Differential Regulation of Lhx9 Expression in Mouse and Chicken Limbs

Abstract

The distal tip of the developing vertebrate limb is covered by a rim of thickened ectoderm called the apical ectodermal ridge (AER). The AER secretes fibroblast growth factors (Fgfs) into the underlying mesoderm to control outgrowth and proximal‐distal patterning. Patterning during outgrowth proceeds from proximal to distal, with the proximal segment or stylopod being laid down first, then the zeugopod, and the distal segment or autopod forming last. Secreted Fgfs from the AER maintain a sub‐AER population of undifferentiated mesoderm, called the progress zone, that provides cells for this progressive differentiation.In mice, Lhx9and its homologue Lhx2are expressed in the sub‐AER mesoderm and appear to play redundant roles in maintaining the expression of other patterning molecules, such as Shh. Interestingly in the chicken, the expression of LHX9 is restricted to the anterior aspect of the sub‐AER mesoderm, while LHX2 retains the full distal expression seen in mice. Recent clinical reports of an LHX9 missense mutation in a patient with hypoplastic/missing thumbs and great toes suggest that LHX9 can play a critical role in normal limb development. We hypothesized that the chicken cis‐regulatory modules (CRMs) regulating Lhx9 might have subtle species‐specific sequence variations compared to the murine CRMs that correlate with this differential expression pattern.To test this hypothesis, potential CRMs were identified near the Lhx9 locus by in silicoanalysis, using sequence conservation and regulatory chromatin markers from published ChIP‐seq data. The potential CRMs were isolated and inserted into CRM‐reporter constructs. The limb‐related activity of a potential CRM was then determined by targeted regional electroporation into the distal chicken limb mesoderm.We identified a highly conserved, transcription‐factor‐rich 623 bp sequence within intron 12 of DENDD1B 132 kbp upstream of the chicken LHX9 transcription start site. The potential CRM was associated with H3k4me2, H3k27ac, p300 and H3k27me3. The associated CRM‐reporter construct displayed activity in both anterior and posterior sub‐AER limb mesoderm overlapping LHX9 and LHX2 expression. These findings are consistent with a distal sub‐AER LHX9enhancer, but do not account for the lack of posterior expression. This suggests that additional CRMs contribute to the final expression pattern of LHX9 in the limb. Twenty‐two additional potential CRMs were identified and will be further characterized to determine their role in restricting LHX9expression from the posterior mesoderm in chickens. Characterizing the differential regulation of Lhx9 during limb development may provide insight into the species‐specific variation of limb patterning.