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A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

ABSTRACT: 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.

SUBMITTER: Goldsmith TM

PROVIDER: S-EPMC7815324 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

Goldsmith Taylor M TM Sakib Sadman S Webster Dennis D Carlson Daniel F DF Van der Hoorn Frans F Dobrinski Ina I

Cell and tissue research 20200103 1

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 CRIS ...[more]

PMID: 31900662

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Project description:Background:Primary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. These small organelles serve as sensory structures sampling the extracellular environment and reprogramming the transcriptional machinery in response to environmental change. Primary cilia are decorated with a variety of receptor proteins and are necessary for specific signaling cascades such as the Sonic hedgehog (Shh) pathway. Disrupting cilia structure or function results in a spectrum of diseases collectively referred to as ciliopathies. Common to human ciliopathies is cognitive impairment, a symptom also observed in Alzheimer's disease (AD). One hallmark of AD is accumulation of senile plaques composed of neurotoxic Amyloid-? (A?) peptide. The A? peptide is generated by the proteolytic cleavage of the amyloid precursor protein (APP). We set out to determine if A? affects primary cilia structure and the Shh signaling cascade. Methods:We utilized in vitro cell-based assays in combination with fluorescent confocal microscopy to address our study goals. Shh signaling and cilia structure was studied using two different cell lines, mouse NIH3T3 and human HeLa cells. To investigate how A? levels affect Shh signaling and cilia structure in these cells, we utilized naturally secreted A? as well as synthetic A?. Effects on Shh signaling were assessed by luciferase activity while cilia structure was analyzed by fluorescent microscopy. Results:Here, we report that APP localizes to primary cilia and A? treatment results in distorted primary cilia structure. In addition, we demonstrate that A? treatment interrupts canonical Shh signal transduction. Conclusions:Overall, our study illustrates that A? can alter primary cilia structure suggesting that elevated A? levels, like those observed in AD patients, could have similar effects on neuronal primary cilia in the brain. Additionally, our study suggests that A? impairs the Shh signaling pathway. Together our findings shed light on two novel targets for future AD therapeutics.

Dataset Information

A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

Publications

A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

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