Randall B. Widelitz

Wnt signaling through canonical and non-canonical pathways: Recent progress

The Wnt–beta-catenin pathway regulates cell adhesion, morphology, proliferation, migration and structural remodeling. The aspects of the canonical and non-canonical pathways are reviewed here. The major components of this network are the Wnt ligands which bind to frizzled receptors at the cell surface. Activation of Wnt signaling down regulates the intracellular beta-catenin degradation component, allowing beta-catenin levels to accumulate within the cell. At normal levels, beta-catenin associates at the intracellular side of the membrane with cadherins to promote cell adhesion and with the actin microfilament cytoskeletal network to control cell shape. If beta-catenin levels become elevated, it can begin to accumulate within the cell nucleus and activate transcription in conjunction with co-transcription factors Lefs/Tcfs. Cell populations can regulate neighboring populations via the paracrine action of growth factors and through the action of cell adhesion molecules. Many examples of these interactions exist. The major players in Wnt signaling and its downstream canonical and non-canonical partners are reviewed here. For more details visit the World Wide Web Wnt Homepage (http://www.stanford.edu/∼ rnusse/wntwindow.html).

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.

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.

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.

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

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.

Morphoregulation of avian beaks: Comparative mapping of growth zone activities and morphological evolution

Avian beak diversity is a classic example of morphological evolution. Recently, we showed that localized cell proliferation mediated by bone morphogenetic protein 4 (BMP4) can explain the different shapes of chicken and duck beaks (Wu et al. [2004] Science 305:1465). Here, we compare further growth activities among chicken (conical and slightly curved), duck (straight and long), and cockatiel (highly curved) developing beak primordia. We found differential growth activities among different facial prominences and within one prominence. The duck has a wider frontal nasal mass (FNM), and more sustained fibroblast growth factor 8 activity. The cockatiel has a thicker FNM that grows more vertically and a relatively reduced mandibular prominence. In each prominence the number, size, and position of localized growth zones can vary: it is positioned more rostrally in the duck and more posteriorly in the cockatiel FNM, correlating with beak curvature. BMP4 is enriched in these localized growth zones. When BMP activity is experimentally altered in all prominences, beak size was enlarged or reduced proportionally. When only specific prominences were altered, the prototypic conical shaped chicken beaks were converted into an array of beak shapes mimicking those in nature. These results suggest that the size of beaks can be modulated by the overall activity of the BMP pathway, which mediates the growth. The shape of the beaks can be fine‐tuned by localized BMP activity, which mediates the range, level, and duration of locally enhanced growth. Implications of topobiology vs. molecular blueprint concepts in the Evo–Devo of avian beak forms are discussed. Developmental Dynamics 235:1400–1412, 2006.

Sonic hedgehog signaling pathway in vertebrate epithelial appendage morphogenesis: perspectives in development and evolution.

Abstract. Vertebrate epithelial appendages are elaborate topological transformations of flat epithelia into complex organs that either protrude out of external (integument) and internal (oral cavity, gut) epithelia, or invaginate into the surrounding mesenchyme. Although they have specific structures and diverse functions, most epithelial appendages share similar developmental stages, including induction, morphogenesis, differentiation and cycling. The roles of the SHH pathway are analyzed in exemplary organs including feather, hair, tooth, tongue papilla, lung and foregut. SHH is not essential for induction and differentiation, but is involved heavily in morphogenetic processes including cell proliferation (size regulation), branching morphogenesis, mesenchymal condensation, fate determination (segmentation), polarizing activities and so on. Through differential activation of these processes by SHH in a spatiotemporal-specific fashion, organs of different shape and size are laid down. During evolution, new links of developmental pathways may occur and novel forms of epithelial appendages may emerge,upon which evolutionary selections can act. Sites of major variations have progressed from the body plan to the limb plan to the epithelial appendage plan. With its powerful morphogenetic activities, the SHH pathway would likely continue to play a major role in the evolution of novel epithelial appendages.

Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation

Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell–dermal papilla-based epithelial–mesenchymal interaction layer and the hair follicle–intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation.

Organ-Level Quorum Sensing Directs Regeneration in Hair Stem Cell Populations

Coordinated organ behavior is crucial for an effective response to environmental stimuli. By studying regeneration of hair follicles in response to patterned hair plucking, we demonstrate that organ-level quorum sensing allows coordinated responses to skin injury. Plucking hair at different densities leads to a regeneration of up to five times more neighboring, unplucked resting hairs, indicating activation of a collective decision-making process. Through data modeling, the range of the quorum signal was estimated to be on the order of 1 mm, greater than expected for a diffusible molecular cue. Molecular and genetic analysis uncovered a two-step mechanism, where release of CCL2 from injured hairs leads to recruitment of TNF-α-secreting macrophages, which accumulate and signal to both plucked and unplucked follicles. By coupling immune response with regeneration, this mechanism allows skin to respond predictively to distress, disregarding mild injury, while meeting stronger injury with full-scale cooperative activation of stem cells.