Supporting data for “Elucidating the Role of PHF5A-SF3B1-DLC1 Splicing Complex in Avian Trunk Neural Crest Specification”

This dataset contains multiple qualitative and quantitative analyses mentioned below and two oral/poster presentation files related to the study of splicing complex PHF5A-SF3B1-DLC1 on the expression of specifier genes in avian trunk neural crest development. Acquisition of neural crest cell (NCC) fate within the neural plate border is coordinated by a gene regulatory network (GRN) involving multiple signals and transcriptional factors that endow the neural crest (NC) with a unique regulatory state, which is distinct from the neural progenitor and non-neural ectoderm. These NC progenitors in the dorsal neural tube undergo epithelium mesenchymal transition (EMT), migration and multipotency to generate multiple functional cell types (neurons and glial of the peripheral nervous system, craniofacial skeleton and melanocytes for skin pigmentation) during embryogenesis. A few NC specifiers genes have been well characterized for the transcriptional regulation of their expression and function in specifying NC identity. Notably, two intron-containing genes involve an underlying additional level of post-transcriptional control of their expression in the generation of their functional proteins. However, components of the spliceosome complex regulating splicing of NC specifier pre-mRNAs remain largely unclear. To address this issue, in situ hybridization and immunofluorescence analyses were performed on stage 10 chick embryos electroporated with Cas9 + gRNA of PHF5A/SF3B1/DLC1 individually or Cas9+Ctrl gRNA. Data reveal increased neural progenitor expression in the dorsal neural tube where reduced expression of three NC specifiers genes were detected, followed by abolishment of HNK-1 marked migratory NCCs in treatment group compared with Ctrl group. Since PHF5A-SF3B1 is core component of ubiquitously expressed U2 spliceosome, customized primers flanking the junction of exon and intron of NC specifier genes were designed, qPCR analysis shows retained introns of two NC specifier genes in the absence of PHF5A, SF3B1 or DLC1. Concomitant with the association of the three components of the complex analyzed by immunoprecipitation (IP), these data suggest that perturbed splicing regulation of the complex on the expression of two intron-containing NC specifier genes leads to the loss of NC progenitors in the pre-migratory dorsal neural tube. Above results were further supported by analyses using chemical splicing modulator. Underlying mechanisms were also deciphered using IP and RNA-immunoprecipitation. Collectively, the results unravel how tissue-specific splicing perturbation leads to the abnormal state of distinct intronic sequence of NC specifier genes, shedding light on the therapeutics of NC-associated diseases.