The Molecular Circuit Regulating Tooth Development in Crocodilians.

Alligators have robust regenerative potential for tooth renewal. In contrast, extant mammals can either renew their teeth once (diphyodont dentition, as found in humans) or not at all (monophyodont dentition, present in mice). Previously, the authors used multiple mitotic labeling to map putative stem cells in alligator dental laminae, which contain quiescent odontogenic progenitors. The authors demonstrated that alligator tooth cycle initiation is related to ß-catenin/Wnt pathway activity in the dental lamina bulge. However, the molecular circuitry underlying the developmental progression of polyphyodont teeth remains elusive. Here, the authors used transcriptomic analyses to examine the additional molecular pathways related to the process of alligator tooth development. The authors collected juvenile alligator dental laminae at different developmental stages and performed RNA-seq. This data shows that Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) pathways are activated at the transition from pre-initiation stage (bud) to initiation stage (cap). Intriguingly, the activation of Wnt ligands, receptors and co-activators accompanies the inactivation of Wnt antagonists. In addition, the authors identified the molecular circuitry at different stages of tooth development. The authors conclude that multiple pathways are associated with specific stages of tooth development in the alligator. This data shows that Wnt pathway activation may play the most important role in the initiation of tooth development. This result may offer insight into ways to modulate the genetic controls involved in mammalian tooth renewal.

Topology of feather melanocyte progenitor niche allows complex pigment patterns to emerge.

Color patterns of bird plumage affect animal behavior and speciation. Diverse patterns are present in different species and within the individual. Here, we study the cellular and molecular basis of feather pigment pattern formation. Melanocyte progenitors are distributed as a horizontal ring in the proximal follicle, sending melanocytes vertically up into the epithelial cylinder, which gradually emerges as feathers grow. Different pigment patterns form by modulating the presence, arrangement, or differentiation of melanocytes. A layer of peripheral pulp further regulates pigmentation via patterned agouti expression. Lifetime feather cyclic regeneration resets pigment patterns for physiological needs. Thus, the evolution of stem cell niche topology allows complex pigment patterning through combinatorial co-option of simple regulatory mechanisms.

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

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.

beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.

We explored the role of beta-catenin in chicken skin morphogenesis. Initially beta-catenin mRNA was expressed at homogeneous levels in the epithelia over a skin appendage tract field which became transformed into a periodic pattern corresponding to individual primordia. The importance of periodic patterning was shown in scaleless mutants, in which beta-catenin was initially expressed normally, but failed to make a punctuated pattern. To test beta-catenin function, a truncated armadillo fragment was expressed in developing chicken skin from the RCAS retrovirus. This produced a variety of phenotypic changes during epithelial appendage morphogenesis. In apteric and scale-producing regions, new feather buds with normal-appearing follicle sheaths, dermal papillae, and barb ridges were induced. In feather tracts, short, wide, and curled feather buds with abnormal morphology and random orientation formed. Epidermal invaginations and placode-like structures formed in the scale epidermis. PCNA staining and the distribution of molecular markers (SHH, NCAM, Tenascin-C) were characteristic of feather buds. These results suggest that the beta-catenin pathway is involved in modulating epithelial morphogenesis and that increased beta-catenin pathway activity can increase the activity of skin appendage phenotypes. Analogies between regulated and deregulated new growths are discussed.

In vivo analysis of cancerous gene expression by RNA-polymerase chain reaction.

An easy and routine procedure to amplify messenger RNA (mRNA) libraries from a few tissue cells can provide molecular gene expression profiles at high resolution. A novel PCR-like method, the RNA-PCR, was developed to generate high quality and quantity mRNAs from as few as 20 cells (2 pg mRNAs). The principle relies upon the cycling steps of promoter-linked double-stranded cDNA synthesis and promoter-driven transcription to amplify mRNAs up to 250-fold/cycle with good representation of high and low copy mRNAs. The amplified mRNA libraries were shown to possess high fidelity, purity, specificity and reproducibility for in vivo analyses of cancerous gene expression in human prostate cancers.

Self-organization of periodic patterns by dissociated feather mesenchymal cells and the regulation of size, number and spacing of primordia.

Periodic patterning is a fundamental organizing process in biology. Using a feather reconstitution assay, we traced back to the initial stage of the patterning process. Cells started from an equivalent state and self-organized into a periodic pattern without previous cues or sequential propagation. When different numbers of dissociated mesenchymal cells were confronted with a piece of same-sized epithelium, the size of feather primordia remained constant, not the number or interbud spacing, suggesting size determination is intrinsic to dissociated cells. Increasing bone morphogenetic protein (BMP) receptor expression in mesenchymal cells decreased the size of primordia while antagonizing the BMP pathway with Noggin increased the size of primordia. A threshold number of mesenchymal cells with a basal level of adhesion molecules such as NCAM were sufficient to trigger the patterning process. The process is best visualized by the progressive restriction of beta-catenin transcripts in the epidermis. Therefore, feather size, number and spacing are modulated through the available morphogen ligands and receptors in the system.

Epidermal dysplasia and abnormal hair follicles in transgenic mice overexpressing homeobox gene MSX-2.

The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.

Wnt-7a in feather morphogenesis: involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated with an in vitro reconstitution model.

How do vertebrate epithelial appendages form from the flat epithelia? Following the formation of feather placodes, the previously radially symmetrical primordia become anterior-posterior (A-P) asymmetrical and develop a proximo-distal (P-D) axis. Analysis of the molecular heterogeneity revealed a surprising parallel of molecular profiles in the A-P feather buds and the ventral-dorsal (V-D) Drosophila appendage imaginal discs. The functional significance was tested with an in vitro feather reconstitution model. Wnt-7a expression initiated all over the feather tract epithelium, intensifying as it became restricted first to the primordia domain, then to an accentuated ring pattern within the primordia border, and finally to the posterior bud. In contrast, sonic hedgehog expression was induced later as a dot within the primordia. RCAS was used to overexpress Wnt-7a in reconstituted feather explants derived from stage 29 dorsal skin to further test its function in feather formation. Control skin formed normal elongated, slender buds with A-P orientation, but Wnt-7a overexpression led to plateau-like skin appendages lacking an A-P axis. Feathers in the Wnt-7a overexpressing skin also had inhibited elongation of the P-D axes. This was not due to a lack of cell proliferation, which actually was increased although randomly distributed. While morphogenesis was perturbed, differentiation proceeded as indicated by the formation of barb ridges. Wnt-7a buds have reduced expression of anterior (Tenascin) bud markers. Middle (Notch-1) and posterior bud markers including Delta-1 and Serrate-1 were diffusely expressed. The results showed that ectopic Wnt-7a expression enhanced properties characteristic of the middle and posterior feather buds and suggest that P-D elongation of vertebrate skin appendages requires balanced interactions between the anterior and posterior buds.

Msx-2 and the regulation of organ size: epidermal thickness and hair length.

During organogenesis, the issue of size regulation is as important as shape and differentiation. We propose that the regulation of the dimensions of the epithelium and its appendages (length, width, thickness) are based on regulation of cell numbers in specific sites, reflecting the input and output of cells in that region. This process is in turn regulated by the flow from the domain of proliferating cells to the domain of postmitotic differentiated cells. When the homeobox gene Msx-2 is over-expressed in transgenic mice under the control of the CMV promoter, the epidermis is thickened with hyperproliferation and hyperkeratosis. Hairs are shorter and the matrix region is shrunken. We suggest that Msx-2 may be one of the regulators involved in the control of organ size, and the above phenotypes are the manifestations of an increased cellular flow from proliferation domain to differentiation domain in the tissue.

Early events in skin appendage formation: induction of epithelial placodes and condensation of dermal mesenchyme.

The formation of skin appendages represents a morphogenetic process through which a homogeneous system is converted into a patterned system. We have pursued molecules involved in the early placode induction and mesenchymal condensation stages of this process. We found that intracellular and extracellular signaling molecules collaborate to position the location of feather primordia and initiate mesenchymal condensations mediated by adhesion molecules. During the inductive stage, cells interact in a fashion best described by a reaction-diffusion mechanism. Thus in early feather morphogenesis, low level adhesion molecules drive cell interactions. The interactions were modulated by extracellular signaling molecules, which eventually increase the level of signaling molecules at sites of feather initiation and subsequently the level of adhesion molecules (Jiang et al, 1999a). These physico-chemical events lead to the formation of dermal condensations and epithelial placodes at sites of feather primordia, thus achieving the earliest and most fundamental events of skin appendage formation: induction.

Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning; however, no molecular candidates have been identified. Here we show that the feather tract is initiated by a continuous stripe of Shh, Fgf-4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, SHH and FGF-4, which induce feather formation. Both FGF-4 and SHH induce local expression of Bmp-4, while BMP-4 suppresses local expression of both. FGF-4 also induces Shh. Based on these findings, we propose a model that involves (1) homogeneously distributed global activators that define the field, (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented.

Lineage and pluripotentiality of epithelial precursor cells in developing chicken skin.

How do epithelial cells in developing skin accommodate the constantly growing embryo? Where do cells in skin appendages come from? Are they derivatives of a single appendage stem cell, or are they polyclonal? Here we analyze these issues in developing chicken skin using a replication-defective virus carrying beta-galactosidase and DiI microinjections. The results demonstrate that in early skin, epithelial cells labelled near the spine show a parallel linear stripe distribution pattern that is perpendicular to the midline of the trunk. This is similar to the human lines of Blaschko, a linear pattern on the skin, which many skin nevoid or acquired disorders follow. In later skin, feather buds form and contain a mixture of labeled and unlabeled cells, attesting to their polyclonal origin. When cells are traced for shorter time intervals, the labeled progeny appear to follow certain rules. The degree of cell dispersion and mixing increases with a longer incubation period between the time of labeling and detection. The spatial maturation sequence of skin appendages is not regulated by the order in which epithelial cells are generated. Epithelial cells at this developmental stage are pluripotent and competent to respond to new signals to assume appropriate fates according to their micro-environment. The results suggest that local interactions act upon the originally linearly deposited pluripotential epithelial cells to form skin appendages.

Isolation and characterization of chicken beta-catenin.

beta-catenin interacts with a number of proteins in different important biological processes, including cell adhesion through cadherins, actin organization through fascin, body axis determination through Wnt signaling, tumor suppression through APC, and transcriptional activation through LEF-1. To examine its function in chicken embryogenesis, we isolated the chicken homolog of beta-catenin from a chicken embryo cDNA library. The sequence is highly conserved at the amino acid level between chicken, mouse (99%), human (99%) and Xenopus (97%). In-situ hybridization and immunostaining showed that in the developing limb, it is specifically expressed in the apical ectodermal ridge, suggesting a role in epithelial-mesenchymal interactions.

Molecular histology in skin appendage morphogenesis.

Classical histological studies have demonstrated the cellular organization of skin appendages and helped us appreciate the intricate structures and function of skin appendages. At this juncture, questions can be directed to determine how these cellular organizations are achieved. How do cells rearrange themselves to form the complex cyto-architecture of skin appendages? What are the molecular bases of the morphogenesis and histogenesis of skin appendages? Recently, many new molecules expressed in a spatial and temporal specific manner during the formation of skin appendages were identified by molecular biological approaches. In this review, novel molecular techniques that are useful in skin appendage research are discussed. The distribution of exemplary molecules from different categories including growth factors, intracellular signaling molecules, homeobox genes, adhesion molecules, and extracellular matrix molecules are summarized in a diagram using feather and hair as models. We hope that these results will serve as the ground work for completing the molecular mapping of skin appendages which will refine and re-define our understanding of the developmental process beyond relying on morphological criteria. We also hope that the listed protocols will help those who are interested in this venture. This new molecular histology of skin appendages is the foundation for forming new hypotheses on how molecules are mechanistically involved in skin appendage development and for designing experiments to test them. This may also lead to the modulation of healing and regeneration processes in future treatment modalities.

FGF induces new feather buds from developing avian skin.

Induction of skin appendages involves a cascade of molecular events. The fibroblast growth factor (FGF) family of peptide growth factors is involved in cell proliferation and morphogenesis. We explored the role of the FGFs during skin appendage induction using developing chicken feather buds as a model. FGF-1, FGF-2, or FGF-4 was added directly to the culture medium or was released from pre-soaked Affigel blue beads. Near the midline, FGFs led to fusion of developing feather buds, representing FGFs' ability to expand feather bud domains in developing skin. In lateral regions of the explant where feather placodes have not formed, FGF treatment produces a zone of condensation and a region with an increased number of feather buds. In ventral epidermis that is normally apteric (without feathers), FGFs can also induce new feather buds. Like normal feather buds, the newly induced buds express Shh. The expression of Grb, Ras, Raf, and Erk, intracellular signaling molecules known to be downstream to tyrosine kinase receptors such as the FGF receptor, was enriched in feather bud domains. Genistein, an inhibitor of tyrosine kinase, suppressed feather bud formation and the effect of FGF. These results indicate that there are varied responses to FGFs depending on epithelial competence. All the phenotypic responses, however, show that FGFs facilitate the formation of skin appendage domains.

Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance.

Early molecular events during the development and regeneration of skin appendages were studied using cultured chicken skin explants with epithelial-mesenchymal recombination. The explant epithelium was separated from the mesenchyme, rotated 90 degrees or 180 degrees, recombined with the mesenchyme, and cultured. After this procedure, existing feather buds disappeared and new buds were regenerated. The location of the new buds is determined by the original dermal condensations, whereas the orientation is dictated by the original epithelium. The temporal expression of key morphogenetic molecules was examined 3, 6, and 20 h after recombination by whole-mount in situ hybridization and immunostaining. The results showed the following. (i) Placode formation and the expression of wingless-int (Wnt) 7a and Msx-1 in the placode epithelium are mesenchyme dependent. (ii) Hox C6 and neural cell adhesion molecule (NCAM) expression in the anterior mesenchyme is placode epithelium dependent. (iii) Bone morphogenetic protein (BMP)-2, BMP-4, and fibroblast growth factor (FGF)-4 expression in the original dermal condensations was unaffected by recombination. (iv) Old dermal condensations can induce new placodes with new Wnt 7a, sonic hedgehog (Shh), and Msx-1 and -2 expression. (v) The new placode epithelium can then induce new Hox C6 and NCAM microgradients in the feather bud mesenchyme. (vi) The order of appearance can be classified into four groups in the following order: BMP-2, BMP-4, and FGF-4 (peptide growth factors); Wnt 7a and Shh (Drosophila segment polarity gene homologs); Msx-1 and Msx-2 (Msx class homeobox genes); and then Hox C6 (Hox class homeobox genes) and NCAM (adhesion molecules). These results suggest an order for the molecular cascade during the inductive phase of skin appendage development.

cDCC (chicken homologue to a gene deleted in colorectal carcinoma) is an epithelial adhesion molecule expressed in the basal cells and involved in epithelial-mesenchymal interaction.

Cloning of human DCC (deleted in colorectal carcinoma, Fearon et al., 1990) showed that it is an immunoglobulin superfamily member homologous to neural cell adhesion molecules (N-CAM). To explore the normal function of this molecule, we have cloned a chicken homologue to DCC (cDCC) and raised an antibody to DCC. cDCC is a protein of 160 kDa with an expression pattern distinct from those of other immunoglobulin family members including N-CAM and Ng-CAM. Transgene expression of cDCC in fibroblasts led to increased cell-cell adhesion. Localization studies in chicken and mouse embryos showed that DCC is expressed in the epithelia of skin, gut, lung, and bladder. In adult, the expression of DCC is limited to the basal layer of stratified epithelium in skin, crypt regions of intestinal villi, and stem cells in mammary duct. Cell aggregation assay using embryonic chicken skin epithelial cells and antibody to DCC showed it is a Ca2+ independent cell adhesion molecule. In epithelial-mesenchymal interactions during feather morphogenesis, antibody to DCC suppressed the formation of dermal condensations and the polarized localization of N-CAM and fibronectin. These results implied that DCC is an epithelial cell adhesion molecule required for mediating critical functions in epithelial-epithelial and epithelial-mesenchymal interactions.

Adhesion molecules in skeletogenesis: II. Neural cell adhesion molecules mediate precartilaginous mesenchymal condensations and enhance chondrogenesis.

Neural cell adhesion molecules (NCAM) was expressed transiently by mesenchymal cells in precartilaginous condensations of the embryonic chicken limb but was lost upon differentiation into cartilage. Consequently, NCAM was present in the periphery of the limb anlagen but was absent in the cartilaginous center of the growing limb. To determine NCAM function in limb bud chondrogenesis we incubated dissociated stage 22/23 distal mesenchymal limb bud cells with Fab' fragments of antibodies to NCAM. Cell aggregation was inhibited by incubating the cells with anti-NCAM Fab'. These results suggest that NCAM may mediate the formation of precartilaginous condensations. This hypothesis was further tested using micromass cultures. NCAM expression in micromass cultures in vitro recapitulated that in vivo. NCAM was enriched in condensations of 2 day cultures, but was diminished and concentrically distributed around cartilage nodules in 4 day cultures. Anti-NCAM Fab' fragments reduced the area occupied by precartilaginous condensations and the degree of chondrogenic differentiation. Control antibody against chicken embryo fibroblasts had no effect. The effect of overexpressing NCAM was analyzed by electroporating expression vectors directing the synthesis of chicken NCAM. Limb bud cells cultured after electroporation with an NCAM expression vector displayed larger cartilage nodules and greater chondrogenic differentiation than cells electroporated with vector alone. The expression of NCAM in electroporated cells also increased. Control experiments using plasmids encoding beta-galactosidase indicated that approximately 10% of the limb bud cells were transfected under these conditions. The results suggest that NCAM is involved in the chondrogenesis pathway by mediating the formation of precartilaginous condensations.

Adhesion molecules and homeoproteins in the phenotypic determination of skin appendages.

We examined the roles of adhesion molecules and homeoproteins in the morphogenesis of skin appendages using feather as a model. The expression pattern of these molecules in different stages of feather development were very dynamic. For example, neural cell adhesion molecules are present first in the dermal condensations, then in distal bud epithelium, then in the dermal papilla, and finally in the marginal and axial plates. Tenascin is present first in the placode, then in the anterior bud epithelium and mesoderm, and then in the dermal papilla. The expression patterns suggest that the adhesion molecules are involved in forming the boundary of cell groups that interact to form skin appendages. Antibody perturbation of embryonic skin-explant cultures showed that liver cell adhesion molecules are involved in establishing the hexagonal pattern, neural cell adhesion molecules are involved in the formation of dermal condensations, tenascin appears to be involved in the growth of feather buds, and integrin is essential for epithelial-mesenchymal interactions. Using antibodies to XlHbox 1 (similar to Hox 3.3 or C6) and Hox 4.2 (or D4), we showed that there is a homeoprotein gradient within the feather buds, and that the expression pattern is position-specific. It is hypothesized that Hox codes, derived from the combined expression pattern of homeoproteins, determine the phenotypes and orientation of skin appendages. Experiments using retinoids in the media or retinoid-soaked beads to create a local retinoid gradient are consistent with this hypothesis. As demonstrated here, feather development provides an excellent opportunity to analyze the molecular cascade of skin-appendage morphogenesis.