Identification and developmental expression analysis of a novel homeobox gene closely linked to the mouse Twirler mutation

Abstract

The Twirler mutation arose spontaneously and causes inner ear defects in heterozygous and cleft lip and/or cleft palate in homozygous mutant mice, providing a unique animal model for investigating the molecular mechanisms of inner ear and craniofacial development. Here, we report the identification of a novel homeobox gene, Iroquois-related homeobox like-1 (Irxl1), from the Twirler locus. Irxl1 encodes a TALE-family homeodomain protein with its homeodomain exhibiting the highest amino acid sequence identity (54%) to those of invertebrate Iroquois and vertebrate Irx subfamily members. The putative Irxl1 protein lacks the Iro-box, a conserved motif in all known members of the Irx subfamily. Searching the databases showed that Irxl1 orthologs exist in Xenopus, chick, and mammals. In situ hybridization analyses of mouse embryos at various developmental stages showed that Irxl1 mRNA is highly expressed in the frontonasal process and palatal mesenchyme during primary and secondary palate development. In addition, Irxl1 mRNA is strongly expressed in mesenchyme surrounding the developing inner ear, in discrete regions of the developing mandible, in the dermamyotome during somite differentiation, and in a subset of muscular structures in late embryonic stages. The developmental expression pattern indicates that Irxl1 is a good candidate gene for the Twirler gene.

Fig. 1

Irxl1 encodes a new TALE family homeodomain protein in vertebrates. (A) Alignment of the mouse Irxl1 protein sequence with the products of the human C10orf48 gene and the Xenopus MGC82772 full-length cDNA. Numbers to the right represent numbers of the last residue in that line. Dots represent identities to the mouse Irxl1 protein and dashes represent gaps. The homeodomain sequences (underlined) are identical in all three species. The overall high amino acid sequence identity indicates that these are orthologs in different species. (B) Alignment of the homeodomain sequence of the mouse Irxl1 protein with those of Drosophila (Dm ara), Caenorhabditis elegans (Ce 1k114), and mouse Irx proteins. The residues of the three helices of the homeodomain are overlined and marked on top. For comparison, the typical homeodomain of Drosophila Antennapedia protein (Dm Antp) is shown at the bottom. Dots represent identities to the Drosophila ara sequence and dashes represent gaps. Numbers in the bottom denote amino acid positions in the typical homeodomain. The TALE family of homeodomain proteins has additional three amino acids between the first and second helices, compared with the typical hemeodomain of 60 amino acid residues. The proline-tyrosine-proline tripeptide residues (PYP, underlined) are conserved in all TALE homeodomains (Burglin, 1997).

Fig. 2

Expression pattern of Irxl1 mRNA in early mouse embryos. mRNA signals detected by whole mount in situ hybridization appear purple (A–F) and those detected by section in situ hybridization are shown in red on blue-counterstained sections (G–K). (A) Irxl1 mRNA was detected at E10.0 in the dermamyotome of rostral somites (arrowheads). (B) Dorsal view of an E10.0 embryo showing strong Irxl1 mRNA expression in the dermamyotome of somites around the forelimb level. (C) At E11.5, Irxl1 mRNA was expressed in the ventral domain of dermamyotome caudal to the forelimb bud (arrow) and in all dorsal dermamyotome. Strong Irxl1 expression was also detected at the base of the forelimb buds. (D) At E11.75, strong Irxl1 mRNA expression was detected in discrete domains in the fore- and hindlimb bunds. (E) High magnification lateral view of the forehead region of an E11.75 embryo showing strong expression of Irxl1 mRNA ventral to the telencephalic vesicle (arrow). (F) An E12.5 forelimb bud showing Irxl1 mRNA expression in the digital mesenchyme. (G) In situ hybridization of sagittal sections of E9.5 embryos showed specific Irxl1 mRNA expression in the newly formed dermamyotome of rostral somites (arrow). (H) Sagittal section of an E10.5 embryo showing Irxl1 mRNA expression in the frontonasal process and in the yolk sac, in addition to the dermomyotome. (I, J) Sagittal sections of E11.5 embryos showing Irxl1 mRNA expression in the medial nasal process (arrow in I), head mesenchyme including mesenchyme surrounding the developing otocyst, as well as in the ventral domain of the dermamyotome (arrowhead in J). (K) Frontal section of an E11.5 embryo showing Irxl1 mRNA expression in the medial and lateral nasal processes, the dermamyotome and the myogenic mesenchyme of the forelimb buds. dm, dermamyotome; fl, forelimb bud; fnp, frontonasal process; hl, hindlimb; hm, head mesenchyme; lnp, lateral nasal process; mnp, medial nasal process; ot, otic vesicle; tv, telencephalic vesicle; ys, yolk sac.

Fig. 3

Dynamic expression of Irxl1 mRNA during craniofacial and skeletal muscle development. mRNA signals are shown in red on blue-counterstained sections. (A) Frontal section of an E12.5 embryo showing strong Irxl1 mRNA expression in the neural crest-derived head mesenchyme surrounding the brain(arrowheads), in the secondary palate, in the mesenchymal precusor cells of the mandibular bone surrounding the Meckel’s cartilages, and in a subset of skeletal muscle cells (arrows). (B) Frontal section of an E13.5 embryo showing strong Irxl1 mRNA expression in the mesenchyme of the palate shelves and mandible, periocular muscles (arrows), in a subset of cells in the muscle of the tongue (arrowheads). (C, D) Frontal (C) and sagittal (D) sections of E14.5 embryos showing that strong Irxl1 mRNA expression persists in the palatal shelves after elevation and fusion, as well as in the periocular muscles (arrow in C) and in the mesenchyme surrounding the mandibular ossification centers. (E) Sagittal section of an E14.5 embryo showing highly restricted expression of Irxl1 mRNA in the intervertebral discs (arrowheads) and in the tail muscles (arrows). The asterisk in E marks an air bubble with autofluorescence. No Irxl1 mRNA expression in the liver was detected by in situ hybridization. e, eye; gt, genital tubercle; li, liver; lu, lung; mb, mandible; mc, Meckel’s cartilage; mi, mandibular incisor; nc, nasal cavity; oc, ossification center in the mandible; p, palate; po, preossification mesenchyme in the mandible; pp, primary palate; t, tongue; tl, tail.