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.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras

The quail-chick marker system has been used to study the early developmental stages of the ganglia located along cranial nerves VII, IX, and X. The streams of neural crest cells arising from the rhombencephalic-vagal neural crest were followed from the onset of their migration up to the localization of crest cells in the trunk and root ganglia of these nerves. It was shown that two different populations of crest cells are segregated early as a result of morphogenetic movements in the hypobranchial region. The dorsal population gives rise to the root ganglia of nerves IX and X located close to the encephalic vesicles, where the crest cells differentiate both into neurons and into glia. In contrast, the ventral stream of neural crest cells contributes together with cells from epibranchial placodes to the trunk ganglia (geniculate, petrous, and nodose ganglia) of cranial nerves VII, IX, and X. The successive steps of the invasion of the placodal anlage by crest cells can be followed owing to the selective labeling of the neural crest cells. It appears that the latter give rise to the satellite cells of the geniculate, petrous, and nodose ganglia while the large sensory neurons originate from the placodes. The nodose ganglion has been the subject of further studies aimed to investigate whether neuronal potentialities can be elicited in the neural crest-derived cells that it contains. The ability to label selectively either the neurons or the glia by the quail nuclear marker made this investigation possible in the particular case of the nodose ganglion whose neurons and satellite cells have a different embryonic origin. By the technique already described (N. M. Le Douarin, M. A. Teillet, C. Ziller, and J. Smith, 1978, Proc. Nat. Acad. Sci. USA75, 2030–2034) of back-transplantation into the neural crest migration pathway of a younger host, it was shown that the presumptive glial cells of the nodose ganglion are able to remigrate when transplanted into a 2-day chick host and to differentiate into autonomic structures (sympathetic ganglion cells, adrenomedullary cells, and enteric ganglia). It is proposed as a working hypothesis that neuronal potentialities contained in the neural crest cells which invade the placodal primordium of the nodose ganglion are repressed through cell-cell interactions occurring between placodal and crest cells.     

Embryonic origin of substance P containing neurons in cranial and spinal sensory ganglia of the avian embryo

The ontogeny of the neurons exhibiting substance P-like immunoreactivity (SPLI) was examined in the spinal and cranial sensory ganglia of chick and quail embryos. It was shown that in dorsal root ganglia (DRG) virtually all neuronal somas occupying the mediodorsal (MD) region of the ganglia are SPLI-positive while the larger neurons of the lateroventral (LV) area are SPLI-negative. In the cranial nerve ganglia, both types of neurons coexist in the trigeminal ganglion but with a different distribution: small neurons with SPLI are proximal while large neurons without SPLI occupy the maxillomandibular and ophthalmic lobes. The distal ganglia of nerves VII and IX (i.e., geniculate, petrosal) do not show cell bodies with SPLI in the two species considered. A few of them only (about 12%) are found in the nodose (distal ganglion of nerve X). The proximal ganglia of nerves IX and X (i.e., superior-jugular complex) are composed of small neurons which virtually all exhibit SPLI. Chimaeric cranial sensory ganglia were constructed by grafting the quail hind-brain primordium into chick embryos. Revelation of SPLI was combined with acridine orange staining on the same sections in order to ascertain the placodal (chick host) or neural crest (quail donor) origin of the SP-positive neurons in each type of ganglion. We found that all the neurons showing SPLI are derived from the neural crest in the trigeminal and in the superior and jugular ganglia. In the geniculate, petrosal, and nodose all the neurons are derived from the placodal ectoderm. The small number of SPLI-positive cells of the nodose ganglia are not an exception to this rule. Therefore, generally speaking, the sensory neurons of the cranial ganglia that express the SP phenotype are derived from the crest, with the exception of some neurons present in the nodose of both quail and chick embryos and which are of placodal origin. The vast majority of placode-derived neurons do not have amounts of SP that can be detected under the conditions of the present study.     

The control of avian cephalic neural crest cytodifferentiation. II. Neural tissues.

A series of neural crest transplantations has been performed to (1) analyze whether avian premigratory cranial neural crest cells are pluripotential or restricted to specific developmental pathways and (2) examine the ability of trunk neural crest cells to develop in an environment usually occupied by cranial crest cells. Quail embryos, the cells of which have a unique nuclear marker, were used as donors and chick embryos as hosts. Hindbrain crest cells grafted in the place of diencephalic crest cells failed to form neurons in all but one case, in which a small ectopic ganglion was found. In the reciprocal transplants, neural crest cells emigrating from a segment of forebrain crest tissue grafted in the place of metencephalic crest cells produced trigeminal and ciliary ganglia which were completely normal. Thus, crest cells which normally never form ganglionic neurons will do so if placed in a suitable neurogenic environment. These results prove that premigratory avian cranial crest cells are not restricted to specific developmental pathways, but are initially pluripotential. Trunk crest cells grafted in the place of metencephalic crest cells form neuronal ganglia along the proximal trigeminal motor roots but do not form normal trigeminal ganglia. These root ganglia do not display normal peripheral projections, and placode cells, a normal component of the trigeminal ganglion, form ganglia in ectopic locations. Thus, while trunk crest cells respond to the metencephalic environment and form neurons, their response is different from that of cranial crest cells in the same location. Whether this is due to differences in developmental potential or in initial population size is not known.     

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.     

Temporal restriction of migratory and lineage potential in rhombomere 1 and 2 neural crest

Migratory cranial neural crest cells differentiate into a wide range of cell types, such as ectomesenchymal tissue (bone and connective tissues) ventrally in the branchial arches and neural tissue (neurons and glia) dorsally. We investigated spatial and temporal changes of migration and differentiation potential in neural crest populations derived from caudal midbrain and rhombomeres 1 and 2 by back-transplanting cells destined for the first branchial arch and trigeminal ganglion from HH8-HH19 quail into HH7-HH11 chicks. Branchial arch cells differentiated down ectomesenchymal lineages but largely lost both the ability to localize to the trigeminal position and neurogenic differentiation capacity by HH12-HH13, even before the arch is visible, and lost long distance migratory ability around HH17. In contrast, neural crest-derived cells from trigeminal ganglia lost ectomesechymal differentiation potential by HH17. Despite this, they retain the ability to migrate into the branchial arches until at least HH19. However, many of the neural crest-derived trigeminal ganglia cells in the branchial arch localized to the non-neural crest core of the arch from HH13 and older donors. These results suggest that long distance migration ability, finer scale localization, and lineage restriction may not be coordinately regulated in the cranial neural crest population.     

Cranial Nerve Development Requires Co-Ordinated Shh and Canonical Wnt Signaling

Cranial nerves govern sensory and motor information exchange between the brain and tissues of the head and neck. The cranial nerves are derived from two specialized populations of cells, cranial neural crest cells and ectodermal placode cells. Defects in either cell type can result in cranial nerve developmental defects. Although several signaling pathways are known to regulate cranial nerve formation our understanding of how intercellular signaling between neural crest cells and placode cells is coordinated during cranial ganglia morphogenesis is poorly understood. Sonic Hedgehog (Shh) signaling is one key pathway that regulates multiple aspects of craniofacial development, but whether it co-ordinates cranial neural crest cell and placodal cell interactions during cranial ganglia formation remains unclear. In this study we examined a new Patched1 (Ptch1) loss-of-function mouse mutant and characterized the role of Ptch1 in regulating Shh signaling during cranial ganglia development. Ptch1^{Wig/ Wig} mutants exhibit elevated Shh signaling in concert with disorganization of the trigeminal and facial nerves. Importantly, we discovered that enhanced Shh signaling suppressed canonical Wnt signaling in the cranial nerve region. This critically affected the survival and migration of cranial neural crest cells and the development of placodal cells as well as the integration between neural crest and placodes. Collectively, our findings highlight a novel and critical role for Shh signaling in cranial nerve development via the cross regulation of canonical Wnt signaling.     

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.     

Altered neuronal lineages in the facial ganglia of Hoxa2 mutant mice

Neurons of cranial sensory ganglia are derived from the neural crest and ectodermal placodes, but the mechanisms that control the relative contributions of each are not understood. Crest cells of the second branchial arch generate few facial ganglion neurons and no vestibuloacoustic ganglion neurons, but crest cells in other branchial arches generate many sensory neurons. Here we report that the facial ganglia of Hoxa2 mutant mice contain a large population of crest-derived neurons, suggesting that Hoxa2 normally represses the neurogenic potential of second arch crest cells. This may represent an anterior transformation of second arch neural crest cells toward a fate resembling that of first arch neural crest cells, which normally do not express Hoxa2 or any other Hox gene. We additionally found that overexpressing Hoxa2 in cultures of P19 embryonal carcinoma cells reduced the frequency of spontaneous neuronal differentiation, but only in the presence of cotransfected Pbx and Meis Hox cofactors. Finally, expression of Hoxa2 and the cofactors in chick neural crest cells populating the trigeminal ganglion also reduced the frequency of neurogenesis in the intact embryo. These data suggest an unanticipated role for Hox genes in controlling the neurogenic potential of at least some cranial neural crest cells.     

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.     

Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve

The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke's pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves.     

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.     

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.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

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.     

Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis

Neural crest cells arising from different rostrocaudal axial levels form different sets of derivatives as diverse as ganglia, cartilage and cornea. These variations may be due to intrinsic properties of the cell populations, different environmental factors encountered during migration or some combination thereof. We test the relative roles of intrinsic versus extrinsic factors by challenging the developmental potential of cardiac and trunk neural crest cells via transplantation into an ectopic midbrain environment. We then assess long-term survival and differentiation into diverse derivatives, including cornea, trigeminal ganglion and branchial arch cartilage. Despite their ability to migrate to the periocular region, neither cardiac nor trunk neural crest contribute appropriately to the cornea, with cardiac crest cells often forming ectopic masses on the corneal surface. Similarly, the potential of trunk and cardiac neural crest to form somatosensory neurons in the trigeminal ganglion was significantly reduced compared with control midbrain grafts. Cardiac neural crest exhibited a reduced capacity to form cartilage, contributing only nominally to Meckle's cartilage, whereas trunk neural crest formed no cartilage after transplantation, even when grafted directly into the first branchial arch. These results suggest that neural crest cells along the rostrocaudal axis display a graded loss in developmental potential to form somatosensory neurons and cartilage even after transplantation to a permissive environment. Hox gene expression was transiently maintained in the cardiac neural tube and neural crest at 12 hours post-transplantation to the midbrain, but was subsequently downregulated. This suggests that long-term differences in Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case.     

The basic framework of VE-cadherin junctions revealed by cryo-EM

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.