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Hypomorphic Smo mutant with inefficient ciliary enrichment disrupts the highest level of vertebrate Hedgehog response.

ABSTRACT: Smoothened (Smo) is the essential transducer of Sonic hedgehog (Shh) signaling, which regulates cell fate and proliferation during embryogenesis. We identified a novel mouse mutant, cabbie (cbb), and found that its cause is a missense mutation in Smo. We showed the Smocbb mutation is insensitive to the Shh agonist SAG, perhaps due to the disruption of SAG binding. We characterized Smocbb for defects in craniofacial and skeletal development, as well as neural tube patterning, and revealed Smocbb affected processes that require the highest levels of Shh activity. Smo is normally enriched in cilia upon Shh stimulation; however, we detected inefficient enrichment of Smo in Smocbb mutants whether we stimulated with Shh or SAG. Taken together, our data suggest that the highest levels of vertebrate Hedgehog signaling activity require efficient Smo ciliary enrichment.

SUBMITTER: Gigante ED

PROVIDER: S-EPMC5903954 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Project description:UnlabelledBackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.ResultsPACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer's vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.

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Hypomorphic Smo mutant with inefficient ciliary enrichment disrupts the highest level of vertebrate Hedgehog response.

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