Figure S5. CRISPR-Cas9 mediated editing of pitx2c gene perturbs stomach asymmetry. Xenopus embryos were injected with 2 ng synthetic Cas9 mRNA plus 300 pg control tyrosinase (tyr) gRNA (A) or pitx2c gRNA (B), and allowed to develop until stage 42. The graph (C) indicates the percentage of embryos in which the greater curvature of the stomach is normal (arrowhead in A) or reduced/absent (* in B) after injection with the tyr gRNA or two different pitx2c gRNAs (#1 and #2). D) Genomic DNA from 10 embryos injected with each gRNA was pooled and PCR amplified using exon 1-specific primers, and then tested for CRISPR/Cas9-induced mutations by T7 endonuclease I assay. The asterisk (*) indicates the 500 bp amplicon not cut in un-injected or tyr gRNA-injected control embryos. Arrowheads indicate bands resulting from mismatches (inferred mutations) in amplicons cleaved by T7 endonuclease I. E) Sequencing of a subset of individual clones validates the presence of deleterious mutations in pitx2c gRNA injected embryos. For pitx2c gRNA #1, 9/17 mutations were likely nulls and 3/17 were predicted to result in compromised function; for pitx2c gRNA #2: 2/18 mutations were likely nulls and 4/17 were likely to result in compromised function. F-N) Sections through stomachs of Cas9 control and pitx2c gRNA (#1) injected embryos (Stage 39) were stained for integrin (green) and false color-coded as in Fig. 1 to highlight the relevant tissue layers (RE, right endoderm; LE, left endoderm; RM, right mesoderm; LM, left mesoderm). Controls show normal asymmetric expansion of the left stomach wall (F), but this is eliminated in embryos injected with pitx2c gRNA (G-H). Normal asymmetries in the lengths of the left and right stomach walls (I), and the widths of the endoderm (Endo) and mesoderm (Meso) layers (J-K), are also significantly perturbed by CRISPR-Cas editing of pitx2c; * denotes p<0.01. Compared to controls (L), left endoderm tissue architecture is severely disrupted, and apicobasal polarity is reduced (M), and/or disorganized (N) in embryos with CRISPR-mediated mutations in pitx2c; arrowheads indicate the expression of the apical marker aPKC (red). Scalebars= 100 ïM.
Image published in: Davis A et al. (2017)
Copyright © 2017. Image reproduced with permission of the Publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
| Gene | Synonyms | Species | Stage(s) | Tissue |
|---|---|---|---|---|
| itgb1.L | Beta 1 integrin, cd29, fnrb, gpiia, itgb1-a, itgb1-b, mdf2, msk12, vlab, vla-beta | X. laevis | Throughout NF stage 39 | stomach left right epithelium endoderm mesoderm |
| prkcz.L | PKC zeta, pkc-zeta, PKCzeta, PKMz, TPKC | X. laevis | Throughout NF stage 39 | stomach left right epithelium endoderm mesoderm |
Image source: Published
| Experiment + Assay | Source | Phenotypes and Disease | ||
|---|---|---|---|---|
| Xla Wt + pitx2 gRNA1 CRISPR + NF42 (whole-mount microscopy) | Fig. S5. BC |
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| Xla Wt + pitx2 gRNA2 CRISPR + NF42 (whole-mount microscopy) | Fig. S5 C |
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| Xla Wt + pitx2 gRNA1 CRISPR + NF42 (immunohistochemistry) | Fig. S5. GHJKMN |
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