Cheng-Ming Chuong

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic β-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/β-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought ‘chalone’ inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.

An Integrated Gene Regulatory Network Controls Stem Cell Proliferation in Teeth

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

The morphogenesis of feathers.

Feathers are highly ordered, hierarchical branched structures that confer birds with the ability of flight. Discoveries of fossilized dinosaurs in China bearing ‘feather-like’ structures have prompted interest in the origin and evolution of feathers. However, there is uncertainty about whether the irregularly branched integumentary fibres on dinosaurs such as Sinornithosaurus are truly feathers, and whether an integumentary appendage with a major central shaft and notched edges is a non-avian feather or a proto-feather. Here, we use a developmental approach to analyse molecular mechanisms in feather-branching morphogenesis. We have used the replication-competent avian sarcoma retrovirus to deliver exogenous genes to regenerating flight feather follicles of chickens. We show that the antagonistic balance between noggin and bone morphogenetic protein 4 (BMP4) has a critical role in feather branching, with BMP4 promoting rachis formation and barb fusion, and noggin enhancing rachis and barb branching. Furthermore, we show that sonic hedgehog (Shh) is essential for inducing apoptosis of the marginal plate epithelia, which results in spaces between barbs. Our analyses identify the molecular pathways underlying the topological transformation of feathers from cylindrical epithelia to the hierarchical branched structures, and provide insights on the possible developmental mechanisms in the evolution of feather forms.

Morphoregulation of teeth: modulating the number, size, shape and differentiation by tuning Bmp activity

Summary During development and evolution, the morphology of ectodermal organs can be modulated so that an organism can adapt to different environments. We have proposed that morphoregulation can be achieved by simply tilting the balance of molecular activity. We test the principles by analyzing the effects of partial downregulation of Bmp signaling in oral and dental epithelia of the keratin 14‐Noggin transgenic mouse. We observed a wide spectrum of tooth phenotypes. The dental formula changed from 1.0.0.3/1.0.0.3 to 1.0.0.2(1)/1.0.0.0. All mandibular and M3 maxillary molars were selectively lost because of the developmental block at the early bud stage. First and second maxillary molars were reduced in size, exhibited altered crown patterns, and failed to form multiple roots. In these mice, incisors were not transformed into molars. Histogenesis and differentiation of ameloblasts and odontoblasts in molars and incisors were abnormal. Lack of enamel caused misocclusion of incisors, leading to deformation and enlargement in size. Therefore, subtle differences in the level, distribution, and timing of signaling molecules can have major morphoregulatory consequences. Modulation of Bmp signaling exemplifies morphoregulation hypothesis: simple alteration of key signaling pathways can be used to transform a prototypical conical‐shaped tooth into one with complex morphology. The involvement of related pathways and the implication of morphoregulation in tooth evolution are discussed.

‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously — although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.

Self-Organizing and Stochastic Behaviors During the Regeneration of Hair Stem Cells

Cycling of active and quiescent states of the hair follicle integrates activator and inhibitor signals for patterning. Stem cells cycle through active and quiescent states. Large populations of stem cells in an organ may cycle randomly or in a coordinated manner. Although stem cell cycling within single hair follicles has been studied, less is known about regenerative behavior in a hair follicle population. By combining predictive mathematical modeling with in vivo studies in mice and rabbits, we show that a follicle progresses through cycling stages by continuous integration of inputs from intrinsic follicular and extrinsic environmental signals based on universal patterning principles. Signaling from the WNT/bone morphogenetic protein activator/inhibitor pair is coopted to mediate interactions among follicles in the population. This regenerative strategy is robust and versatile because relative activator/inhibitor strengths can be modulated easily, adapting the organism to different physiological and evolutionary needs.

Sonic hedgehogin feather morphogenesis: Induction of mesenchymal condensation and association with cell death

Sonic hedgehog is involved in vertebrate tissue interactions during development. During early feather development, Sonic hedgehog appears very early in epithelial placodes. During late feather development, Sonic hedgehog expression precedes the development of the marginal plates and is specifically localized in the marginal plate epithelium, which will later undergo cell death. By using retroviral vectors, exogenous Sonic hedgehog overexpression in developing feathers induced enlarged feather buds that have either lost their anterior‐posterior polarity or exhibited reverse orientation. The enlarged dermal condensations may be mediated through broader TGF‐β2 expression and reduced protein kinase C (PKC) expression. Reciprocal mesenchymal interaction is required for the induction and maintenance of Sonic hedgehog in the epithelial placodes. In scaleless mutant, Sonic hedgehog is absent in the apteric region and aberrantly expressed in the mesenchyme of the abnormal feather ridge. These findings suggest that Sonic hedgehog mediates key interactions between the epithelium and mesenchyme during feather morphogenesis.

Evo-Devo of feathers and scales: building complex epithelial appendages

The vertebrate body is covered by either scales, feathers or fur to provide warmth and protection. Comparing and contrasting the formation of these different integument appendages may provide insights into their common embryonic origin as well as evolutionary divergence. The reptile integument is mainly made of scales [1]. In birds, there are two major integument appendages: scales on the foot and feathers on most of the rest of the body [2••]. Scales provide protection and prevent water loss. The major innovation of the avian integument was the evolution of feathers, which provide novel functions such as insulation, display (communication), and flight. Chickens have three major types of scales, which are morphologically similar to reptile scales (Figure 1a,b [1,3]). Reticulate scales are found on the foot pad: they are radially symmetric and express α-keratin only. Scutate scales are large and rectangular and are the major type found on the anterior meta-tarsal shank and dorsal part of the toes. Scutella scales are distributed lateral to the scutate scales and are smaller in size but are also rectangular. Both scutate and scutella scales have anterior–posterior polarity, with an outer surface composed of β-keratin and an inner surface and a hinge region composed of α-keratin. Cell proliferation is distributed diffusely in scales [4•] without a localized growth zone (e.g. hair matrix or feather collar), dermal papillae, or follicular structures. Figure 1 Morphology of scales and feathers: (a) reptile scales; (b) avian foot scales; and (c) avian feathers. Avian reticulate scales are similar in shape to reptile tuberculate scales. Avian scutate and scutella scales are similar in shape to reptile overlapping ... Feathers are arranged in specific tracts over the body which are divided by apteric zones (regions without feathers [2••]). The base of each feather follicle contains protected tissues, permitting the epithelial–mesenchymal interactions (epidermal collar and dermal papillae) that provide a source for continuous feather elongation and molting. Epithelial and dermal sheaths lie along the exterior part of the feather, whereas pulp is found within the epithelial cylinder during development. A typical feather is composed of a rachis (primary shaft), barbs (secondary branches), and barbules (tertiary branches; Figure 1c). The variation in feather size, shape and texture is complex. With regard to size, feathers of the same bird are of different length and diameter, and often distributed in a gradient. For shape, types range from down feathers that are mainly radially symmetric (the rachis is either absent or very short) and contour feathers the symmetry of which is mainly bilateral. Flight feathers are bilaterally asymmetric (Figure 1c). For texture, feathers can either be fluffy or form a firm vane. The barbules can be bilaterally symmetric to each other and therefore fluffy (plumulaceous), or the distal barbule can form a hooklet enabling it to interweave with the proximal barbule of the next barb in a ‘velcro-like’ mechanism (pennaceous). The calamus is the region of a shaft without barbs. A feather can have different ratios of these structures, thus providing an enormous number of permutations of structural and functional variations [2••,5]. The feather is the most complex vertebrate integument appendage ever evolved. How is a flat piece of epidermis transformed into a three level branched structure? Here we present ten complexity levels of integument appendages that correspond to developmental stages of chicken skin and feather precursors recently identified in dinosaur/primitive bird fossils. Cellular and molecular events that convert one complexity level to the next are discussed, including those converting avian foot scales to feathers.

Morpho-Regulation of Ectodermal Organs : Integument Pathology and Phenotypic Variations in K14-Noggin Engineered Mice through Modulation of Bone Morphogenic Protein Pathway

Ectodermal organs are composed of keratinocytes organized in different ways during induction, morphogenesis, differentiation, and regenerative stages. We hypothesize that an imbalance of fundamental signaling pathways should affect multiple ectodermal organs in a spatio-temporal-dependent manner. We produced a K14-Noggin transgenic mouse to modulate bone morphogenic protein (BMP) activity and test the extent of this hypothesis. We observed thickened skin epidermis, increased hair density, altered hair types, faster anagen re-entry, and formation of compound vibrissa follicles. The eyelid opening was smaller and ectopic cilia formed at the expense of Meibomian glands. In the distal limb, there were agenesis and hyperpigmentation of claws, interdigital webbing, reduced footpads, and trans-differentiation of sweat glands into hairs. The size of external genitalia increased in both sexes, but they remained fertile. We conclude that modulation of BMP activity can affect the number of ectodermal organs by acting during induction stages, influence the size and shape by acting during morphogenesis stages, change phenotypes by acting during differentiation stages, and facilitate new growth by acting during regeneration stages. Therefore during organogenesis, BMP antagonists can produce a spectrum of phenotypes in a stage-dependent manner by adjusting the level of BMP activity. The distinction between phenotypic variations and pathological changes is discussed.

Mapping stem cell activities in the feather follicle

It is important to know how different organs ‘manage’ their stem cells. Both hair and feather follicles show robust regenerative powers that episodically renew the epithelial organ. However, the evolution of feathers (from reptiles to birds) and hairs (from reptiles to mammals) are independent events and their follicular structures result from convergent evolution. Because feathers do not have the anatomical equivalent of a hair follicle bulge, we are interested in determining where their stem cells are localized. By applying long-term label retention, transplantation and DiI tracing to map stem cell activities, here we show that feather follicles contain slow-cycling long-term label-retaining cells (LRCs), transient amplifying cells and differentiating keratinocytes. Each population, located in anatomically distinct regions, undergoes dynamic homeostasis during the feather cycle. In the growing follicle, LRCs are enriched in a ‘collar bulge’ niche. In the moulting follicle, LRCs shift to populate a papillar ectoderm niche near the dermal papilla. On transplantation, LRCs show multipotentiality. In a three-dimensional view, LRCs are configured as a ring that is horizontally placed in radially symmetric feathers but tilted in bilaterally symmetric feathers. The changing topology of stem cell activities may contribute to the construction of complex feather forms.