Sonic Hedgehog-signalling patterns the developing chicken comb as revealed by exploration of the pea-comb mutation

Abstract

The genetic basis and mechanisms behind the morphological variation observed throughout the animal kingdom is still relatively unknown. In the present work we have focused on the establishment of the chicken comb-morphology by exploring the Pea-comb mutant. The wild-type single-comb is reduced in size and distorted in the Pea-comb mutant. Pea-comb is formed by a lateral expansion of the central comb anlage into three ridges and is caused by a mutation in SOX5, which induces ectopic expression of the SOX5 transcription factor in mesenchyme under the developing comb. Analysis of differential gene expression identified decreased Sonic hedgehog (SHH) receptor expression in Pea-comb mesenchyme. By experimentally blocking SHH with cyclopamine, the wild-type single-comb was transformed into a Pea-comb-like phenotype. The results show that the patterning of the chicken comb is under the control of SHH and suggest that ectopic SOX5 expression in the Pea-comb change the response of mesenchyme to SHH signalling with altered comb morphogenesis as a result. A role for the mesenchyme during comb morphogenesis is further supported by the recent finding that another comb-mutant (Rose-comb), is caused by ectopic expression of a transcription factor in comb mesenchyme. The present study does not only give knowledge about how the chicken comb is formed, it also adds to our understanding how mutations or genetic polymorphisms may contribute to inherited variations in the human face.

Figure 1. Comparison of Pea- and single-comb with respect to comb morphology, Alcian blue cartilage staining and SOX5 expression.

(A–D) Morphology of E12 and E18, Pea- and single-combs. (E–H) Alcian blue stained cross section of E12 and E18 Pea- and single-combs to visualize cartilaginous structures. Note that in spite of the differences in Pea- and single-comb morphology the underlying cartilage structures are normal. The sections are not exactly on the same level. (I) Bar graph with qRT-PCR results and fluorescence micrographs of immunohistological analysis of SOX5 mRNA levels. Bar graph data are normalized to the ß-actin mRNA levels and is related to the ß-actin mRNA level. Bars±s.e.m., ANOVA, n>4 combs per sample * p<0.05, **p<0.005. (J–S) Photographs of Pea- (K, L, N–Q) and single combed (J, M, R, S) chicken of the sex and ages as indicated in the figure. E; embryonic day, pc; Pea-comb, sc; single-comb, w; weeks. Scale bars are 250 µm.

Figure 2. Candidate gene expression analysis in Pea- and single-comb tissue.

Bar graphs with qRT-PCR analysis data for (A) RUNX2, (B) ETS1, (C) PAX3, (D) COL1A2, (E) ITGB3, (F) IHH, (G) SHH, (H) PTCH1, (I) SMO, (J) GLI1 and (K) GLI3. Bar graph data are normalized to ß-actin mRNA levels and is relative to the ß-actin mRNA level in Pea- and single-comb tissue respectively. Bar graphs are mean±s.e.m., ANOVA, n = 6 (single-comb) n = 5 (Pea-comb). * p<0.05, **p<0.001.

Figure 3. ETS1, SOX5 and PTCH1 expression in E9 Pea- and single-comb.

Micrographs depicting immunohistochemical analysis of SOX5 and ETS1 expression and in situ hybridization analysis for PTCH1 mRNA in the E9 comb-region. (A) Low magnification fluorescence micrograph showing the single-comb region with SOX5 in nasal cartilage and ETS1 in the dermal mesenchyme. DAPI staining visualises the mesenchymal condensation under the comb-ridge. Sub-dermal mesenchyme is indicated by the dashed straight line. (B) Single-comb-ridge with staining for SOX5 (red) and ETS1 (green). The mesenchyme condensation is delineated by the dashed line and correlates with lower ETS1 expression. (C, D) Separate red and green fluorescence signals for image shown in B. (E) Pea-comb-ridge with staining for SOX5 (red) and ETS1 (green). (F, G) Separation of fluorescence signals shown in E. (H, I) PTCH1 in situ hybridisation analysis of (H) single- and (I) Pea-comb-ridge. ect; ectoderm, mes; dermal mesenchyme, nac; nasal cartilage, pc; Pea-comb, sc; single-comb. Scale bars in A, I are 100 µm and in G 50 µm also valid for B–F.

Figure 4. SHH expression and effects of cyclopamine treatment during comb formation.

SHH expression was analysed by using immunohistochemistry of Pea- and single-comb E4 embryos. Fluorescence micrographs of (A) sagittal section of a single-combed and (B) a Pea-combed E4 head labelled for SHH. (C) Schematic illustration of the region shown in sections depicted in A–B. (D) Frontal view of the facial region of an E4 single-combed chicken head with the plane of sections in A–C indicated by a dashed line. (E) Dorsal view of the forehead of an E18 single-combed chicken treated with cyclopamine at E5. The beak is pointing down in the image. Note the comb that is split in three rows of serration in the caudal part. Some feather anlagen were removed to better display the comb. (F) Magnification of the affected comb-region depicted in E. (G) E18 single-comb control chicken. (H–J) Fluorescence micrograph of DAPI stained cross section of (H) the cyclopamin-treated comb depicted in E, (I) an E18 single-comb and (J) a E18 Pea-comb. (K) Table with the number of animals affected when treated at E4– E7 by cyclopamine or control with split comb or affected serrations. ^# Number of individually distinguishable points or serrations. ^## Two control embryos were affected by other head and intestine malformations. Side-view of serrations of E15 single-combs treated at E7 with (L) HBC/PBS control and (M) cyclopamine. Arrow indicates the posterior part of the comb with a lateral expansion. (N) Bar-graph showing the effect on serration seen at E15 in cyclopamine or HBC/PBS control-treated single-combs at E7. Non-parametric Mann-Whitney U-test, n as indicated in the figure * p<0.05 ** p<0.01, *** p<0.001. Scale bar in C is 100 µm also valid for A and B, bar in G is 1000 µm also valid for E, bar in J is 400 µm also valid for H and I, bar in M is 250 µm also valid for L. E; embryonic day, Ctrl; Control, Cyclop; Cyclopamine, or; optic recess, pc; Pea-comb; sc; single-comb, SHH; Sonic hedgehog, t; telencephalon.