Abstract
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.
Highlights
Disorders in the development of reproductive organs are the most common birth defects worldwide
While it is known that gene regulatory networks critical for bone/limb development play important roles in regulating phallus development and urethral closure [3,4,5,6], it is yet to be determined how these factors are coordinated on a tissue level to execute the process of urethral closure itself
Far less is known about the role of Indian hedgehog (IHH) in this process, though its expression is positively regulated by androgens, and knockout of IHH results in a failure of proper phallus masculinisation in the mouse [13]
Summary
Disorders in the development of reproductive organs are the most common birth defects worldwide. SHH plays important roles in the development of the genital tubercle from which the mouse phallus forms [7,8,9,10] as well as later masculinisation events and the formation of the penile urethra [8,11]. Far less is known about the role of IHH in this process, though its expression is positively regulated by androgens, and knockout of IHH results in a failure of proper phallus masculinisation in the mouse [13]. Whether these hedgehog factors have distinct functions in urethral closure has never been investigated
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