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.

Evolution and functional significance of derived sternal ossification patterns in ornithothoracine birds.

The midline pattern of sternal ossification characteristic of the Cretaceous enantiornithine birds is unique among the Ornithodira, the group containing birds, nonavian dinosaurs and pterosaurs. This has been suggested to indicate that Enantiornithes is not the sister group of Ornithuromorpha, the clade that includes living birds and their close relatives, which would imply rampant convergence in many nonsternal features between enantiornithines and ornithuromorphs. However, detailed comparisons reveal greater similarity between neornithine (i.e. crown group bird) and enantiornithine modes of sternal ossification than previously recognized. Furthermore, a new subadult enantiornithine specimen demonstrates that sternal ossification followed a more typically ornithodiran pattern in basal members of the clade. This new specimen, referable to the Pengornithidae, indicates that the unique ossification pattern observed in other juvenile enantiornithines is derived within Enantiornithes. A similar but clearly distinct pattern appears to have evolved in parallel in the ornithuromorph lineage. The atypical mode of sternal ossification in some derived enantiornithines should be regarded as an autapomorphic condition rather than an indication that enantiornithines are not close relatives of ornithuromorphs. Based on what is known about molecular mechanisms for morphogenesis and the possible selective advantages, the parallel shifts to midline ossification that took place in derived enantiornithines and living neognathous birds appear to have been related to the development of a large ventral keel, which is only present in ornithuromorphs and enantiornithines. Midline ossification can serve to medially reinforce the sternum at a relatively early ontogenetic stage, which would have been especially beneficial during the protracted development of the superprecocial Cretaceous enantiornithines.

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.

What is the biological basis of pattern formation of skin lesions?

Pattern recognition is at the heart of clinical dermatology and dermatopathology. Yet, while every practitioner of the art of dermatological diagnosis recognizes the supreme value of diagnostic cues provided by defined patterns of 'efflorescences', few contemplate on the biological basis of pattern formation in and of skin lesions. Vice versa, developmental and theoretical biologists, who would be best prepared to study skin lesion patterns, are lamentably slow to discover this field as a uniquely instructive testing ground for probing theoretical concepts on pattern generation in the human system. As a result, we have at best scraped the surface of understanding the biological basis of pattern formation of skin lesions, and widely open questions dominate over definitive answer. As a symmetry-breaking force, pattern formation represents one of the most fundamental principles that nature enlists for system organization. Thus, the peculiar and often characteristic arrangements that skin lesions display provide a unique opportunity to reflect upon--and to experimentally dissect--the powerful organizing principles at the crossroads of developmental, skin and theoretical biology, genetics, and clinical dermatology that underlie these--increasingly less enigmatic--phenomena. The current 'Controversies' feature offers a range of different perspectives on how pattern formation of skin lesions can be approached. With this, we hope to encourage more systematic interdisciplinary research efforts geared at unraveling the many unsolved, yet utterly fascinating mysteries of dermatological pattern formation. In short: never a dull pattern!

What is the 'true' function of skin?

Conventional textbook wisdom portrays the skin as an organ that literally enwraps whatever each of us stands for as a more or less functional, individual member of the mammalian species, and has it that the skin primarily establishes, controls and transmits contacts with the external world. In addition, the skin has long been recognized to protect the organism from deleterious environmental impacts (physical, chemical,microbiological), and is well-known as crucial for the maintenance of temperature, electrolyte and fluid balance. Now, ever more studies are being published that show the skin to also operate as a huge and highly active biofactory for the synthesis,processing and/or metabolism of an astounding range of e.g. structural proteins, glycans, lipids and signaling molecules. Increasingly, it becomes appreciated that the skin, furthermore, is an integral component of the immune, nervous and endocrine systems, with numerous lines of cross-talk between these systems established intracutaneously (e.g. Ann NY Acad Sci Vol 885, 1999; Endocrine Rev 21:457-487, 2000; Physiol Rev 80:980-1020, 2001; Exp Dermatol 10: 349-367, 2001). All these emerging cutaneous functions beyond the classical image of the skin as a barrier and sensory organ are immediately relevant for many of the quandaries that clinical dermatology, dermatopathology, and dermatopharmacology are still struggling with to-date, and offer the practising dermatologist attractive new targets for therapeutic intervention. Yet, many of these skin functions are not even mentioned in dermatology textbooks and await systematic therapeutic targeting. Following a suggestion by Enno Christophers, the current 'Controversies' feature brings together an unusually diverse council of biologists and clinicians, who share their thought-provoking views with the readers and allow us to peek into the future of research in cutaneous biology, not the least by reminding us of the -- often ignored -- evolutionary and embryonal origins of our favorite organ. Hopefully, this unique discussion feature will foster an understanding of the 'true' skin functions that is both more comprehensive and more profound than conventional teaching on this topic, and will stimulate more than 'skin-deep' reflections on the full range of skin functions.

Immunohistochemical localization of deleted-in-colon-cancer (DCC) protein in human epithelial, neural, and neuro-endocrine tissues in paraffin sections with antigen retrieval.

A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin superfamily with similarity to the N-CAM transmembrane proteins, and it is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is know to occur, resulting in different isoforms of the protein. Using a polyclonal antibody against the DCC gene product, we have demonstrated, by antigen retrieval immunostaining, the presence of a DCC protein isoform on the cell surface of goblet cells in the G-I tract, cytoplasm of squamous epithelium in the skin & esophagus and transitional epithelium in the urinary bladder, ductal glandular epithelium of endometrium, endocervix, prostate, gall bladder and breast, cytoplasm of neuron in the cerebral cortex and Purkinje cells in the cerebellum. In addition, we also demonstrated DCC protein expression in neuroendocrine cells including argentaffin cells of the stomach, small intestine, appendix and colon, C cells of thyroid gland, chromaffin cells of the adrenal gland, islets of Langerhans in the pancreas and neurons of the sympathetic ganglion. This restrictive distribution suggests the DCC gene products may be abundant expression in neuroendocrine cells of human tissue.

Dinosaur's feather and chicken's tooth? Tissue engineering of the integument.

The integument forms the interface between animals and the environment. During evolution, diverse integument and integument appendages have evolved to adapt animals to different niches. The formation of these different integument forms is based on the acquisition of novel developmental mechanisms. This is the way Nature does her tissue/organ engineering and experiments. To do tissue engineering of the integument in the new century for medical applications, we need to learn more principles from developmental and evolutionary studies. A novel diagram showing the evolution and development of integument complexity is presented, and the molecular pathways involved discussed. We then discuss two examples in which the gain and loss of appendages are modulated: transformation of avian scale epidermis into feathers with mutated beta catenin, and induction of chicken tooth like appendages with FGF, BMP and feather mesenchyme.

A Novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells.

The templates required for inducing posttranscriptional gene silencing (PTGS) effects have been investigated in human prostate cancer LNCaP cells. Transfection of a mRNA-cDNA hybrid construct was found to result in a relatively long-term interference of specific gene expression. Androgen-stimulated expression of bcl-2 has been reported to increase the tumorigenic and metastatic potentials of human prostate cancer LNCaP cells, as well as their resistance to many apoptotic stimuli. The addition of bcl-2 antisense oligonucleotides, however, restored apoptosis. Our studies demonstrate gene silencing effects of the mRNA-cDNA transfection that is similar to those of PTGS/RNAi in this in vitro prostate cancer cell model. A potential RNA-directed RNA polymerase activity was also detected which is alpha-amanitin-sensitive. These findings indicate that a novel gene silencing system may exist in mammalian cells.

D-RNAi (messenger RNA-antisense DNA interference) as a novel defense system against cancer and viral infections.

D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4(+) T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was shown to knock out more than 99% endogenous beta-catenin gene expression, while the in cell transfection of HIV-1 D-RNAi homolog rejected viral gene replication completely. D-RNAi was found to have long-term gene knockout effects resulting from a posttranscriptional gene silencing mechanism that may involve the homologous recombination between intracellular mRNA and the mRNA components of a D-RNAi construct. These findings provide a potential intracellular defense system against cancer and viral infections.

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.

Conservation of early odontogenic signaling pathways in Aves.

Teeth have been missing from birds (Aves) for at least 60 million years. However, in the chick oral cavity a rudiment forms that resembles the lamina stage of the mammalian molar tooth germ. We have addressed the molecular basis for this secondary loss of tooth formation in Aves by analyzing in chick embryos the status of molecular pathways known to regulate mouse tooth development. Similar to the mouse dental lamina, expression of Fgf8, Pitx2, Barx1, and Pax9 defines a potential chick odontogenic region. However, the expression of three molecules involved in tooth initiation, Bmp4, Msx1, and Msx2, are absent from the presumptive chick dental lamina. In chick mandibles, exogenous bone morphogenetic protein (BMP) induces Msx expression and together with fibroblast growth factor promotes the development of Sonic hedgehog expressing epithelial structures. Distinct epithelial appendages also were induced when chick mandibular epithelium was recombined with a tissue source of BMPs and fibroblast growth factors, chick skin mesenchyme. These results show that, although latent, the early signaling pathways involved in odontogenesis remain inducible in Aves and suggest that loss of odontogenic Bmp4 expression may be responsible for the early arrest of tooth development in living birds.

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.

Dynamic expression of lunatic fringe during feather morphogenesis: a switch from medial-lateral to anterior-posterior asymmetry.

Expression of Lunatic fringe mRNA was studied during feather morphogenesis and showed three stages of dynamic expression pattern. (1) Lunatic fringe was first expressed in the epithelium as a ring bordering the feather primordium when it was initially induced. (2) Shortly after, it showed a polarized pattern, first toward the lateral side of the feather primordium and then made a 90 degrees C switch toward the posterior side of the short bud. It then becomes weakly expressed in the long bud stage. (3) Finally, it is expressed in the marginal plate epithelia of feather filaments. In contrast, Radical fringe is weakly expressed in the feather bud, but is also present in the marginal plate epithelia of feather filaments.

Suppression of activin-induced apoptosis by novel antisense strategy in human prostate cancer cells.

Apoptosin, a novel gene encoding a mitotic kinase-motif protein, is stimulated by activin, a member of TGF-beta family, in human LNCaP prostate cancer cells and in patient tissues. We employed a gene knockout methodology based on the covalent bonding of chemically modified antisense probes to apoptosin mRNAs in LNCaP cells. The mRNA-antisense hybrid duplexes were neither translated nor post-transcriptionally modified, resulting in no protein synthesis. Introducing antisense apoptosin into activin-induced apoptotic LNCaP cells prevented apoptosis, interfered with genomic DNA fragmentation and released cell cycle checkpoint. These findings suggest that the apoptosin, in addition to p53, is important in apoptotic regulation of human prostate cancers.

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.