in many aspects of embryonic organ development and tumorigenesis in adult animals. Detailed information on the Hh pathway in mammals can be found in other reviews (Varjosalo and Taipale, 2008; Kasper et al., 2009) and only a general overview is provided here. In mammals, three Hh orthologs have been identified: Desert hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic hedgehog (Shh). Although these three Hh genes have diverse expression patterns and functions, they are thought to induce a common signal transduction pathway in the target cells. Hh proteins bind to a receptor complex consisting of Patched family receptors (PTCH1 and PTCH2) and Smoothened (SMO) on the membrane. PTCH1 and PTCH2 have similar ligands binding affinity but their expression patterns are not fully overlapped (Carpenter et al., 1998). Hh-receiving cells are enriched with cilia where PTCH inhibits SMO activity by preventing its accumulation within cilia (Rohatgi et al., 2007). Binding of Hh to PTCH1 releases the inhibitory effect of PTCH1 on SMO, therefore allowing SMO to activate the downstream signaling components that involves transcription factors GLI1, GLI2, and GLI3 (Ingham and McMahon, 2001; Chari and McDonnell, 2007). In the absence of Hh, GLI2 and GLI3 are targeted for proteasomal processing (Sheng et al., 2006; Tempe et al., 2006; Wang and Li, 2006). The processed GLI2 and GLI3 serve as repressors that inhibit target gene transcription. In the presence of Hh, activated SMO inhibits proteolysis of GLI2 and GLI3, leading to the production of full-length transcriptional activator forms of these proteins. GLI1, on the other hand, contains only the activation domain and acts solely as a transcriptional activator (Dai et al., 1999). Although Hh ligands probably act through a common pathway, they elicit different effects in the tissue-specific manner, probably due to interaction with other signaling pathways (McMahon et al., 2003).
In the fetal mouse adrenal, Shh is the only Hh ligand present and its expression is restricted to SF1-positive adrenocortical cells underneath the adrenal capsule (Bitgood and McMahon, 1995; Kim et al., 2009). On the other hand, Ptch1, Gli1, and Gli2 expressions are found in the adrenal capsule, which is negative for SF1 (Ching and Vilain, 2009; King et al., 2009; Huang et al., 2010). We and others develop a conditional knockout mouse model that Shh is inactivated specifically in the SF1-positive adrenocortical cells (Ching and Vilain, 2009; King et al., 2009; Huang et al., 2010). Loss of Shh in the SF1-positive cells leads to severely stunted adrenal cortex and hypoplasia of the SF1-negative/Ptch1-positive adrenal capsule. The adrenal capsule and the underlying subcapsular cells are postulated to be the sources of progenitor/stem cells for the adrenocortex (Kim and Hammer, 2007). This concept is first raised from the result of cytological studies using the trypan blue dye. Trypan blue-labeled cells are restricted in the capsule right after the injection of the dye. Later on, the dye-labeled cells are found in the zona glomerulosa while the capsule becomes free of dye (Salmon and Zwemer, 1941). In adult rats, when the adrenal parenchyma is removed (a process called enucleation), a functional cortex is restored within 30 days, presumably due to regeneration of the remaining capsule and subcapsular cells (Skelton, 1959; Perrone et al., 1986). Using 3H-thymidine pulse-chase to trace proliferating cells, it was found that cortical cells migrate centripetally from outer to inner layers (Bertholet, 1980; Zajicek et al., 1986). An “escalator” hypothesis was therefore proposed such that the progenitor cells derived from the capsule migrate centripetally and become a part of adrenal cortex (Zwemer et al., 1938; Jones, 1948). The centripetal movement also occurs in the differentiated adrenocortical cells, which are aligned in columns (Ford and Young, 1963; Iannaccone et al., 2003). Using a genetic fate-mapping system that permanently marks Hh-responding Gli1-positive cells, we and others found that the Gli1-positive capsular cells indeed migrate centripetally and transform into SF1-positive adrenocortical cells (Ching and Vilain, 2009; King et al., 2009; Huang et al., 2010).
Shh derived from the SF1-positive adrenocortical cells apparently controls the expansion of progenitor cells in the capsule. This is based on the fact that both capsular thickness and the number of capsular cells are significantly reduced in the Shh knockout adrenal. We presented these results at the 2008 Endocrine Society Annual Meeting (ENDO) and the XII Adrenal Cortex Conference, and proposed the model that Shh from the SF1-positive cells regulates adrenal growth through proliferation of the progenitor cells in the capsule (Huang et al., 2008a,b). Despite significant underdevelopment, the Shh conditional knockout adrenocortex undergo proper zonation, indicating that Shh is dispensable for differentiation of adrenocortex. In addition, this observation also suggests two possible sources of adrenocortical cells: (1) the SF1-positive cells from the adrenal primordium, which forms the foundation of the organ and (2) SF1-negative capsular cells, which contribute to further growth of the adrenocortex (Fig. 2). The lineage tracing experiment performed by the Morohashi group demonstrates that the adult adrenocortex could derive from not only the SF1-positive cells in the fetal adrenal but also from unknown cell populations that are negative for SF1 (Zubair et al., 2008).
In addition to Shh, Gli3 is another gene in the Hh pathway that plays a role in adrenal development. Gli3 mutant mice display a wide range of developmental abnormalities including absence of adrenal glands (Bose et al., 2002). In humans, Pallister–Hall syndrome (PHS) represents autosomal-dominant disorders associated with central polydactyly, imperforate anus, hypothalamic hamartoma, and other malformations (Clarren et al., 1980; Hall et al., 1980). A mutation in the Gli3 gene that results in a truncated form of GLI3 protein is found in some human PHS patients with adrenal hypoplasia and kidney malformation (Kang et al., 1997). It remains to be determined if Gli3 belongs to a component of the Shh pathway in the adrenal.
In the fetal mouse testis, Sertoli cells start to produce Dhh at 11.5 dpc via a coordinate action of SF1 and DAX1, right after the onset of testis differentiation (Bitgood and McMahon, 1995; Bitgood et al., 1996; Park et al., 2005). The Hh receptor Ptch1 is expressed in the interstitial compartment 24 hr after Dhh expression (Bitgood et al., 1996) and Gli1 and Gli2 expression mirror the pattern of Ptch1 (unpublished results). The downregulation of Ptch1 in Dhh null mutants indicates that Dhh could be the primary Hh ligand in action in fetal testes (Bitgood et al., 1996).
Inactivation of the Dhh gene in mice with the 129/Sv inbred background results in properly formed testes; however, these male are infertile, with a complete absence of mature sperm (Bitgood et al., 1996). Interestingly, when the Dhh null alleles are introduced to a mixed genetic background, severe testis dysgenesis phenotypes arise in the fetal testis, including apolar Sertoli cells and anastomotic testis cords (Clark et al., 2000; Pierucci-Alves et al., 2001). In addition to testis cord dysgenesis, both Dhh knockout male mouse and human patients with DHH mutations exhibit male pseudohermaphroditism with underdevelopment of internal male accessory organs and feminized external genitalia due to insufficient production of androgens (Umehara et al., 2000; Canto et al., 2004, 2005). It was later found that loss of Dhh leads to a decreased number of fetal Leydig cells, the major source of androgens (Yao et al., 2002). However, a few fetal Leydig cells remain in the Dhh knockout testis, suggesting that other pathways such as Pdgf could partially compensate for the loss of Dhh (Brennan et al., 2003). When fetal testes are cultured ex vivo with the general Hh inhibitor cyclopamine prior to the appearance of fetal Leydig cells, no fetal Leydig cells are detected (Yao and Capel, 2002; Yao et al., 2002). However, cyclopamine has no effects on fetal Leydig cells when they are already present in the testis (Yao and Capel, 2002; Yao et al., 2002). To investigate if the Hh pathway alone is sufficient to induce fetal Leydig cell differentiation, we activated the Hh pathway ectopically in SF1-positive cells in the fetal ovary, where the Hh pathway is normally silent. Fully differentiated fetal Leydig cells appear in the fetal ovary in response to Hh activation. These ectopic fetal Leydig cells in the ovary are functional, producing enough testosterone to masculinize the female embryos (Barsoum et al., 2009). The effects of Hh activation on the transformation of SF1-positive cells to fetal Leydig cells in the ovary is direct, as evident by the absence of Sertoli cells and other testicular factors. These results together indicate that (1) the Hh pathway is the primary facilitator of fetal Leydig cell differentiation and (2) the Hh pathway is responsible for initiation, rather than maintenance, of fetal Leydig cell differentiation.
DHH triggers fetal Leydig cell differentiation by upregulating Sf1 expression in the precursors of fetal Leydig cells (Yao et al., 2002; Barsoum and Yao, 2009). Ectopic activation of the Hh pathway in the fetal ovary also induces expression of SF1 (Barsoum et al., 2009). In humans, SF1 haploinsufficiency is associated with impaired Leydig cell function (Jameson, 2004). Haploinsufficiency of Sf1 in mouse embryos causes delayed expression of both Sertoli and Leydig cell markers such as Amh, Cyp11a1, and Cyp17 (Park et al., 2005). Loss of one Sf1 allele in the Dhh knockout background (Sf1+/−;Dhh−/−) abolished the fetal Leydig cell population compared to the reduced fetal Leydig cell population in Sf1+/+;Dhh−/− males (Park et al., 2007). Several conserved Hedgehog responsive elements (HRE, TGGGTGGTC) are present in the mouse Sf1 promoter region, suggesting a direct role of the Hh signaling on Sf1 transcriptional regulation.
In summary, in the fetal testis, the SF1-positive precursor cells differentiate into at least two distinct somatic cell populations: DHH-producing Sertoli cells and DHH-responsive fetal Leydig cells. The main function of this paracrine crosstalk is to ensure the proper appearance of fetal Leydig cells and production of androgens (Fig. 3). In addition to Dhh, we have found that Shh mRNA is also expressed in the developing testis. However, contrary to the Dhh knockout, mice that lack Shh specifically in the SF1-positive cells in the testis are fertile, indicating that Shh alone is dispensable for testis development (unpublished results).
In the fetal mouse ovary, the Hh pathway is presumably inactive based on the fact that expression of Dhh, Ptch1, Gli1, and Gli2 is absent (Bitgood and McMahon, 1995; Yao et al., 2002). However, the Hh pathway appears to be activated in the adult mouse ovary. In situ hybridization results reveal the cellular sources of Dhh and Ihh as granulosa cells and the sources of Ptch1 and Gli1 as the adjacent theca cell compartment in mouse ovaries (Wijgerde et al., 2005; Russell et al., 2007). During preovulatory stages after superovulation induction, both Ptch1 and Gli1 in the theca cell compartment are gradually decreased along with the loss of expression of Ihh and Dhh in granulosa cells (Wijgerde et al., 2005). In vitro study shows that treatment of follicles with SHH increases proliferation of granulosa cells, while treatment with the Hh inhibitor cyclopamine increases progesterone production (Russell et al., 2007). Bovine theca cells express Ptch1 and Smo and treatment with SHH induces proliferation and androstenedione production in these cells in culture (Spicer et al., 2009). These data indicate a possible paracrine crosstalk between granulosa (source of Hh) and theca cells (Hh-responding cells) in steroidogenesis and cell proliferation (Fig. 4). Dhh knockout female mice have no obvious phenotypes and are fertile (Bitgood et al., 1996). In addition, inactivation of Shh in the SF1-positive cells in the ovary yields no ovarian phenotypes (unpublished results). Although other Hh ligands may compensate for the lack of Dhh or Shh in these cases, the exact role of the Hh pathway in ovarian development remains to be determined.
We have recently begun a study on roles of the Hh pathway in fetal ovary development. A distinct pattern of Gli1 and Gli2 expression begins to emerge at 14.5 dpc. Gli1 expression is absent in the ovary throughout fetal life; however, after birth, strings of Gli1-positive cells enter the ovarian medulla via the ovarian vasculature and later populate the stroma surrounding the follicle. In contrast to Gli1, Gli2 expression becomes detectable in the fetal ovary at 14.5 dpc in a subset of somatic cells (probably endothelial cells of the vasculature) surrounding the germ cell nest. These unpublished findings suggest that Gli1 and Gli2 play different roles in the ovary. We are in the process of defining their roles and investigating if these two Gli factors become active in an Hh-dependent or independent manner.
Expression of SHH, DHH, PTCH1, SMO, and GLI1 proteins is increased stepwise in benign, borderline, and malignant ovarian tumors (Chen et al., 2007; Bhattacharya et al., 2008). Inhibition of the Hh pathway by cyclopamine or Gli1 siRNA results in decreased cell proliferation of ovarian carcinoma cells (Chen et al., 2007). In addition, ectopic activation of the Hh pathway in mouse granulosa cells leads to infertility, reduced ovulation, and delayed luteinization (Ren et al., 2009). These observations indicate that aberrant activation of the Hh pathway may be responsible for ovarian abnormality and carcinogenesis.
The Hh family of proteins is one of the highly conserved paracrine/autocrine morphogens from metazoans to mammals. In the developing adrenals and gonads, evidence of paracrine crosstalk via the Hh pathway is emerging. The Hh ligands are produced by the earliest differentiated SF1-positive cells such as adrenocortical cells in the adrenal (Shh), Sertoli cells in the testis (Dhh), and granulosa cells in the ovary (Dhh and Ihh). These tissue-specific ligands then act upon Hh-responding cells located mainly in the mesenchyme, including capsular cells in the fetal adrenal, fetal Leydig cells in the testis, and theca cells in the adult ovary. Although their mode of paracrine action seems conserved, these Hh ligands induce tissue-specific cellular responses in these three organs. In the fetal adrenal, SHH controls adrenocortex expansion by inducing proliferation of the SF1-negative progenitor cells in the capsule. In the developing testis, DHH is the primary regulator that initiates fetal Leydig cell differentiation. In the adult ovary, multiple Hh ligands appear to be involved in steroidogenesis of the theca layer. It is interesting to note that most of the Hh-producing cells are SF1-positive and the Hh-receiving cells respond to the Hh ligand by upregulating SF1 expression. This suggests that Hh activation could be a universal mechanism for the induction of SF1 in certain cell lineages. More studies are needed to determine how certain cells in the adrenal and gonads acquire the ability to produce Hh and how a tissue-specific regulatory pattern is established. Furthermore, the role of the Hh pathway in adult adrenal and gonads require further investigation (Szczepny et al., 2009). A better understanding of the involvement of the Hh pathway will shed new light onto the normal process of organogenesis and the potential causes for neoplasia in these steroidogenic organs.
This study was supported by National Institute of Health (HD46861 to H.H.Y.) and Billie Field Memorial Scholarship (C.-C.J.H.). We also like to dedicate this review to our collaborator Dr. Keith Parker, who passed away on December 13, 2008.