An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos

Abstract

Myogenic regulatory factors (MRFs) are known to have essential roles in both the establishment and differentiation of the skeletal muscle cell lineage. MyoD is expressed early in the Xenopus mesoderm where it is present and active several hours before the activation of muscle differentiation genes. Previous studies in cultured cells and in Xenopus laevis have identified sets of genes that require MyoD prior to differentiation of skeletal muscle. Here we report results from experiments using CRISPR/Cas9 to target the MyoD gene in the diploid frog Xenopus tropicalis, that are analysed by RNA-seq at gastrula stages. We further investigate our data using cluster analysis to compare developmental expression profiles with that of MyoD and α-cardiac actin, reference genes for skeletal muscle determination and differentiation. Our findings provide an assessment of using founder (F0) Xenopus embryos from CRISPR/Cas9 protocols for transcriptomic analyses and we conclude that although targeted F0 embryos are genetically mosaic for MyoD, there is significant disruption in the expression of a specific set of genes. We discuss candidate target genes in context of their role in the sub-programs of MyoD regulated transcription.

Gli2 is required for the induction and migration of Xenopus laevis neural crest

“…The expression of gli2 in the optic territory during tailbud stages concurs with the role of NC in ocular development, and also agrees with the role that Hh plays during the development of Xenopus retinal pigment epithelium (Perron et al, 2003). Finally, gli2 was also detected in the developing somites, which is consistent with the fact that gli2 is a direct target of the myogenic regulatory factor myoD in Xenopus development (McQueen and Pownall, 2017).…”

Quantitative analysis of transcriptome dynamics provides novel insights into developmental state transitions

“…For example, Ca14 , Pygm and Tmcc1 were among genes upregulated in stage 12 animal caps in response to Wnt and Nodal2 [ 109 ] and Ca14 , Tmcc1 , and Nptx2 were upregulated in stage 11 embryos in response to somatic cell nuclear transfer from an endoderm cell [ 110 ]. Similarly, P ygm has been identified as a target of Myod [ 111 ]. Thus, using limma analysis as an unbiased approach for detecting genes sets that share expression pattern dynamics allows identification of potential new members of developmental GRNs using our data sets.…”

Quantitative Analysis of Transcriptome Dynamics Provides Novel Insights into Developmental State Transitions

“…For example, ca14 , pygm and tmcc1 were among genes upregulated in stage 12 animal caps in response to wnt and nodal2 (Ding et al 2018) and ca14 , tmcc1 , and nptx2 were upregulated in stage 11 embryos in response to somatic cell nuclear transfer from an endoderm cell (Hormanseder et al 2017). Similarly, p ygm has been identified as a target of myod (McQueen and Pownall 2017). Thus, using limma analysis as an unbiased approach for detecting genes sets that share expression pattern dynamics allows identification of potential new members of developmental GRNs using our data sets.…”