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. 2021 Dec 23;10:e74336. doi: 10.7554/eLife.74336

Figure 3. Generation of pou-iv null mutant sea anemones.

(A, B) Diagrams of the pou-iv locus (A) and the disrupted mutant allele (pou-iv-; B). Blue bars show predicted translation start sites; red bars show predicted translation termination sites. In A, filled boxes indicate exons, and the regions that encode the POU- and homeo-domains are highlighted in orange (‘POU’) and yellow (‘HD’), respectively. Purple arrowheads show single guide RNA (sgRNA) target sites. The region that encodes peptides targeted by the antibody generated in this study is indicated by a green line. In B, deletion mutation is boxed in dotted orange lines, and blue arrows mark regions targeted in the PCR analysis shown in C; reverse primers are numbered (1)–(2). (C) Genotyping PCR. Note that the wildtype allele-specific primer (1) generates a 689 bp PCR product from the wildtype allele ('+') but cannot bind to the pou-iv- allele due to deletion mutation. The primer (2) generates a 558 bp PCR product from the pou-iv- allele, and a 1312 bp PCR product from the wildtype allele. (D) Western blotting with an antibody against Nematostella vectensis POU-IV. An antibody against acetylated α-tubulin (‘α-tubulin’; ca. 52 kDa) was used as a loading control. The anti-POU-IV reacts with a protein of expected size for wildtype POU-IV (35.2 kDa) in wildtype (+/+) polyp extracts, but not in pou-iv mutant (-/-) polyp extracts (black arrowhead). Also note that the antibody’s reactivity with a protein of expected size for mutant POU-IV lacking DNA-binding domains (18.7 kDa) is detectable in mutant (-/-) extracts, but not in wildtype (+/+) extracts (purple arrowhead). The band just below the expected size of the wildtype POU-IV occur in both wildtype and mutant protein extracts, and therefore represents non-POU-IV protein(s) that are immunoreactive with the anti-POU-IV antibody.

Figure 3—source data 1. An original gel image used to generate Figure 3C and the original image with relevant lanes labeled.
Figure 3—source data 2. An original western blot image used to generate Figure 3D (top; anti-Nematostella vectensis POU-IV) and the original image with relevant lanes labeled.
Figure 3—source data 3. An original western blot image used to generate Figure 3D (bottom; anti-acetylated α-tubulin) and the original image with relevant lanes labeled.

Figure 3.

Figure 3—figure supplement 1. Generation of pou-iv F0 mosaic mutants by CRISPR-Cas9-mediated mutagenesis in Nematostella vectensis.

Figure 3—figure supplement 1.

A schematic view of the pou-iv locus (left), and genomic DNA PCR results of an uninjected wildtype (WT) embryo (‘WT’) and an F0 embryo injected with locus-specific sgRNAs and Cas9 (‘pou-iv F0’) (right). In the schematic depicting the pou-i locus, filled boxes indicate exons. A blue bar shows the predicted translation start site, and a red bar shows the predicted translation termination site. The regions that encode the POU- and homeo-domains are highlighted in orange (‘POU’) and yellow (‘HD’), respectively. Purple arrowheads show single guide RNA (sgRNA) target sites. Blue arrows mark regions targeted in the PCR analysis shown to the right. Note that genomic PCR of the WT embryo shows expected sizes of PCR fragments (1495 bp) for primary PCR (‘a’), and 1263 bp for secondary nested PCR (‘b’), while F0 embryos show additional bands of smaller sizes (arrowheads), indicating that targeted deletions of different sizes have occurred mosaically in each embryo. DNA sequencing of the < 500 bp band (lower arrowhead) indicated that this mutant allele harbored 981 bp deletion encompassing the POU- and homeo-domain-encoding regions.
Figure 3—figure supplement 1—source data 1. An original gel image used to generate Figure 3—figure supplement 1 (right) and the original image with relevant lanes labeled.
Figure 3—figure supplement 2. Sequence alignment of wildtype and mutant pou-iv alleles.

Figure 3—figure supplement 2.

(A) Diagrams of the pou-iv locus and the disrupted mutant allele (pou-iv-). Blue bars show predicted translation start sites; red bars show predicted translation termination sites. In the schematic depicting the pou-iv locus, filled boxes indicate exons, and the regions that encode the POU- and homeo-domains are highlighted in orange (‘POU’) and yellow (‘HD’), respectively. In the schematic of the pou-iv- allele, deletion mutation is boxed in orange dotted lines. (B) Alignment of nucleotide and translated amino acid sequences of wildtype (‘+’) and mutant (‘pou-iv-’) alleles boxed in A. POU and homeo-domains are boxed in orange and yellow, respectively. Predicted translation termination sites are boxed in red, and 705 bp deletion mutation is boxed in dotted blue lines. Note that a sequence encoding all but the first four residues of the POU domain and the entire homeodomain is deleted in the pou-iv- allele.