Open AccessA reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids
Author(s) -
Taylor Goldsmith,
Sadman Sakib,
Dennis A. Webster,
Daniel F. Carlson,
Frans van der Hoorn,
Ina Dobrinski
Publication year - 2020
Publication title -
cell and tissue research/cell and tissue research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.64
H-Index - 137
eISSN - 1432-0878
pISSN - 0302-766X
DOI - 10.1007/s00441-019-03121-8
Subject(s) - cilium , microbiology and biotechnology , morphogenesis , biology , hedgehog , hedgehog signaling pathway , smoothened , gene knockdown , cyclopamine , intraflagellar transport , signal transduction , cell culture , genetics , gene , flagellum
Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9-mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.