Prospective Epidermis Research Articles

From neural crest cells to melanocytes: cellular plasticity during development and beyond.

Here, we review melanocyte development and how the embryonic melanoblast, although specified to become a melanocyte, is prone to cellular plasticity and is not fully committed to the melanocyte lineage. Even fully differentiated and pigment-producing melanocytes do not always have a stable phenotype. The gradual lineage restriction of neural crest cells toward the melanocyte lineage is determined by both cell-intrinsic and extracellular signals in which differentiation and pathfinding ability reciprocally influence each other. These signals are leveraged by subtle differences in timing and axial positioning. The most extensively studied migration route is the dorsolateral path between the dermomyotome and the prospective epidermis, restricted to melanoblasts. In addition, the embryonic origin of the skin dermis through which neural crest derivatives migrate may also affect the segregation between melanogenic and neurogenic cells in embryos. It is widely accepted that, irrespective of the model organism studied, the immediate precursor of both melanoblast and neurogenic populations is a glial-melanogenic bipotent progenitor. Upon exposure to different conditions, melanoblasts may differentiate into other neural crest-derived lineages such as neuronal cells and vice versa. Key factors that regulate melanoblast migration and patterning will regulate melanocyte homeostasis during different stages of hair cycling in postnatal hair follicles.

Abstract 1541: The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation

Abstract Introduction: Our lab discovered that mutations in the tumor suppressor BAP1 are strongly associated with metastasis and death in patients with uveal melanoma. Subsequently, other cancers have been found to harbor BAP1 mutations, including skin melanoma, kidney cancer, mesothelioma and others. Germline BAP1 mutations are responsible for a newly described genetic cancer syndrome. Therapeutic molecules that reverse the effects of BAP1 mutations could represent a potent new treatment strategy for BAP1-mutant cancers. Unfortunately, there are several obstacles to developing such therapies. First, BAP1 is a tumor suppressor that is inactivated by mutations, such that targeted therapy would need to be directed against downstream effectors that are deregulated by BAP1 loss. Second, the effectors of BAP1 that are relevant to cancer are not known. Interestingly, most known proteins that interact with Bap1 are developmental epigenetic regulators such as Asxl1/2, Cbx1/3 and Kdm1b. Third, BAP1 is difficult to study in cultured cells because BAP1 loss results in cell cycle exit and stem cell-like behavior. These obstacles led us to shift to Xenopus laevis as an in vivo developmental model to study the functions of BAP1. Results and conclusions: Loss of BAP1 during embryo development results in a failure to turn off pluripotency genes such as ventx 1, ventx2 (Xenopus orthologues of the mammal gene nanog), oct 25, oct 91 (Xenopus orthologues of the mammal gene oct3/4) and pax3, and a failure to induce lineage specification genes such as the prospective epidermis marker keratin1 and the melanocyte precursor marker sox10. This block in the shift from pluripotency to differentiation programs results in a delay in gastrulation, neural crest specification and migration, mesodermal differentiation and other phenotypes. The BAP1-deficient phenotype can be rescued by Xenopus or human wildtype BAP1 or by the histone deacetylase inhibitor SAHA (vorinostat). We conclude that BAP1 is a fundamental regulator of multiple developmental lineages, including the neural crest from which melanomas arises, and that this in vivo model can be used to screen for novel therapeutic compounds that reverse the phenotypic effects of BAP1 loss. Citation Format: Jeffim N. Kuznetsov, Tristan Aguero, Stefan Kurtenbach, Matthew G. Field, Mary Lou King, J William Harbour. The tumor suppressor BAP1 promotes a developmental switch from pluripotency to differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1541. doi:10.1158/1538-7445.AM2017-1541

Cadherin interplay during neural crest segregation from the non-neural ectoderm and neural tube in the early chick embryo.

In the avian embryo, neural crest (NC) progenitors arise in the neuroectoderm during gastrulation, long before their dissemination. Although the gene regulatory network involved in NC specification has been deciphered, the mechanisms involved in their segregation from the other neuroectoderm-derived progenitors, notably the epidermis and neural tube, are unknown. Because cadherins mediate cell recognition and sorting, we scrutinized their expression profiles during NC specification and delamination. We found that the NC territory is defined precociously by the robust expression of Cadherin-6B in cells initially scattered among other cells uniformly expressing E-cadherin, and that NC progenitors are progressively sorted and regrouped into a discrete domain between the prospective epidermis and neural tube. At completion of NC specification, the epidermis, NC, and neural tube are fully segregated in contiguous compartments characterized by distinct cadherin repertoires. We also found that Cadherin-6B down-regulation constitutes a major event during NC delamination and that, with the exception of the caudal part of the embryo, N-cadherin is unlikely to control NC emigration. Our results indicate that partition of the neuroectoderm is mediated by cadherin interplays and ascribes a key role to Cadherin-6B in the specification and delamination of the NC population. Developmental Dynamics 246:550-565, 2017. © 2017 Wiley Periodicals, Inc.

Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos

The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W(sh)/W(sh)), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases.

The neural crest.

The neural crest (NC) is a highly migratory multipotent cell population that forms at the interface between the neuroepithelium and the prospective epidermis of a developing embryo. Following extensive migration throughout the embryo, NC cells eventually settle to differentiate into multiple cell types, ranging from neurons and glial cells of the peripheral nervous system to pigment cells, fibroblasts to smooth muscle cells, and odontoblasts to adipocytes. NC cells migrate in large numbers and their migration is regulated by multiple mechanisms, including chemotaxis, contact-inhibition of locomotion and cell sorting. Here, we provide an overview of NC formation, differentiation and migration, highlighting the molecular mechanisms governing NC migration.

Embryology of a planktonic tunicate reveals traces of sessility.

A key problem in understanding deuterostome evolution has been the origin of the chordate body plan. A biphasic life cycle with a sessile adult and a free-swimming larva is traditionally considered ancestral in chordates with subsequent neotenic loss of the sessile adult stage. Molecular phylogenies challenged this view, suggesting that the primitive life cycle in chordates was entirely free-living as in modern day larvaceans. Here, we report the precise cell lineage and fate map in the normal embryo of the larvacean Oikopleura dioica, using 4D microscopy technique and transmission electron microscopy. We document the extraordinary rapidity of cleavage and morphogenetic events until hatching and demonstrate that--compared with ascidians--fate restriction occurs considerably earlier in O. dioica and that clonal organization of the cell lineage is more tightly coupled to tissue fate. We show that epidermal cells in the trunk migrate through 90 degrees, reminiscent of events in ascidian metamorphosis and that the axis of bilateral symmetry in the tail rotates in relation to the trunk. We argue that part of the tail muscle cells are ectomesodermal, because they are more closely associated with prospective epidermis than with other tissues in the cell lineage. Cladistic comparison with other deuterostomes suggests that these traits are derived within tunicates strengthening the hypothesis that the last common ancestor of tunicates had a sessile adult and thus support traditional morphology-derived scenarios. Our results allow hypothesizing that molecular developmental mechanisms known from ascidian models are restricted to fewer, yet identifiable, cells in O. dioica.

CDermo-1 misexpression induces dense dermis, feathers, and scales

Reciprocal epithelio-mesenchymal interactions between the prospective epidermis and the underlying dermis are the major driving forces in the development of skin appendages. Feather development is initiated by a still unknown signal from the dermis in feather-forming skin. The morphological response of the ectoderm to this signal is the formation of an epidermal placode, which signals back to the mesenchyme to induce dermal condensations. Together, epidermal and dermal components constitute the outgrowing feather bud. The bHLH transcription factor cDermo-1 is expressed in developing dermis and is the earliest known marker of prospective feather tracts. To test its function during feather development, we forced cDermo-1 expression in embryonic chicken dermis using a retroviral expression vector. In featherless (apteric) regions, cDermo-1 misexpression induced dense, thickened dermis normally observed in feathered skin (pterylae), and leads to the development of regularly spaced and normally shaped ectopic feather buds. In pterylae, cDermo-1 misexpression enhanced feather growth. In hindlimb skin, according to the local skin identity, misexpression of cDermo-1 induced ectopic scale formation. Thus, we show that forced cDermo-1 expression in developing dermis is sufficient to launch the developmental program leading to skin appendage formation. We propose a role of cDermo-1 at the initial stages of feather induction upstream of FGF10.

Molecular mechanisms of neural crest induction.

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.

Transcription Factor AP-2 Is an Essential and Direct Regulator of Epidermal Development in Xenopus

Expression of the Xenopus homolog of the mammalian transcription factor AP-2α (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo.

Calcium signaling during convergent extension in Xenopus.

During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.

Avian neural crest cell fate decisions: a diffusible signal mediates induction of neural crest by the ectoderm

During neurulation, a region of central ectoderm becomes thickened to form the neural plate which then folds upon itself to generate the neural tube, from which all neurons and glia cells of the central nervous system arise. Neural crest cells form at the border of the neural plate, where it abuts the prospective epidermis. The neural crest is a transient population of cells that undergo an epithelial-mesenchymal transition, become highly migratory and subsequently differentiate into most of the peripheral nervous systems as well as numerous other derivatives. The origin of neural crest cells at the epidermal–neural plate border suggests that an interaction between these two tissues may be involved in neural crest formation. By experimentally juxtaposing prospective epidermis with naive neural plate, we previously showed that an inductive interaction between these tissues can generate neural crest cells. Here, we further characterize the nature of this inductive interaction by co-culturing isolated neural plate and prospective epidermis on opposing sides of polycarbonate filters with differing pore sizes. We find that neural crest cells are generated even when epidermis and neural plate are separated by filters that do not allow cell contact. These results suggest that the epidermal inducer is a diffusible, secreted molecule. We discuss the developmental potential of neural crest precursors and lineage decisions that effect their differentiation into numerous derivatives.

Snail-Related Transcriptional Repressors Are Required in Xenopus for both the Induction of the Neural Crest and Its Subsequent Migration

The neural crest is a transient population of precursor cells that arises at the border between the neural plate and prospective epidermis in vertebrate embryos. The earliest known response to neural-crest-inducing signals is the expression of the zinc-finger transcription factors slug and snail. Although it is widely believed that these transcription factors play an essential role in neural crest development, relatively little is understood about their mechanism of action during this process. We have previously shown that overexpression of XSlug leads to expanded expression of neural crest markers and an excess of at least one neural crest derivative, melanocytes. In order to further investigate XSlug function, we overexpressed mutant constructs in which the DNA-binding domain was fused to either the activation domain from Gal4 or the repressor domain from Drosophila Engrailed. The Engrailed repressor fusion was found to mimic the effects of wild-type XSlug, indicating that XSlug functions as a transcriptional repressor during neural crest formation. In contrast, overexpression of either the activation domain fusion or the DNA-binding domain alone was found to inhibit XSlug function. Using a hormone-inducible inhibitory mutant, we show that inhibition of XSlug function at early stages prevents the formation of neural crest precursors, while inhibition at later stages interferes with neural crest migration, demonstrating for the first time that this transcriptional repressor is required during multiple stages of neural crest development.

Mechanisms of early neural crest development: From cell specification to migration

The neural crest is a group of embryonic progenitors that forms during the process of neurulation by interactions that take place between the prospective epidermis and the specified neuroectoderm. Although initially an integral part of the neuroepithelium, neural crest cells separate from the central nervous system primordium by a process of epitheliomesenchymal transition and become a motile cell population. These mesenchymal cells then migrate through stereotypic pathways, some of which are common and others unique to various vertebrate species. Furthermore, the availability of distinct migratory pathways also differs according to embryonic stage and axial level. Studies have begun to address the molecular basis of neural crest specification, delamination, and migration. The present review summarizes some major advances in our understanding of the nature of the intercellular interactions and the molecules that mediate them during early phases of neural crest ontogeny.

Ectodermal patterning in the avian embryo: epidermis versus neural plate.

Ectodermal patterning of the chick embryo begins in the uterus and continues during gastrulation, when cells with a neural fate become restricted to the neural plate around the primitive streak, and cells fated to become the epidermis to the periphery. The prospective epidermis at early stages is characterized by the expression of the homeobox gene DLX5, which remains an epidermal marker during gastrulation and neurulation. Later, some DLX5-expressing cells become internalized into the ventral forebrain and the neural crest at the hindbrain level. We studied the mechanism of ectodermal patterning by transplantation of Hensen's nodes and prechordal plates. The DLX5 marker indicates that not only a neural plate, but also a surrounding epidermis is induced in such operations. Similar effects can be obtained with neural plate grafts. These experiments demonstrate that the induction of a DLX5-positive epidermis is triggered by the midline, and the effect is transferred via the neural plate to the periphery. By repeated extirpations of the endoderm we suppressed the formation of an endoderm/mesoderm layer under the epiblast. This led to the generation of epidermis, and to the inhibition of neuroepithelium in the naked ectoderm. This suggests a signal necessary for neural, but inhibitory for epidermal development, normally coming from the lower layers. Finally, we demonstrate that BMP4, as well as BMP2, is capable of inducing epidermal fate by distorting the epidermis-neural plate boundary. This, however, does not happen independently within the neural plate or outside the normal DLX5 domain. In the area opaca, the co-transplantation of a BMP4 bead with a node graft leads to the induction of DLX5, thus indicating the cooperation of two factors. We conclude that ectodermal patterning is achieved by signalling both from the midline and from the periphery, within the upper but also from the lower layers.

Geminin, a neuralizing molecule that demarcates the future neural plate at the onset of gastrulation.

In an expression cloning screen in Xenopus embryos, we identified a gene that when overexpressed expanded the neural plate at the expense of adjacent neural crest and epidermis. This gene, which we named geminin, had no sequence similarity to known gene families. We later discovered that geminin's neuralizing domain was part of a bifunctional protein whose C-terminal coiled-coil domain may play a role in regulating DNA replication. We report here on the neuralizing function of geminin. The localization, effect of misexpression and activity of a dominant negative geminin suggest that the product of this gene has an essential early role in specifying neural cell fate in vertebrates. Maternal geminin mRNA is found throughout the animal hemisphere from oocyte through late blastula. At the early gastrula, however, expression is restricted to a dorsal ectodermal territory that prefigures the neural plate. Misexpression of geminin in gastrula ectoderm suppresses BMP4 expression and converts prospective epidermis into neural tissue. In ectodermal explants, geminin induces expression of the early proneural gene neurogenin-related 1 although not itself being induced by that gene. Later, embryos expressing geminin have an expanded dorsal neural territory and ventral ectoderm is converted to neurons. A putative dominant negative geminin lacking the neuralizing domain suppresses neural differentiation and, when misexpressed dorsally, produces islands of epidermal gene expression within the neurectodermal territory, effects that are rescued by coexpression of the full-length molecule. Taken together, these data indicate that geminin plays an early role in establishing a neural domain during gastrulation.

7 Neural Crest Diversification

This chapter discusses some of the basic principles that have emerged from the study of neural crest biology. It discusses the origin of the neural crest in the embryo and considers some of the general principles underlying neural crest diversification. Finally, the chapter discusses what is known about how specific derivatives of the neural crest are generated. The neural crest has captured the imagination of developmental biologists for over a century. There are two principal reasons for this. First, it is a highly migratory cell population and very little is known about the mechanisms that precisely guide neural crest cells to specific sites in the embryo. Second, the neural crest gives rise to an enormous diversity of cell types, including the entire peripheral nervous system, melanocytes, endocrine cells, and much of the connective tissue, bone, and cartilage of the face and skull. Neural crest cells arise at the junction between the prospective epidermis and the prospective neural plate in all vertebrates, regardless of whether the neural crest ultimately migrates from the open neural folds, the closed neural tube, or an ectodermal thickening at the neural plate-epidermis boundary. Although a number of models have been proposed for how neural crest cells are generated, the weight of evidence at present suggests that neural crest arises from local cell-cell interactions between the epidermis and the neural plate.

A Member of the Met/HGF-Receptor Family Is Expressed in a BMP-4-like Pattern in the Ectoderm ofXenopusGastrulae

The importance and involvement of growth factors and their corresponding receptors in embryonic induction has been more and more recognized during the past decade, in particular by loss-of-function experiments using dominant negative receptors. Here, we report the isolation of XHR, aXenopusreceptor-type tyrosine kinase, with homology to members of the Met/hepatocyte growth factor (HGF)-receptor family. Sequence comparison of XHR with other members of the Met/HGF-receptor family as well as in situ expression analyses suggest that XHR represents a novel member of this family of receptor-type tyrosine kinases. As could be shown by whole-mount in situ analysis, XHR transcripts are first expressed in the entire ectoderm at the onset of gastrulation. As gastrulation proceeds, XHR-transcription is turned off in cells induced by dorsal mesoderm to form neural tissue and thus, becomes predominantly confined to prospective epidermis. The strikingly similar expression patterns of XHR and Bone Morphogenetic Protein-4 (BMP-4), an inducer of epidermis and inhibitor of neural development, suggest an involvement of XHR signalling in the early cell-fate decision of ectodermal cells to form either neural derivatives or epidermis.

Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.

During gastrulation of Xenopus embryos the prospective mesoderm is induced initially with domains of dorsal and ventral fate, then further patterned to generate somitic mesoderm by signals from the gastrula organizer. Although Xwnt-8 is expressed in future ventrolateral mesoderm and induces prospective epidermis to differentiate in vitro as ventral mesoderm, no loss-of-function studies have demonstrated a requirement for Wnt signaling for the normal expression of mesodermal genes in the gastrula. We report development of a dominant-negative Wnt (dnXwnt-8) that inhibits embryonic responses to Wnt signaling in a cell-nonautonomous fashion. By expressing dnXwnt-8 in Xenopus embryos, we uncover a requirement of Wnt signaling for localized expression in prospective mesoderm of XMyoDa and Xenopus-posterior (Xpo). Because ectopic expression of functional Xwnt-8 in the dorsal marginal zone of the gastrula induces ectopic XMyoDa and Xpo, both gain-of-function and loss-of-function experiments support a model in which endogenous Xwnt-8 functions to induce expression of genes involved in specification of ventral and somitic mesoderm.

Origins of the avian neural crest: the role of neural plate-epidermal interactions

We have investigated the lineage and tissue interactions that result in avian neural crest cell formation from the ectoderm. Presumptive neural plate was grafted adjacent to non-neural ectoderm in whole embryo culture to examine the role of tissue interactions in ontogeny of the neural crest. Our results show that juxtaposition of non-neural ectoderm and presumptive neural plate induces the formation of neural crest cells. Quail/chick recombinations demonstrate that both the prospective neural plate and the prospective epidermis can contribute to the neural crest. When similar neural plate/epidermal confrontations are performed in tissue culture to look at the formation of neural crest derivatives, juxtaposition of epidermis with either early (stages 4-5) or later (stages 6-10) neural plate results in the generation of both melanocytes and sympathoadrenal cells. Interestingly, neural plates isolated from early stages form no neural crest cells, whereas those isolated later give rise to melanocytes but not crest-derived sympathoadrenal cells. Single cell lineage analysis was performed to determine the time at which the neural crest lineage diverges from the epidermal lineage and to elucidate the timing of neural plate/epidermis interactions during normal development. Our results from stage 8 to 10+ embryos show that the neural plate/neural crest lineage segregates from the epidermis around the time of neural tube closure, suggesting that neural induction is still underway at open neural plate stages.

The development of the neural crest in amphibians

Our review deals with the development of the neural crest (NC) in amphibians. We will consider relevant aspects of evolution, ontogeny, migration and differentiation, and investigate principal problems such as the regulation of NC cell determination, pathway selection and destination recognition. Earlier data and more recent findings will be presented. The NC probably evolved about 440 million years ago from the anlagen of epidermal nerve plexuses in protochordates. In urodele amphibians, the prospective NC is already present in the early gastrula as a narrow band of ectodermal cells between the prospective epidermis and the prospective neural plate. The NC proper develops later from the apices of the neural folds and forms, after neural fold fusion, a transient cellular ridge on the dorsal surface of the neural tube. NC cells migrate extensively into various body regions and give rise to a wide variety of derivatives including the mesenchymal elements of the skull, the neural and glial precursors of the peripheral nervous system and the pigment cells. NC cell migration is stimulated by components of the extracellular matrix (ECM) and may conveniently be analyzed in the system of wild-type (dark) and white mutant axolotl embryos. Skeletal elements of the head derive from cranial NC cells following an interaction with pharyngeal endoderm. Other derivatives of the NC are the ganglia of the peripheral nervous system in the head (sensory ganglia; mostly mixed origin with placodal material) and trunk (spinal, sympathetic and enteric ganglia). Pigment cells also derive from the NC and become arranged into uniform or banded pigment patterns.