Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

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.     

Fine-grained fate maps for the ophthalmic and maxillomandibular trigeminal placodes in the chick embryo

Vertebrate cranial ectodermal placodes are transient, paired thickenings of embryonic head ectoderm that are crucial for the formation of the peripheral sensory nervous system: they give rise to the paired peripheral sense organs (olfactory organs, inner ears and anamniote lateral line system), as well as the eye lenses, and most cranial sensory neurons. Here, we present the first detailed spatiotemporal fate-maps in any vertebrate for the ophthalmic trigeminal (opV) and maxillomandibular trigeminal (mmV) placodes, which give rise to cutaneous sensory neurons in the ophthalmic and maxillomandibular lobes of the trigeminal ganglion. We used focal DiI and DiO labelling to produce eight detailed fate-maps of chick embryonic head ectoderm over approximately 24 h of development, from 0-16 somites. OpV and mmV placode precursors arise from a partially overlapping territory; indeed, some individual dyespots labelled both opV and mmV placode-derived cells. OpV and mmV placode precursors are initially scattered within a relatively large region of ectoderm adjacent to the neural folds, intermingled both with each other and with future epidermal cells, and with geniculate and otic placode precursors. Although the degree of segregation increases with time, there is no clear border between the opV and mmV placodes even at the 16-somite stage, long after neurogenesis has begun in the opV placode, and when neurogenesis is just beginning in the mmV placode. Finally, we find that occasional cells in the border region between the opV placode and mmV placode express both Pax3 (an opV placode specific marker) and Neurogenin1 (an mmV placode specific marker), suggesting that a few cells are responding to both opV and mmV placode-inducing signals. Overall, our results fill a large gap in our knowledge of the early stages of development of both the opV and mmV placodes, providing an essential framework for subsequent studies of the molecular control of their development.     

A fate-map for cranial sensory ganglia in the sea lamprey

Cranial neurogenic placodes and the neural crest make essential contributions to key adult characteristics of all vertebrates, including the paired peripheral sense organs and craniofacial skeleton. Neurogenic placode development has been extensively characterized in representative jawed vertebrates (gnathostomes) but not in jawless fishes (agnathans). Here, we use in vivo lineage tracing with DiI, together with neuronal differentiation markers, to establish the first detailed fate-map for placode-derived sensory neurons in a jawless fish, the sea lamprey Petromyzon marinus, and to confirm that neural crest cells in the lamprey contribute to the cranial sensory ganglia. We also show that a pan-Pax3/7 antibody labels ophthalmic trigeminal (opV, profundal) placode-derived but not maxillomandibular trigeminal (mmV) placode-derived neurons, mirroring the expression of gnathostome Pax3 and suggesting that Pax3 (and its single Pax3/7 lamprey ortholog) is a pan-vertebrate marker for opV placode-derived neurons. Unexpectedly, however, our data reveal that mmV neuron precursors are located in two separate domains at neurula stages, with opV neuron precursors sandwiched between them. The different branches of the mmV nerve are not comparable between lampreys and gnatho-stomes, and spatial segregation of mmV neuron precursor territories may be a derived feature of lampreys. Nevertheless, maxillary and mandibular neurons are spatially segregated within gnathostome mmV ganglia, suggesting that a more detailed investigation of gnathostome mmV placode development would be worthwhile. Overall, however, our results highlight the conservation of cranial peripheral sensory nervous system development across vertebrates, yielding insight into ancestral vertebrate traits.     

Dual embryonic origin of the mammalian otic vesicle forming the inner ear

The inner ear and cochleovestibular ganglion (CVG) derive from a specialized region of head ectoderm termed the otic placode. During embryogenesis, the otic placode invaginates into the head to form the otic vesicle (OV), the primordium of the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV is required for inner ear morphogenesis and neurogenesis. In this study, we show that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute directly to the OV from the neural tube. Using Wnt1-Cre, Pax3(Cre/+) and Hoxb1(Cre/+) mice to label and fate map cranial NEC lineages, we have demonstrated that cells from the neural tube incorporate into the otic epithelium after otic placode induction has occurred. Pax3(Cre/+) labeled a more extensive population of NEC derivatives in the OV than did Wnt1-Cre. NEC derivatives constitute a significant population of the OV and, moreover, are regionalized specifically to proneurosensory domains. Descendents of Pax3(Cre/+) and Wnt1-Cre labeled cells are localized within sensory epithelia of the saccule, utricle and cochlea throughout development and into adulthood, where they differentiate into hair cells and supporting cells. Some NEC derivatives give rise to neuroblasts in the OV and CVG, in addition to their known contribution to glial cells. This study defines a dual cellular origin of the inner ear from sensory placode ectoderm and NECs, and changes the current paradigm of inner ear neurosensory development.     

Molecular analysis of neurogenic placode development in a basal ray-finned fish

Neurogenic placodes are transient, thickened patches of embryonic vertebrate head ectoderm that give rise to the paired peripheral sense organs and most neurons in cranial sensory ganglia. We present the first analysis of gene expression during neurogenic placode development in a basal actinopterygian (ray-finned fish), the North American paddlefish (Polyodon spathula). Pax3 expression in the profundal placode confirms its homology with the ophthalmic trigeminal placode of amniotes. We report the conservation of expression of Pax2 and Pax8 in the otic and/or epibranchial placodes, Phox2b in epibranchial placode-derived neurons, Sox3 during epibranchial and lateral line placode development, and NeuroD in developing cranial sensory ganglia. We identify Sox3 as a novel marker for developing fields of electrosensory ampullary organs and for ampullary organs themselves. Sox3 is also the first molecular marker for actinopterygian ampullary organs. This is consistent with, though does not prove, a lateral line placode origin for actinopterygian ampullary organs.     

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.     

Lens morphogenesis is dependent on Pax6‐mediated inhibition of the canonical Wnt/beta‐catenin signaling in the lens surface ectoderm

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens‐specific gene expression in the surface‐ectoderm. Supression of canonical Wnt/β‐catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down‐regulation of canonical Wnt signaling in the presumptive lens ectoderm. genesis 48:86–95, 2010. © 2009 Wiley‐Liss, Inc.     

Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus

Induction of the otic placode, the rudiment of the inner ear, is believed to depend on signals derived from surrounding tissues, the head mesoderm and the prospective hindbrain. Here we report the first attempt to define the specific contribution of the neuroectoderm to this inductive process in Xenopus. To this end we tested the ability of segments of the neural plate (NP), isolated from different axial levels, to induce the otic marker Pax8 when recombined with blastula stage animal caps. We found that one single domain of the NP, corresponding to the prospective anterior hindbrain, had Pax8-inducing activity in this assay. Surprisingly, more than half of these recombinants formed otic vesicle-like structures. Lineage tracing experiments indicate that these vesicle-like structures are entirely derived from the animal cap and express several pan-otic markers. Pax8 activation in these recombinants requires active Fgf and canonical Wnt signaling, as interference with either pathway blocks Pax8 induction. Furthermore, we demonstrate that Fgf and canonical Wnt signaling cooperate to activate Pax8 expression in isolated animal caps. We propose that in the absence of mesoderm cues the combined activity of hindbrain-derived Wnt and Fgf signals specifies the otic placode in Xenopus, and promotes its morphogenesis into an otocyst.     

The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm

In the current analysis, we have investigated both the cytoskeletal and signaling roles of beta-catenin during the early phases of lens development using conditional loss- and gain-of-function strategies. Conditional loss of beta-catenin in the presumptive lens does not perturb the normal sequential appearance of lens fate markers but results in a dramatic failure of the coordinated epithelial cell behavior that constitutes lens morphogenesis. Similarly, loss-of-function for Lrp6, the Wnt pathway coreceptor expressed in the eye primordium, does not prevent expression of lens induction markers. Surprisingly, conditional deletion of beta-catenin in periocular ectoderm results in the formation of Prox-1 and beta-crystallin-positive ectopic lentoid bodies. Combined with the observation that the Wnt pathway reporter TOPGAL is expressed in nasal periocular ectoderm, these data suggest that, in this location, the canonical Wnt signaling pathway normally suppresses lens fate in favor of other structures. Consistent with this proposal, a dominant-active form of beta-catenin causes a loss of lens fate and a complete absence of lens development when expressed in the presumptive lens ectoderm.     

BMP7 acts in murine lens placode development

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.     

Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.