Expression profiles of cIRF6, cLHX6 and cLHX7 in the facial primordia suggest specific roles during primary palatogenesis

Abstract

Background: The LIM-homeodomain transcription factors LHX7 and LHX6 have been implicated in palatogenesis in mice and thus may also contribute to the incidence of isolated palatal clefts and/or clefts of the lip and primary palate (CL/P) in humans. Causative mutations in the transcription factor IRF6 have also been identified in two allelic CL/P syndromes and common polymorphisms in the same gene are significantly associated with non-syndromal CL/P in different populations.

Results: Here we report the isolation of chick orthologues of LHX7, LHX6 and IRF6 and the first characterisation of their profiles of expression during morphogenesis of the midface with emphasis on the period around formation of the primary palate. LHX7 and LHX6 expression was restricted to the ectomesenchyme immediately underlying the ectoderm of the maxillary and mandibular primordia as well as to the lateral globular projections of the medial nasal process, again underlying the pre-fusion primary palatal epithelia. In contrast, IRF6 expression was restricted to surface epithelia, with elevated levels around the frontonasal process, the maxillary primordia, and the nasal pits. Elsewhere, high expression was also evident in the egg tooth primordium and in the apical ectodermal ridge of the developing limbs.

Conclusion: The restricted expression of both LHX genes and IRF6 in the facial primordia suggests roles for these gene products in promoting directed outgrowth and fusion of the primary palate. The manipulability, minimal cost and susceptibility of chicks to CL/P will enable more detailed investigations into how perturbations of IRF6, LHX6 and LHX7 contribute to common orofacial clefts.

Figure 1

Protein alignments of mouse, chick and human LHX7 (a), LHX6 (b) and IRF6 (c). cLHX7 displays 89% and 95% identity with mouse Lhx7 and human LHX7, respectively. cLHX6 displays 94% identity and 99% similarity to both human LHX6 and mouse Lhx6. cIRF6 displays 83% identity, 99% similarity with human IRF6 and mouse Irf6. Legend: The LIM domains of LHX6/7 and DNA-binding domain of IRF6 are boxed. The homeodomain of LHX6/7 is underlined.

Figure 2

Expression pattern of cLHX7 and cLHX6 in the developing chick embryo. cLHX7 (top panel) and cLHX6 (bottom panel) were restricted to the ventral extremities of the maxillary primordia and the rostral tip of the mandibular primordia before and after fusion of the maxillary primordia and medial nasal process during formation of the primary palate (a – i, k). From around HH27, cLHX6 expression was dispersed throughout the mandibular primordia (k). cLHX7 and cLHX6 expression was detected in the pre-fusion zone of the medial nasal process, prior to fusion with the maxillary primordia (e, f, g, h). The expression in the medial nasal process remained in the mesenchymal bridge of the beak after fusion (i, k). cLHX7 and cLHX6 expression was detected in the mesenchyme throughout the palatal shelves at HH30 (m, n). cLHX7 specifically displayed increased expression on the anterior tips of the developing shelves (m). Both cLHX7 and cLHX6 expression was detected in the otic vesicle from HH25 to HH30 (j, l). Abbreviations: max: maxillary primordia; man: mandibular primordia; mnp: medial nasal process; ov: otic vesicle; ps: palatal shelves.

Figure 3

Vibratome sections of whole-mount in situ hybridization embryos. Sectioning of stage HH23 whole mount in situ hybridization embryos indicates that both LHX7 (left column) and LHX6 (right column) show expression in the neural crest-derived mesenchyme of the first branchial arch (maxillary primordia shown) (c, d) and the lateral globular masses at the edges of the medial nasal process (e, f) restricted to the region directly subjacent to the ectoderm. Abbreviations: max mes: maxillary primordia mesenchyme; ep: epithelium; mnp: medial nasal process

Figure 4

Expression of IRF6 in the developing chick embryo. Expression is restricted to facial ectoderm. Whole-mount in situ hybridisation (a, c, e) and vibratome sections of whole-mount embryos (b, d) revealed notable IRF6 expression in the epithelia surrounding the frontonasal process, the maxillary primordia, and the nasal pits. IRF6 expression was also detected in the ectoderm of the leading edges of the palatal shelves and in the ridges of the primitive oral cavity at HH30 (h). High IRF6 expression was also found in the apical ectodermal ridge of the limb buds (g) and in the egg tooth primordium (f).