Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering

Abstract

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body.

Figure 1. The Naked neck phenotype is caused by a cis-regulatory mutation that results in elevated BMP12 expression.

(A) Adult Na/Na. Feathers are absent on the neck and head, excepting the crown. (B) E8.5 embryos hybridized with a β-catenin probe to mark the patterning field and feather primordia. Punctate expression of β-catenin in feather placodes is seen on the body but not the neck of the mutant. WT, wild type; Na/Na, Naked neck. (C) E12.5 embryos showing limited lateral tract expansion (arrows) in Na/Na, reducing body feather coverage. (D) Quantitative RT-PCR determination of BMP12 expression in body and neck skin of E7.5 and E8.5 wild type and Na/Na embryos. (E,F) In situ hybridization detecting BMP12 in wild type and Na/Na embryos at (E) E7.5 and (F) E8.5. Wild type and mutant embryos were hybridized and photographed together. Na/Na embryos have elevated and diffuse expression of BMP12 in the skin. (G) Sequence traces of PCR products from E8.5 Na/+. Genomic DNA PCR products display double peaks following a TA indel polymorphism in the BMP12 3′UTR. RT-PCR products from neck and body skin show a single trace throughout, indicating predominant expression of the Naked neck BMP12 allele, while both alleles are detected in RT-PCR products from other tissues. (H) Schematic showing insertion of chromosome 1 sequences into chromosome 3 at the Naked neck locus. Chromosome coordinates, the Naked neck identical by descent segment, gene names, exons, untranslated regions, and non-coding elements conserved between chicken and human genomes, based on the ENSEMBL genome viewer, are indicated.

Figure 2. Naked neck skin displays elevated BMP signaling.

(A) Application of recombinant BMP12 to cultured skin for 15 h leads to elevation of SOSTDC1 expression, determined by quantitative RT-PCR. (B–E) Detection of SOSTDC1 expression by in situ hybridization. (B) At E7.5 wild type embryos have two rows of feather placodes running up the neck. SOSTDC1 is expressed at the periphery of the placodes and is not detected in the medial region between the lateral rows of placodes. (C) By E8.5 the medial region of the neck is populated by feather placodes. (D) E7.5 Na/Na embryos have placodes on the dorsum, but widespread SOSTDC1 expression on the neck, including the medial region. (E) At E8.5 the Naked neck skin maintains a high level of widespread SOSTDC1 expression, with peri-placode expression visible on the body. (F) Ex vivo rescue of the Naked neck phenotype by suppression of BMP signaling. E7.0 Na/Na skin was cultured in the presence of dorsomorphin (DM, used at 8 µM) and SB203580 (SB, used at 5 µM), pharmacological inhibitors of BMP signal transduction, for 48 h. This permitted feather development across most of the mutant neck skin.

Figure 3. Differential sensitivity to BMP signals alters neck patterning while maintaining body feather placode periodicity and size.

(A,B) β-catenin in situ hybridization revealing the effects of recombinant BMP12 application on feather periodicity and regional distribution in wild type skin after 48 h. (C,D) Dose effects of BMP12 on the number of feather placode rows on the spinal tract of the body. Feather primordia are visualized by β-catenin in situ hybridization. (E) SOSTDC1 expression on control and 80 ng/ml BMP12 treated skin explants. Feather placodes express SOSTDC1 at their periphery on both body and neck. Upon application of BMP12, the non-placode skin of the neck expresses a higher level of SOSTDC1 than does the body (compare signal intensity in the red boxed area to that of the blue boxed area). (F) Schematic of reaction-diffusion regulatory interactions. Adjacent numbering refers to mathematical terms in the supporting methods. C_I represents the constitutive, ubiquitous production of the Inhibitor. (G) Quantification of periodicity of Activator foci in simulated neck and body with differential sensitivities to Inhibitor. C_I increases along the x-axis. (H) Pattern outcomes from reaction-diffusion dynamics in a field with graded sensitivity to the Inhibitor. Abolition of Activator foci in the more sensitive part of the field is achieved with little effect on periodic spacing in the remainder of the field, producing a macropatttern that matches the effects of BMP12 treatment on cultured skin. Colors denote local Activator concentrations, with black representing the highest and white the lowest Activator levels. Areas with high Activator concentration represent placodes.

Figure 4. Regional disparity in pattern behavior upon suppression of BMP signal transduction.

(A) Simulated pattern outcomes upon reduction of Inhibitor potency. Transition from production of circular foci to a striped pattern occurs, first on the less sensitive (simulated body) region, followed by stripe formation on the more sensitive domain (simulated neck) at higher levels of signal suppression. (B) Quantification of pattern characteristics from simulation of diminished Inhibitor potency. The proportion of total Activator positive area that is represented by circular foci is plotted. (C) β-catenin in situ hybridization detecting placode pattern upon suppression of BMP signal transduction in cultured E7.0 chicken skin. SB, p38 MAPK inhibitor SB203580; DM, Smad1/5/8 inhibitor dorsomorphin. Inhibition of p38 MAPK has little effect on the placode pattern, but yielded a robust effect in concert with suppression of Smad function. At low doses of DM stripes begin to form first on the body, then at higher doses on the neck. High doses cause β-catenin expression throughout the skin. (D) Quantification of the proportion of total β-catenin positive area that is represented by circular placodes in cultured skin treated with BMP inhibitors. Statistically significant p values are indicated above data points.

Figure 5. Retinoic acid production and signaling in neck skin distinguishes this region from the body.

(A,B) Detection of RALDH2 expression in E7.0 and E8.0 embryos by whole mount in situ hybridization. RALDH2 is expressed more strongly in neck skin than in body skin and is also detected in the neural tube (midline). (C,D) RALDH3 is expressed broadly in neck skin at E7.0 and moves laterally by E8.0. (E) Expression of the RA target gene DHRS3 in skin cultured from E7.0 for 2 d in the presence or absence of 5 µM RA. Both neck and body skin respond to RA. (F,G) In vivo DHRS3 is expressed on the neck, but not the feather tract of the body. (H) Quantitative RT-PCR detecting RALDH2, RALDH3, and DHRS3 expression in neck and body skin from E6 to E10. The disparity between neck and body skin is greatest at E7 and E8, when feather patterning is taking place. DHRS3 levels track RALDH2 expression dynamics more closely than those of RALDH3. (I) Quantitative RT-PCR detection of RALDH2, RALDH3, and DHRS3 expression in separated epidermis (Epi) and dermis (Derm) at E7.0. The RA producing enzymes are expressed in the dermis, while RA target gene expression is activated in the epidermis.

Figure 6. Retinoic acid potentiates BMP inhibition of feather patterning.

(A) RA administration reduces the density of placodes, which are detected by β-catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM SB203580 rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.

Figure 7. Schematic of periodic pattern formation neck and body skin.

A single core periodic patterning system based on a reaction-diffusion mechanism operates across the body and neck. Such a system operating in isolation has a single characteristic wavelength, thus producing placodes at a single density (right). Sensitization of neck skin to BMP signals as a result of RA production in this region alters the output of the patterning mechanism, allowing a reduction in feather density or the abolition of neck feathering, depending on the global level of BMP at the onset of patterning.