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Expression of capn8 is significantly dysregulated in dact1/2−/− mutants. (A) Single-cell RNAseq gene expression analysis of capn8 in wildtype and dact1/2−/− mutants. In wildtype embryos, capn8 expression is restricted predominantly to the epidermis whereas capn8 is widely expressed throughout the embryo in dact1/2−/− mutants, especially in the mesoderm. (B) Wholemount in situ hybridization of capn8 expression in wildtype and dact1/2−/− mutant embryos at 2 ss. Staining corroborates the single-cell RNAseq data, with expanded ectopic expression of capn8 throughout the embryo. Flat mounts are oriented anterior to the left. Scale bar: 100 μm. (C) Brightfield images and Alcian blue staining of the ethmoid plate show ectopic expression of capn8 mRNA (200 pg) at the 1 cell stage in dact1+/-,dact2+/- embryos recapitulates the dact1/2−/− compound mutant craniofacial phenotype. The mutant craniofacial phenotype did not manifest in gfp mRNA (200 pg) injected 1 cell-stage dact1+/-,dact2+/- embryos. Scale bar: 100 μm (D) Quantification of mutant and normal craniofacial phenotype in 4 dpf larvae after mRNA injection at the 1 cell stage. Larvae were derived from dact1/2+/- interbreeding. Larvae were uninjected or injected with 200 pg gfp control or capn8 mRNA. A Fisher exact test showed a significant effect of capn8 mRNA injecting in the dact1/2 double heterozygotes. Asterisk indicates a significant difference between conditions (p = 0.013).
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