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. 2017 Aug 15;144(16):2951–2960. doi: 10.1242/dev.145839

Fig. 3.

Fig. 3.

Pharyngeal development and ectodermal cell polarity, but not cnidocyte differentiation of surface ectodermal cells, are disrupted after Nvbra excision. (A-D) Control embryos were injected with Cas9 only. (A,B) At 48 h post-fertilization (hpf) the blastopore is visible (arrow) and a well-defined epithelial endoderm has formed (dotted line). Ectodermal cells protrude into the archenteron to form the pharynx rudiment (arrowhead). (C,D) By 72 hpf the ectoderm has extended well into the blastocoel (arrowhead). (E-H) In Nvbra/Cas9 embryos, the pharynx fails to form. (E,F) At 48 hpf, the blastopore is visible (arrow) but the endoderm is thicker than in controls (compare dotted lines in B and F) and appears disorganized. No pharyngeal ectoderm extends into the archenteron. (G,H) By 72 hpf, the blastopore is still evident but no pharynx has formed. The endoderm is highly disorganized with cells filling the blastocoel, and embryos appear rounded when compared with the elongated phenotype of control embryos. (I-L) Cas9 control embryos develop cnidocytes normally. At 48 hpf (I) and 72 hpf (J), cnidocytes (red, anti-mcol4) are abundant throughout the ectoderm. Control embryos (72 hpf) also exhibit ectodermal expression of (K) Lgl and highly polarized expression of (L) aPKC (arrowhead) in the apical cortex of ectodermal cells. (M-P) Cnidocytes are present in the ectoderm of Nvbra/Cas9 embryos at both (M) 48 hpf and (N) 72 hpf. (O) Lgl is still basolateral and restricted to the ectodermal cells; there is abrupt cessation of Lgl staining at the blastopore (arrow, compare with K). Expression of aPKC (P) is no longer apically restricted, spreading into the basal regions of ectodermal cells (arrowhead). Ratios in the bottom left corner indicate the number of embryos showing the indicated phenotype (wild type or abnormal)/total number of embryos counted; only embryos with proper ectoderm formation were counted in order to exclude dead or clearly abnormal embryos. Phenotypes of 72 hpf Nvbra/Cas9 embryos are quantified in more detail in Fig. S5. The blastopore is on the left of each image, indicated by an asterisk in the first column. Images A-H and K,L,O,P are single optical sections; images I,J,M,N are 3D rendered from z-stacks.