Molecular identification of colour pattern genes in birds

Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cis-regulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the up-regulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes

Identification de gènes de dessin de plumage chez les oiseaux

Le plumage des oiseaux montre une diversité de coloration spectaculaire. L’objectif de cette thèse est d’élucider les mécanismes génétiques contribuant à cette diversité en étudiant plus particulièrement la formation de dessins sur la plume et sur le corps. Les études I et II sont consacrées à deux dessins de barrure chez le poulet : la barrure autosomale et la barrure liée au sexe. En réalisant un croisement en retour à partir de la race Fayoumi, nous avons pu démontrer que la barrure autosomale est associée à une région du chromosome 11 et porte le gène de pigmentation MC1R. L’analyse fonctionnelle met en évidence qu’une mutation faux-sens, et non une modification de régulation, a le rôle d’une mutation causale. Dans l’étude II, nous démontrons que la barrure liée au sexe est créée par la combinaison de mutations de régulation en cis et de mutations faux-sens dans le gène CDKN2A. L’activation de l’expression de CDKN2A par deux mutations non-codantes détermine un phénotype de barrure très dilué. Des tests fonctionnels in vitro révèlent que les deux mutations faux-sens identifiées dans le même gène restreignent sa fonction et diminuent l’effet de dilution des mutations non-codantes. Seule la combinaison des mutations de régulation et d’une des mutation faux-sens, produit la barrure bien nette observée dans les races de poule actuelles. Les études III et IV précisent les mécanismes contrôlant la variation des dessins de plumage chez les oiseaux sauvages. Le chevalier combattant Philomachus pugnax présente trois formes reproductives chez le mâle, dénommées ‘indépendant’, ‘satellite’ et ‘faeder’. Nous avons d’abord identifié une inversion de 4.5 Mb chez les mâles satellite et faeder, qui diffèrent nettement entre eux, comme de l’indépendant, par leur comportement, leur stratégie reproductive et l’aspect de leur plumage. Un examen plus précis de la structure de l’inversion a montré que les points de cassure interrompaient le gène CENPN ce qui rend l’inversion létale à l’état homozygote. Nous avons identifié un grand nombre de variants contribuant assez probablement au phénotype et avons découvert quatre SNPs dans l’allèle MC1R du mâle satellite. L’étude IV a consisté à analyser la contribution de ces mutations de MC1R au plumage clair du mâle satellite pendant la reproduction. Nos données montrent que MC1R est activé dans les plumes colorées du mâle satellite en raison d’une plus forte expression de l’allèle indépendant. L’étude de la signalisation de MC1R en culture cellulaire a ensuite révélé que les mutations altèrent les propriétés du récepteur, mais suggère aussi que l’évaluation fonctionnelle du MC1R aviaire en cellules mammaliennes ne rend pas complètement compte du rôle complexe que MC1R joue dans les mélanocytes aviaires.Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cisregulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the upregulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes

Identification de gènes de dessin de plumage chez les oiseaux

Birds display a spectacular range of plumage pigmentation. The purpose of this thesis was to elucidate genetic mechanisms that contribute to pattern formation on individual feathers and the body. In study I and II, we investigated two barring patterns in chicken. We show that in the Fayoumi breed autosomal barring is associated with a 1Mb un-recombined region on chromosome 11, which contains the MC1R gene. Our functional analysis strongly suggests that autosomal barring is primarily caused by activating MC1R mutations and that other loci contribute to the appearance of the pattern. In study II, we demonstrate that sex-linked barring is created by a combination of cisregulatory and missense mutations in the CDKN2A/ ARF gene. We demonstrate that the upregulation of CDKN2A expression is caused by non-coding mutation(s) and is resulting in a dilute barring pattern. Functional testing revealed that the two missense mutations in ARF hamper its function and restrict the diluting effect of the non-coding mutations. Only the combination of both regulatory and missense mutations generates clear barring pattern as observed e.g. in the Barred Plymouth Rock. In study III and IV, we investigated the genetic mechanisms driving pigment pattern variation in the ruff (Philomachus pugnax). We first identified a 4.5 Mb inversion to be associated with the two male reproductive morphs called satellite and faeder. These morphs differ substantially in behavior, reproductive strategy, body size and plumage appearance between each other as well as from the third, more prevalent morph, the independent. The inversion disrupts the CENPN gene making this genomic re-arrangement homozygous lethal. We identified a large set of variants; among them four missense mutations in MC1R associated with the Satellite allele. In study IV, we explored whether these MC1R mutations are contributing to the light display plumage of the satellite morph. Our data shows that MC1R is up-regulated in all coloured satellite feathers and that this is due to a higher expression of the Independent allele. Evaluation of MC1R signaling in cell culture models subsequently revealed that the mutations alter receptor properties such as cAMP production, sensitization and surface expression but also suggests that transfection assays using mammalian cells might not reveal the complex function MC1R is most likely having in avian melanocytes.Le plumage des oiseaux montre une diversité de coloration spectaculaire. L’objectif de cette thèse est d’élucider les mécanismes génétiques contribuant à cette diversité en étudiant plus particulièrement la formation de dessins sur la plume et sur le corps. Les études I et II sont consacrées à deux dessins de barrure chez le poulet : la barrure autosomale et la barrure liée au sexe. En réalisant un croisement en retour à partir de la race Fayoumi, nous avons pu démontrer que la barrure autosomale est associée à une région du chromosome 11 et porte le gène de pigmentation MC1R. L’analyse fonctionnelle met en évidence qu’une mutation faux-sens, et non une modification de régulation, a le rôle d’une mutation causale. Dans l’étude II, nous démontrons que la barrure liée au sexe est créée par la combinaison de mutations de régulation en cis et de mutations faux-sens dans le gène CDKN2A. L’activation de l’expression de CDKN2A par deux mutations non-codantes détermine un phénotype de barrure très dilué. Des tests fonctionnels in vitro révèlent que les deux mutations faux-sens identifiées dans le même gène restreignent sa fonction et diminuent l’effet de dilution des mutations non-codantes. Seule la combinaison des mutations de régulation et d’une des mutation faux-sens, produit la barrure bien nette observée dans les races de poule actuelles. Les études III et IV précisent les mécanismes contrôlant la variation des dessins de plumage chez les oiseaux sauvages. Le chevalier combattant Philomachus pugnax présente trois formes reproductives chez le mâle, dénommées ‘indépendant’, ‘satellite’ et ‘faeder’. Nous avons d’abord identifié une inversion de 4.5 Mb chez les mâles satellite et faeder, qui diffèrent nettement entre eux, comme de l’indépendant, par leur comportement, leur stratégie reproductive et l’aspect de leur plumage. Un examen plus précis de la structure de l’inversion a montré que les points de cassure interrompaient le gène CENPN ce qui rend l’inversion létale à l’état homozygote. Nous avons identifié un grand nombre de variants contribuant assez probablement au phénotype et avons découvert quatre SNPs dans l’allèle MC1R du mâle satellite. L’étude IV a consisté à analyser la contribution de ces mutations de MC1R au plumage clair du mâle satellite pendant la reproduction. Nos données montrent que MC1R est activé dans les plumes colorées du mâle satellite en raison d’une plus forte expression de l’allèle indépendant. L’étude de la signalisation de MC1R en culture cellulaire a ensuite révélé que les mutations altèrent les propriétés du récepteur, mais suggère aussi que l’évaluation fonctionnelle du MC1R aviaire en cellules mammaliennes ne rend pas complètement compte du rôle complexe que MC1R joue dans les mélanocytes aviaires

Efficient recovery of whole blood RNA - a comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species

Conclusion: By carefully choosing the appropriate RNA extraction method, whole blood can become a valuable source for high-throughput applications like expression arrays or transcriptome sequencing from natural populations. Additionally, candidate genes showing signs of selection could subsequently be genotyped in large population samples using whole blood as a source for RNA without harming individuals from rare or endangered species

The evolution of Sex-linked barring alleles in chickens involves both regulatory and coding changes in CDKN2A

Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the CDKN2A tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The B0 allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the B1 and B2 alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are cis-regulatory mutations causing a higher CDKN2A expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that CDKN2A is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner

Alleles and phenotypes at the <i>Sex-linked barring</i> (<i>CDKN2A</i>) locus in chicken.

<p>(A) Female and male Coucou de Rennes chicken with separately depicted feather illustrating the iconic Sex-linked barring phenotype caused by the <i>B1</i> allele. (B) <i>Sex-linked barring</i> alleles and associated sequence variants. SNP1 and SNP2 are non-coding while SNP3 and SNP4 constitute non-synonymous changes in the region encoding the MDM2 binding domain. (C) Sex-linked dilution phenotype caused by the <i>B2</i> allele. Note how the homozygous male has an almost white appearance whereas the hemizygous female as well as the heterozygous male show a Sex-linked barring pattern. (D) Phenotype with individual feathers from <i>N/N</i>, <i>B2/N</i> and <i>B0/N</i> chicken. Photo credits: (A) Hervé Ronné, Ecomusée du pays de Rennes, (C) Susanne Kerje, (D) Dominic Wright and Doreen Schwochow-Thalmann.</p

Functional characterization of the V9D and R10C substitutions in chicken ARF.

<p>(A) Far-UV circular dichroism (CD) is measured in mDeg and monitors protein secondary structure. Spectra of MDM2_204-298, ARF_1-14^WT, and complexes of ARF_1-14^WT/MDM2_204-298, ARF_1-14^V9D/MDM2_204-298, and ARF_1-14^R10C/MDM2_204-298. MDM2_204-298 was used at 10 μM and all peptides at 64 μM final concentrations. Spectra of all peptides in free form and at 25 μM concentration in complex with MDM2_204-298 are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006665#pgen.1006665.s008" target="_blank">S5 Fig</a>. (B) Isothermal titration calorimetry (ITC) experiments in which the heat (enthalpy) associated with binding is recorded as μcal/sec for each titration point (top panel) and integrated and normalized against the concentrations of ARF peptide and MDM2_204-298 to obtain a binding isotherm (expressed as kcal mol^-1 versus molar ratio; bottom panel). In the experiment, ARF peptides (WT, V9D, and R10C, respectively) were titrated into 100 μM MDM2_204-298 with the peptide concentration increasing by approximately 10 μM in each titration point. Peaks (top) and integrated energies (bottom) corrected for heats of dilution are shown. Raw data are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006665#pgen.1006665.s009" target="_blank">S6 Fig</a>. (C) Assessment of the effect of the two coding mutations on the interaction between ARF and MDM2 based on a luciferase assay. Reduced luciferase activity implies weaker interaction between ARF and MDM2, monitored as a decreased ability of ARF to protect the transcription factor p53 from degradation (Student’s t-test; *<i>P</i><0.05, ***<i>P</i><0.001).</p

Characterization of the expression of MITF, MART1, <i>TYR</i> and <i>CDKN2A</i> in melanocyte progenitors and differentiated pigment cells in feathers from different genotypes at the <i>Sex-linked barring</i> locus.

<p>(A) Anatomy of growing feather, papilla ectoderm (PE), lower bulge (LB), middle bulge (MB), upper bulge (UB), ramogenic zone (RGZ), and barb (BA). (B) Distribution of cells from the melanocyte lineage across different parts of the feather in different genotypes detected by immunohistochemistry (MITF and MART) and <i>in-situ</i> hybridization (<i>TYR</i> and <i>CDKN2A</i>). (C) Average number of MITF+, MART1+, <i>TYR+</i> and <i>CDKN2A+</i> positive cells in the barbs of chickens with different genotypes. Significant differences are indicated by stars (One-way ANOVA, Tukey’s multi-comparison post-hoc test; * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Differential expression of <i>CDKN2A</i> in feathers.

<p>(A) Relative expression of <i>CDKN2A</i> in Sex-linked barred chickens carrying the <i>B0</i> or <i>B2</i> allele and non-barred control feathers. Expression data was normalized using <i>EEF2</i> and <i>UB</i>. (B) Allele-specific expression of <i>CDKN2A</i> in <i>B2/N</i> feathers, skin and liver. Left panel: cDNA data using tissue samples from four <i>B2/N</i> chickens. Right panel: Genomic DNA from the different genotypes was used as control. The relative expression of the two alleles was determined by pyrosequencing. (Student’s t-test; * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001).</p