Figure 5

Xenopus tropicalis (acbd6) crispants have gastrulation, movement, craniofacial, brain and eye defects together with microcephaly. (A) The gene structure of human (ACBD6) and X. tropicalis (acbd6) reveals eight exons. (B) Gastrulation defects, including failure of blastopore closure and anterior posterior defects, were observed in F₀X. tropicalis embryos injected with two different CRISPR/Cas9 constructs (sgRNA-68 and sgRNA-71) disrupting exon 1 of acbd6. (C) Those animals surviving to free-feeding stages presented with microcephaly, craniofacial dysmorphism and eye abnormalities. (D) The differences in head size between the uninjected control (2.07 ± 0.36 mm) and acbd6 crispant tadpoles (1.52 ± 0.27 mm, sgRNA-68) were found to be significant, t(34) = 5.183, P < 0.001. (E and F) Alcian blue staining marking the cartilaginous structures in the head and neck show equivalent structures between control (E) and acbd6 crispant tadpoles (F), revealing no gross morphological abnormalities. (G) Detailed structural analysis in higher resolution microCT imaging (1% phosphotungstic acid contrast stain) revealed significant structural abnormalities in the facial musculature (red arrows; G), abnormalities of the eye (microphthalmia, anophthalmia; yellow arrows, G) and structural abnormalities in the brain most pronounced in the midbrain regions (blue arrow, G). (H) Locomotion analysis at NF44/45 revealed that crispants moved significantly less than control tadpoles. (I) The Kaplan–Meier survival analysis of 65 control and crispant tadpoles shows two periods of crispant-specific decline, the first at gastrula stages (Day 0–1) and the second with post-feeding [Day 8, Nieuwkoop and Faber (NF) stage 47].