Wound Healing and Scale Modelling in Zebrafish.

We propose to study the wound healing in Zebrafish by using firstly a differential approach for modelling morphogens diffusion and cell chemotactic motion, and secondly a hybrid model of tissue regeneration, where cells are considered as individual objects and molecular concentrations are described by partial differential equations.

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!

Dorsal dermis development depends on a signal from the dorsal neural tube, which can be substituted by Wnt-1.

To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or of cells producing Shh led to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, triggered the formation of a feather-inducing dermis. These results show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least mimicked by Wnt-1.

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli.

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.

Mediolateral somitic origin of ribs and dermis determined by quail-chick chimeras.

Somites are transient mesodermal structures giving rise to all skeletal muscles of the body, the axial skeleton and the dermis of the back. Somites arise from successive segmentation of the presomitic mesoderm (PSM). They appear first as epithelial spheres that rapidly differentiate into a ventral mesenchyme, the sclerotome, and a dorsal epithelial dermomyotome. The sclerotome gives rise to vertebrae and ribs while the dermomyotome is the source of all skeletal muscles and the dorsal dermis. Quail-chick fate mapping and diI-labeling experiments have demonstrated that the epithelial somite can be further subdivided into a medial and a lateral moiety. These two subdomains are derived from different regions of the primitive streak and give rise to different sets of muscles. The lateral somitic cells migrate to form the musculature of the limbs and body wall, known as the hypaxial muscles, while the medial somite gives rise to the vertebrae and the associated epaxial muscles. The respective contribution of the medial and lateral somitic compartments to the other somitic derivatives, namely the dermis and the ribs has not been addressed and therefore remains unknown. We have created quail-chick chimeras of either the medial or lateral part of the PSM to examine the origin of the dorsal dermis and the ribs. We demonstrate that the whole dorsal dermis and the proximal ribs exclusively originates from the medial somitic compartment, whereas the distal ribs derive from the lateral compartment.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Expression of DM-GRASP/BEN in the developing mouse spinal cord and various epithelia.

The expression pattern of the immunoglobulin DM-GRASP/BEN gene was studied in the mouse embryo using in situ hybridization. DM-GRASP/BEN is expressed in the spinal cord in a subset of motoneurons expressing Islet-1, and non homogeneously in the dorsal root ganglia (DRG). In contrast, it's expression is homogeneous in the vestibulo-cochlear and trigemminal ganglia. DM-GRASP/BEN is also expressed in various epithelia of ectodermal or endodermal origin like the nasal, buccopharyngal and lung epithelia. In upper lip, DM-GRASP/BEN transcripts are present in the epidermal cells of the developing hair vibrissa follicles. First detected in the hair placode, DM-GRASP/BEN expression is localized in the central cells of the epithelial hair peg and then in a thin layer of cells crushed against the outer root sheath by the outgrowth of the hair shaft.

Epithelial stem cells in the skin: definition, markers, localization and functions.

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?

Differential expression pattern of the three Fringe genes is associated with epidermal differentiation.

Epidermal differentiation, as keratinocytes go through different layers to the skin surface, may imply a differential activation of Notch transmembrane proteins. In mouse, as recently shown in Drosophila, Notch activation by its ligands may be modulated by Fringe secreted proteins. Therefore, we cloned the mouse homolog of Radical-fng, synthesized riboprobes for Lunatic-fng, Manic-fng, and Radical-fng, and examined their expression during epidermal differentiation. Expression of all three genes is differentially activated during embryonic epidermal stratification. Manic-fng and Lunatic-fng are expressed in the basal layer, whereas Lunatic-fng is expressed in the granular layer and Radical-fng is restricted to the most differentiated nucleated layer. This expression decreases by a few days postnatally and can be reactivated by retinoic acid treatment, which triggers a new distribution of Fringe transcripts and a thickening of the granular layer. Therefore, Manic, Lunatic, and Radical Fringe by modulating the Notch pathway may play a key role in defining the different steps of keratinocyte differentiation.

Both retinoic acid receptors alpha (RARalpha) and gamma (RARgamma) are able to initiate mouse upper-lip skin glandular metaplasia.

Embryonic mouse upper-lip skin explants treated with 16.7 microM all-trans retinoic acid (tRA) give rise to a glandular metaplasia of hair vibrissa follicles; however, at this concentration, tRA can activate not only the three retinoic acid receptors (RARalpha, beta, and gamma), but also the retinoid X receptors (RXRalpha, beta, and gamma) as a consequence of its isomerization to 9-cis retinoic acid. We therefore studied the respective roles of the RXR and RAR by treating RARalpha(-/-), beta(-/-), and gamma(-/-) skin explants with tRA and wild-type explants with synthetic retinoids specific for RXR or for each of the RAR. The null mutation of the RARalpha, RARbeta, and RARgamma genes did not prevent tRA-induced hair glandular metaplasia, but RARgamma inactivation dramatically reduced its ratio. As demonstrated by treating explants with a RAR- or a RXR-specific panagonist (CD367 and Ro25-7386, respectively), RAR are primarily responsible for this metaplasia. The use of two retinoids (Ro40-6055, 8 x 10(-3) microM, or CD437, 7.7 x 10(-2) microM) that are believed to act, respectively, as a RARalpha- or a RARgamma-specific agonist showed that both these receptors can initiate a metaplasia. In contrast, BMS453, a RARbeta-specific agonist, was unable to give rise to any metaplasia. Nevertheless, the highest degrees and ratios of metaplasia were only obtained after treatment with the CD367 RAR panagonist, or with either Ro40-6055 or CD437 at a concentration sufficient to allow the activation of the three RAR, suggesting that RARbeta activation is required for a metaplasia of all vibrissae.

Chick Delta-1 gene expression and the formation of the feather primordia.

The chick dermis is known to control the formation of feathers and interfeathery skin in a hexagonal pattern. The evidence that the segregation of two types of fibroblasts involves Delta/Notch signalling is based on three facts. Rings of C-Delta-1-expressing fibroblasts precede and delimit the forming feather primordia. C-Delta-1 is uniformly expressed in the dermis of the scaleless mutant, which is almost entirely devoid of feathers. Feather development is inhibited by overexpression of C-Delta-1 in wild type dermis using a retroviral construct. We also show that the distribution of C-Delta-1 in the mutant dermis can be rescued by its association with a wild type epidermis, which acts as a permissive inducer, or by epidermal secreted proteins like FGF2.

CHOXC-8 and CHOXD-13 expression in embryonic chick skin and cutaneous appendage specification.

We studied the expression of two distantly clustered Hox genes which could, respectively, be involved in specification of dorsal feather- and foot scale-forming skin in the chick embryo: cHoxc-8, a median paralog, and cHoxd-13, located at the 5' extremity of the HoxD cluster. The cHoxc-8 transcripts are present at embryonic day 3.5 (E3.5) in the somitic cells, which give rise to the dorsal dermis by E5, and at E6.5-8.5 in the dorsal dermal and epidermal cells during the first stages of feather morphogenesis. The cHoxd-13 transcripts are present at E4.5-9.5 in the autopodial mesenchyme and at E10.5-12.5 in the plantar dermis during the initiation of reticulate scale morphogenesis. Both the cHoxc-8 and cHoxd-13 transcripts are no longer detectable after the anlagen stage of cutaneous appendage morphogenesis. Furthermore, heterotopic dermal-epidermal recombinations of dorsal, plantar, and apteric tissues revealed that the epidermal ability or inability to form feathers is already established by the time of skin formation. Retinoic acid (RA) treatment at E11 induces after 12 hr an inhibition of cHoxd-13 expression in the plantar dermis, followed by the formation of feather filaments on the reticulate scales. When E7.5 dorsal explants are treated with RA for 6 days, they form scale-like structures where the Hox transcripts are no more detectable. Protein analysis revealed that the plantar filaments, made up of feather beta-keratins, corresponded to a homeotic transformation, whereas the scale-like structures, composed also of feather beta-keratins, were teratoid. These results strengthen the hypothesis that different homeobox genes play a significant role in specifying the regional identity of the different epidermal territories.

Adult epidermal keratinocytes are endowed with pilosebaceous forming abilities.

Pluristratified epithelia of adult vertebrate skin continuously regenerate from stem cells, and the question still arises as to whether those cells are committed to the production of only one cell lineage, or in contrast they conserve their embryonic pluripotentiality. In order to investigate the abilities of adult cultured as well as wound healing epidermis, heterospecific fibroblast-keratinocyte recombinations were performed, which allow unquestionable identification of the cells implicated in the structures that differentiate. Adult human cultured breast epidermal cells and full-thickness wound healing from human facial skin and foreskin were associated with either rabbit embryonic trichogenic dermis or cultured dermal papilla cells of adult rat, before grafting onto nude mice for two weeks to one month. In situ hybridization with a human specific sequence Alu probe labeled the human cells, whereas implanted rabbit or rat and host mouse cells were distinguished by the Hoechst staining of their nuclei. The results show that human adult cultured breast epidermal cells are able to form hair buds and to participate in hair follicle formation, while adult healing epidermis from a sparsely hairy skin as the human face or the dorsal skin of nude mouse, or even from a glabrous epidermis as the human foreskin, are able to differentiate pilosebaceous units. Although a follicular origin of the involved keratinocytes cannot be excluded in the three first cases, the formation of hair and sebaceous glands by foreskin keratinocytes of children 2 to 10 years-old establishes the cutaneous appendage ability of the interfollicular epidermal stem cells. The formation of interspecies mosaic follicles also highlights the fact that there must be a significant level of commonality in the interactive signaling molecules used by epithelial cells from different species.

Isolation and characterization of genomic clones of human sequences presumably coding for hair cysteine-rich proteins.

The major biochemical components of the mammalian hair are the intermediate filaments or keratins and the keratin associated proteins. Keratin associated proteins are classified into two groups (high-cysteine and high glycine-tyrosine-rich polypeptides) according to the content of these amino acids. Cysteine-rich group contains high sulphur (16-24% cysteine) and ultra-high sulphur (> 30% cysteine) proteins. We report here the identification of a human sequence presumably coding for a new ultra-high sulphur protein (hUHSp21) and the isolation and characterization of four genomic clones containing six related sequences. We also discuss the possibility that all the genes encoding keratin associated proteins are evolutionary related. These human clones should provide useful molecular tools for studies of hair differentiation and understanding of the molecular basis of human trichothiodystrophy.

A longitudinal study of a harlequin infant presenting clinically as non-bullous congenital ichthyosiform erythroderma.

Over the past 8 years, we have followed a child born as a harlequin baby, who survived due to treatment with retinoids. His condition evolved clinically towards the erythrodermic form of lamellar ichthyosis (non-bullous congenital ichthyosiform erythroderma, NBCIE). According to ultrastructural and biochemical criteria, our patient originally presented with type II harlequin ichthyosis. Investigations showed an abnormal keratinosome structure and extrusion, a keratin pattern characteristic for epidermal hyperproliferation, and an absence of conversion of profilaggrin to filaggrin. Persisting keratinocyte hyperproliferation, associated with the presence of a dermal infiltrate, is in agreement with the present clinical picture of severe NBCIE. However, abnormal lamellar body production and defective filaggrin processing, which is not one of the diagnostic criteria of NBCIE, persist in the patient's skin. Further studies of the epidermal lipid composition, and of possible mutations of the keratinocyte transglutaminase gene performed on epidermal cell cultures of harlequin ichthyosis, will be necessary before type II harlequin ichthyosis can be accepted as an extremely severe form of NBCIE.

Characterization of cDNAs encoding two chick retinoic acid receptor alpha isoforms and distribution of retinoic acid receptor alpha, beta and gamma transcripts during chick skin development.

The amino acid sequence of the retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) can be divided into six functional domains (A-F), different isoforms arising from the presence of different A domains by differential splicing. In order to address the respective roles of the different RARs during skin morphogenesis in birds, cDNAs encoding two chick RAR alpha isoforms (alpha1 and alpha2) have been isolated. While the A1 and B-F domains of the RAR alpha are highly conserved across species, the chick A2 domain contains 50% specific amino acids. The three RAR alpha, beta and gamma genes display specific patterns of expression during chick skin morphogenesis. As in mouse, RAR alpha and gamma transcripts are present in both the dermis and epidermis during the first stages of skin appendage formation. Furthermore, Northern blot analysis suggests that different RAR alpha and gamma isoforms could be successively required during feather formation. The RAR gamma gene, continuously expressed in the epidermal cells in both chick and mouse, is thus likely to play a similar role in skin development in these two species. However, RAR alpha transcripts, only transiently detected during mouse skin development, still accumulate in epidermis during the later stages of chick skin differentiation. Furthermore, RAR beta transcripts, never detected during normal development in mouse skin, are actually present at the early stages of chick skin morphogenesis. Thus, our results suggest that the role of the three RAR in skin development has not been strictly conserved in the different classes of vertebrates.

Retinoic acid-mediated increase in TrkA expression is sufficient to elicit NGF-dependent survival of sympathetic neurons.

Sympathetic neurons depend on the classical neurotrophin NGF for survival by the time they innervate their targets, but the mechanisms controlling the onset of NGF responsiveness in developing neuroblasts have not been defined. Immature chick sympathetic neurons are unresponsive to NGF, but express low mRNA levels of the high-affinity NGF receptor trkA. Treatment with retinoic acid (RA) leads to increased levels of both trkA mRNA and protein, a response mediated through retinoic acid receptor alpha (RAR alpha). Ectopic expression of trkA in these cells results in the ability to survive with NGF, suggesting that RA-induced trkA expression is sufficient to elicit NGF-dependent survival. Our data establish a mechanism controlling NGF responsiveness and implicate a function for RA at defined late stages of neuron development.

Differential expression of two different homeobox gene families during mouse tegument morphogenesis.

The expression of six genes belonging to two different homeobox gene families was studied during the embryonic and postnatal morphogenesis of head and body regions of the mouse integument. The first family included the Otx1 and Otx2 genes, both related to the orthodenticle Drosophila gene and the second was represented by four members of the Antennapedia class HOX genes: Hoxc8 and three Hoxd genes, d9, d11 and d13. In situ hybridizations with 35S labeled antisense RNA probes were performed on head serial frontonasal sections, as well as entire embryo and postnatal tail longitudinal sections. The expression of these genes shows a differential spatiotemporal pattern along the cephalo-caudal axis. In 12.5-day and 15.5-day embryos, the Otx2 gene expression is restricted to the nasal epithelium and its associated glands, while the Otx1 transcripts are present in both nasal and facial integuments, including nasal glands and hair vibrissa follicles. The Hoxc8 expression first appears in skin at 14.5 days of gestation in the sternal region and is extended at 16.5 days to the thoracic ventral and lumbar dorsal regions. The Hoxd9 and Hoxd11 genes are only expressed in the caudal skin from 14.5 days of gestation. The Hoxd13 transcripts are the last to appear, 2 days after birth, and are limited to the last epidermal cells to differentiate, i.e. those of the hair matrix of the caudal pelage hair follicles. Taken together, these observations strengthen the hypothesis that different homeobox gene families specify the regional identity of the skin in the cephalic and body regions.

Characterization of cDNAs encoding the chick retinoic acid receptor gamma 2 and preferential distribution of retinoic acid receptor gamma transcripts during chick skin development.

Retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) are ligand-inductible transcriptional activators which belong to the steroid/thyroid hormone receptor superfamily. At least two major isoforms (1 and 2) of each RAR arise by differential use of two promoters and alternative splicing. In mouse, the three RAR genes are expressed in stage- and tissue-specific patterns during embryonic development. In order to understand the role of the different RARs in chick, RAR gamma 2 cDNAs were isolated from an 8.5-day (stage 35 of Hamburger and Hamilton) chick embryo skin library. The deduced chick RAR gamma 2 amino acid sequence displays uncommon features such as 21 specific amino acid replacements, 12 of them being clustered in the amino-terminal region (domains A2 and B), and a truncated acidic carboxy-terminal region (F domain). However, the pattern of RAR gamma expression in chick embryo resembles that reported in mouse, particularly in skin where RAR gamma expression occurs in both the dermal and epidermal layers at the beginning of feather formation, and is subsequently restricted to the differentiating epidermal cells. Northern blot analysis suggests that different RAR gamma isoforms could be successively required during chick development.

Retinoic acid and mouse skin morphogenesis. II. Role of epidermal competence in hair glandular metaplasia.

Retinoic acid (RA) has marked effects on mouse upper-lip skin morphogenesis, leading to the development of glomerular gland instead of hair vibrissa follicle, but does not apparently change the dorsal pelage hair developmental program. In order to test the hypothesis that an up-regulation of the beta retinoic acid nuclear receptor (RAR beta) may be implicated in the alteration of the dermal-epidermal interactions which occur during cutaneous appendage development, RA-treated and untreated skin explants, controls as well as heterotopic recombinants, were made among nasal, upper-lip, and dorsal mouse embryonic tissues. They were analyzed by in situ hybridization with RAR beta 35S-labeled probe after 48 hr of in vitro culture as well as by identification of the morphological phenotype of cutaneous appendages after 6 additional days of culture on the chick chorioallantoic membrane. The results show that only mesenchyme from the facial region can express the RAR beta gene either normally or after RA treatment, depending on its nasal or upper-lip origin. However, the RAR beta up-regulation is unrelated to hair glandular metaplasia, which depends both on a glandular bias of the upper-lip epidermis and on the weakening of hair follicle-inducing dermal properties. The latter occurs in both the upper-lip and dorsal dermis as a consequence of RA treatment.