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. 2020 Sep 23;9:e57779. doi: 10.7554/eLife.57779

Figure 5. Ectopic expression of Hmga1 or Pax7 rescues cranial neural crest specification.

Figure 5.

(A) Plasmid construct used to rescue Hmga1. An independent ribosome entry site (IRES) controls translation of nuclear RFP in transfected cells. The electroporation strategy for knocking out Hmga1 using CRISPR-Cas9 in gastrula stage chick embryos. (B) Embryos were electroporated with the ‘rescue’ construct on the right side and a control nuclear RFP plasmid on the left side. (C) Exogenous expression of the Hmga1 coding sequence under the regulation of a ubiquitous enhancer/promoter combination causes cranial neural crest migration defects. (D–G’’’) Overexpression of the coding sequence (G’’’) of Hmga1 compensates for its loss of function, rescuing proper cranial neural crest migration (D), as assayed by number of Pax7-positive neural crest cells (E), and expression of the neural crest specifier gene Sox10 (F) in migratory cranial neural crest (G,G’) and Pax7 in the dorsal neural tube (G’’). Electroporated embryos were allowed to develop until HH9+ and screened for the expression of H2B-RFP. (H–H’) The coding sequence for Pax7 was ectopically expressed in an Hmga1-knockout background. Transverse section through a representative embryo shows the comparison between endogenous (left) and overexpressed (right; arrow) Pax7 transcript levels in the dorsal neural tube. I-K. Ectopic expression of Pax7 rescued neural crest specification defects caused by the loss of Hmga1 as assayed by the expression of neural crest specifier genes Tfap2b (I) and Snai2 (I’) in transverse cross-sections through the hindbrain (J,K). nt, neural tube; nc, notochord. See also Figure 5—source data 1.

Figure 5—source data 1. Pax7-positive cell counts in transverse sections through the hindbrain compared between Control and Hmga1-rescue sides in panel E.