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.

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.

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.

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.

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.

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 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 and mouse skin morphogenesis. I. Expression pattern of retinoic acid receptor genes during hair vibrissa follicle, plantar, and nasal gland development.

The spatial and temporal expression of the nuclear retinoic acid receptors alpha, beta, and gamma (RAR-alpha, beta, and gamma) was compared by in situ hybridization during hair vibrissa follicle and nasal and plantar eccrine gland morphogenesis in mouse embryo. The RAR-alpha and RAR-gamma transcripts are abundant in the dermal papilla cells of the hair vibrissa when these cells elicit epidermal hair placode (12.5-d embryos) and hair follicle (13.5-d embryos) formation. Both these transcripts are also abundant in the dermal cells of the plantar foot pad at the initiation stage (17.5-d embryos) of glandular morphogenesis. In epidermal cells, the distribution of RAR-gamma transcripts increases in parallel with hair vibrissa follicle and sweat gland differentiation, and thus may be part of the epidermal response to the dermal instructions. The RAR-beta signal is barely above control level during both hair vibrissa and plantar gland morphogenesis. By contrast, during nasal gland formation (12.5- to 15.5-d embryos), the RAR-beta signal reaches a high level in mesenchymal cells, whereas the RAR-alpha-transcripts are present in both epithelial and mesenchymal cells. These results suggest a role for RAR-alpha and RAR-gamma in the epidermal-dermal interactions that lead to hair follicle and plantar gland morphogenesis, whereas the nasal gland development implies RAR-alpha and RAR-beta gene expression. This should be correlated with the expression of the RAR-beta gene that was previously shown to be linked to the RA-induced glandular metaplasia of hair vibrissa follicles.

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.

Structure-activity relationships of retinoids in the morphogenesis of cutaneous appendages in the chick embryo.

We have evaluated the efficacy of 13 analogues of retinoic acid (retinoids) in producing in vivo feathers on the normally scaly foot integument (ptilopody) of the chick embryo. The retinoids were dissolved in dimethylsulfoxide and injected at doses from 1.25-250 micrograms into the amniotic cavity of embryos of 10 days of incubation. The results showed that the production of ptilopody requires specific retinoids, namely, those incorporating a free trans carboxylic end group. Indeed, the esterification of this acid end group (retinoid ethyl ester) leads to a delay in the effect of the product. Its substitution by an alcohol (retinol) or an aldehyde group (retinal), or its isomerization, leads to inactive products. The esterification by acetic acid of the alcohol terminal group of retinol leads to a moderately active compound (retinyl acetate). By contrast, modifications of the ring do not interfere with the efficacy of the retinoid, and modifications of the side chain by addition of one or two supplementary rings lead to an increase of the activity.

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.

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.

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!

[Genetic expression and morphogenesis of the skin in vertebrates]. / Expression génique et morphogenèse de la peau des vertébrés.

Retinoic acid is an exception amongst teratogenic agents: indeed, it not only interferes with morphogenesis, but it also alters positional values, which leads to the formation of additional structures. The latter effect, similar to that of homeotic mutations, which have been characterized in drosophila, is the most instructive way to study morphogenetic processes. Intramniotic injection of retinoic acid into 10-day-old chick embryos causes feather formation on the scales of the anterior side of tarsometatarsus. Likewise, when the upper-lip skin of 13.5-day mouse embryos is treated in vitro with retinoic acid, there is a development of mouse vibrissa hair buds into glands. The expression of retinoic acid nuclear receptors (RARs) has been studied by in situ hybridization during normal morphogenesis of hair vibrissa follicles as well as during retinoic acid-induced glandular metaplasia. During the normal development of hair follicle, only RAR alpha and RAR gamma genes are transcribed, whereas RAR beta gene expression remains undetected by the techniques used. Retinoic acid skin treatment brings about RAR beta expression in the dermis, leading to the formation of glomerular glands. Now, hair, gland, scale or feather differentiation implies dermal-epidermal interactions which have been extensively studied. However, little is known about the language of communication used by the two tissues. Even if the inductive role of the dermis seems dominant, the competence of the epidermis must not be neglected: the latter determines in some cases the type of cutaneous appendage which is formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Feather-forming capacities of the avian extra-embryonic somatopleure.

Blocks of 12.5- or 13.5-day embryonic mouse upper-lip dermis were introduced under the ectoderm of the extra-embryonic area of 2- to 3-day chick or duck embryos. Two kinds of ectopic cutaneous appendages were produced: either arrested feathers alone, or arrested feathers and full-grown feathers. The former developed in the ectoderm overlying the implanted mouse dermal cells, the latter formed in their close vicinity, but contained host dermal cells exclusively. Thus, avian extra-embryonic somatopleure, both ectoderm and mesoderm, possesses the information for feather development: the extra-embryonic ectoderm, if it is brought in contace with an appendage-forming dermis, is able to respond to the dermal induction by initiating feather morphogenesis; the extra-embryonic mesoderm, if it is experimentally transformed into a dense dermis, can express its feather-forming capacity by specifying feather tract morphology and barb-ridge number, thus leading to the acievement of feather morphogenesis.

[Feather- and hair-forming properties of dermal cells of glabrous skin from bird and mammals]. / Propriétés phanérogènes des cellules dermiques de peau glabre d'Oiseau ou de Mammifère

The absence of cutaneous appendages in glabrous skin areas of birds and mammals rises from the incapacity of the dermis to trigger off the development of feather and hair buds. The dermal cells from avian or mammalian glabrous regions are however able to induce the continuation and completion of feather or hair morphogenesis respectively, provided the initiation of appendage bud formation has been triggered off by dermis from feather- or hair-forming regions.

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.

Use of retinoic acid for the analysis of dermal-epidermal interactions in the tarsometatarsal skin of the chick embryo.

Feet of chicks are normally covered with scales. Injection of retinoic acid into the amniotic cavity of 10-day chick embryos causes the formation of feathers on the foot scales. To elucidate whether retinoic acid affects primarily the epidermis or the dermis, heterotypic dermal-epidermal recombinants of tarsometatarsal skin were tested as to their morphogenetic capacity, when grafted to the chick chorioallantoic membrane. Recombinants involving treated epidermis and untreated dermis formed feathered scales, while the reverse recombinants of untreated epidermis and treated dermis led to the formation of scales only. Likewise the association of treated tarsometatarsal dermis with untreated epidermis from a non-appendage-forming region (the midventral apterium) resulted in the formation of scales only. These results show that retinoic acid affects primarily the epidermis. Further insight into the mechanism of dermal-epidermal interaction was gained by heterotopic recombinations of early (8.5- and 10-day) untreated tarsometatarsal dermis with epidermis from the midventral apterium. These recombinants formed scales, proving that tarsometatarsal dermis is endowed with scale-forming properties as early as 8.5 days of incubation. Finally, it is concluded that retinoic acid acts on the chick foot epidermal cells by temporarily inhibiting their scale placode-forming properties, allowing their latent feather placode-forming properties to be expressed.