Epidermal Cell Fate Research Articles

Plant Proteases: From Phenotypes to Molecular Mechanisms

Plant genomes encode hundreds of proteases, which represent dozens of unrelated families. The biological role of these proteases is mostly unknown, but mutant alleles, gene silencing, and overexpression studies have provided phenotypes for a growing number of proteases. The aim of this review is to show the diversity of the processes that are regulated by proteases, and to summarize the current knowledge of the underlying molecular mechanisms. The emerging picture is that plant proteases are key regulators of a striking variety of biological processes, including meiosis, gametophyte survival, embryogenesis, seed coat formation, cuticle deposition, epidermal cell fate, stomata development, chloroplast biogenesis, and local and systemic defense responses. The functional diversity correlates with the molecular data: Proteases are specifically expressed in time and space and accumulate in different subcellular compartments. Their substrates and activation mechanisms are elusive, however, and represent a challenging topic for further research.

Thérapie génique cutanée

Prospects of ex vivo cutaneous gene therapy rely on stable corrective gene transfer in epidermal stem cells followed by engraftment of corrected cells in patients. In the case of cancer prone genodermatoses, such as xeroderma pigmentosum, cells that received the corrective gene must be selected. However, this step is potentially harmful and can increase risks of immune rejection of grafts. These obstacles have recently been overcome thanks to the labeling of genetically modified stem cells using a small epidermal protein naturally absent in stem cells. This approach was shown to be respectful of the fate of epidermal stem cells that retained full growth and differentiation capacities, as well as their potential to regenerate normal human skin when grafted in a mouse model in the long term. These progresses now open realistic avenues towards ex vivo cutaneous gene therapy of cancer prone genodermatoses such as xeroderma pigmentosum. However, major technical improvements are still necessary to preserve skin appendages which would contribute to aesthetic features and comfort of patients.

From priming to plasticity: the changing fate of rhizodermic cells

The fate of root epidermal cells is controlled by a complex interplay of transcriptional regulators, generating a genetically determined, position-biased arrangement of root hair cells. This pattern is altered during postembryonic development and in response to environmental signals to confer developmental plasticity that acclimates the plant to the prevailing conditions. Based on the hypothesis that events downstream of this initial mechanism can modulate the pattern installed during embryogenesis, we have developed a reaction diffusion model that reproduces the root hair patterning previously observed experimentally. Under all growth conditions, an almost equal spacing between root hair forming cells was observed both in vitro and in silico, indicating that long-range intercellular communication is crucial for the trichoblasts' decision to form a root hair. We assume that a hair growth promoter (HGP) is upregulated in root-hair-forming cells by a trichoblast-specific component. Once established, HGP production is self-enhancing. The autocatalytic regulation of HGP is antagonized by an HGP-produced hair growth inhibitor (HGI). HGI is exported from trichoblasts and diffuses to neighboring cells, where it inhibits further HGP production and promotes the non-hair cell fate. Under conditions of phosphate deficiency, we hypothesise that HGP production is increased and HGI diffusion rate is reduced, leading to a position-independent formation of extra root hairs.

シロイヌナズナの表皮細胞分化に関与するCPC LIKE MYB遺伝子の解析

シロイヌナズナの表皮細胞分化に関与するCPC LIKE MYB遺伝子の解析

PAR1 specifies ciliated cells in vertebrate ectoderm downstream of aPKC.

Partitioning-defective 1 (PAR1) and atypical protein kinase C (aPKC) are conserved serine/threonine protein kinases implicated in the establishment of cell polarity in many species from yeast to humans. Here we investigate the roles of these protein kinases in cell fate determination in Xenopus epidermis. Early asymmetric cell divisions at blastula and gastrula stages give rise to the superficial (apical) and the deep (basal) cell layers of epidermal ectoderm. These two layers consist of cells with different intrinsic developmental potential, including superficial epidermal cells and deep ciliated cells. Our gain- and loss-of-function studies demonstrate that aPKC inhibits ciliated cell differentiation in Xenopus ectoderm and promotes superficial cell fates. We find that the crucial molecular substrate for aPKC is PAR1, which is localized in a complementary domain in superficial ectoderm cells. We show that PAR1 acts downstream of aPKC and is sufficient to stimulate ciliated cell differentiation and inhibit superficial epidermal cell fates. Our results suggest that aPKC and PAR1 function sequentially in a conserved molecular pathway that links apical-basal cell polarity to Notch signaling and cell fate determination. The observed patterning mechanism may operate in a wide range of epithelial tissues in many species.

Water-extractable humic substances alter root development and epidermal cell pattern in Arabidopsis

The effect of a low-molecular weight, water-extractable fraction of humic substances (WEHS) derived from sphagnum peat on post-embryonic plant development has been studied using Arabidopsis roots. Application of humic substances caused an array of changes in root morphology, such as an increase in root hair length and density, formation of ectopic root hairs, and an increase in cell proliferation in the root ground tissue. Application of WEHS affected genes involved in epidermal cell fate specification, suggesting that humic substances can alter developmental programs at an early stage of root cell differentiation. The WEREWOLF and GLABRA2 genes, encoding negative regulators of the root hair cell fate, were significantly down-regulated in the presence of WEHS. Thus, the presence of humic substances caused an ordered remodeling of the root morphology, leading to an increased absorptive surface of the root. Growth in the presence of WEHS did not rescue the phenotype of the root hair defective rhd6 mutant. Analyzing BA3:uidA and DR5:uidA transgenic plants, carrying auxin response elements, and monitoring the expression of the auxin-responsive GH3 gene by real-time RT-PCR did not provide evidence for a WEHS-induced expression of auxin-related genes. It is concluded that WEHS do not exert their effects in an auxin-like manner.

The Xenopus POU class V transcription factor XOct-25 inhibits ectodermal competence to respond to bone morphogenetic protein-mediated embryonic induction

Bone morphogenetic proteins (BMPs) have been shown to play a key role in controlling ectodermal cell fates by inducing epidermis at the expense of neural tissue during gastrulation. Here, we present evidence that the Xenopus POU class V transcription factor XOct-25 regulates ectodermal cell fate decisions by inhibiting the competence of ectodermal cells to respond to BMP during Xenopus embryogenesis. When overexpressed in the ectoderm after the blastula stage, XOct-25 suppressed early BMP responses of ectodermal cells downstream of BMP receptor activation and promoted neural induction while suppressing epidermal differentiation. In contrast, inhibition of XOct-25 function in the prospective neuroectoderm resulted in expansion of epidermal ectoderm at the expense of neuroectoderm. The reduction of neural tissue by inhibition of XOct-25 function could be rescued by decreasing endogenous BMP signaling, suggesting that XOct-25 plays a role in the formation of neural tissue at least in part by inhibiting BMP-mediated epidermal induction (neural inhibition). This hypothesis is supported by the observation that ectodermal cells from XOct-25 morphants were more sensitive to BMP signaling than cells from controls in inducing both immediate early BMP target genes and epidermis at the expense of neural tissue, while cells overexpressing XOct-25 are less competent to respond to BMP-mediated induction. These results document an essential role for XOct-25 in commitment to neural or epidermal cell fates in the ectoderm and highlight the importance of a regulatory mechanism that limits competence to respond to BMP-mediated embryonic induction.

Epidermal stem cell fate: what can we learn from embryonic stem cells?

Because of its constant renewal and high propensity for repair, the epidermis is, together with the gut and the hematopoietic system, a tissue of choice to explore stem cell biology. Previous research over many years has revealed the complexity of the epidermis: the heterogeneity of the stem cell compartment, with its rare, slowly cycling, multipotent, hair-follicle, "bulge" stem cells and the more restricted interfollicular, follicle-matrix, and sebaceous-gland stem cells, which in turn generate the large pool of transit-amplifying progeny. Stem cell activity has been used for some considerable time to repair skin injuries, but ex-vivo keratinocyte amplification has its limitations, and grafted skin homeostasis is not totally satisfactory. Human embryonic stem cells raise the hope that the understanding of the developmental steps leading to the generation of epidermal stem cells and the characterization of the key signaling pathways involved in skin morphogenesis (such as p63) will be translated into therapeutic benefit. Our recent results suggest the feasibility not only of identifying but also of amplifying human ES cells, early ectodermal progenitors with an intact multipotent potential that might improve the quality and functionality of grafts, provided that preclinical in vivo studies confirm our expectations from in vitro analysis.

Thepax-3gene is involved in vulva formation inPristionchus pacificusand is a target of the Hox genelin-39

The Hox gene lin-39 plays a crucial role in the establishment of the nematode vulva equivalence group. Mutations in lin-39 in Caenorhabditis elegans and Pristionchus pacificus result in a vulvaless phenotype because presumptive vulva precursor cells adopt non-vulval fates. Interestingly, the non-vulval fate of anterior and posterior epidermal cells differs between Caenorhabditis and Pristionchus; in C. elegans, non-vulval cells fuse with the hypodermis, whereas, in P. pacificus, they die as a result of programmed cell death. C. elegans lin-39 (Cel-lin-39) indirectly controls the cell fusion gene eff-1 by regulating the GATA transcription factors egl-18 and elt-6. In P. pacificus, the genetic context of its lin-39 (Ppa-lin-39) function was unknown. Here, we describe the isolation and characterization of gev-2, a second generation-vulvaless mutant in P. pacificus. We show that gev-2 is the Ppa-pax-3 gene and that it has distinct functions in the cell fate specification of epidermal cells. Whereas Ppa-pax-3 regulates cell survival of the presumptive vulval precursor cells, it controls cell death of posterior epidermal cells. Molecular studies indicate that Ppa-pax-3 is a direct target of Ppa-LIN-39. Thus, we describe the first specific developmental defect of a nematode pax-3 gene and our data reveal different regulatory networks for the specification of the vulva equivalence group.

Integration of cytokinin and gibberellin signalling byArabidopsistranscription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate

The effective integration of hormone signals is essential to normal plant growth and development. Gibberellins (GA) and cytokinins act antagonistically in leaf formation and meristem maintenance and GA counteract some of the effects of cytokinins on epidermal differentiation. However, both can stimulate the initiation of defensive epidermal structures called trichomes. To understand how their relative influence on epidermal cell fate is modulated, we investigated the molecular mechanisms through which they regulate trichome initiation in Arabidopsis. The control by cytokinins of trichome production requires two genes expressed in late inflorescence organs, ZFP8 and GIS2, which encode C2H2 transcription factors related to GLABROUS INFLORESCENCE STEMS (GIS). Cytokinin-inducible GIS2 plays a prominent role in the cytokinin response, in which it acts downstream of SPINDLY and upstream of GLABROUS1. In addition, GIS2 and ZFP8 mediate, like GIS, the regulation of trichome initiation by gibberellins. By contrast, GIS does not play a significant role in the cytokinin response. Collectively, GIS, ZFP8 and GIS2, which encode proteins that are largely equivalent in function, play partially redundant and essential roles in inflorescence trichome initiation and in its regulation by GA and cytokinins. These roles are consistent with their pattern of expression and with the regional influence of GA and cytokinins on epidermal differentiation. Our findings show that functional specialization within a transcription factor gene family can facilitate the integration of different developmental cues in the regulation of plant cell differentiation.

A Functional Enhancer of Keratin14 Is a Direct Transcriptional Target of ΔNp63

Keratin14 (K14) is a prototypic marker of dividing basal keratinocytes where its gene is transcribed at high levels. Transcriptional regulation of K14 is governed by an evolutionarily conserved functional enhancer marked by DNase 1 hypersensitive sites present upstream of the gene. This enhancer is sufficient to confer epidermal-specific gene expression, which is mediated in part by binding of members of activator protein-2 (AP)-2, AP-1, Ets, and Sp1 families of transcription factors. Here we provide evidence that a keratinocyte-specific nuclear protein identified as deltaNp63 binds to a conserved motif within this enhancer. Interestingly, the selective expression profile of deltaNp63 in various cell lines correlates with both the nuclear complex and the expression of K14. Biochemical studies reveal that deltaNp63 can bind to a specific DNA sequence present in the K14 enhancer and this binding leads to transactivation. In addition, chromatin immunoprecipitation experiments with deltaNp63-specific antibodies demonstrate that the enhancer is occupied by deltaNp63 in cultured keratinocytes and in mouse skin epidermal cells in vivo. Finally, we show that ectopic expression of either p63 isoform (deltaN or TA) can induce de novo expression of K14. These studies provide a potential mechanism by which deltaNp63 directly governs the expression of K14 in a keratinocyte-specific manner.

Reprogramming of root epidermal cells in response to nutrient deficiency

Post-embryonic development of the root system is highly plastic to environmental cues, compensating for the sessile lifestyle of plants. The fate of epidermal cells of Arabidopsis roots is particularly responsive to nutritional signals, leading to an increase in the root's surface area in the absence of the essential but immobile minerals iron, phosphate and manganese. The resulting phenotype is characteristic of the respective condition. Growth under nutrient starvation affects the expression of genes involved in cell specification, indicating that environmental signals are perceived at an early stage of cell development. Cell fate decisions are controlled at different levels, probably integrated at the level of chromatin organization.

Transcriptome and phenotypic analysis reveals Gata3-dependent signalling pathways in murine hair follicles

The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. Here, we describe a conditional Gata3-/- mouse (K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates.

Jagged 1 is a β-catenin target gene required for ectopic hair follicle formation in adult epidermis

The Wnt and Notch signalling pathways regulate hair follicle maintenance, but how they intersect is unknown. We show that Notch signalling is active in the hair follicle pre-cortex, a region of high Wnt activity, where commitment to hair lineages occurs. Deletion of jagged 1 (Jag1) results in inhibition of the hair growth cycle and conversion of hair follicles into cysts of cells undergoing interfollicular epidermal differentiation. Conversely, activation of Notch in adult epidermis triggers expansion of the base of the hair follicle, sebaceous gland enlargement and abnormal clumping of the follicles. In adult epidermis, the induction of new hair follicle formation by beta-catenin is prevented by blocking Notch signalling pharmacologically or through Jag1 deletion. Conversely, activation of both pathways accelerates growth and differentiation of ectopic follicles. beta-catenin stimulates Notch signalling by inducing Jag1 transcription. We conclude that the Notch pathway acts downstream of the Wnt/beta-catenin pathway to determine epidermal cell fate.

BMPs: Conserved Morphogens in Neural Patterning

BMPs: Conserved Morphogens in Neural Patterning

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize

Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.

Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development.

Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.

TheCaenorhabditis elegansGATA factorelt-1is essential for differentiation and maintenance of hypodermal seam cells and for normal locomotion

The Caenorhabditis elegans GATA transcription factor elt-1 has previously been shown to have a central role in the specification of hypodermal (epidermal) cell fates and acts several cell divisions before the birth of hypodermal cells. Here we report that elt-1 also has essential functions during subsequent development. Reporter gene studies show that elt-1 expression is maintained in lateral seam cells throughout development and elt-1 RNA interference experiments support an essential role for elt-1 in the differentiation of lateral seam cells in the embryo. The maintenance of seam-cell fates in all larval stages including L2d and dauer also requires elt-1. The elt-1 RNAi phenotype shows that seam cells are essential for the structural integrity of adult hermaphrodites in the vulval region and for diametric shrinkage during dauer larval formation. By contrast, severe seam-cell loss in the larval stages has little effect on moulting, indicating that the presence of these cells is not essential for this process. The elt-1 reporter gene is also expressed in neurones of the locomotory circuit. Loss of elt-1 function during postembryonic development results in a hypermotility phenotype whereas overexpression of elt-1 leads to a reciprocal phenotype of reduced motility and paralysis. These results suggest that elt-1 is a key regulator of neuronal function in larvae and adult worms.

Following the fate of murine epidermal stem cells in a syngeneic dermal equivalent in vivo

Background Stem cells in healthy epidermis have the capacity to produce structures, such as interfollicular epidermis, hair follicle, and sebaceous glands. Embryonic stem (ES) cells can be induced to differentiate into epidermal stem cells in vitro. These ESCs (epidermal or epidermoid stem cells derived from ES cells) have the potential to be an ideal replacement of those stem cells destroyed in severe injury, such as in deep and extensive burn. The aim of this study was to follow the fate of murine ESCs which were seeded in a syngeneic dermal equivalent and implanted into syngeneic recipient mice subcutaneously. Methods ES cells were induced in vitro to differentiate into ESCs. Stained with a fluorescent dye Hoechst 33342, these ESCs were seeded into a fibroblast–collagen–gelatin sponge complex, functioning as a dermal equivalent model, and then implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells. Results These ESCs were clearly visible in the implant by fluorescent microscopy 3 weeks or longer after implantation. These cells remained viable, differentiating into hair follicle-like structures, glandular structures, and gave rise to additional structures resembling native dermis. A number of markers were expressed in the differentiated structures, including CD29 (integrin β1 subunit) and cytokeratin 18 (CK18). No apparent rejection or severe side effects were observed at least during the 10 weeks following implantation. Conclusions Now that ESCs can survive in vivo in this dermal equivalent model and differentiate into hair follicle-like structures as well as glandular structures, it is feasible to use these cells as seed cells in studies to fabricate dermal equivalents that have the potential to develop dermal appendages.

The WEREWOLF MYB protein directly regulatesCAPRICEtranscription during cell fate specification in theArabidopsisroot epidermis

The Arabidopsis root epidermis is composed of two types of cells, hair cells and non-hair cells, and their fate is determined in a position-dependent manner. WEREWOLF (WER), a R2R3 MYB protein, has been shown genetically to function as a master regulator to control both of the epidermal cell fates. To directly test the proposed role of WER in this system, we examined its subcellular localization and defined its transcriptional activation properties. We show that a WER-GFP fusion protein is functional and accumulates in the nucleus of the N-position cells in the Arabidopsis root epidermis, as expected for a transcriptional regulator. We also find that a modified WER protein with a strong activation domain (WER-VP16) promotes the formation of both epidermal cell types, supporting the view that WER specifies both cell fates. In addition, we used the glucocorticoid receptor (GR) inducible system to show that CPC transcription is regulated directly by WER. Using EMSA, we found two WER-binding sites (WBSs; WBSI and WBSII) in the CPC promoter. WER-WBSI binding was confirmed in vivo using the yeast one-hybrid assay. Binding between the WER protein and both WBSs (WBSI and WBSII), and the importance of the two WBSs in CPC promoter activity were confirmed in Arabidopsis. These results provide experimental support for the proposed role of WER as an activator of gene transcription during the specification of both epidermal cell fates.