Hoxc13-dependent gene expression in toes of Xenopus tropicalis

The transcription factor Hoxc13 is essential for the growth of hair and nails in mammals, but its role in cornified skin appendages of non-mammalian vertebrates are not known. CRISPR/Cas9-mediated genome editing was applied to generate hoxc13 mosaic mutants of the western clawed frog (Xenopus tropicalis) and, upon further breeding, hoxc13 knockout (KO) frogs. In wildtype frogs (WT1-WT3), cornified claws developed on toes I, II and III of the hind limbs, here referred to as hind limb inner (HI) toes, whereas toes IV and V of the hind limbs, here referred to as hind limb outer (HO) toes, lacked claws. The deletion of hoxc13 (out-of-frame mutations leading to premature stop of translation) abrogated claws on HI toes (frogs KO1 and KO2). In a mosaic mutant frog, HI toes carrying 2 knockout alleles of hoxc13 (KO3_mosaic) lacked claws, whereas HI toes carrying one functional hoxc13 allele with an R80M point mutation (WT_mosaic_R80M) developed claws. RNA was prepared from 3 biological replicates of hoxc13-expressing (WT) HI toes with claws and 3 biological replicates of hoxc13-deficient (KO) HI toes that were clawless. For comparison, RNA was also prepared from the clawless HO toes of one WT frog (WT2) and one KO frog (KO2). The RNAs were subjected to RNA-seq analysis.