Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.

Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis.

Merkel cells (MCs) are located in the touch-sensitive area of the epidermis and mediate mechanotransduction in the skin. Whether MCs originate from embryonic epidermal or neural crest progenitors has been a matter of intense controversy since their discovery >130 yr ago. In addition, how MCs are maintained during adulthood is currently unknown. In this study, using lineage-tracing experiments, we show that MCs arise through the differentiation of epidermal progenitors during embryonic development. In adults, MCs undergo slow turnover and are replaced by cells originating from epidermal stem cells, not through the proliferation of differentiated MCs. Conditional deletion of the Atoh1/Math1 transcription factor in epidermal progenitors results in the absence of MCs in all body locations, including the whisker region. Our study demonstrates that MCs arise from the epidermis by an Atoh1-dependent mechanism and opens new avenues for study of MC functions in sensory perception, neuroendocrine signaling, and MC carcinoma.

KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin.

A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioning future hair follicle epithelium from interfollicular epidermis. Although many developmental signalling pathways are implicated in follicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7) receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, the underlying mechanisms have not been characterised. Here we show that receptors for EGF and KGF undergo marked downregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persist throughout hair follicle initiation. Using embryonic skin organ culture, we show that when skin from the sites of primary pelage and whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. We then used downstream molecular markers and microarray profiling to provide evidence that, in response to KGF and EGF signalling, epidermal differentiation is promoted at the expense of hair follicle fate. We propose that hair follicle initiation in placodes requires downregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicular epidermis. We have also uncovered a previously unrecognised role for KGF signalling in the formation of hair follicles in the mouse.

A mutation in hairless dogs implicates FOXI3 in ectodermal development.

Mexican and Peruvian hairless dogs and Chinese crested dogs are characterized by missing hair and teeth, a phenotype termed canine ectodermal dysplasia (CED). CED is inherited as a monogenic autosomal semidominant trait. With genomewide association analysis we mapped the CED mutation to a 102-kilo-base pair interval on chromosome 17. The associated interval contains a previously uncharacterized member of the forkhead box transcription factor family (FOXI3), which is specifically expressed in developing hair and teeth. Mutation analysis revealed a frameshift mutation within the FOXI3 coding sequence in hairless dogs. Thus, we have identified FOXI3 as a regulator of ectodermal development.

Protein distribution of Kcnq1, Kcnh2, and Kcne3 potassium channel subunits during mouse embryonic development.

Voltage-dependent potassium channels consist of a pore-forming alpha-subunit, which is modulated by additional beta-ancillary or regulatory subunits. Kcnq1 and Kcnh2 alpha-channel subunits play pivotal roles in the developing and adult heart. However, Kcnq1 and Kcnh2 have a much wider expression profile than strictly confined to the myocardium, similar to their putative regulatory Kcne1-5 beta-subunits. At present, the distribution of distinct potassium channel subunits has been partially mapped in adult tissues, whereas almost no information is available during embryonic development. In this study, we report a detailed analysis of Kcnq1, Kcnh2, and Kcne3 protein expression during mouse embryogenesis. Our results demonstrate that Kcnq1 and Kcnh2 are widely distributed. Coexpression of both alpha-subunits is observed in a wide variety of organs, such as heart and the skeletal muscle, whereas others display unique Kcnq1 or Knch2 expression. Interestingly, Kcne3 expression is also widely observed in distinct tissue layers during embryogenesis, supporting the notion that an exquisite balance of alpha- and beta-subunit expression is required for modulating potassium conductance in distinct organs and tissue layers.

Characterization of Lef-1 promoter segments that facilitate inductive developmental expression in skin.

Lymphoid Enhancer Factor 1 (Lef-1) is an important developmental transcription factor required for the inductive formation of several epithelial-derived organs including hair follicles. Inductive expression of Lef-1 mRNA is tightly regulated during embryo development, suggesting the involvement of a highly regulated promoter. In vitro analysis of the Lef-1 gene has demonstrated the existence of at least two spatially distinct promoters with multiple transcriptional start sites that are responsive to the canonical Wnt/beta-catenin pathway. Regions of the Lef-1 promoter required for inductive regulation in vivo, however, have yet to be determined. To this end, we utilized LacZ-reporter transgenic mice to define segments of the human Lef-1 promoter capable of reproducing mesenchymal- or epithelial-restricted transcriptional patterns of Lef-1 expression during hair and vibrissa follicle development. These studies have revealed that a 110 bp Wnt/beta-catenin-responsive element, contained within a minimal 2.5 kb Lef-1 promoter, plays an important role in regulating mesenchymal, and potentially epithelial, expression during follicle development in mouse embryos. This 2.5 kb Lef-1 promoter also demonstrated inductive mesenchymal expression during postnatal anagen stage hair-follicle cycling. Additionally, analysis of Lef-1 promoter expression revealed previously uncharacterized regions of endogenous Lef-1 expression seen in the sebaceous glands of vibrissa and hair follicles in transgenic lines harboring the minimal Lef-1 promoter and additional intronic sequences. In summary, these studies have begun to dissect the transcriptional diversity of the human Lef-1 promoter during the hair/vibrissa follicle and sebaceous gland formation.

Altered whisker patterns induced by ectopic expression of Shh are topographically represented by barrels.

Barrels in the somatosensory cortex are segregated columns, which somatotopically relate to facial whiskers. The barrel pattern is assumed to be determined by an extrinsic mechanism (the domino theory). This theory is based on whisker lesion experiments and developmental observations regarding the serial establishment of the somatotopic pattern in which pattern formations are relayed from the periphery to the central nervous system. However, the barrel pattern is possibly determined by an intrinsic mechanism, especially in its primitive form. In order to investigate the definitive mechanism, we established an experimental system in which the cortical barrel pattern can be altered, not by using a lesion paradigm, but by epigenetically changing the whisker pattern. Sonic hedgehog (Shh) plays a pivotal role in whisker development. We transfected an adenovirus harboring chicken Shh (Ad-cShh) to mouse embryos (E9.5-E11.5) using an in utero surgical technique. When Ad-cShh was expressed in the epidermis, Bmp4, Ptch, Ptch2 and Gli1 were induced ectopically in the interfollicular region. In contrast, the expression of Bmp2 and Shh itself was unaltered. At a suitable dose of Ad-cShh, some pups displayed supernumerary whiskers or a disordered whisker pattern. The barrel patterns of these mice after the critical period were topographic representations of the contralateral side of the new whisker patterns when visualized by a cytochrome oxidase or Nissle staining method, supporting the instructive role of the extrinsic mechanism.

Dynamic expression and nuclear accumulation of beta-catenin during the development of hair follicle-derived structures.

Beta-catenin has a dual role in the cell. At the membrane, it connects E-cadherin to the actin cytoskeleton, while in the nucleus, it controls gene expression in concert with Tcf-like transcription factors. Nuclear translocation of beta-catenin is induced by the Wnt signal transduction pathway. Control of this process is essential since elevated beta-catenin levels interfere with differentiation and development, and can initiate cancer in many tissues. An important role for beta-catenin during hair follicle related development and tumorigenesis has recently been established, though little is known of its endogenous expression during the development of these structures. Here, we have investigated the expression of beta-catenin in relation to markers for proliferation, differentiation and Wnt signaling during the development of three hair follicle related structures, i.e. whiskers, normal body hair and the preputial gland, and a hair follicle-derived tumor, the epidermal cyst. We observed nuclear accumulation of beta-catenin, the hallmark of Wnt signaling, in the upper matrix, the dermal papilla, the developing ringwulst of the whisker and in the tumor, though it was never in association with proliferation or terminal differentiation. Co-localization of nuclear beta-catenin with Tcf-3/4 was found only in the dermal papilla and the developing ringwulst of the whisker, but not in the upper matrix or in the tumor. These results further elucidate the role of the Wnt signal transduction pathway during hair follicle related development and tumorigenesis and illustrate the dynamic role of beta-catenin in signal transduction and cell-adhesion.

Effects of retinoic acid exposure in utero on mouse vibrissal follicle development.

It is known that topical all-trans-retinoic acid (RA) modulates growth and differentiation of skin and its cutaneous appendages. To examine whether a pre-natal exposure to a potentially non-teratogenic dosage of all-trans-RA had any effect on vibrissal follicle development, the histologic and immunohistochemical responses to RA during its morphogenesis in NMRI mouse were investigated. After a single oral dose of 30 mg/kg body weight of all-trans-RA on day 11.5 of gestation, no fetal malformations were detected and the histological features and the distribution of keratin (K) proteins in comparable stages of vibrissal development were similar for the untreated, vehicle-treated and RA-treated mice. The absence of teratogenic response and of adverse effects on the vibrissae under the experimental conditions indicates that this protocol may be useful for investigation of the effects of pre-natal exposure to RA on the post-natal development of experimental tumours in the mouse skin.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

Epidermal growth factor receptor as a mediator of developmental toxicity of dioxin in mouse embryonic teeth.

We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.

In situ hybridization analysis of ZPK gene expression during murine embryogenesis.

ZPK is a recently described protein serine/threonine kinase that has been originally identified from a human teratocarcinoma cell line by the polymerase chain reaction and whose function in signal transduction has not yet been elucidated. To investigate the potential role of this protein kinase in developmental processes, we have analyzed the spatial and temporal patterns of expression of the ZPK gene in mouse embryos of different gestational ages. Northern blot analysis revealed a single mRNA species of about 3.5 KB from Day 11 of gestation onwards. In situ hybridization studies demonstrated strong expression of ZPK mRNA in brain and in a variety of embryonic organs that rely on epitheliomesenchymal interactions for their development, including skin, intestine, pancreas, and kidney. In these tissues, the ZPK mRNA was localized primarily in areas composed of specific types of differentiating cells, and this expression appeared to be upregulated at a time concomitant with the onset of terminal differentiation. Taken together, these observations raise the possibility that the ZPK gene product is involved in the establishment and/or maintenance of a fully cytodifferentiated state in a variety of cell lineages.

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.

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.

[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)