Neural ectoderm,neural crest,and placodes: Contribution of the otic placode to the ectodermal lining of the embryonic opercular cavity in Atlantic cod (Teleostei)

Development of neural ectoderm, neural crest, and otic placode with special reference to a new placodal derivative, the ectodermal lining of the opercular cavity, is described in a teleost fish, the Atlantic cod Gadus morhua, from a stage-by-stage examination of embryonic development. The ectodermal lining of the opercular cavity forms by invagination of the otic placode. The neural plate “infolds” by a wave of cellular rearrangement that transforms the neural plate into a neural rod. This transformation creates a distinct dorsal ectodermal cell layer. When the neural rod is arranged as monostratified columnar cells in the forebrain and midbrain, dorsal ectoderm at the midbrain level thickens lateral to the neural rod to form a cell cluster—the presumptive neural crest and placode. Upon migration of the neural crest from the postoptic midbrain, the dorsolateral area of the dorsal ectoderm thickens and segregates from the neural crest as a placode that is continuous with the presumptive lens placode. As the neural crest migrates from the hindbrain, this placode extends along the hindbrain as a single continuous cluster of cells. At the onset of formation of the lens placode, this continuous placode becomes the placode in the postoptic area of the midbrain and separates into the otic placode at the hindbrain. The otic placode gives rise to the otic neuromast and probably the otic lateral line nerves rostrally and to the ectodermal cell lining of the opercular cavity and otic vesicles caudally. The opercular cavity forms by invagination of the otic placode, creating an internal lumen lined by ectoderm that becomes continuous with evaginated endodermal pharyngeal cells. Free neuromasts are observed along the trailing edge of the external opening of the opercular cavity, which lies horizontally, ventral to the otic vesicles. As embryos develop to hatching, the opening rotates and takes up a vertical position. The adult opercular apparatus, including associated bones and muscles, forms during larval stages. The otic neuromast may be a remnant of neuromasts in the spiracle organ. The spiracle opening lies between the mandibular and hyoid arches, whereas the opercular cavity opens between the hyoid and the first branchial arches. The spiracle opening is, therefore, not homologous with the external opening of the opercular cavity, although the cell lining of the spiracle opening may be of placodal origin. J Morphol 231:231–252, 1997. © 1997 Wiley-Liss, Inc.     

YY1 regulates the neural crest-associated slug gene in Xenopus laevis

slug gene expression is associated with the specification and migration of neural crest cells in the African clawed frog Xenopus laevis. We provide evidence that the protein Ying-Yang 1 (YY1) regulates the slug gene expression both indirectly and directly, via a YY1 cis-element in the slug promoter, during Xenopus development. The ability of the YY1 to bind this YY1 cis-element was confirmed by electromobility shift assays and reporter assays. YY1 was detected in the nuclei of ectodermal cells contemporaneously with the process of neural crest specification. The injection of anti-YY1 morpholino, which targeted both YY1alpha and YY1beta gene products, depleted YY1 expression below 20% and was lethal at gastrulation. Sublethal depletion of YY1 reduced the length of the anterior-posterior axis and severely inhibited the expression of the neural marker Nrp1 and of the slug gene. Overexpression of YY1 or mutation of the YY1 cis-element reduced the restricted spatial expression of the slug reporter gene in the neural ectoderm border and provoked its expression in the nonneural ectoderm. Chromatin immunoprecipitation indicated that endogenous YY1 interacts directly with the YY1 cis-element of the endogenous slug gene and with the slug gene reporter sequence injected into embryos. The results suggest that YY1 is essential for Xenopus development; is necessary for neural ectoderm differentiation, a prerequisite for neural crest specification; and restricts which cells can form neural crest mesenchyme through directly blocking slug gene activity.     

Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm

A number of regulatory genes have been implicated in neural crest development. However, the molecular mechanism of how neural crest determination is initiated in the exact ectodermal location still remains elusive. Here, we show that the cooperative function of Pax3 and Zic1 determines the neural crest fate in the amphibian ectoderm. Pax3 and Zic1 are expressed in an overlapping manner in the presumptive neural crest area of the Xenopus gastrula, even prior to the onset of the expression of the early bona fide neural crest marker genes Foxd3 and Slug. Misexpression of both Pax3 and Zic1 together efficiently induces ectopic neural crest differentiation in the ventral ectoderm, whereas overexpression of either one of them only expands the expression of neural crest markers within the dorsolateral ectoderm. The induction of neural crest differentiation by Pax3 and Zic1 requires Wnt signaling. Loss-of-function studies in vivo and in the animal cap show that co-presence of Pax3 and Zic1 is essential for the initiation of neural crest differentiation. Thus, co-activation of Pax3 and Zic1, in concert with Wnt, plays a decisive role for early neural crest determination in the correct place of the Xenopus ectoderm.     

Indian hedgehog signaling is required for proper formation, maintenance and migration of Xenopus neural crest

Neural crest induction is the result of the combined action at the neural plate border of FGF, BMP, and Wnt signals from the neural plate, mesoderm and nonneural ectoderm. In this work we show that the expression of Indian hedgehog (Ihh, formerly named Banded hedgehog) and members of the Hedgehog pathway occurs at the prospective neural fold, in the premigratory and migratory neural crest. We performed a functional analysis that revealed the requirement of Ihh signaling in neural crest development. During the early steps of neural crest induction loss of function experiments with antisense morpholino or locally grafted cyclopamine-loaded beads suppressed the expression of early neural crest markers concomitant with the increase in neural and epidermal markers. We showed that changes in Ihh activity produced no alterations in either cell proliferation or apoptosis, suggesting that this signal involves cell fate decisions. A temporal analysis showed that Hedgehog is continuously required not only in the early and late specification but also during the migration of the neural crest. We also established that the mesodermal source of Ihh is important to maintain specification and also to support the migratory process. By a combination of embryological and molecular approaches our results demonstrated that Ihh signaling drives in the migration of neural crest cells by autocrine or paracrine mechanisms. Finally, the abrogation of Ihh signaling strongly affected only the formation of cartilages derived from the neural crest, while no effects were observed on melanocytes. Taken together, our results provide insights into the role of the Ihh cell signaling pathway during the early steps of neural crest development.     

Origins and Developmental Potential of the Neural Crest

Neural crest cells are a migratory population that forms most of the peripheral nervous system, facial skeleton, and numerous other derivatives. These cells arise from the neural ectoderm and are first recognizable as discrete cells after neural tube closure. In this review, I summarize the results of studies from our laboratory on neural crest cell lineage and origin. Our recent experiments demonstrate that interactions between the presumptive neural plate and the nonneural ectoderm are likely to be instrumental in the induction of the avian neural crest. Juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. However, neural crest cells do not appear to be segregated from other neuroepithelial cells; cell lineage studies have demonstrated that individual precursor cells within the neural tube can give rise to both neural crest and neural tube derivatives as diverse as sensory, commissural, and motor neurons. This suggests that individual neuroectodermal cells are multipotent, such that a precursor within the neural tube has the ability to form both neural tube (central nervous system) and neural crest (peripheral nervous system and other) derivatives. Further support for flexibility in the developmental program of neuroepithelial cells comes from experiments in which the cranial neural folds are ablated; this results in regulation by the remaining ventral neural tube cells to form neural crest cells after the endogenous neural crest is removed. At later stage of development, this regulative capacity is lost. Following their emigration from the neural tube, neural crest cells become progressively restricted to defined embryonic states. Taken together, these experiments demonstrate that: (1) the neural crest is an induced population that arises by interactions within the ectoderm; (2) initially, progenitor cells are multipotent, having the potential to form multiple neural crest and neural tube derivatives; and (3) with time, the precursors become progressively restricted to form neural crest derivatives and eventually to individual phenotypes.     

Conditional BMP inhibition in Xenopus reveals stage-specific roles for BMPs in neural and neural crest induction

Bone morphogenetic protein (BMP) inhibition has been proposed as the primary determinant of neural cell fate in the developing Xenopus ectoderm. The evidence supporting this hypothesis comes from experiments in explanted "animal cap" ectoderm and in intact embryos using BMP antagonists that are unregulated and active well before gastrulation. While informative, these experiments cannot answer questions regarding the timing of signals and the behavior of cells in the more complex environment of the embryo. To examine the effects of BMP antagonism at defined times in intact embryos, we have generated a novel, two-component system for conditional BMP inhibition. We find that while blocking BMP signals induces ectopic neural tissue both in animal caps and in vivo, in intact embryos, it can only do so prior to late blastula stage (stage 9), well before the onset of gastrulation. Later inhibition does not induce neural identity, but does induce ectopic neural crest, suggesting that BMP antagonists play temporally distinct roles in establishing neural and neural crest identity. By combining BMP inhibition with fibroblast growth factor (FGF) activation, the neural inductive response in whole embryos is greatly enhanced and is no longer limited to pre-gastrula ectoderm. Thus, BMP inhibition during gastrulation is insufficient for neural induction in intact embryos, arguing against a BMP gradient as the sole determinant of ectodermal cell fate in the frog.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.     

Molecular and tissue interactions governing induction of cranial ectodermal placodes

Whereas neural crest cells are the source of the peripheral nervous system in the trunk of vertebrates, the “ectodermal placodes,” together with neural crest, form the peripheral nervous system of the head. Cranial ectodermal placodes are thickenings in the ectoderm that subsequently ingress or invaginate to make important contributions to cranial ganglia, including epibranchial and trigeminal ganglia, and sensory structures, the ear, nose, lens, and adenohypophysis. Recent studies have uncovered a number of molecular signals mediating induction and differentiation of placodal cells. Here, we described recent advances in understanding the tissue interactions and signals underlying induction and neurogenesis of placodes, with emphasis on the trigeminal and epibranchial. Important roles of Fibroblast Growth Factors, Platelet Derived Growth Factors, Sonic Hedgehog, TGFβ superfamily members, and Wnts are discussed.     

A balance of FGF, BMP and WNT signalling positions the future placode territory in the head

The sensory nervous system in the vertebrate head arises from two different cell populations: neural crest and placodal cells. By contrast, in the trunk it originates from neural crest only. How do placode precursors become restricted exclusively to the head and how do multipotent ectodermal cells make the decision to become placodes or neural crest? At neural plate stages, future placode cells are confined to a narrow band in the head ectoderm, the pre-placodal region (PPR). Here, we identify the head mesoderm as the source of PPR inducing signals, reinforced by factors from the neural plate. We show that several independent signals are needed: attenuation of BMP and WNT is required for PPR formation. Together with activation of the FGF pathway, BMP and WNT antagonists can induce the PPR in na?ve ectoderm. We also show that WNT signalling plays a crucial role in restricting placode formation to the head. Finally, we demonstrate that the decision of multipotent cells to become placode or neural crest precursors is mediated by WNT proteins: activation of the WNT pathway promotes the generation of neural crest at the expense of placodes. This mechanism explains how the placode territory becomes confined to the head, and how neural crest and placode fates diversify.     

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.     

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.     

Essential role for PDGF signaling in ophthalmic trigeminal placode induction

Much of the peripheral nervous system of the head is derived from ectodermal thickenings, called placodes, that delaminate or invaginate to form cranial ganglia and sense organs. The trigeminal ganglion, which arises lateral to the midbrain, forms via interactions between the neural tube and adjacent ectoderm. This induction triggers expression of Pax3, ingression of placode cells and their differentiation into neurons. However, the molecular nature of the underlying signals remains unknown. Here, we investigate the role of PDGF signaling in ophthalmic trigeminal placode induction. By in situ hybridization, PDGF receptor beta is expressed in the cranial ectoderm at the time of trigeminal placode formation, with the ligand PDGFD expressed in the midbrain neural folds. Blocking PDGF signaling in vitro results in a dose-dependent abrogation of Pax3 expression in recombinants of quail ectoderm with chick neural tube that recapitulate placode induction. In ovo microinjection of PDGF inhibitor causes a similar loss of Pax3 as well as the later placodal marker, CD151, and failure of neuronal differentiation. Conversely, microinjection of exogenous PDGFD increases the number of Pax3+ cells in the trigeminal placode and neurons in the condensing ganglia. Our results provide the first evidence for a signaling pathway involved in ophthalmic (opV) trigeminal placode induction.