Med23 Regulates Sox9 Expression during Craniofacial Development

Trainor

2022

Development

Self Cite

Ribosomal RNA (rRNA) transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies; however, the cellular and molecular reasons for these defects are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. ncl mutant (ncl−/−) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observed that ncl−/− mutants exhibited decreased rRNA synthesis and p53-dependent apoptosis, consistent with a role in ribosome biogenesis. However, we found that Nucleolin also performs functions not associated with ribosome biogenesis. We discovered that the half-life of fgf8a mRNA was reduced in ncl−/− mutants, which perturbed Fgf signaling, resulting in misregulated Sox9a-mediated chondrogenesis and Runx2-mediated osteogenesis. Consistent with this model, exogenous FGF8 treatment significantly rescued the cranioskeletal phenotype in ncl−/− zebrafish, suggesting that Nucleolin regulates osteochondroprogenitor differentiation. Our work has therefore uncovered tissue-specific functions for Nucleolin in rRNA transcription and post-transcriptional regulation of growth factor signaling during embryonic craniofacial development.

Section: Methodsmentioning

confidence: 99%

Nucleolin loss of function leads to aberrant Fibroblast Growth Factor signaling and craniofacial anomalies

Trainor

2022

Development

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“…MEFs treated with tamoxifen were lysed using lysis buffer and western blot was performed using standard protocols as described previously (15). Protein quantity was estimated via a BCA assay.…”

Section: Methodsmentioning

confidence: 99%

“…MEFs were cultured on a T75 plate and harvested following tamoxifen treatment. Immunoprecipitation was performed as previously described (15). Briefly, the cells were homogenized in 500 µl lysis buffer containing Tris pH 8.0, Sodium Chloride, SDS, Sodium deoxycholate, NP-40 and protease inhibitor.…”

Section: Methodsmentioning

confidence: 99%

Dynamic regulation and requirement for ribosomal RNA transcription during mammalian development

Falcon

Watt

et al. 2021

Preprint

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Ribosomal RNA (rRNA) transcription by RNA Polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies which frequently affect craniofacial development. Here, we present a cellular and molecular mechanism to explain the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCC), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCC particulalry sensitive to rRNA synthesis defects. Underpinning these findings, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which causes an imbalance between rRNA and ribosomal proteins. This leads to increased ribosomal protein binding to Mdm2 and concomitantly diminished Mdm2 binding to p53. Consequently, p53 protein accumulates, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbates the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins Syndrome and Acrofacial Dysostosis Cincinnati Type. Our novel results therefore demonstrate the dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of craniofacial congenital diseases.

“…Protein samples consisting of 5 fish/sample were collected at appropriate stages. Embryos were homogenized and suspended in sample buffer containing Tris pH 8.0, Sodium Chloride, SDS, Sodium deoxycholate, NP-40 and protease inhibitor and used for Western blotting according to standard protocols 74 . Protein quantity was estimated via a BCA assay.…”

Section: Western Blotmentioning

confidence: 99%

Nucleolin loss-of-function leads to aberrant FGF signaling and craniofacial anomalies

Trainor

2021

Preprint

Self Cite

rRNA transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies, however the cellular and molecular reasons for this are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. We discovered that Nucleolin is dynamically expressed during embryonic development with high enrichment in the craniofacial tissues. Consistent with this pattern of expression, ncl homozygous mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observe that ncl-/- mutants exhibit decreased rRNA synthesis and p53-dependent neuroepithelial cell death. In addition, the half-life of fgf8a mRNA is reduced in ncl-/- mutants, which perturbs Fgf signaling, resulting in misregulation of Sox9a mediated chondrogenesis and Runx2 mediated osteogenesis. Exogenous addition of human recombinant FGF8 to the mutant zebrafish significantly rescues the cranioskeletal phenotype, suggesting that Nucleolin regulates osteochondroprogenitor differentiation during craniofacial development by post-transcriptionally regulating Fgf signaling. Our work has therefore uncovered a novel tissue-specific function for Nucleolin in rRNA transcription and growth factor signaling during embryonic craniofacial development.