Fig. 9.

aura encodes Mid1ip1l. (A) Mid1ip1l protein in wild-type and aura mutant alleles. Red boxes indicate predicted alpha helices. The aur^t9792 mutation generates a premature stop that truncates the last conserved helix. The CRISPR/Cas9-generated mid1ip1l^uw39 allele results in a frameshift translated region (dark red box) followed by an early stop. (B) DNA sequencing trace of the aur^t9792 allele, which creates a stop codon in amino acid 142. (C) Amino acid sequence comparison between various mid1ip1 homologs in the aur^t9792 mutation site region. The mutation in aur^t9792 occurs at a highly conserved lysine. Red stars (A,C) indicate amino acid directly affected by the mutations. D.r., Danio rerio; X.t., Xenopus tropicalis; H.s., Homo sapiens; G.g., Gallus gallus; M.m., Mus Musculus. Bottom row numbers are the Consistency Score according to the PRALINE protein alignment program. (D-G′) The CRISPR/Cas9-generated mid1ip1l^uw39 allele does not complement aur^t9792. (D,E) All embryos from aur^t9792/mid1ip1l^uw39 transheterozygous females (E) exhibit reduced membrane integrity (asterisk), reduced yolk coalescence (arrowhead) and regressed furrows (arrow; see also Fig. S1A,B), whereas control embryos from heterozygous siblings (D) are wild type in appearance. (F,G) 8-cell embryos from transheterozygous females exhibit reduced β-Catenin accumulation in mature furrows (27/27; G,G′, arrowheads), ectopic CGs throughout the cortex (G) and apparently stabilized FMA (G′, arrow), compared with control siblings (0/25; F,F′). (F′,G′) Higher magnifications of boxed regions in F,G. Scale bars: 100 μm in F,G; 10 μm in F′,G′.