Severe limb defects in Hypodactyly mice result from the expression of a novel, mutant HOXA13 protein.
Hypodactyly (Hoxa13(Hd)) mice have a 50-bp deletion in the coding region of exon 1 of the Hoxa13 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 Hoxa13(Hd/Hd) but not Hoxa13(-/-) limb buds. In addition, compound heterozygotes for one Hd 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 the Hd mutation. The Hoxa13(Hd) deletion does not interfere with steady-state mRNA levels; however, its consequences on translation are unknown. In this paper, we characterize the Hoxa13 transcription initiation site in limbs and determine the initiator methionine of HOXA13. We show that the Hoxa13(Hd) 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 (HOXA13(Hd)) 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, HOXA13(Hd) 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 HOXA13(Hd) could alter limb morphogenesis, we misexpressed the mutant mRNA throughout the developing limb bud using a Prx-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 HOXA13(Hd) plays a role in the profound failure of digit formation in Hoxa13(Hd/Hd) mice and explains the morphologic differences between these two Hoxa13 alleles.
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Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, 48109, USA.
Margulies
E H
EH
Kuo
A
A
Innis
J W
JW
D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000737:chemistry; D005121:Extremities / Q000196:embryology*; D016368:Frameshift Mutation; D017353:Gene Deletion; D018398:Homeodomain Proteins / Q000235:genetics*; D018878:Limb Buds / Q000196:embryology*; D017880:Limb Deformities, Congenital / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D017384:Sequence Deletion; D014158:Transcription, Genetic
D018398:Homeodomain Proteins; C099959:homeobox protein HOXA13