Severe Limb Defects in Hypodactyly Mice Result from the Expression of a Novel, Mutant HOXA13 Protein

Abstract

Hypodactyly (Hoxa13Hd) mice have a 50-bp deletion in the coding region of exon 1 of theHoxa13 gene and have more severe limb defects than mice with an engineered deletion of the entire gene (Hoxa13−/−). Increased cell death is observed in the autopod of Hoxa13Hd/Hd but not Hoxa13−/− limb buds. In addition, compound heterozygotes for oneHd allele and a Hoxa13− allele have a more severe limb phenotype than mice homozygous for the engineered null allele, suggesting a dominant-negative effect of theHd mutation. The Hoxa13Hd deletion does not interfere with steady-state mRNA levels; however, its consequences on translation are unknown. In this paper, we characterize theHoxa13 transcription initiation site in limbs and determine the initiator methionine of HOXA13. We show that the Hoxa13Hd deletion results in a translational frame shift that leads to the loss of wild-type HOXA13 protein and the simultaneous production of a novel, stable protein in the limb buds of mutant mice. The mutant Hd protein (HOXA13Hd) consists of the first 25 amino acids of wild-type HOXA13 sequence, followed by 275 amino acids of arginine- and lysine-rich, novel sequence, and lacks the homeodomain. Like wild-type HOXA13, HOXA13Hd is localized to the nucleus in transfected COS-7 cells, perhaps mediated by the arginine- and lysine-rich peptide sequences created by the translational frame shift. To determine whether HOXA13Hd could alter limb morphogenesis, we misexpressed the mutant mRNA throughout the developing limb bud using aPrx-1 promoter–Hd gene construct in transgenic mice. Three of 15 transgenic founder animals displayed reduction or absence of proximal and distal limb structures. We propose that the expression of HOXA13Hd plays a role in the profound failure of digit formation in Hoxa13Hd/Hd mice and explains the morphologic differences between these twoHoxa13 alleles.