RNA Polymerase III subunit Polr3a is required for craniofacial cartilage and bone development in zebrafish

Abstract Transcription by RNA Polymerase III (Pol III) is essential for ribosome biogenesis and translation in all cells, but pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific phenotypes including craniofacial differences. To understand the function of Pol III in craniofacial development, we examined polr3a mutant zebrafish. These mutants display hypoplasia of the neural crest cell-derived craniofacial cartilage and bone but, surprisingly, no significant changes were observed in neural crest cell proliferation or survival during embryogenesis. At larval stages, increased cell death was observed throughout the head, including in the craniofacial cartilage. These changes coincide with reduced transcription of transfer RNAs and reduced ribosome biogenesis in polr3a mutant zebrafish. To determine tissue-specific transcriptional changes, we performed single-cell RNA-sequencing. Analysis revealed both global and cartilage-specific changes, including upregulation of tp53. However, Tp53 inhibition alone was not sufficient to rescue craniofacial cartilage and bone, indicating that additional factors are important to support cartilage and bone growth in polr3a mutants. Altogether, our study provides new mechanistic insights into the functions of Pol III in craniofacial development. Author Summary Craniofacial anomalies account for around 33% of all congenital birth defects and many of these are associated with defects in neural crest cell development. Disruptions in RNA Polymerase III, which plays important roles in ribosome biogenesis and translation, can result in tissue-specific phenotypes including craniofacial anomalies. However, the mechanisms underlying these craniofacial anomalies are not well understood. Here, we establish a zebrafish model to understand how a mutation in Pol III subunit polr3a affects craniofacial development. We observe hypoplasia of the craniofacial cartilage and bone in polr3a mutant zebrafish along with diminished transcription of transfer RNAs and reduced ribosome biogenesis. This leads to reduced proliferation and increased cell death throughout the head, but we do not detect any differences in early neural crest cell development. Using single-cell RNA-sequencing, we examine the transcriptional changes both broadly throughout the head and specifically within the craniofacial cartilage and identify upregulation of the Tp53 pathway. Inhibition of tp53 only partially rescues cartilage and bone development, suggesting that Tp53-independent mechanisms contribute to cranioskeletal development in polr3a mutant zebrafish. Altogether, these studies highlight the critical function of Pol III during development and specifically in the differentiation and growth of craniofacial cartilage and bone. Competing Interest Statement The authors have declared no competing interest. Footnotes Figure 1 was updated to incorporate more information on the location of the mutation; Figure 2 and 3 were revised to incorporate additional measurements; Figure 8 was revised to include additional experiments; Figure 9 was added; Supplemental files were updated; the author list was updated; author affiliations were updated to reflect a department name change.