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.     

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.     

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode‐derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD‐A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near‐total loss of cranial sensory neurons. Taken together, our cell‐lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.     

Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral sensory nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a useful model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the signaling pathways governing opV placode development have not. Here we tested in chick whether the canonical Wnt signaling pathway regulates opV placode development. By introducing a Wnt reporter into embryonic chick head ectoderm, we show that the canonical pathway is active in Pax3+ opV placode cells as, or shortly after, they are induced to express Pax3. Blocking the canonical Wnt pathway resulted in the failure of targeted cells to adopt or maintain an opV fate, as assayed by the expression of various markers including Pax3, FGFR4, Eya2, and the neuronal differentiation markers Islet1, neurofilament, and NeuN, although, surprisingly, it led to upregulation of Neurogenin2, both in the opV placode and elsewhere in the ectoderm. Activating the canonical Wnt signaling pathway, however, was not sufficient to induce Pax3, the earliest specific marker of the opV placode. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells toward an opV placode fate.     

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.     

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.     

Otx expression during lamprey embryogenesis provides insights into the evolution of the vertebrate head and jaw

Agnathan or jawless vertebrates, such as lampreys, occupy a critical phylogenetic position between the gnathostome or jawed vertebrates and the cephalochordates, represented by amphioxus. In order to gain insight into the evolution of the vertebrate head, we have cloned and characterized a homolog of the head-specific gene Otx from the lamprey Petromyzon marinus. This lamprey Otx gene is a clear phylogenetic outgroup to both the gnathostome Otx1 and Otx2 genes. Like its gnathostome counterparts, lamprey Otx is expressed throughout the presumptive forebrain and midbrain. Together, these results indicate that the divergence of Otx1 and Otx2 took place after the gnathostome/agnathan divergence and does not correlate with the origin of the vertebrate brain. Intriguingly, Otx is also expressed in the cephalic neural crest cells as well as mesenchymal and endodermal components of the first pharyngeal arch in lampreys, providing molecular evidence of homology with the gnathostome mandibular arch and insights into the evolution of the gnathostome jaw.     

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.     

Influence of mesodermal Fgf8 on the differentiation of neural crest-derived postganglionic neurons

The interaction between the cranial neural crest (NC) and the epibranchial placode is critical for the formation of parasympathetic and visceral sensory ganglia, respectively. However, the molecular mechanism that controls this intercellular interaction is unknown. Here we show that the spatiotemporal expression of Fibroblast growth factor 8 (Fgf8) is strategically poised to control this cellular relationship. A global reduction of Fgf8 in hypomorph embryos leads to an early loss of placode-derived sensory ganglia and reduced number of NC-derived postganglionic (PG) neurons. The latter finding is associated with the early loss of NC cells by apoptosis. This loss occurs concurrent with the interaction between the NC and placode-derived ganglia. Conditional knockout of Fgf8 in the anterior mesoderm shows that this tissue source of Fgf8 has a specific influence on the formation of PG neurons. Unlike the global reduction of Fgf8, mesodermal loss of Fgf8 leads to a deficiency in PG neurons that is independent of NC apoptosis or defects in placode-derived ganglia. We further examined the differentiation of PG precursors by using a quantitative approach to measure the intensity of Phox2b, a PG neuronal determinant. We found reduced numbers and immature state of PG precursors emerging from the placode-derived ganglia en route to their terminal target areas. Our findings support the view that global expression of Fgf8 is required for early NC survival and differentiation of placode-derived sensory neurons, and reveal a novel role for mesodermal Fgf8 on the early differentiation of the NC along the parasympathetic PG lineage.     

Cadherin‐7 mediates proper neural crest cell–placodal neuron interactions during trigeminal ganglion assembly

The cranial trigeminal ganglia play a vital role in the peripheral nervous system through their relay of sensory information from the vertebrate head to the brain. These ganglia are generated from the intermixing and coalescence of two distinct cell populations: cranial neural crest cells and placodal neurons. Trigeminal ganglion assembly requires the formation of cadherin‐based adherens junctions within the neural crest cell and placodal neuron populations; however, the molecular composition of these adherens junctions is still unknown. Herein, we aimed to define the spatio‐temporal expression pattern and function of Cadherin‐7 during early chick trigeminal ganglion formation. Our data reveal that Cadherin‐7 is expressed exclusively in migratory cranial neural crest cells and is absent from trigeminal neurons. Using molecular perturbation experiments, we demonstrate that modulation of Cadherin‐7 in neural crest cells influences trigeminal ganglion assembly, including the organization of neural crest cells and placodal neurons within the ganglionic anlage. Moreover, alterations in Cadherin‐7 levels lead to changes in the morphology of trigeminal neurons. Taken together, these findings provide additional insight into the role of cadherin‐based adhesion in trigeminal ganglion formation, and, more broadly, the molecular mechanisms that orchestrate the cellular interactions essential for cranial gangliogenesis.     

Embryonic origin of avian corneal sensory nerves.

Sensory nerves play a vital role in maintaining corneal transparency. They originate in the trigeminal ganglion, which is derived from two embryonic cell populations (cranial neural crest and ectodermal placode). Nonetheless, it is unclear whether corneal nerves arise from neural crest, from placode, or from both. Quail-chick chimeras and species-specific antibodies allowed tracing quail-derived neural crest or placode cells during trigeminal ganglion and corneal development, and after ablation of either neural crest or placode. Neural crest chimeras showed quail nuclei in the proximal part of the trigeminal ganglion, and quail nerves in the pericorneal nerve ring and in the cornea. In sharp contrast, placode chimeras showed quail nuclei in the distal part of the trigeminal ganglion, but no quail nerves in the cornea or in the pericorneal nerve ring. Quail placode-derived nerves were present, however, in the eyelids. Neural crest ablation between stages 8 and 9 resulted in diminished trigeminal ganglia and absence of corneal innervation. Ablation of placode after stage 11 resulted in loss of the ophthalmic branch of the trigeminal ganglion and reduced corneal innervation. Noninnervated corneas still became transparent. These results indicate for the first time that although both neural crest and placode contribute to the trigeminal ganglion, corneal innervation is entirely neural crest-derived. Nonetheless, proper corneal innervation requires presence of both cell types in the embryonic trigeminal ganglion. Also, complete lack of innervation has no discernible effect on development of corneal transparency or cell densities.