CRISPR-Cas9 mediated generation of a grhl3 deletion zebrafish model. (a–f) ISH expression of grhl3 at 3–5 hpf, shown in lateral (a–c) and dorsal (d–f) views. grhl3 is not maternally deposited (a,d), but is strongly and specifically expressed within all EVL cells at ~4 hpf, corresponding temporally to the maternal-zygotic transition (MZT; b–e). grhl3 mRNA is rapidly downregulated, remaining primarily localised at the leading-edge margins (asterisk, c). (g) Sequence details of the exon 4 region of grhl3, showing a loss of 3 bp and insertion of a STOP codon cassette comprising 17 bases, resulting in a net gain (genomic lesion) of 14 bp in the grhl3 mutant. (h) Schematic of the zebrafish grhl3 protein (to scale; amino acids 1–557), showing trans-activation (TA), DNA-binding (DNA), and dimerisation (DIM) domains, indicating site of genomic lesion at codon 143. (i) Sequencing of WT and grhl3 −/−(+14bp) embryos confirming presence of the genomic lesion. The red underline indicates (in WT) the position of the three deleted nucleotides, green underline shows insertion of 17 bp of STOP-cassette, black underline shows the resultant in-frame stop codon, yellow arrowhead indicates the CRISPR/Cas9-cut site. (j) RT-PCR showing ~50% loss of grhl3 transcript in grhl3 −/−(+14bp) embryos relative to controls at 8 hpf; a full-length gel photo is presented in Supplementary Figure S8. (k–l) Epiboly is delayed in grhl3 −/−(+14bp) embryos (l) relative to WT (k), resulting in premature contraction of the leading-edge actin ring (arrows in L) at ~75% epiboly. (m,n) WT embryo at the onset of somitogenesis showing normal development (m), and grhl3 −/−(+14bp) embryo (n) showing rupture of the EVL resulting in lethality (arrow in n). (O) Q-RT-PCR showing decreased grhl3 expression, no change in grhl2a, and slightly elevated expression of grhl2b at ~75% epiboly in grhl3 −/−(+14bp) embryos relative to WT controls.
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