Hypospadias is a failure of appropriate urethral positioning within the phallus (penis) during fetal development, and represents the second most common birth defect in Victoria. While modifications in androgen, oestrogen and hedgehog signalling are all implicated in the aetiology of hypospadias, the underlying tissue and molecular mechanisms controlling normal urethral closure are yet to be fully revealed. This study aimed to identify the fundamental signalling networks that regulate urethral closure, and develop a new model for this developmental process.
Marsupials give birth to altricial young, with penis development and urethral closure occurring postnatally. This provides a unique system to directly manipulate the mechanisms controlling urethral closure. Male and female wallaby pouch young were treated orally with estrogens or androgens, respectively, during a critical period of urethral closure. Samples were subjected to RNAseq for differential gene expression and gene-ontology analyses. The distribution of the hedgehog proteins, Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, was assessed in normal wallaby phallus tissue using immunofluorescence. Lastly, normal phallus tissue culture explants were treated with SHH or IHH and analysed for AR, ESR1, SOX9, IHH and SHH gene expression by qPCR.
Gene ontology showed enrichment for chondrocyte proliferation/differentiation in male samples compared with either female samples, or samples from males that received oestradiol. The expression of SHH and IHH localised to discrete regions of the phallus during normal development similar to their independent, compartmentalised expression in developing cartilage. Treatment of phallus explants induced the expression of genes associated with chondrocyte proliferation/differentiation in response to SHH or IHH. Results of this study reveal a new developmental interaction involved in urethral development that mimics chondrocyte differentiation, providing insight into the potential causes of hypospadias.