BOR - Papers in Press, published online ahead of print
April 2, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.015941
BIOLOGY OF REPRODUCTION 69, 8–14 (2003)
DOI: 10.1095/biolreprod.103.015941
© 2003 by the Society for the Study of Reproduction, Inc.
Emerging Roles for Hedgehog-Patched-Gli Signal Transduction in Reproduction1
David O. Walterhouse2,
Marilyn L.G. Lamm,
Elisabeth Villavicencio, and
Philip M. Iannaccone
Children's Memorial Hospital and the Children's Memorial Institute for Education and Research, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60614
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ABSTRACT
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Hedgehog (Hh) proteins are expressed during vertebrate development in some tissues with inductive properties and at epithelial-mesenchymal boundaries in several developing organs, including the lung, gut, hair follicle, and tooth. The Hh signaling pathway is highly conserved, and important clues to understanding the mechanism of Hh signal transduction in vertebrates have come from studies in Drosophila. In recent years, Hh signaling has been recognized during embryonic development and in some cases during postnatal life in several mammalian tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the prostate and mammary gland. The role of the pathway in these tissues is highly reminiscent of its role at epithelial-mesenchymal-stromal boundaries in other organ systems, which has provided a framework within which to explore Hh signaling in tissues that function in reproduction. Some features unique to these tissues are emerging, including a role in proliferation and differentiation of male germline cells in mammals and apparent influences of sex steroids on Hh signaling. However, many questions remain about the function of Hh signaling in the gonads, uterus, prostate, and mammary gland, including factors regulating the signal transduction pathway, identification of downstream target genes, and roles for Hh signaling in diseases involving these tissues.
developmental biology, embryo, mammary glands, prostate, testis
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INTRODUCTION
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Embryogenesis is highly regulated by complex signaling cascades. One such signaling pathway is initiated through the secreted Hedgehog (Hh) proteins that are expressed during very early vertebrate development in tissues with inductive properties, including the notochord, floor plate, and the zone of polarizing activity in the limb [1–6]. Hh proteins are also expressed at epithelial-mesenchymal boundaries in developing organs, including the lung, gut, hair follicle, and tooth [7, 8]. Hh signaling drives cell proliferation, promotes cell survival, and directs cell differentiation during embryonic development. Dysregulation of the pathway during development has been associated with significant human birth defects, including holoprosencephaly, basal cell nevus syndrome, and polydactyly [9–15].
A more limited role for Hh signaling has been described in postnatal life, largely in tissues requiring cell renewal. Hh signaling occurs at epithelial-mesenchymal boundaries during development and at epithelial-stromal boundaries in postnatal life in some tissues whose functions are essential for reproduction, including the gonads, uterus, and hormonally responsive accessory sex glands such as the mammary gland and prostate. Dysregulation of the pathway during postnatal life has been associated with human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas, supporting its critical role in the regulation of cell proliferation [16–21].
After a brief review of the Hh signaling pathway and some of its inductive properties at epithelial-mesenchymal boundaries, we focus on the emerging roles for Hh signaling in the gonads, uterus, and accessory sex glands during embryogenesis and postnatal development.
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Hh-Ptc-Gli SIGNALING
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Details of the Hh signaling pathway were first identified in the fruit fly Drosophila melanogaster (Table 1). The Drosophila hedgehog gene (hh) is critical in establishing segment polarity during embryogenesis and in patterning the wing and eye imaginal discs later in development [22–24]. The Hh precursor protein undergoes autocatalytic cleavage, which yields an amino terminal peptide that, upon further lipid modifications in the form of covalent coupling with cholesterol and palmitoylation, participates in both short- and long-range signaling [25–27].
The amino terminal Hh ligand interacts with a 12-span transmembrane receptor protein encoded in Drosophila by a single gene called patched (ptc). The interaction between the Hh and Ptc proteins relieves Ptc-mediated inhibition of the activity of a G protein-coupled seven-span transmembrane protein called Smoothened (Smo). The nature of the Ptc-Smo interaction remains unclear, with suggestions that Ptc acts indirectly to regulate Smo phosphorylation and hence its activity [28]. Some evidence in Drosophila has indicated that Hh can also signal to cells in a Ptc-independent process, which raises the possibility that Smo activity is controlled by some other protein [29]. When the Hh signal is absent or present at low levels in Drosophila imaginal discs, Ptc inhibits signal transduction through Smo. In this situation, the transcription factor Cubitus interruptus (Ci), which mediates Hh signaling, is sequestered in the cytoplasm, bound to microtubules in a protein complex that includes the kinesin-related protein Costal-2 (Cos-2), the putative serine-threonine kinase Fused (Fu), and a third protein called Supressor of Fused (Su(Fu)) [30–32]. Protein kinase A (PKA), which inhibits expression of Hh target genes [33], induces phosphorylation of the sequestered 155-kDa full-length Ci protein, which targets this protein for Slimb-mediated processing to a 75-kDa repressor form [34, 35]. The 75-kDa form of Ci, which retains the zinc finger DNA binding domain but lacks the carboxy terminal transactivation domain, enters the nucleus to repress transcription of target genes [34, 36].
In the presence of high-level Hh signaling in Drosophila, the 155-kDa form of Ci is released from the microtubules [31], translocates to the nucleus, binds to the coactivator CREB Binding Protein (CBP), and activates transcription of target genes, including hh, wingless (wg), decapentaplegic (dpp), and ptc [36–38]. Upregulation of ptc provides a mechanism for negative feedback in the pathway because Ptc inhibits further Hh signaling.
Elements of the Drosophila Hh signaling pathway and their general functions in the pathway are highly conserved in vertebrates, albeit with increased levels of complexity. Whereas Drosophila has single genes for hh, ptc, and the Gli family transcription factor ci, there are multiple homologues of these genes in vertebrates: three Hedgehog (Hh) genes (Sonic hedgehog, Shh; Indian hedgehog, Ihh; and Desert hedgehog, Dhh), two Patched genes (Ptc1 and Ptc2), and three Gli genes (Gli1, Gli2, and Gli3). Vertebrate homologues of Fu and Su(fu) have recently been cloned and characterized [39–42].
Roles of the multiple homologous genes in the vertebrate Hedgehog pathway remain somewhat unclear, although specific and redundant functions have been defined for some components of the pathway. Shh is widely utilized throughout the developing embryo, Dhh appears to be restricted to the testis, and Ihh seems to exert specific and overlapping functions with Shh in some developing organ systems [8, 43]. It appears that Ptc1 and Ptc2 can each interact with Smo to form a Ptc1-Smo or Ptc2-Smo receptor complex that mediates the action of the three mammalian Hh proteins [44]. Nevertheless, Ptc2 is missing a long C-terminal tail present in Ptc1 that could translate to a possible specificity of signaling function [44].
Although Gli family transcription factors mediate Hh signaling in vertebrates and Drosophila, different regulatory mechanisms have been described in the two systems. First, Hh control of Ci function in Drosophila occurs only via posttranslational modification of the 155-kDa form, whereas the regulation of Gli transcription factors occurs at both the transcription and translation levels [45–47]. Second, Ci acts as both a transcription activator and a repressor of Hh signaling; however, only Gli2 and Gli3 appear to undergo proteolytic cleavage that allows them to function as either transcription activators or repressors [46–49]. There is no evidence as yet that Gli1 undergoes cleavage. Third, although Ci binds the coactivator CBP in the nucleus, only Gli3 similarly binds CBP, whereas Gli1 binds the coactivator TAFII31, suggesting different mechanisms of action [46–50].
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HEDGEHOG SIGNALING AT EPITHELIAL-MESENCHYMAL BOUNDARIES
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Signaling interactions between epithelium and mesenchyme play important roles during organogenesis. Although early tissue grafting experiments supported an inductive role for the mesenchyme, more recent studies have indicated that the epithelium is a source of signaling ligands that target neighboring mesenchyme. Components of the Hh signaling pathway have been localized at numerous epithelial-mesenchymal boundaries in the developing embryo, including the lungs, gut, hair follicles, and teeth. Furthermore, functional and mutational analyses show that Hh signaling between epithelium and mesenchyme is critical in the development of these organ systems [51–61].
Through these studies, a model has emerged in which Hh signal originates in the epithelium, targets the mesenchyme primarily although not exclusively, and promotes mesenchymal cell proliferation and differentiation through Ptc receptors and Gli family transcription factors that are expressed in the mesenchyme (Fig. 1). The mesenchyme then provides for the most part unknown signals, presumably targets of the Gli family transcription factors, that promote epithelial proliferation. Evidence for direct effects of Hh signaling on the epithelium, which expresses Ptc receptors not mediated through mesenchymal interactions, exists in some situations.
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FIG. 1. Model of hedgehog (Hh) signaling and its effects on cell proliferation and differentiation at epithelial-mesenchymal boundaries
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Many questions, however, remain to be answered. The significance of Shh versus Ihh signaling, Ptc1 versus Ptc2 utilization, and transcriptional regulatory effects of Gli1, Gli2, and Gli3 remain unclear in most settings. For the most part, factors regulating the expression and function of these genes and proteins remain unknown. In addition, downstream targets of the pathway that contribute to cell proliferation, including cell cycle regulators and growth factors, await identification, which will be critical to understanding the significance and function of Hh signaling in different settings. Presumably, such a system provides signaling between epithelium and mesenchyme during development that ultimately ensures coordinated normal function.
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SIGNALING IN THE GONADS, UTERUS, AND ACCESSORY SEX GLANDS
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Studies during the last 10 years have steadily provided evidence for a critical requirement for Hh signaling in the development and differentiation of the gonads and accessory sex glands (Table 2). Members of the Hh signaling pathway are expressed in these organs, and Hh signaling between the epithelial-mesenchymal-stromal tissue compartments has either been inferred or identified based on functional and mutational analyses. Although many questions remain about the specific function of Hh signaling in reproductive organs, current studies suggest that the Hh signaling mechanism identified at other epithelial-mesenchymal boundaries during development is utilized as a cassette in the gonads, uterus, and accessory sex glands as well. In the gonads, Hh signaling appears to play the additional role of signaling growth and/or differentiation of germ cells.
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HEDGEHOG SIGNALING IN THE TESTIS
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Hh signaling plays an essential role in spermatogenesis in the adult and in the development of Leydig cells, peritubular cells, and seminiferous tubules. Dhh is expressed by the epithelium-derived Sertoli cells [7, 43]. Its expression in Sertoli cell precursors at Embryonic Day 11.5 in the mouse, shortly after activation of the sex-determining gene Sry, represents one of the earliest indications of male gonadal development. Expression persists in the adult testis. The intimate association of Sertoli cells with primitive spermatogonia adjacent to the basement membrane of the seminiferous tubule and with meiotic spermatocytes and maturing spermatids toward the tubular lumen provides a spatial arrangement that strongly favors a role for Dhh signaling during germ cell maturation. Male mice homozygous for a Dhh-null mutation lack mature sperm [43]. Examination of the developing testis following crosses of the Dhh-null mutation into different genetic backgrounds suggests that Dhh regulates both the early and late stages of spermatogenesis. Ptc2 is expressed in primary and secondary spermatocytes, where it colocalizes with Fu, indicating that these germ cells are direct targets of Dhh signaling [44]. Deletion of the Ptc2 locus on human chromosome 1p32–34 has been described in seminomas, suggesting that Ptc2 may function as a tumor suppressor gene in the testis [62].
Gli1 and Gli3 mRNA are expressed in proliferating spermatogonial cells, with expression decreasing to low or undetectable levels at later developmental stages [63]. Gli1 protein, however, has been demonstrated only in later germ cell types, with expression beginning in pachytene cells and persisting through elongating spermatids [64]. The discordance of Gli1 transcript and protein expression patterns, which has also been found in the developing gastrointestinal tract [65], demonstrates the need to conduct studies of Gli family transcription factors at the protein level to precisely determine where and when during development Hh signals are truly transduced. Gli1 protein has also been localized in Sertoli cells, hinting at possible autocrine regulation of these cells by Dhh signaling [64], although inhibition of Hh signaling does not seem to affect Sertoli cell differentiation [66]. The expression pattern of Gli1 protein in germ cells exhibits a shift from cytoplasmic to nuclear and then back to cytoplasmic localization in a spermatogenesis stage-specific manner [64]. This expression pattern suggests that nuclear import of Gli1 is highly regulated, reminiscent of Drosophila Ci, and suggests stage-specific signaling through Gli1 during spermatogenesis. A line of transgenic mice overexpressing human Gli1 exhibited male sterility with spermatogenesis blocked at the pachytene stage, suggesting that overexpression of Gli1 upsets a delicate balance of Gli1-related signals that are required for completion of spermatogenesis [64]. Detailed functional studies are needed to identify specific germ cell targets of Sertoli cell-derived Dhh signaling. Moreover, the unique and shared roles of Gli1 and Gli3 in spermatogenesis remain to be determined.
In addition to restricted spermatogenesis, Dhh-null mice lack mesenchyme-derived testosterone-producing Leydig cells, demonstrate interstitial fibrosis, and display an abnormal basal lamina between the peritubular myoid cells and Sertoli cells, which is believed to contribute to the development of irregular and anastomotic seminiferous tubules [67, 68]. Ptc1 is expressed by testicular interstitial cells, including the Leydig cells, peritubular myoid cells, and endothelial cells, indicating that these cells are also targets of Dhh signaling from the Sertoli cells [43, 67, 69]. Inhibition of Dhh signaling by cyclopamine, a specific Hh signaling inhibitor [70–72], downregulates Ptc1 expression in all interstitial cells and leads to defects in fetal Leydig cell differentiation, as indicated by a significant loss in expression of a marker protein, the P450 side-chain cleavage (Scc) enzyme, which echoes the loss of both Ptc1 and Scc expression in Dhh-null XY gonads [43, 66, 69].
It remains unclear whether the phenotypic effects on spermatogenesis in the Dhh-null mice are direct or are the result of testosterone deficiency secondary to the Leydig cell abnormalities. However, Dhh signaling plays specific roles during the development of Leydig cells and testicular peritubular tissues that are independent of androgenic support; androgen insensitive Tfm mice exhibit abundant adult type Leydig cells and an intact basal lamina [67]. Therefore, as has been described at other epithelial-mesenchymal boundaries, Hh signaling by epithelium-derived cells (Sertoli cells) once again appears to target mesenchyme-derived cells (Leydig cells) and influences their proliferation and differentiation. The role of mesenchyme-derived signals on the proliferation and/or function of Sertoli cells is less clear.
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HEDGEHOG SIGNALING IN THE PROSTATE
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The prostate develops from the male urogenital sinus, a tubular structure composed of a multilayered epithelium surrounded by a mesenchymal sheath. The outgrowth of the urogenital sinus epithelium into the surrounding mesenchyme to form buds is the earliest morphological evidence of prostate development [73]. Ductal bud formation, which begins in late gestation (Embryonic Day 17.5 in the mouse), is a testosterone-dependent process that involves epithelial-mesenchymal interactions [73, 74]. Shh provides a critical signal that promotes embryonic prostate ductal budding. During mouse embryonic prostate development, the expression of Shh, Gli1, Gli2, and Gli3 in the urogenital sinus increases coordinately coincident with ductal budding and declines in the postnatal period to low but detectable levels in the differentiated adult prostate [75, 76]. Shh expression, which is strictly epithelial, shifts from an initial uniform distribution throughout the urogenital sinus epithelium to a more focused localization in the cells of the nascent prostatic epithelial buds [76].
Coincident with the focusing of Shh message in epithelial buds, the expression of Ptc1, Gli1, and Gli2 becomes more restricted in mesenchyme immediately surrounding the prostatic buds, whereas Gli3 expression remains diffuse throughout the mesenchyme. Exogenous human recombinant Shh peptide directly induces the expression of Ptc1 and Gli1 in the isolated urogenital sinus mesenchyme, establishing this tissue compartment as a direct target responsive to Hh signaling in the embryonic prostate [76]. However, Shh-stimulated expression of Gli2 and Gli3 appears to require the presence of both urogenital sinus epithelium and mesenchyme, suggesting that perhaps other secreted factors may be involved in regulating their expression in this setting. Inhibition of Hh signaling by cyclopamine produces a dramatic downregulation of Ptc1 expression and a graded inhibition of Gli expression (Gli1 > Gli2 > Gli3) that is paralleled by severe inhibition of ductal budding and a significant decrease in cell proliferation in both urogenital sinus epithelium and mesenchyme, presumably based on loss of mesenchyme-derived signals [76]. Considering that the homozygous Gli1-null mutation does not produce an overt prostate phenotype, these data collectively hint at possible sharing of transactivating functions among the Gli genes to promote Shh-mediated prostate budding.
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HEDGEHOG SIGNALING IN THE OVARY
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A role for Hh signaling in the vertebrate ovary has not been described. Although Dhh is expressed in the presumptive testis, no such expression is observed in the ovary at the early or later stages of development [43]. Perhaps it should not be surprising that Hh signaling has not been observed in the mature vertebrate ovary where proliferation of germ cells no longer occurs. Studies addressing roles for Hh signaling during oogenesis prior to the entry of primary oocytes into their first meiotic division during embryonic development have not been reported. In contrast, the role of Hh signaling in the adult Drosophila ovary is well characterized, where Hh drives proliferation of somatic and germline stem cells.
Drosophila oocytes develop in a structure within the ovary called the germarium. The germarium is a tubular structure and consists of maturing germline stem cells and an epithelium of somatic follicle cells. As the oocytes mature from germline stem cells, they migrate posteriorly in germline cysts within the germarium. At the anterior tip of the germarium, adjacent to the germline stem cells, there are nonproliferating epithelial somatic cells called terminal filament cells and cap cells. In adult Drosophila ovaries, hh is expressed by terminal filament cells and cap cells and appears to affect development of the germline stem cells indirectly and the somatic follicle cells directly [77]. Maintenance and proliferation of the germline stem cells, which localize to the anterior end of the germarium adjacent to the hh-expressing terminal filament cells and cap cells, is regulated directly by dpp [78]. The dpp signal is believed to derive from the surrounding somatic follicle cells and to be induced by Hh. Overexpression of dpp produces ovarian stem cell tumors.
The hh gene expressed by terminal filament cells and cap cells drives proliferation of the more posteriorly located somatic follicle cells in the germarium, some of which lie several cell diameters away [77]. Experimentally induced loss of ptc activity, which is in effect similar to ectopic Hh signaling, results in the production of excessive somatic follicle cells, which in turn may cause abnormal positioning of oocytes within the egg chambers [79]. Continued proliferation of somatic follicle cells induced by loss of ptc activity is also believed to delay differentiation programs in these cells as they move posteriorly, leading to defective differentiation of the posterior follicle cells. This defective differentiation in turn may disrupt polarity within the more posteriorly located maturing oocytes, presumably based on aberrant signals received from the disrupted follicle cells [79]. Therefore, Hh signaling in the Drosophila ovary appears to drive proliferation of somatic follicle cells, to play a role in their differentiation, and to indirectly contribute to germline stem cell proliferation.
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HEDGEHOG SIGNALING IN THE MAMMARY GLAND
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The mammary gland, a specialized skin derivative, consists of a system of ducts whose growth, branching morphogenesis, and functional differentiation require epithelial-epithelial and epithelial-mesenchymal-stromal interactions tightly regulated by hormonal influences. Several components of the Hh signaling network are expressed in the mouse mammary gland, suggestive of a role for this regulatory pathway in mediating cell-cell interactions during mammary gland development, pregnancy, and lactation. Ihh expression has been localized by in situ hybridization exclusively to the epithelial lining of developing mammary ducts, including the undifferentiated epithelial "body cells" at the tips of terminal end buds of elongating ducts during puberty [80]. Ihh expression appears to be hormonally regulated because levels increase in mammary ductal epithelium and alveoli during pregnancy and lactation, become undetectable in the early days of involution, and revert to low levels characteristic of the adult mammary gland by about 2 wk of involution [80]. The absence of detectable Shh expression by in situ hybridization at any stage of mammary gland development coupled with the lack of an overt phenotype in females with homozygous Dhh-null mutation indicate that Ihh is the primary active Hh signaling protein in mammary development [43, 80].
As with Ihh, temporal expression of Ptc1 is linked to the reproductive status of the animal: level of expression progressively increases during pregnancy and lactation and then during involution reverts to a low level typical of the basal state [80]. Throughout the different stages of mammary gland development, Ptc1 is expressed in both the epithelium and surrounding mesenchyme/periductal stroma, suggestive of both autocrine and paracrine Hh signaling mechanisms in the mammary gland [80, 81]. Ptc1 appears to regulate epithelial proliferation, and dysplasias observed in mice with Ptc1 haploinsufficiency suggest a potential role for Ptc1 as a mammary tumor suppressor gene [80].
The spatial localization of Gli2 expression in the mammary gland also appears to be developmentally regulated [81]. Gli2 expression is confined to the stroma surrounding mammary ducts during the virgin stages of mouse development. During pregnancy and lactation, expression becomes localized in both stromal and epithelial compartments with the highest expression in the alveoli of lactating glands. The functional relevance of the highest expression levels in the epithelium during pregnancy and lactation remains to be elucidated; however, this level of expression suggests direct epithelial-epithelial Hh signaling under these circumstances. Additionally, the precise influences of specific hormones on Ihh signaling during mammary gland development, pregnancy, and lactation remain to be identified. There is some evidence that Ptc1 and Gli2 in the stroma are required for normal ductal morphogenesis, presumably providing necessary mesenchyme-derived signals [81]. Effects of Hh signaling on the proliferation and differentiation of the mammary gland mesenchyme have not yet been described.
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HEDGEHOG SIGNALING IN THE UTERUS
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The mammalian uterus undergoes hormonally induced changes in cell proliferation and differentiation during the estrous cycle and pregnancy [82]. Implantation requires cell-cell interactions between the uterine epithelium and the underlying stroma, which occur in part in response to the ovary-derived steroid hormones progesterone and estrogen and unidentified factors from the blastocyst [83]. Recent evidence suggests that at least some of the progesterone-related effects on uterine cell-cell interactions during implantation may be mediated through Ihh signaling.
Progesterone-dependent upregulated Ihh expression has been localized by in situ hybridization to both the luminal and glandular epithelium of the mouse endometrium during implantation [84–86]. Progesterone, whose receptor is expressed in the epithelium, stimulates uterine stromal cell proliferation [83, 87]. Because recombinant Shh protein (presumably mimicking endogenous Ihh action) promotes cell proliferation in isolated uterine mesenchyme explants obtained from early pregnant mice [85] and expression levels of Ptc1, Gli1, Gli2, and Gli3 increase in the uterine stroma during implantation, Ihh signaling might mediate, at least in part, the proliferative action of progesterone on the uterine stroma by a paracrine pathway similar to that utilized at other epithelial-stromal boundaries [84–86]. Curiously, Ptc1 and Gli3 are expressed in the uterine epithelium prior to implantation. Because Gli3 suppresses the expression of Hh target genes in some mammalian tissues [8], this uterine expression pattern suggests that Gli3 may mediate autocrine inhibition of epithelial cell proliferation in early pregnancy [85]. The elucidation of the precise role of Hh signaling, identification of downstream target genes, and resolution of shared and/or antagonistic functions of the Gli transcription factors in the uterus, both during pregnancy and during the estrous cycle, are important areas of future research.
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CONCLUSIONS
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The role of Hh signaling in the gonads and uterus and at epithelial-mesenchymal-stromal boundaries of accessory sex glands is highly reminiscent of its roles in lung, gut, hair follicle, and tooth development. In these tissues, the Hh signal also originates in epithelium-derived cells, whether in the gonad, uterus, or the accessory sex glands. In gonads, Hh signaling seems to have been conserved across widely divergent species as a means of mediating interactions between the germline and the somatic cells. In the testis, the mesenchyme-derived Leydig cells and the germ cells appear to be targets of Hh signaling, which appears to affect their proliferation and differentiation. In the prostate and mammary gland, epithelium-derived Hh signaling targets the neighboring mesenchyme and ultimately provides feedback to drive proliferation of the gland epithelium. There is also evidence for direct epithelial-epithelial Hh signaling in the mammary gland during pregnancy and lactation. In the uterus, early evidence suggests a role for epithelium-derived Hh signaling in proliferation of mesenchyme during implantation. A role for Hh signaling during development of the external genitalia is just beginning to be defined.
Based on the specific roles of Hh signaling in the gonads, uterus, and accessory sex glands, several ideas arise that might have general relevance for a better understanding of the roles of Hh signaling in development and in diseases. First, roles for all three vertebrate Hh proteins have been described in these organs, and current evidence suggests that each signals in a similar manner through shared downstream elements including Ptc1, Ptc2, and Gli family transcription factors. In most settings, however, details of the complete signal transduction pathway and its regulation remain unknown. Identification of downstream targets will be critical to fully understand the roles of Hh signaling. Second, understanding the roles of the three vertebrate Hh proteins, which likely arose by gene duplication, in different organisms and tissues, including sexually dimorphic tissues, may have relevance to understanding the evolutionary origins of species, organs, and sexual differentiation. Third, several observations suggest hormonal regulation of Hh signaling, including testosterone-dependent Shh expression in the male urogenital sinus and enhanced Ihh expression during pregnancy and lactation in the mammary gland and during implantation in the uterus. The influence of sex steroids on Hh signaling remains to be clearly defined. Fourth, Hh signaling during adult life in the vertebrate testis, Drosophila ovary, and accessory sex glands appears to be related to the need for continued cell renewal in these tissues and raises the possibility that dysregulation of this pathway may play a role in the development of cancers in these tissues.
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FOOTNOTES
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1 This study was supported by National Institutes of Health USPHS grant P01 ES10549. 
2 Correspondence: David O. Walterhouse, Division of Hematology/Oncology, Box 30, Children's Memorial Hospital, Northwestern University Feinberg School of Medicine, 2300 Children's Plaza, Chicago, IL 60614. FAX: 773 880 3223; d-walterhouse{at}northwestern.edu
Received: 30 January 2003.
First decision: 8 February 2003.
Accepted: 7 March 2003.
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