Spots and stripes: Pleomorphic patterning of stem cells via p-ERK-dependent cell chemotaxis shown by feather morphogenesis and mathematical simulation

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction-diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large.     

The formation of the feather pattern in chick skin after a proportion of cells have been killed by X-irradiation

The formation of periodic patterns is of fundamental importance in embryonic development. One of the simplest and most frequently observed patterns is the maintenance of a minimum distance between neighbouring elements, for example between teeth, hair, feathers, digits etc. Theoretical models describing these phenomena have been proposed for feather patterning. However, there has been no detailed quantitative analysis of the relationship between cell population density and feather spacing. To define the relation between these quantities and specifically to test the prediction of a mathematical model, we have examined the formation of the feather pattern after varying proportions of the dermal cells have been killed by X-irradiation. It is known that the development of a feather primordium is normally associated with an increase in cell population density in the dermis. Using X-ray irradiation of the skin in vivo and in vitro, we show that the relation between cell population density and spacing of feather primordia indicates the importance of a threshold number of cells for feather patterning. Moreover, there is a prima facie case for supposing that X-rays act on feather spacing system, reducing the ability of dermal cells to prevent spreading of the pattern. Thus, X-irradiation may have a secondary effect on the spacing of primordia rather than, or as well as, affecting the mechanisms that determine their primary positions.     

BMP2 and BMP7 play antagonistic roles in feather induction

Feathers, like hairs, first appear as primordia consisting of an epidermal placode associated with a dermal condensation that is necessary for the continuation of their differentiation. Previously, the BMPs have been proposed to inhibit skin appendage formation. We show that the function of specific BMPs during feather development is more complex. BMP2 and BMP7, which are expressed in both the epidermis and the dermis, are involved in an antagonistic fashion in regulating the formation of dermal condensations, and thus are both necessary for subsequent feather morphogenesis. BMP7 is expressed earlier and functions as a chemoattractant that recruits cells into the condensation, whereas BMP2 is expressed later, and leads to an arrest of cell migration, likely via its modulation of the EIIIA fibronectin domain and alpha4 integrin expression. Based on the observed cell proliferation, chemotaxis and the timing of BMP2 and BMP7 expression, we propose a mathematical model, a reaction-diffusion system, which not only simulates feather patterning, but which also can account for the negative effects of excess BMP2 or BMP7 on feather formation.     

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.     

Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models

Pattern formation is a fundamental morphogenetic process. Models based on genetic and epigenetic control have been proposed but remain controversial. Here we use feather morphogenesis for further evaluation. Adhesion molecules and/or signaling molecules were first expressed homogenously in feather tracts (restrictive mode, appear earlier) or directly in bud or inter-bud regions ( de novo mode, appear later). They either activate or inhibit bud formation, but paradoxically colocalize in the bud. Using feather bud reconstitution, we showed that completely dissociated cells can reform periodic patterns without reference to previous positional codes. The patterning process has the characteristics of being self-organizing, dynamic and plastic. The final pattern is an equilibrium state reached by competition, and the number and size of buds can be altered based on cell number and activator/inhibitor ratio, respectively. We developed a Digital Hormone Model which consists of (1) competent cells without identity that move randomly in a space, (2) extracellular signaling hormones which diffuse by a reaction-diffusion mechanism and activate or inhibit cell adhesion, and (3) cells which respond with topological stochastic actions manifested as changes in cell adhesion. Based on probability, the results are cell clusters arranged in dots or stripes. Thus genetic control provides combinational molecular information which defines the properties of the cells but not the final pattern. Epigenetic control governs interactions among cells and their environment based on physical-chemical rules (such as those described in the Digital Hormone Model). Complex integument patterning is the sum of these two components of control and that is why integument patterns are usually similar but non-identical. These principles may be shared by other pattern formation processes such as barb ridge formation, fingerprints, pigmentation patterning, etc. The Digital Hormone Model can also be applied to swarming robot navigation, reaching intelligent automata and representing a self-re-configurable type of control rather than a follow-the-instruction type of control.     

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

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.     

Formation of spacing pattern and morphogenesis of chick feather buds is regulated by cytoskeletal structures

Chick feather buds develop sequentially in a hexagonal array. Each feather bud develops with anterior posterior polarity, which is thought to develop in response to signals derived from specialized regions of mesenchymal condensation and epithelial thickening. These developmental processes are performed by cellular mechanisms, such as cell proliferation and migration, which occur during chick feather bud development. In order to understand the mechanisms regulating the formation of mesenchymal condensation and their role in feather bud development, we explanted chick dorsal skin at stage HH29+ with cytochalasin D, which inhibits cytoskeletal formation. We show that the aggregation of mesenchymal cells can be prevented by cytochalasin D treatment in a concentration-dependent manner. Subsequently, cytochalasin D disrupts the spacing pattern and inhibits feather bud axis formation as well. In addition, expression patterns of Bmp-4 and Msx-2, key molecules for early feather bud development, were disturbed by cytochalasin D treatment. Our results fully indicate that both the cytoskeletal structure and cell activity via gene regulation are of fundamental importance in mesenchymal condensation leading to proper morphogenesis of feather bud and spacing pattern formation.     

BMPs induce dermal markers and ectopic feather tracts

Bone morphogenetic protein (BMP) signaling is known to be involved in multiple inductive events during embryogenesis including the development of amniote skin. Here, we demonstrate that early application of BMP-2 to the lateral trunk of chick embryos induces the formation of dense dermis, which is competent to participate in feather development. We show that BMPs induce the dermis markers Msx-1 and cDermo-1 and lead to dermal proliferation, to expression of β-catenin, and eventually to the formation of ectopic feather tracts in originally featherless regions of chick skin. Moreover, we present a detailed analysis of cDermo-1 expression during early feather development. The data implicate that cDermo-1 is located downstream of BMP in a signaling pathway that leads to condensation of dermal cells. The roles of BMP and cDermo-1 during development of dermis and feather primordia are discussed.     

FGF signaling is required for initiation of feather placode development

Morphogenesis of hairs and feathers is initiated by an as yet unknown dermal signal that induces placode formation in the overlying ectoderm. To determine whether FGF signals are required for this process we over-expressed soluble versions of FGFR1 or FGFR2 in the skin of chicken embryos. This produced a complete failure of feather formation prior to any morphological or molecular signs of placode development. We further show that Fgf10 is expressed in the dermis of nascent feather primordia, and that anti-FGF10 antibodies block feather placode development in skin explants. In addition we show that FGF10 can induce expression of positive and negative regulators of feather development and can induce its own expression under conditions of low BMP signaling. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and implicate FGF10 as an early dermal signal involved in this process.     

Generation of bioengineered feather buds on a reconstructed chick skin from dissociated epithelial and mesenchymal cells

Various kinds of in vitro culture systems of tissues and organs have been developed, and applied to understand multicellular systems during embryonic organogenesis. In the research field of feather bud development, tissue recombination assays using an intact epithelial tissue and mesenchymal tissue/cells have contributed to our understanding the mechanisms of feather bud formation and development. However, there are few methods to generate a skin and its appendages from single cells of both epithelium and mesenchyme. In this study, we have developed a bioengineering method to reconstruct an embryonic dorsal skin after completely dissociating single epithelial and mesenchymal cells from chick skin. Multiple feather buds can form on the reconstructed skin in a single row in vitro. The bioengineered feather buds develop into long feather buds by transplantation onto a chorioallantoic membrane. The bioengineered bud sizes were similar to those of native embryo. The number of bioengineered buds was increased linearly with the initial contact length of epithelial and mesenchymal cell layers where the epithelial‐mesenchymal interactions occur. In addition, the bioengineered bud formation was also disturbed by the inhibition of major signaling pathways including FGF (fibroblast growth factor), Wnt/β‐catenin, Notch and BMP (bone morphogenetic protein). We expect that our bioengineering technique will motivate further extensive research on multicellular developmental systems, such as the formation and sizing of cutaneous appendages, and their regulatory mechanisms.     

Drm/Gremlin, a BMP antagonist, defines the interbud region during feather development

The pattern of feather buds in a tract is thought to result from the relative ratios between activator and inhibitor signals through a lateral inhibition process. We analyse the role of Drm/Gremlin, a BMPs antagonist expressed during feather pattern formation, in the dermal precursor, the dense dermis, the interbud dermis and in the posterior dermal condensation. We have altered the activity of Drm in embryonic chick skin using retroviral vectors expressing drm/ gremlin and bmps. We show that expression of endogenous drm is under the control of a feedback loop induced by the BMP pathway, and that overexpression of drm results in fusion between adjacent feather buds. We propose that endogenous BMP proteins induce drm expression in the interbud dermis. In turn, the Drm/Gremlin protein limits the inhibitory effect of BMPs, allowing the adjacent row of feathers to form. Thus, the balance between BMPs and its antagonist Drm would regulate the size and spacing of the buds.     

EGF signaling patterns the feather array by promoting the interbud fate

Feather buds form sequentially in a hexagonal array. Bone morphogenetic protein (BMP) signaling from the feather bud inhibits bud formation in the adjacent interbud tissue, but whether interbud fate and patterning is actively promoted by BMP or other factors is unclear. We show that epidermal growth factor (EGF) signaling acts positively to establish interbud identity. EGF and the active EGF receptor (EGFR) are expressed in the interbud regions. Exogenous EGF stimulates epidermal proliferation and expands interbud gene expression, with a concurrent loss of feather bud gene expression and morphology. Conversely, EGFR inhibitors result in the loss of interbud fate and increased acquisition of feather bud fate. EGF signaling acts directly on the epidermis and is independent of BMP signaling. The timing of competence to interpret interbud-promoting signals occurs at an earlier developmental stage than previously anticipated. These data demonstrate that EGFR signaling actively promotes interbud identity.     

Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis

Bone morphogenetic protein 4 (BMP4) and growth/differentiation factor 5 (GDF5) are closely related protein family members and regulate early cartilage patterning and differentiation. In this study, we compared the functional outcome of their actions systematically at various stages of chondrogenesis in mouse embryonic limb bud mesenchyme grown in micromass cultures. Overall, both growth factors enhanced cartilage growth and differentiation in these cultures. Uniquely, BMP4 not only accelerated the formation and maturation of cartilaginous nodules, but also induced internodular mesenchymal cells to express cartilage differentiation markers. On the other hand, GDF5 increased the number of prechondrogenic mesenchymal cell condensation and cartilaginous nodules, without altering the overall pattern of differentiation. In addition, GDF5 caused a more sustained elevated expression level of Sox9 relative to that associated with BMP4. BMP4 accelerated chondrocyte maturation throughout the cultures and sustained an elevated level of Col10 expression, whereas GDF5 caused a transient increase in Col10 expression. Taken together, we conclude that BMP4 is instructive to chondrogenesis and induces mesenchymal cells toward the chondrogenic lineage. Furthermore, BMP4 accelerates the progression of cartilage differentiation to maturation. GDF5 enhances cartilage formation by promoting chondroprogenitor cell aggregation, and amplifying the responses of cartilage differentiation markers. These differences may serve to fine-tune the normal cartilage differentiation program, and can be exploited for the molecular manipulation in biomimetics.     

Morpho-regulation in diverse chicken feather formation: Integrating branching modules and sex hormone-dependent morpho-regulatory modules

Many animals can change the size, shape, texture and color of their regenerated coats in response to different ages, sexes, or seasonal environmental changes. Here, we propose that the feather core branching morphogenesis module can be regulated by sex hormones or other environmental factors to change feather forms, textures or colors, thus generating a large spectrum of complexity for adaptation. We use sexual dimorphisms of the chicken to explore the role of hormones. A long-standing question is whether the sex-dependent feather morphologies are autonomously controlled by the male or female cell types, or extrinsically controlled and reversible. We have recently identified core feather branching molecular modules which control the anterior-posterior (bone morphogenetic orotein [BMP], Wnt gradient), medio-lateral (Retinoic signaling, Gremlin), and proximo-distal (Sprouty, BMP) patterning of feathers. We hypothesize that morpho-regulation, through quantitative modulation of existing parameters, can act on core branching modules to topologically tune the dimension of each parameter during morphogenesis and regeneration. Here, we explore the involvement of hormones in generating sexual dimorphisms using exogenously delivered hormones. Our strategy is to mimic male androgen levels by applying exogenous dihydrotestosterone and aromatase inhibitors to adult females and to mimic female estradiol levels by injecting exogenous estradiol to adult males. We also examine differentially expressed genes in the feathers of wildtype male and female chickens to identify potential downstream modifiers of feather morphogenesis. The data show male and female feather morphology and their color patterns can be modified extrinsically through molting and resetting the stem cell niche during regeneration.     

FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis

Fibroblast growth factors (FGFs) are signals from the apical ectodermal ridge (AER) that are essential for limb pattern formation along the proximodistal (PD) axis. However, how patterning along the PD axis is regulated by AER-FGF signals remains controversial. To further explore the molecular mechanism of FGF functions during limb development, we conditionally inactivated fgf receptor 2 (Fgfr2) in the mouse AER to terminate all AER functions; for comparison, we inactivated both Fgfr1 and Fgfr2 in limb mesenchyme to block mesenchymal AER-FGF signaling. We also re-examined published data in which Fgf4 and Fgf8 were inactivated in the AER. We conclude that limb skeletal phenotypes resulting from loss of AER-FGF signals cannot simply be a consequence of excessive mesenchymal cell death, as suggested by previous studies, but also must be a consequence of reduced mesenchymal proliferation and a failure of mesenchymal differentiation, which occur following loss of both Fgf4 and Fgf8. We further conclude that chondrogenic primordia formation, marked by initial Sox9 expression in limb mesenchyme, is an essential component of the PD patterning process and that a key role for AER-FGF signaling is to facilitate SOX9 function and to ensure progressive establishment of chondrogenic primordia along the PD axis.     

Molecular biology of feather morphogenesis: a testable model for evo-devo research

Darwin's theory describes the principles that are responsible for evolutionary change of organisms and their attributes. The actual mechanisms, however, need to be studied for each species and each organ separately. Here we have investigated the mechanisms underlying these principles in the avian feather. Feathers comprise one of the most complex and diverse epidermal organs as demonstrated by their shape, size, patterned arrangement and pigmentation. Variations can occur at several steps along each level of organization, leading to highly diverse forms and functions. Feathers develop gradually during ontogeny through a series of steps that may correspond to the evolutionary steps that were taken during the phylogeny from a reptilian ancestor to birds. These developmental steps include 1) the formation of feather tract fields on the skin surfaces; 2) periodic patterning of the individual feather primordia within the feather tract fields; 3) feather bud morphogenesis establishing anterio-posterior (along the cranio-caudal axis) and proximo-distal axes; 4) branching morphogenesis to create the rachis, barbs and barbules within a feather bud; and 5) gradual modulations of these basic morphological parameters within a single feather or across a feather tract. Thus, possibilities for variation in form and function of feathers occur at every developmental step. In this paper, principles guiding feather tract formation, distributions of individual feathers within the tracts and variations in feather forms are discussed at a cellular and molecular level.     

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.     

Differentiation of chondrocytes and scleroblasts during dorsal fin skeletogenesis in flounder larvae

In teleosts, the embryonic fin fold consists of a peridermis, an underlying epidermis and a small number of mesenchymal cells. Beginning from such a simple structure, the fin skeletons, including the proximal and distal radials and lepidotrichia (finrays), develop in the dorsal fin fold at the larval stage. Their process of skeletogenesis and embryonic origin are unclear. Using flounder larvae, we report the differentiation process for chondrocytes and scleroblasts prior to fin skeletogenesis and the effects of retinoic acid (RA) on it. In early larvae, the mesenchymal cells grow between the epidermis and spinal cord to form a line of periodical condensations, which are proximal radial primordia, to produce chondrocytes. The prescleroblasts, which ossify the proximal radial cartilages, differentiate in the mesenchymal cells remaining between the cartilages. Then, mesenchymal condensations occur between the distal ends of the proximal radials, forming distal radial primordia, to produce chondrocytes. Simultaneously, condensations occur between the distal radial primordia and peridermis, which are lepidotrichia primordia, to produce prescleroblasts. Exogenous RA specifically inhibits the mesenchymal condensation prior to the proximal radial formation together with the down-regulation of sonic hedgehog (shh) and patched (pta) expression, resulting in the loss of proximal radials. Thus, it was indicated that differentiation of the precursor cells of radials and lepidotrichia begins in the proximal part of the fin fold and that the initial mesenchymal condensation prior to the proximal radial formation is highly susceptible to the effects of RA. Lepidotrichia formation does not occur where proximal radials are absent, indicating that lepidotrichia differentiation requires interaction with the radial cartilages. To examine the suggestion that neural crest cells contribute to the medial fin skeletons, we localized the HNK-1 positive cells in flounder embryos and slug and msxb-positive cells in pufferfish, Fugu rubripes, embryos. That the positive cells commonly arrive at the proximal part of the fin fold does not contradict the suggestion, but their final destiny as radial chondrocytes or lepidotrichia scleroblasts, should be further investigated.