Figure 7—figure supplement 4.

MO and CRISPR approaches to investigate the function of ArhGEF11/PDZ-RhoGEF in the EHT.

(A) Splicing MO interfering with integration of exon 38. Top panel: cartoon representing exons/introns (not drawn at scale) composing the 3-prime region of the gene encoding for ArhGEF11, with the position of the splicing MO. Middle panel: agarose gel showing the two alternative mRNAs encoding for ArhGEF11 in control animals (left track, control) and after injection of the MO at the one cell stage (right track,+MO at 2 and 5 ng). Bottom panel: ArhGEF11 DNA sequences obtained after RT-PCR, cloning and sequencing for 1 control and 2+MO clones (+MO1,+MO3). (B) Morphologies of control and +MO injected 48 hpf embryos. Note the absence of malformations. (C) Top panel: ArhGEF11 wild type and CRISPR-mediated 7 bp deletion in the 3-prime region of exon 38 leading to a frame-shift in the ORF and a downstream premature stop codon; middle panel: CRISPR del/C-ter nucleotide/aa sequence and wild type nucleotide/aa sequence covering the extreme C-ter of the full-length protein; bottom panel: CLUSTAL 2.1 multiple sequence alignment of mouse and zebrafish C-termini highlighting the sequences of spliced variants of potentially similar activity in the regulation of ArhGEF11 activity on RhoA. Accession numbers: NP_001003912.1 and NP_001027010.1 for the mouse and Danio rerio sequence, respectively. (D) Morphologies of control and homozygous ArhGEF11^{CRISPR-Cterdel+/+} 48 hpf embryos ((Kdrl:eGFP-Jam3b; kdrl:ArhGEF11^{CRISPR-Cterdel+/+}) embryos). Note the edema in the cardiac region (35 hpf embryos also exhibited retardation in blood circulation). Raw image of agarose gel showing the 2 alternative mRNAs encoding for ArhGEF11 in control animals (left track, control) and after injection of the MO at the one cell stage (right track, +MO at 2 and 5 ng) are available at https://doi.org/10.5281/zenodo.11355396.