Phenotypic means and standard deviations (in parenthesis) for comb size, comb mass, body weight and RQ values, according to line, sex and Pea-Comb genotype (Ho = homozygous; Het = heterozygous; ND = undetermined carrier status).

<p>Phenotypic means and standard deviations (in parenthesis) for comb size, comb mass, body weight and RQ values, according to line, sex and Pea-Comb genotype (Ho = homozygous; Het = heterozygous; ND = undetermined carrier status).</p

Number of animals recorded per line, sex, trait and Pea-comb genotype.

<p>Adult females were housed in individual cages for precise egg production recording and adult males were housed in a different room, being 1 or 2 in a cage. The mean ambient temperature was set to 21°C. Light duration was 14 hours a day for females and 12 hours a day for males. Animals were fed <i>ad libitum</i> with a layer diet for females, and a maintenance diet for males. The total number of animals included in this study was 178, with 62 males and 116 females (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118706#pone.0118706.t001" target="_blank">Table 1</a>).</p><p>Number of animals recorded per line, sex, trait and Pea-comb genotype.</p

Frequency distribution of comb size measured in arbitrary units for females of each experimental line (CH1, WL-DJ and NOE).

<p>Frequency distribution of comb size measured in arbitrary units for females of each experimental line (CH1, WL-DJ and NOE).</p

Frequency distribution of the number of repeats (RQ values) for the <i>SOX5</i>-CNV in females for each experimental line (CH1, WL-DJ and NOE) according to genotype for the Pea-Comb genotype (Het = heterozygous; Ho = homozygous).

<p>All animals of the CH1 line are homozygous.</p

Significance levels for the different sources of variation in the analysis of comb size (CS) and comb mass (CM), according to the data set, with or without the RQ covariate.

<p>The number of observations used in each analysis is indicated in parenthesis for each variable.</p><p>Significance levels for the different sources of variation in the analysis of comb size (CS) and comb mass (CM), according to the data set, with or without the RQ covariate.</p

Phenotypic correlations between comb size (CS), comb mass (CM), body weight (BW), and the number of CNV repeats (RQ), according to line and sex.

<p>Significance level: the p-value is indicated in parenthesis</p><p>Phenotypic correlations between comb size (CS), comb mass (CM), body weight (BW), and the number of CNV repeats (RQ), according to line and sex.</p

Least squares means and standard error of the means for comb size, comb mass, body weight, and number of repeats (RQ) per line and sex.

<p>*for a given trait, the least squares means showing a different superscript differ at p≤0.01</p><p>Least squares means and standard error of the means for comb size, comb mass, body weight, and number of repeats (RQ) per line and sex.</p

Landmark recording on the picture of the Pea-comb from an adult male (animal #2654); G represents the center of gravity.

<p>Landmark recording on the picture of the Pea-comb from an adult male (animal #2654); G represents the center of gravity.</p

SHH expression and effects of cyclopamine treatment during comb formation.

<p>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 * <i>p</i><0.05 ** <i>p</i><0.01, *** <i>p</i><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.</p

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

<p>Micrographs depicting immunohistochemical analysis of SOX5 and ETS1 expression and <i>in situ</i> 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.</p