Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

BACKGROUND: Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. RESULTS: We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. CONCLUSIONS: The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Exposure to an environmentally relevant concentration of 17α-ethinylestradiol disrupts craniofacial development of juvenile zebrafish.

Endocrine disrupting chemicals (EDCs) can interact with native hormone receptors to interfere with and disrupt hormone signalling that is necessary for a broad range of developmental pathways. EDCs are pervasive in our environment, in particular in our waterways, making aquatic wildlife especially vulnerable to their effects. Many of these EDCs are able to bind to and activate oestrogen receptors, causing aberrant oestrogen signalling. Craniofacial development is an oestrogen-sensitive process, with oestrogen receptors expressed in chondrocytes during critical periods of development. Previous studies have demonstrated a negative effect of high concentrations of oestrogen on early craniofacial patterning in the aquatic model organism, the zebrafish (Danio rerio). In order to determine the impacts of exposure to an oestrogenic EDC, we exposed zebrafish larvae and juveniles to either a high concentration to replicate previous studies, or a low, environmentally relevant concentration of the oestrogenic contaminant, 17α-ethinylestradiol. The prolonged / chronic exposure regimen was used to replicate that seen by many animals in natural waterways. We observed changes to craniofacial morphology in all treatments, and most strikingly in the larvae-juveniles exposed to a low concentration of EE2. In the present study, we have demonstrated that the developmental stage at which exposure occurs can greatly impact phenotypic outcomes, and these results allow us to understand the widespread impact of oestrogenic endocrine disruptors. Given the conservation of key craniofacial development pathways across vertebrates, our model can further be applied in defining the risks of EDCs on mammalian organisms.

Spatiotemporal map of key signaling factors during early penis development.

The formation of the external genitalia is a highly complex developmental process, considering it involves a wide range of cell types and results in sexually dimorphic outcomes. Development is controlled by several secreted signalling factors produced in complex spatiotemporal patterns, including the hedgehog (HH), bone morphogenic protein (BMP), fibroblast growth factor (FGF) and WNT signalling families. Many of these factors act on or are influenced by the actions of the androgen receptor (AR) that is critical to masculinisation. This complexity of expression makes it difficult to conceptualise patterns of potential importance. Mapping expression during key stages of development is needed to develop a comprehensive model of how different cell types interact in formation of external genitalia, and the global regulatory networks at play. This is particularly true in light of the sensitivity of this process to environmental disruption during key stages of development. The goal of this review is to integrate all recent studies on gene expression in early penis development to create a comprehensive spatiotemporal map. This serves as a resource to aid in visualising potentially significant interactions involved in external genital development.

Widespread cis-regulatory convergence between the extinct Tasmanian tiger and gray wolf.

The extinct marsupial Tasmanian tiger, or thylacine, and the eutherian gray wolf are among the most widely recognized examples of convergent evolution in mammals. Despite being distantly related, these large predators independently evolved extremely similar craniofacial morphologies, and evidence suggests that they filled similar ecological niches. Previous analyses revealed little evidence of adaptive convergence between their protein-coding genes. Thus, the genetic basis of their convergence is still unclear. Here, we identified candidate craniofacial cis-regulatory elements across vertebrates and compared their evolutionary rates in the thylacine and wolf, revealing abundant signatures of convergent positive selection. Craniofacial thylacine-wolf accelerated regions were enriched near genes involved in TGF beta (TGFB) and BMP signaling, both of which are key morphological signaling pathways with critical roles in establishing the identities and boundaries between craniofacial tissues. Similarly, enhancers of genes involved in craniofacial nerve development showed convergent selection and involvement in these pathways. Taken together, these results suggest that adaptation in cis-regulators of TGF beta and BMP signaling may provide a mechanism to explain the coevolution of developmentally and functionally integrated craniofacial structures in these species. We also found that despite major structural differences in marsupial and eutherian brains, accelerated regions in both species were common near genes with roles in brain development. Our findings support the hypothesis that, relative to protein-coding genes, positive selection on cis-regulatory elements is likely to be an essential driver of adaptive convergent evolution and may underpin thylacine-wolf phenotypic similarities.

Oestrogen regulates SOX9 bioavailability by rapidly activating ERK1/2 and stabilising microtubules in a human testis-derived cell line.

Nuclear SOX9 is essential for Sertoli cell differentiation and the development of a testis. Exposure of Sertoli cells to exogenous oestrogen causes cytoplasmic retention of SOX9, inhibiting testis development and promoting ovarian development. The cytoplasmic localisation of SOX9 requires a stabilised microtubule network and a key MAPK complex, ERK1/2, is responsive to oestrogen and known to affect the microtubule network. We hypothesised that oestrogen could stabilise microtubules through the activation of ERK1/2 to promote the cytoplasmic retention of SOX9. Treatment of human testis-derived NT2/D1 cells for 30 min with oestrogen rapidly activated ERK1/2, stabilised the microtubule network and increased cytoplasmic localisation of SOX9. The effects of oestrogen on SOX9 and tubulin were blocked by the ERK1/2 inhibitor U0126, demonstrating that ERK1/2 mediates the stabilisation of microtubules and cytoplasmic retention of SOX9 by oestrogen. Together, these data revealed a previously unknown mechanism for oestrogen in impacting MAPK signalling to block SOX9 bioavailability and the differentiation of Sertoli cells.

Annotation of immune genes in the extinct thylacine (Thylacinus cynocephalus).

Advances in genome sequencing technology have enabled genomes of extinct species to be sequenced. However, given the fragmented nature of these genome assemblies, it is not clear whether it is possible to comprehensively annotate highly variable and repetitive genes such as those involved in immunity. As such, immune genes have only been investigated in a handful of extinct genomes, mainly in human lineages. In 2018 the genome of the thylacine (Thylacinus cynocephalus), a carnivorous marsupial from Tasmania that went extinct in 1936, was sequenced. Here we attempt to characterise the immune repertoire of the thylacine and determine similarity to its closest relative with a genome available, the Tasmanian devil (Sarcophilus harrisii), as well as other marsupials. Members from all major immune gene families were identified. However, variable regions could not be characterised, and complex families such as the major histocompatibility complex (MHC) were highly fragmented and located across multiple small scaffolds. As such, at a gene level we were unable to reconstruct full-length coding sequences for the majority of thylacine immune genes. Despite this, we identified genes encoding functionally important receptors and immune effector molecules, which suggests the functional capacity of the thylacine immune system was similar to other mammals. However, the high number of partial immune gene sequences identified limits our ability to reconstruct an accurate picture of the thylacine immune repertoire.

A novel long non-coding RNA, Leat1, causes reduced anogenital distance and fertility in female mice.

Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.

Oestrogen Activates the MAP3K1 Cascade and ß-Catenin to Promote Granulosa-like Cell Fate in a Human Testis-Derived Cell Line.

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate ß-catenin-a factor essential for ovarian development. We show that oestrogen can activate ß-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to ß-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.

Reproduction in a polluted world: implications for wildlife.

Environmental pollution is an increasing problem for wildlife globally. Animals are confronted with many different forms of pollution, including chemicals, light, noise, and heat, and these can disrupt critical biological processes such as reproduction. Impacts on reproductive processes can dramatically reduce the number and quality of offspring produced by exposed individuals, and this can have further repercussions on the ecology and evolution of affected populations. Here, we illustrate how environmental pollutants can affect various components of reproduction in wildlife, including direct impacts on reproductive physiology and development, consequences for gamete quality and function, as well as effects on sexual communication, sexual selection, and parental care. We follow with a discussion of the broader ecological and evolutionary consequences of these effects on reproduction and suggest future directions that may enable us to better understand and address the effects of environmental pollution.

A critical role for estrogen signaling in penis development.

Hypospadias, a developmental defect of the penis, is one of the most common congenital malformations in humans. Its incidence has rapidly increased over recent decades, and this has been largely attributed to our increased exposure to endocrine-disrupting chemicals. Penis development is primarily an androgen-driven process; however, estrogen and xenoestrogens are known to affect penis development in both humans and mice. Here, we investigated the role of estrogen in the developing penis. Using a novel penis culture system, we showed that exogenous estrogen directly targets the developing penis in utero to cause hypospadias. In addition, we also uncovered an unexpected endogenous role for estrogen in normal postnatal penis development and showed that a loss of estrogen signaling results in a mild hypospadias phenotype, the most common manifestation of this disease in humans. Our findings demonstrated that both androgen and estrogen signaling are intrinsically required for normal urethral closure. These findings confirmed that penis development is not an entirely androgen-driven process but one in which endogenous estrogen signaling also plays a critical role.-Govers, L. C., Phillips, T. R., Mattiske, D. M., Rashoo, N., Black, J. R., Sinclair, A., Baskin, L. S., Risbridger, G. P., Pask, A. J. A critical role for estrogen signaling in penis development.

A loss of estrogen signaling in the aromatase deficient mouse penis results in mild hypospadias.

Hypospadias is the abnormal opening of the urethra on the underside of the penis and occurs in approximately 1/125 live male births worldwide. The incidence rate of hypospadias has dramatically increased over the past few decades. This is now attributed, at least in part, to our exposure to endocrine-disrupting chemicals (EDCs) which alter the hormonal signals required for development of the penis. In humans androgens are the main drivers of fusion of the urethral folds to form the urethra within the shaft of the penis, a process required for termination of the urethra in its normal location at the tip of the penis. However, recent research has suggested that estrogen also plays a role in this process. To better understand how EDCs impact urethral development it is essential that we understand the normal function of hormones during development of the penis. To define the role of estrogen in urethral development we examined development of the penis in the aromatase (Cyp19a1) Knockout (ArKO) mouse strain in which endogenous estrogen production is completely ablated. We found that the ArKO penis had a mild hypospadias phenotype. The developing ArKO postnatal penis displayed an early disruption in preputial development, which likely causes the mild hypospadias observed in adults. Using qPCR, we found altered expression of keratin genes and key urethral patterning genes in response to the disrupted estrogen signaling. The hypospadias phenotype was almost identical to that reported for the estrogen receptor α (ERα) knockout confirming that ERα is the predominant receptor for mediating estrogen action during development of the mouse penis. Our results show that estrogen is required for normal prepucial development and placement of the mature urethral opening at the distal aspect of the penis. We also identified several genes which are potential downstream targets of estrogen during normal urethral closure. With this knowledge, we can now better understand how anti-estrogenic as well as estrogenic EDCs disrupt urethral closure to cause mild hypospadias in both mice and humans.

Exogenous Oestrogen Impacts Cell Fate Decision in the Developing Gonads: A Potential Cause of Declining Human Reproductive Health.

The increasing incidence of testicular dysgenesis syndrome-related conditions and overall decline in human fertility has been linked to the prevalence of oestrogenic endocrine disrupting chemicals (EDCs) in the environment. Ectopic activation of oestrogen signalling by EDCs in the gonad can impact testis and ovary function and development. Oestrogen is the critical driver of ovarian differentiation in non-mammalian vertebrates, and in its absence a testis will form. In contrast, oestrogen is not required for mammalian ovarian differentiation, but it is essential for its maintenance, illustrating it is necessary for reinforcing ovarian fate. Interestingly, exposure of the bi-potential gonad to exogenous oestrogen can cause XY sex reversal in marsupials and this is mediated by the cytoplasmic retention of the testis-determining factor SOX9 (sex-determining region Y box transcription factor 9). Oestrogen can similarly suppress SOX9 and activate ovarian genes in both humans and mice, demonstrating it plays an essential role in all mammals in mediating gonad somatic cell fate. Here, we review the molecular control of gonad differentiation and explore the mechanisms through which exogenous oestrogen can influence somatic cell fate to disrupt gonad development and function. Understanding these mechanisms is essential for defining the effects of oestrogenic EDCs on the developing gonads and ultimately their impacts on human reproductive health.

Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Hypospadias is a failure of urethral closure within the penis occurring in 1 in 125 boys at birth and is increasing in frequency. While paracrine hedgehog signalling is implicated in the process of urethral closure, how these factors act on a tissue level to execute closure itself is unknown. This study aimed to understand the role of different hedgehog signalling members in urethral closure. The tammar wallaby (Macropus eugenii) provides a unique system to understand urethral closure as it allows direct treatment of developing offspring because mothers give birth to young before urethral closure begins. Wallaby pouch young were treated with vehicle or oestradiol (known to induce hypospadias in males) and samples subjected to RNAseq for differential expression and gene ontology analyses. Localisation of Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in normal phallus tissue using immunofluorescence. Normal tissue culture explants were treated with SHH or IHH and analysed for AR, ESR1, PTCH1, GLI2, SOX9, IHH and SHH expression by qPCR. Gene ontology analysis showed enrichment for bone differentiation terms in male samples compared with either female samples or males treated with oestradiol. Expression of SHH and IHH localised to specific tissue areas during development, akin to their compartmentalised expression in developing bone. Treatment of phallus explants with SHH or IHH induced factor-specific expression of genes associated with bone differentiation. This reveals a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete hedgehog activity within the developing phallus and phallic urethra.

Of eyes and embryos: subfunctionalization of the CRX homeobox gene in mammalian evolution.

ETCHbox genes are fast-evolving homeobox genes present only in eutherian (placental) mammals which originated by duplication and divergence from a conserved homeobox gene, Cone-rod homeobox (CRX). While expression and function of CRX are restricted to the retina in eutherian mammals, ETCHbox gene expression is specific to preimplantation embryos. This dramatic difference could reflect the acquisition of new functions by duplicated genes or subfunctionalization of pleiotropic roles between CRX and ETCHbox genes. To resolve between these hypotheses, we compared expression, sequence and inferred function between CRX of metatherian (marsupial) mammals and ETCHbox genes of eutherians. We find the metatherian CRX homeobox gene is expressed in early embryos and in eyes, unlike eutherian CRX, and distinct amino acid substitutions were fixed in the metatherian and eutherian evolutionary lineages consistent with altered transcription factor specificity. We find that metatherian CRX is capable of regulating embryonically expressed genes in cultured cells in a comparable way to eutherian ETCHbox. The data are consistent with CRX having a dual role in eyes and embryos of metatherians, providing an early embryonic function comparable to that of eutherian ETCHbox genes; we propose that subfunctionalization of pleiotropic functions occurred after gene duplication along the placental lineage, followed by functional elaboration.

Effects of androgen and oestrogen on IGF pathways controlling phallus growth.

The development of the mammalian phallus involves hormone-dependent mesenchymal-epithelial signalling mechanisms that contribute to urethral closure and regulation of phallus elongation and growth. In marsupials, most differentiation and growth of the phallus occurs post-natally, making them amenable to direct hormone treatment. Expression of IGFs, FGFs, EFNB2, MAFB, DLX5 and AP-1 mRNAs in the phallus at day 50 post-partum (pp) were altered after treatment of tammar wallaby young from day 20 to 40 pp with androgen, oestrogen or after castration at day 25 pp. However, the most interesting changes occurred in the IGF pathway genes. Androgen treatment upregulated IGF1 in female phalluses and oestrogen treatment upregulated IGF1 in male phalluses, but it was downregulated by castration. IGFBP3 was higher in female phalluses and downregulated by androgen. IGF1 expression was higher in all untreated male than in female phalluses from day 50 to 150 pp, but IGFBP3 had the reverse pattern. At day 90 pp, when urethral closure in males is progressing and male phallus growth is accelerating. IGF1 and PCNA protein were only detected in the male urorectal septum, suggesting for the first time that closure and elongation may involve IGF1 activation of cell proliferation specifically in male phalluses. These effects of sex steroids on gene expression and on the IGF1 signalling pathway in particular, suggest that the developing phallus may be especially susceptible to perturbation by exogenous hormones.

Exposure to atrazine during puberty reduces sperm viability, increases weight gain and alters the expression of key metabolic genes in the liver of male mice.

Atrazine (ATZ) is one of the most widely used herbicides worldwide and is a common contaminant in human drinking water. It disrupts metabolic pathways in plants, and has metabolic and reproductive effects in vertebrates, including humans. Few studies have investigated the effects of exposure to low doses of ATZ, especially during sexual development in males. In this study, we exposed C57BL/6J male mice from weaning for 8 weeks to drinking water containing 0.5mgkg-1 bodyweight (BW) day-1 ATZ, the 'no observed effect' level used by the Australian government, or a 10-fold higher dose (5mgkg-1 BW day-1). Mice treated with the low dose of ATZ showed increased total and cumulative weight gain. At 12 weeks of age, there was a significant increase in the percentage of dead spermatozoa in both ATZ-exposed groups, as well as decreased epididymal sperm motility in the low-dose ATZ group. Significant changes in testis and liver gene expression were also observed following ATZ exposure. These data demonstrate that a low dose of ATZ can perturb metabolic and reproductive characteristics in male mice. A chronic reduction in sperm quality and increased weight gain could have negative consequences on the reproductive capacity of males, and further studies should consider the effects of long-term ATZ exposure on male reproductive health.

The X factor: X chromosome dosage compensation in the evolutionarily divergent monotremes and marsupials.

Marsupials and monotremes represent evolutionarily divergent lineages from the majority of extant mammals which are eutherian, or placental, mammals. Monotremes possess multiple X and Y chromosomes that appear to have arisen independently of eutherian and marsupial sex chromosomes. Dosage compensation of X-linked genes occurs in monotremes on a gene-by-gene basis, rather than through chromosome-wide silencing, as is the case in eutherians and marsupials. Specifically, studies in the platypus have shown that for any given X-linked gene, a specific proportion of nuclei within a cell population will silence one locus, with the percentage of cells undergoing inactivation at that locus being highly gene-specific. Hence, it is perhaps not surprising that the expression level of X-linked genes in female platypus is almost double that in males. This is in contrast to the situation in marsupials where one of the two X chromosomes is inactivated in females by the long non-coding RNA RSX, a functional analogue of the eutherian XIST. However, marsupial X chromosome inactivation differs from that seen in eutherians in that it is exclusively the paternal X chromosome that is silenced. In addition, marsupials appear to have globally upregulated X-linked gene expression in both sexes, thus balancing their expression levels with those of the autosomes, a process initially proposed by Ohno in 1967 as being a fundamental component of the X chromosome dosage compensation mechanism but which may not have evolved in eutherians.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Hormone-responsive genes in the SHH and WNT/ß-catenin signaling pathways influence urethral closure and phallus growth.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/ß-catenin. SHH mRNA and ß-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.