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Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis
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Discrete Hedgehog Factor Expression and Action in the Developing Phallus.

Gerard A Tarulli1, Andrew J Pask1, Marilyn B Renfree1

  • 1School of BioSciences, The University of Melbourne, Parkville, VIC 3010, Australia.

International Journal of Molecular Sciences
|February 16, 2020
PubMed
Summary

This study reveals that hedgehog signaling, specifically Sonic Hedgehog (SHH) and Indian Hedgehog (IHH), plays a crucial role in penile urethral closure by mimicking bone differentiation processes during development.

Keywords:
bonehedgehog signallinghypospadiaspenisurethra

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Area of Science:

  • Developmental biology
  • Urology
  • Endocrinology

Background:

  • Hypospadias, a common birth defect involving incomplete urethral closure, is increasing in frequency.
  • Paracrine hedgehog signaling is implicated in urethral closure, but its tissue-level mechanisms remain unclear.

Purpose of the Study:

  • To elucidate the role of different hedgehog signaling members in the process of urethral closure.
  • To investigate the molecular mechanisms underlying hypospadias using a unique animal model.

Main Methods:

  • Utilized the tammar wallaby (Macropus eugenii) for in-vivo studies due to its developmental timeline.
  • Employed RNA sequencing (RNAseq) for differential gene expression and gene ontology analysis.
  • Performed immunofluorescence for protein localization and quantitative PCR (qPCR) for gene expression analysis in tissue explants.

Main Results:

  • Gene ontology analysis revealed enrichment of bone differentiation terms in male samples, which was altered by estradiol treatment.
  • Sonic Hedgehog (SHH) and Indian Hedgehog (IHH) expression localized to specific tissue compartments in the developing phallus.
  • Treatment of phallus explants with SHH or IHH induced factor-specific expression of bone differentiation-associated genes.

Conclusions:

  • Hedgehog signaling pathways (SHH and IHH) are integral to urethral closure, potentially through mechanisms that mimic bone differentiation.
  • This study identifies a novel developmental interaction involving hedgehog signaling in phallic development and urethral closure.