Hypospadias, a failure of appropriate urethral positioning within the penis, is the most common birth defect requiring intervention and can result in lifelong physical and psychological burdens. Surgery is the only therapeutic option for hypospadias, and can fail repeatedly in moderate to severe cases. This is partly because the underlying tissue and molecular signalling networks controlling urethral closure are not fully understood. This study aimed to identify novel networks regulating this process, using a comparative developmental approach.
Marsupials are valuable models to study penis development as gonadal differentiation and subsequent penis development occur after birth. RNAseq, differential expression and gene-ontology analyses were conducted on male and female wallaby penis samples during a critical window of urethral closure, and after treatment with estrogens, androgens and inhibitors of their receptors. The distribution of the hedgehog proteins, Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), as well as the transcription factor SOX9, were assessed in wallaby penis tissue using immunofluorescence. Explants of mouse and wallaby penis were treated with either SHH or IHH, and analysed by qPCR for changes in the expression of genes critical for penis development, to identify any discrete actions of SHH and IHH.
Gene ontology showed enrichment for genes involved in chondrocyte differentiation and bone formation in male samples compared with either female samples, or samples where androgen signalling was interrupted. Key regulators of chondrocyte differentiation, SHH and IHH, are localised to discrete regions of the penis during development, similar to their compartmentalised expression in developing bone. Treatment of explants with SHH or IHH induced distinct expression changes in androgen and Wnt signalling genes important for urethral closure. Thus, SHH and IHH activate distinct developmental networks during urethral closure, and harnessing these distinct actions may lead to novel treatments that improve outcomes of surgery for hypospadias.