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

TY - JOUR

T1 - An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos

AU - Pownall, Mary Elizabeth

AU - McQueen, Caitlin

N1 - This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

PY - 2017/8/15

Y1 - 2017/8/15

N2 - 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 -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.

AB - 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 -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.

KW - Journal Article

U2 - 10.1016/j.mod.2017.05.002

DO - 10.1016/j.mod.2017.05.002

M3 - Article

C2 - 28536000

SN - 0925-4773

VL - 146

SP - 1

EP - 9

JO - Mechanisms of development

JF - Mechanisms of development

IS - 1

ER -