Structure, function and formation of the amniote skin pattern.

From feather and hair dotted arrays to pigmented stripes and spots, the spatial distribution of skin appendages and colouration often forms visible ornaments crucial for fitness in the coat of birds and mammals. These geometrical motifs are extremely diverse in nature. Yet, phenotypic surveys evidenced common themes in variation: the orientation, appendage-specificity or pigmentation of a given region may be conserved across groups or species. Here, we review naturalist observations of natural variation in the anatomy and ecological function of the skin pattern in amniotes. We then describe several decades of genetics, mathematical modelling and experimental embryology work aiming at understanding the molecular and morphogenetic mechanisms responsible for pattern formation. We discuss how these studies provided evidence that the morphological trends and differences representative of the phenotypic landscape of skin patterns in wild amniote species is rooted in the mechanisms controlling the production of distinct compartments in the embryonic skin.

How keratin cortex thickness affects iridescent feather colours.

The bright, saturated iridescent colours of feathers are commonly produced by single and multi-layers of nanostructured melanin granules (melanosomes), air and keratin matrices, surrounded by an outer keratin cortex of varying thicknesses. The role of the keratin cortex in colour production remains unclear, despite its potential to act as a thin film or absorbing layer. We use electron microscopy, optical simulations and oxygen plasma-mediated experimental cortex removal to show that differences in keratin cortex thickness play a significant role in producing colours. The results indicate that keratin cortex thickness determines the position of the major reflectance peak (hue) from nanostructured melanosomes of common pheasant (Phasianus colchicus) feathers. Specifically, the common pheasant has appropriate keratin cortex thickness to produce blue and green structural colours. This finding identifies a general principle of structural colour production and sheds light on the processes that shaped the evolution of brilliant iridescent colours in the common pheasant.

Cell shape anisotropy contributes to self-organized feather pattern fidelity in birds.

Developing tissues can self-organize into a variety of patterned structures through the stabilization of stochastic fluctuations in their molecular and cellular properties. While molecular factors and cell dynamics contributing to self-organization have been identified in vivo, events channeling self-organized systems such that they achieve stable pattern outcomes remain unknown. Here, we described natural variation in the fidelity of self-organized arrays formed by feather follicle precursors in bird embryos. By surveying skin cells prior to and during tissue self-organization and performing species-specific ex vivo drug treatments and mechanical stress tests, we demonstrated that pattern fidelity depends on the initial amplitude of cell anisotropy in regions of the developing dermis competent to produce a pattern. Using live imaging, we showed that cell shape anisotropy is associated with a limited increase in cell motility for sharp and precisely located primordia formation, and thus, proper pattern geometry. These results evidence a mechanism through which initial tissue properties ensure stability in self-organization and thus, reproducible pattern production.

A conserved molecular template underlies color pattern diversity in estrildid finches.

The color patterns that adorn animals' coats not only exhibit extensive diversity linked to various ecological functions but also display recurrences in geometry, orientation, or body location. How processes of pattern formation shape such phenotypic trends remains a mystery. Here, we surveyed plumage color patterns in passerine finches displaying extreme apparent variation and identified a conserved set of color domains. We linked these domains to putative embryonic skin regions instructed by early developmental tissues and outlined by the combinatory expression of few genetic markers. We found that this embryonic prepattern is largely conserved in birds displaying drastic color differences in the adult, interspecies variation resulting from the masking or display of each domain depending on their coloration. This work showed that a simple molecular landscape serves as common spatial template to extensive color pattern variation in finches, revealing that early conserved landmarks and molecular pathways are a major cause of phenotypic trends.

Morphogenesis of Iridescent Feathers in Anna's Hummingbird Calypte anna.

Color is a phenotypic trait of utmost importance, particularly in birds, which are known for their diverse color signals and color-producing mechanisms including pigment-based colors, light scattering from nanostructured feather tissues and combinations thereof. Bright iridescent plumage colors of hummingbirds are caused by light scattering by an organized array of flattened, pigment organelles, containing air-filled vesicles, called melanosomes. These hollow platelets are organized in multilayer arrays that contain numerous sharp air/melanin refractive index interfaces, producing brilliant iridescent colors. Despite their ecological significance and potential for inspiration of new optical materials, how platelets form and spatially arrange in nanostructures in growing feathers remains unknown. Here, we tested the hypothesis that melanosome formation and organization occurs mostly through passive self-assembly processes by assembling a developmental time series of growing hummingbird feathers using optical and electron microscopy. We show that hummingbird platelets contain air bubbles or vesicles upon their formation in pigment-producing cells, melanocytes. When melanosomes are transferred to neighboring keratinocytes (the cells shaping barbule structure) they drastically expand in size; and variation in this enlargement appears to be driven by physical constraints caused by the placement of the melanosomes within the barbule plate and their proximity to other melanosomes. As the barbule elongates and narrows, polymerizing feather corneous beta-protein orients melanosomes unilaterally, forcing them into a stacked configuration. These results reveal potentially novel forces driving the self-assembly of the nanostructures producing some of the brightest colors in nature.

Trends and variation in vertebrate patterns as outcomes of self-organization.

In extant vertebrates, natural motifs such as coat markings, spongy bone structures, neuronal arborescence or collective swarms correspond to diverse pattern types, from fractals to periodic stripes or tessellations, and so on. In this subphylum, evolution produced an apparent paradox: a given pattern may vary tremendously in its extent, periodicity or regularity, but follows general geometrical trends and is produced with meticulous precision. In this review, we discuss the role of self-organization, a patterning strategy in which spontaneous order arises through local interactions without gradient formation, in shaping both natural pattern differences and common themes. Mathematical models evidenced a wide high adaptability of self-organizing dynamics, long-advocating for their contribution to natural pattern diversity. Recent empirical and theoretical approaches taking into account network topologies and natural variation also replaced outcomes of self-organization in more constrained biological contexts, shedding light on mechanisms ensuring pattern fidelity.

The embryonic origin of periodic colour patterns / L'origine embryonnaire des modèles de couleurs périodiques.

Because they vary extensively, the periodic colour motifs that adorn the coat of vertebrates historically served to study the formation and evolution of biological patterns. While two major patterning strategies, namely instructional signalling and self-organisation, have been theorised from numerical and empirical work in model organisms, the origin, nature, and mode of action of factors underlying these strategies in vivo remains unclear. To address this question our laboratory designed a method based on opportunistic surveys of natural variation in periodic plumage motifs. We linked common and varying elements of the striped pattern seen in juvenile poultry birds to early embryonic instruction from the somite and late dose-dependent mechanisms occurring during skin development. These results reconciled patterning theories, showing they combine in a two-step process shaping natural variation in a typical periodic pattern.

Parce qu'ils varient considérablement, les motifs de couleur périodiques qui ornent le pelage des vertébrés ont historiquement servi à étudier la formation et l'évolution des modèles biologiques. Si deux grandes stratégies de formation de motifs, à savoir la signalisation pédagogique et l'auto-organisation, ont été théorisées à partir de travaux numériques et empiriques dans des organismes modèles, l'origine, la nature et le mode d'action des facteurs qui sous-tendent ces stratégies in vivo restent flous. Pour répondre à cette question, notre laboratoire a conçu une méthode basée sur des études opportunistes de la variation naturelle des motifs périodiques du plumage. Nous avons établi un lien entre les éléments communs et variables du motif rayé observé chez les jeunes volailles et l'instruction embryonnaire précoce provenant du somite et les mécanismes tardifs dépendant de la dose qui se produisent pendant le développement de la peau. Ces résultats ont permis de réconcilier les théories sur les motifs, en montrant qu'elles se combinent dans un processus en deux étapes façonnant la variation naturelle d'un motif périodique typique.

A "Numerical Evo-Devo" Synthesis for the Identification of Pattern-Forming Factors.

Animals display extensive diversity in motifs adorning their coat, yet these patterns have reproducible orientation and periodicity within species or groups. Morphological variation has been traditionally used to dissect the genetic basis of evolutionary change, while pattern conservation and stability in both mathematical and organismal models has served to identify core developmental events. Two patterning theories, namely instruction and self-organisation, emerged from this work. Combined, they provide an appealing explanation for how natural patterns form and evolve, but in vivo factors underlying these mechanisms remain elusive. By bridging developmental biology and mathematics, novel frameworks recently allowed breakthroughs in our understanding of pattern establishment, unveiling how patterning strategies combine in space and time, or the importance of tissue morphogenesis in generating positional information. Adding results from surveys of natural variation to these empirical-modelling dialogues improves model inference, analysis, and in vivo testing. In this evo-devo-numerical synthesis, mathematical models have to reproduce not only given stable patterns but also the dynamics of their emergence, and the extent of inter-species variation in these dynamics through minimal parameter change. This integrative approach can help in disentangling molecular, cellular and mechanical interaction during pattern establishment.

The embryonic origin of periodic color patterns.

The periodic color motifs such as the spots or stripes that adorn the coat of vertebrates have served as emblematic systems in empirical and theoretical studies of pattern formation, because they vary extensively between taxa but often have conserved orientation and are highly reproducible within species. Two major patterning theories have been proposed, namely instructional signaling, in which positional information is encoded as a program, and self-organization, in which position is spontaneously acquired within the developing tissue. We review here recent empirical evidence that supports both theories in vertebrates: with the advent of new molecular techniques and functional approaches, researchers nowadays take advantage of natural populations of mammals, birds and fish species, closely-related to model organisms and varying in periodic patterns. As a whole, results strongly suggest that instruction and self-organization act in combination in space and time. The orientation and reproducibility of periodic patterns relies on initial foundations provided by early developmental landmarks while their periodicity and natural variation are shaped by late-acting self-organizing processes.

Symmetry breaking in the embryonic skin triggers directional and sequential plumage patterning.

The development of an organism involves the formation of patterns from initially homogeneous surfaces in a reproducible manner. Simulations of various theoretical models recapitulate final states of natural patterns, yet drawing testable hypotheses from those often remains difficult. Consequently, little is known about pattern-forming events. Here, we surveyed plumage patterns and their emergence in Galliformes, ratites, passerines, and penguins, together representing the three major taxa of the avian phylogeny, and built a unified model that not only reproduces final patterns but also intrinsically generates shared and varying directionality, sequence, and duration of patterning. We used in vivo and ex vivo experiments to test its parameter-based predictions. We showed that directional and sequential pattern progression depends on a species-specific prepattern: an initial break in surface symmetry launches a travelling front of sharply defined, oriented domains with self-organising capacity. This front propagates through the timely transfer of increased cell density mediated by cell proliferation, which controls overall patterning duration. These results show that universal mechanisms combining prepatterning and self-organisation govern the timely emergence of the plumage pattern in birds.

The periodic coloration in birds forms through a prepattern of somite origin.

The periodic stripes and spots that often adorn animals' coats have been largely viewed as self-organizing patterns, forming through dynamics such as Turing's reaction-diffusion within the developing skin. Whether preexisting positional information also contributes to the periodicity and orientation of these patterns has, however, remained unclear. We used natural variation in colored stripes of juvenile galliform birds to show that stripes form in a two-step process. Autonomous signaling from the somite sets stripe position by forming a composite prepattern marked by the expression profile of agouti Subsequently, agouti regulates stripe width through dose-dependent control of local pigment production. These results reveal that early developmental landmarks can shape periodic patterns upstream of late local dynamics, and thus constrain their evolution.

Recurrent DCC gene losses during bird evolution.

During development, midline crossing by axons brings into play highly conserved families of receptors and ligands. The interaction between the secreted ligand Netrin-1 and its receptor Deleted in Colorectal Carcinoma (DCC) is thought to control midline attraction of crossing axons. Here, we studied the evolution of this ligand/receptor couple in birds taking advantage of a wealth of newly sequenced genomes. From phylogeny and synteny analyses we can infer that the DCC gene has been conserved in most extant bird species, while two independent events have led to its loss in two avian groups, passeriformes and galliformes. These convergent accidental gene loss events are likely related to chromosome Z rearrangement. We show, using whole-mount immunostaining and 3Disco clearing, that in the nervous system of all birds that have a DCC gene, DCC protein expression pattern is similar to other vertebrates. Surprisingly, we show that the early developmental pattern of commissural tracts is comparable in all birds, whether or not they have a DCC receptor. Interestingly, only 4 of the 5 genes encoding secreted netrins, the DCC ligands in vertebrates, were found in birds, but Netrin-5 was absent. Together, these results support a remarkable plasticity of commissural axon guidance mechanisms in birds.

Developmental mechanisms of stripe patterns in rodents.

Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.

Developmental genetics in emerging rodent models: case studies and perspectives.

For decades, mammalian developmental genetic studies have focused almost entirely on two laboratory models: Mus and Rattus, species that breed readily in the laboratory and for which a wealth of molecular and genetic resources exist. These species alone, however, do not capture the remarkable diversity of morphological, behavioural and physiological traits seen across rodents, a group that represents >40% of all mammal species. Due to new advances in molecular tools and genomic technologies, studying the developmental events underlying natural variation in a wide range of species for a wide range of traits has become increasingly feasible. Here we review several recent studies and discuss how they not only provided technical resources for newly emerging rodent models in developmental genetics but also are instrumental in further encouraging scientists, from a wide range of research fields, to capitalize on the great diversity in development that has evolved among rodents.

The developmental role of Agouti in color pattern evolution.

Animal color patterns can affect fitness in the wild; however, little is known about the mechanisms that control their formation and subsequent evolution. We took advantage of two locally camouflaged populations of Peromyscus mice to show that the negative regulator of adult pigmentation, Agouti, also plays a key developmental role in color pattern evolution. Genetic and functional analyses showed that ventral-specific embryonic expression of Agouti establishes a prepattern by delaying the terminal differentiation of ventral melanocytes. Moreover, a skin-specific increase in both the level and spatial domain of Agouti expression prevents melanocyte maturation in a regionalized manner, resulting in a novel and adaptive color pattern. Thus, natural selection favors late-acting, tissue-specific changes in embryonic Agouti expression to produce large changes in adult color pattern.

Convergence in pigmentation at multiple levels: mutations, genes and function.

Convergence--the independent evolution of the same trait by two or more taxa--has long been of interest to evolutionary biologists, but only recently has the molecular basis of phenotypic convergence been identified. Here, we highlight studies of rapid evolution of cryptic coloration in vertebrates to demonstrate that phenotypic convergence can occur at multiple levels: mutations, genes and gene function. We first show that different genes can be responsible for convergent phenotypes even among closely related populations, for example, in the pale beach mice inhabiting Florida's Gulf and Atlantic coasts. By contrast, the exact same mutation can create similar phenotypes in distantly related species such as mice and mammoths. Next, we show that different mutations in the same gene need not be functionally equivalent to produce similar phenotypes. For example, separate mutations produce divergent protein function but convergent pale coloration in two lizard species. Similarly, mutations that alter the expression of a gene in different ways can, nevertheless, result in similar phenotypes, as demonstrated by sister species of deer mice. Together these studies underscore the importance of identifying not only the genes, but also the precise mutations and their effects on protein function, that contribute to adaptation and highlight how convergence can occur at different genetic levels.

Melanism in peromyscus is caused by independent mutations in agouti.

Identifying the molecular basis of phenotypes that have evolved independently can provide insight into the ways genetic and developmental constraints influence the maintenance of phenotypic diversity. Melanic (darkly pigmented) phenotypes in mammals provide a potent system in which to study the genetic basis of naturally occurring mutant phenotypes because melanism occurs in many mammals, and the mammalian pigmentation pathway is well understood. Spontaneous alleles of a few key pigmentation loci are known to cause melanism in domestic or laboratory populations of mammals, but in natural populations, mutations at one gene, the melanocortin-1 receptor (Mc1r), have been implicated in the vast majority of cases, possibly due to its minimal pleiotropic effects. To investigate whether mutations in this or other genes cause melanism in the wild, we investigated the genetic basis of melanism in the rodent genus Peromyscus, in which melanic mice have been reported in several populations. We focused on two genes known to cause melanism in other taxa, Mc1r and its antagonist, the agouti signaling protein (Agouti). While variation in the Mc1r coding region does not correlate with melanism in any population, in a New Hampshire population, we find that a 125-kb deletion, which includes the upstream regulatory region and exons 1 and 2 of Agouti, results in a loss of Agouti expression and is perfectly associated with melanic color. In a second population from Alaska, we find that a premature stop codon in exon 3 of Agouti is associated with a similar melanic phenotype. These results show that melanism has evolved independently in these populations through mutations in the same gene, and suggest that melanism produced by mutations in genes other than Mc1r may be more common than previously thought.

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program.

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. We now show that Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3' of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation.

Myostatin promotes the terminal differentiation of embryonic muscle progenitors.

Myostatin, a TGF-beta family member, is an important regulator of adult muscle size. While extensively studied in vitro, the mechanisms by which this molecule mediates its effect in vivo are poorly understood. We addressed this question using chick and mouse embryos. We show that while myostatin overexpression in chick leads to an exhaustion of the muscle progenitor population that ultimately results in muscle hypotrophy, myostatin loss of function in chick and mouse provokes an expansion of this population. Our data demonstrate that myostatin acts in vivo to regulate the balance between proliferation and differentiation of embryonic muscle progenitors by promoting their terminal differentiation through the activation of p21 and MyoD. Previous studies have suggested that myostatin imposes quiescence on muscle progenitors. Our data suggest that myostatin's effect on muscle progenitors is more complex than previously realized and is likely to be context-dependent. We propose a novel model for myostatin mode of action in vivo, in which myostatin affects the balance between proliferation and differentiation of embryonic muscle progenitors by enhancing their differentiation.