Epibranchial Placodes Research Articles

Fibroblast growth factor 8 promotes in vitro neurite outgrowth of placode-derived petrosal and nodose ganglia to varying degrees

Fibroblast growth factors (FGFs) are required for the specification and formation of the epibranchial placodes, which give rise to the distal part of the cranial sensory ganglia. However, it remains unclear whether FGFs play a role in regulating the neurite outgrowth of the epibranchial placode-derived ganglia during further development. Previous studies have shown that Fibroblast growth factor 8 (FGF8) promotes neurite outgrowth from the statoacoustic ganglion in vitro. However, these studies did not distinguish between the neural crest- and placode-derived components of the sensory ganglia. In this study, we focused on the petrosal and nodose ganglia as representatives of the epibranchial ganglia and investigated their axonal outgrowth under the influence of FGF8 signaling protein in vitro. To precisely isolate the placode-derived ganglion part, we labeled the placode and its derivatives with enhanced green fluorescent protein (EGFP) through electroporation. The isolated ganglia were then collected for qRT-PCR assay and cultured in a collagen gel with and without FGF8 protein. Our findings revealed that both placode-derived petrosal and nodose ganglia expressed FGFR1 and FGFR2. In culture, FGF8 exerted a neural trophic effect on the axon outgrowth of both ganglia. While the expression levels of FGFR1/2 were similar between the two ganglia, the petrosal ganglion exhibited greater sensitivity to FGF8 compared to the nodose ganglion. This indicates that the placode-derived ganglia have differential responsiveness to FGF8 signaling during axonal extension. Thus, FGF8 is not only required for the early development of the epibranchial placode, as shown in previous studies, but also promotes neurite outgrowth of placode-derived ganglia.

Molecular Characterization of Nodose Ganglia Development Reveals a Novel Population of Phox2b+ Glial Progenitors in Mice.

The vagal ganglia, comprised of the superior (jugular) and inferior (nodose) ganglia of the vagus nerve, receive somatosensory information from the head and neck or viscerosensory information from the inner organs, respectively. Developmentally, the cranial neural crest gives rise to all vagal glial cells and to neurons of the jugular ganglia, while the epibranchial placode gives rise to neurons of the nodose ganglia. Crest-derived nodose glial progenitors can additionally generate autonomic neurons in the peripheral nervous system, but how these progenitors generate neurons is unknown. Here, we found that some Sox10+ neural crest-derived cells in, and surrounding, the nodose ganglion transiently expressed Phox2b, a master regulator of autonomic nervous system development, during early embryonic life. Our genetic lineage-tracing analysis in mice of either sex revealed that despite their common developmental origin and extreme spatial proximity, a substantial proportion of glial cells in the nodose, but not in the neighboring jugular ganglia, have a history of Phox2b expression. We used single-cell RNA-sequencing to demonstrate that these progenitors give rise to all major glial subtypes in the nodose ganglia, including Schwann cells, satellite glia, and glial precursors, and mapped their spatial distribution by in situ hybridization. Lastly, integration analysis revealed transcriptomic similarities between nodose and dorsal root ganglia glial subtypes and revealed immature nodose glial subtypes. Our work demonstrates that these crest-derived nodose glial progenitors transiently express Phox2b, give rise to the entire complement of nodose glial cells, and display a transcriptional program that may underlie their bipotent nature.

Exosome-shuttled miR-126 mediates ethanol-induced disruption of neural crest cell-placode cell interaction by targeting SDF1.

During embryonic development, 2 populations of multipotent stem cells, cranial neural crest cells (NCCs) and epibranchial placode cells (PCs), are anatomically adjacent to each other. The coordinated migration of NCCs and PCs plays a major role in the morphogenesis of craniofacial skeletons and cranial nerves. It is known that ethanol-induced dysfunction of NCCs and PCs is a key contributor to the defects of craniofacial skeletons and cranial nerves implicated in fetal alcohol spectrum disorder (FASD). However, how ethanol disrupts the coordinated interaction between NCCs and PCs was not elucidated. To fill in this gap, we established a well-designed cell coculture system to investigate the reciprocal interaction between human NCCs (hNCCs) and human PCs (hPCs), and also monitored the migration behavior of NCCs and PCs in zebrafish embryos. We found that ethanol exposure resulted in a disruption of coordinated hNCCs-hPCs interaction, as well as in zebrafish embryos. Treating hNCCs-hPCs with exosomes derived from ethanol-exposed hNCCs (ExoEtOH) mimicked ethanol-induced impairment of hNCCs-hPCs interaction. We also observed that SDF1, a chemoattractant, was downregulated in ethanol-treated hPCs and zebrafish embryos. Meanwhile, miR-126 level in ExoEtOH was significantly higher than that in control exosomes (ExoCon). We further validated that ExoEtOH-encapsulated miR-126 from hNCCs can be transferred to hPCs to suppress SDF1 expression in hPCs. Knockdown of SDF1 replicated ethanol-induced abnormalities either in vitro or in zebrafish embryos. On the contrary, overexpression of SDF1 or inhibiting miR-126 strongly rescued ethanol-induced impairment of hNCCs-hPCs interaction and developmental defects.

Patterns of senescence and apoptosis during development of branchial arches, epibranchial placodes, and pharyngeal pouches.

Many developmental processes are coregulated by apoptosis and senescence. However, there is a lack of data on the development of branchial arches, epibranchial placodes, and pharyngeal pouches, which harbor epibranchial signaling centers. Using immunohistochemical, histochemical, and 3D reconstruction methods, we show that in mice, senescence and apoptosis together may contribute to the invagination of the branchial clefts and the deepening of the cervical sinus floor, in antagonism to the proliferation acting in the evaginating branchial arches. The concomitant apoptotic elimination of lateral line rudiments occurs in the absence of senescence. In the epibranchial placodes, senescence and apoptosis appear to (1) support invagination or at least indentation by immobilizing the margins of the centrally proliferating pit, (2) coregulate the number and fate of Pax8+ precursors, (3) progressively narrow neuroblast delamination sites, and (4) contribute to placode regression. Putative epibranchial signaling centers in the pharyngeal pouches are likely deactivated by rostral senescence and caudal apoptosis. Our results reveal a plethora of novel patterns of apoptosis and senescence, some overlapping, some complementary, whose functional contributions to the development of the branchial region, including the epibranchial placodes and their signaling centers, can now be tested experimentally.

Early Steps towards Hearing: Placodes and Sensory Development.

Sensorineural hearing loss is the most prevalent sensory deficit in humans. Most cases of hearing loss are due to the degeneration of key structures of the sensory pathway in the cochlea, such as the sensory hair cells, the primary auditory neurons, and their synaptic connection to the hair cells. Different cell-based strategies to replace damaged inner ear neurosensory tissue aiming at the restoration of regeneration or functional recovery are currently the subject of intensive research. Most of these cell-based treatment approaches require experimental in vitro models that rely on a fine understanding of the earliest morphogenetic steps that underlie the in vivo development of the inner ear since its initial induction from a common otic-epibranchial territory. This knowledge will be applied to various proposed experimental cell replacement strategies to either address the feasibility or identify novel therapeutic options for sensorineural hearing loss. In this review, we describe how ear and epibranchial placode development can be recapitulated by focusing on the cellular transformations that occur as the inner ear is converted from a thickening of the surface ectoderm next to the hindbrain known as the otic placode to an otocyst embedded in the head mesenchyme. Finally, we will highlight otic and epibranchial placode development and morphogenetic events towards progenitors of the inner ear and their neurosensory cell derivatives.

Responses of Epibranchial Placodes to Disruptions of the FGF and BMP Signaling Pathways in Embryonic Mice.

Placodes are ectodermal thickenings of the embryonic vertebrate head. Their descendants contribute to sensory organ development, but also give rise to sensory neurons of the cranial nerves. In mammals, the signaling pathways which regulate the morphogenesis and neurogenesis of epibranchial placodes, localized dorsocaudally to the pharyngeal clefts, are poorly understood. Therefore, we performed mouse whole embryo culture experiments to assess the impact of pan-fibroblast growth factor receptor (FGFR) inhibitors, anti-FGFR3 neutralizing antibodies or the pan-bone morphogenetic protein receptor (BMPR) inhibitor LDN193189 on epibranchial development. We demonstrate that each of the three paired epibranchial placodes is regulated by a unique combination of FGF and/or bone morphogenetic protein (BMP) signaling. Thus, neurogenesis depends on fibroblast growth factor (FGF) signals, albeit to different degrees, in all epibranchial placodes (EP), whereas only EP1 and EP3 significantly rely on neurogenic BMP signals. Furthermore, individual epibranchial placodes vary in the extent to which FGF and/or BMP signals (1) have access to certain receptor subtypes, (2) affect the production of Neurogenin (Ngn)2+ and/or Ngn1+ neuroblasts, and (3) regulate either neurogenesis alone or together with structural maintenance. In EP2 and EP3, all FGF-dependent production of Ngn2+ neuroblasts is mediated via FGFR3 whereas, in EP1, it depends on FGFR1 and FGFR3. Differently, production of FGF-dependent Ngn1+ neuroblasts almost completely depends on FGFR3 in EP1 and EP2, but not in EP3. Finally, FGF signals turned out to be responsible for the maintenance of both placodal thickening and neurogenesis in all epibranchial placodes, whereas administration of the pan-BMPR inhibitor, apart from its negative neurogenic effects in EP1 and EP3, causes only decreases in the thickness of EP3. Experimentally applied inhibitors most probably not only blocked receptors in the epibranchial placodes, but also endodermal receptors in the pharyngeal pouches, which act as epibranchial signaling centers. While high doses of pan-FGFR inhibitors impaired the development of all pharyngeal pouches, high doses of the pan-BMPR inhibitor negatively affected only the pharyngeal pouches 3 and 4. In combination with partly concordant, partly divergent findings in other vertebrate classes our observations open up new approaches for research into the complex regulation of neurogenic placode development.

Hoxb3 Regulates Jag1 Expression in Pharyngeal Epithelium and Affects Interaction With Neural Crest Cells.

Craniofacial morphogenesis depends on proper migration of neural crest cells and their interactions with placodes and other cell types. Hox genes provide positional information and are important in patterning the neural crest and pharyngeal arches (PAs) for coordinated formation of craniofacial structures. Hox genes are expressed in the surface ectoderm and epibranchial placodes, their roles in the pharyngeal epithelium and their downstream targets in regulating PA morphogenesis have not been established. We altered the Hox code in the pharyngeal region of the Hoxb3Tg/+ mutant, in which Hoxb3 is driven to ectopically expressed in Hoxb2 domain in the second pharyngeal arch (PA2). In the transgenic mutant, ectopic Hoxb3 expression was restricted to the surface ectoderm, including the proximal epibranchial placodal region and the distal pharyngeal epithelium. The Hoxb3Tg/+ mutants displayed hypoplasia of PA2, multiple neural crest-derived facial skeletal and nerve defects. Interestingly, we found that in the Hoxb3Tg/+ mutant, expression of the Notch ligand Jag1 was specifically up-regulated in the ectodermal pharyngeal epithelial cells of PA2. By molecular experiments, we demonstrated that Hoxb3 could bind to an upstream genomic site S2 and directly regulate Jag1 expression. In the Hoxb3Tg/+ mutant, elevated expression of Jag1 in the pharyngeal epithelium led to abnormal cellular interaction and deficiency of neural crest cells migrating into PA2. In summary, we showed that Hoxb3 regulates Jag1 expression and proposed a model of pharyngeal epithelium and neural crest interaction during pharyngeal arch development.

Molecular identity, anatomy, gene expression and function of neural crest vs. placode-derived nociceptors in the lower airways

The lower airways (larynx to alveoli) are protected by a complex array of neural networks that regulate respiration and airway function. Harmful stimuli trigger defensive responses such as apnea, cough and bronchospasm by activating a subpopulation of sensory afferent nerves (termed nociceptors) which are found throughout the airways. Airway nociceptive fibers are projected from the nodose vagal ganglia, the jugular vagal ganglia and the dorsal root ganglia, which are derived from distinct embryological sources: the former from the epibranchial placodes, the latter two from the neural crest. Embryological source determines nociceptive gene expression of receptors and neurotransmitters and recent evidence suggests that placode- and neural crest-derived nociceptors have distinct stimuli sensitivity, innervation patterns and functions. Improved understanding of the function of each subset in specific reflexes has substantial implications for therapeutic targeting of the neuronal components of airway disease such as asthma, viral infections and chronic obstructive pulmonary disease.

The Role of the Placodes in the Development of the Glossopharyngeal, Vagal, and Trigeminal Ganglia

The epibranchial placodes combine with the neural crest to form the inferior and superior ganglia of the glossopharyngeal and vagal cranial nerves, respectively. By comparison, the single trigeminal ganglion is composed of both neural crest and placodal cells. The steps that lead up to these events include gastrulation and the embryology of the notochord, neural crest, and the placodes. Each of these steps is reviewed in some detail. In previous reviews in this series, the embryology related to the olfactory, otic, and lens placodes, and to the geniculate ganglia has been discussed.1-3 However, the somewhat unusual embryology of the 2 ganglia of cranial nerves IX and X was only briefly mentioned as was the development of the trigeminal ganglion.4 This present review revisits these events and specifically focuses on how these ganglia develop.Learning Objective: The reader will learn the unusual development of the superior and inferior glossopharyngeal and the vagal ganglia as well as review the varied steps in the embryology that proceeds these events. By comparison, the development of the single trigeminal ganglion is presented and the differences in its development from that of the ganglia of cranial nerves IX and X are emphasized.

SPARC: The Structure and Location of Vagal Nociceptive Nerve Fibers in the Mouse Trachea

Vagal afferent innervation of the airways and lungs comprises of afferent fibers from vagal nodose neurons (developmentally derived from epibranchial placodes) and vagal jugular neurons (derived from neural crest). Here we evaluated the structure and the location in tracheal wall of afferent nerve fibers innervating the trachea focusing on the nociceptive TRPV1‐positive fibers (putative C‐fibers). We used genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of the trachea were immunostained for fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (in the vagal system the marker of TRPV1‐positive fibers derived from neural crest) we observed dense innervation of the trachea. In the anterolateral tracheal wall the TRPV1+Tac1+ fibers were located beneath the epithelium in the mucosa above tracheal ligaments (which connect tracheal rings) running transversally (i.e. in parallel with tracheal rings). These fibers ramified richly and repetitively. In the dorsal (membranous) tracheal wall the TRPV1+Tac1+ fibers were also located beneath the epithelium in the mucosa above tracheal smooth muscle, running craniocaudally and often branching. These fibers occasionally penetrated through the tracheal smooth muscle. Some TRPV1+Tac1+ fibers were also observed in the epithelium in the sublaryngeal region of the trachea. In the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP or AAV9‐FLEX‐tdTomato virus vector into vagal ganglia (N=4), in which only the vagal TRPV1‐positive fibers express GFP or tdTomato, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with expected lower density and patchy distribution. Similarly, in the Tac1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP vector into vagal ganglia (N=3) in which only the vagal Tac1‐positive fibers express GFP had the same pattern. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of vagal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the trachea. However, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEXEGFP vector into vagal ganglia (N=2) in which GFP is selectively expressed in vagal P2X2− positive fibers, the P2X2+ fibers had distinct distribution from TRPV1+Tac1+ and often located in the tracheal smooth muscle and tracheal epithelium. Our data indicate that vagal afferent nociceptive (TRPV1+) nerve fibers (putative C‐fibers) innervating the mouse trachea originate almost exclusively from jugular ganglion neurons. In contrast, vagal nodose ganglia project only TRPV1‐fibers into the trachea. Our results are in agreement with electrophysiological studies in other species.Support or Funding InformationNIH SPARC

Initial axon growth rate from embryonic sensory neurons is correlated with birth date

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later‐born neurons within each population have further to extend their axons to reach their targets than early‐born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.

Notch signalling regulates epibranchial placode patterning and segregation.

ABSTRACTEpibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

Phenotypic distinctions between the nodose and jugular TRPV1-positive vagal sensory neurons in the cynomolgus monkey.

Vagal capsaicin-sensitive afferent C-fibers play an important role in the maintenance of visceral homeostasis and contribute to symptoms in visceral diseases. Based on their developmental origin two functionally distinct types of vagal C-fibers are recognized: those with neurons in the vagal nodose ganglia (derived from epibranchial placodes) and in the vagal jugular ganglia (from neural crest). Studies in nonprimate species demonstrated that the vagal nodose and jugular C-fibers differ in activation profile, neurotrophic regulation, and expression of neurotransmitters. We hypothesized that the expression of selected markers related to key phenotypic properties of vagal C-fibers in the cynomolgus monkey is similar to that reported in nonprimate species. We performed single-cell RT-PCR on nodose and jugular putative C-fiber (TRPV1-positive) neurons isolated from the cynomolgus monkey. We found that the expression of purinergic P2X receptors that underlie selective responsiveness of nodose C-fiber terminals to ATP was conserved in that P2X2 and P2X3 subunits were expressed in nodose, but only P2X3 subunit was expressed in jugular TRPV1-positive neurons. Also conserved was the preferential expression of neurotrophic receptor TrkB in the nodose and preprotachykinin-A in the jugular TRPV1-positive neurons. Several key distinctions in gene expression between nodose and jugular TRPV1-positive (C-fiber) neurons that have been noted in mice, rats, and guinea pigs, are conserved in the cynomolgus monkey. Our results support the translatability of distinct vagal C-fiber phenotypes to primates.

Chicken embryos share mammalian patterns of apoptosis in the posterior placodal area.

In the posterior placodal area (PPA) of C57BL/6N mice and primate-related Tupaia belangeri (Scandentia), apoptosis helps to establish morphologically separated otic and epibranchial placodes. Here, we demonstrate that basically identical patterns of apoptosis pass rostrocaudally through the Pax2+ PPA of chicken embryos. Interplacodal apoptosis eliminates unneeded cells either between the otic anlage and the epibranchial placodes 1, 2 and/or 3, respectively (type A), or between neighbouring epibranchial placodes (type B). These observations support the idea that in chicken embryos, as in mammals, interplacodal apoptosis serves to remove vestigial lateral line placodes (Washausen & Knabe, 2018, Biol Open 7, bio031815). A special case represents the recently discovered Pax2- /Sox2+ paratympanic organ (PTO) placode that has been postulated to be molecularly distinct from and developmentally independent of the ventrally adjacent first epibranchial (or 'geniculate') placode (O'Neill etal. 2012, Nat Commun 3, 1041). We show that Sox2+ (PTO placodal) cells seem to segregate from the Pax2+ geniculate placode, and that absence of Pax2 in the mature PTO placode is due to secondary loss. We further report that, between Hamburger-Hamilton (HH) stages HH14 and HH26, apoptosis in the combined anlage of the first epibranchial and PTO placodes is almost exclusively found within and/or immediately adjacent to the dorsally located PTO placode. Hence, apoptosis appears to support decision-making processes among precursor cells of the early developing PTO placode and, later, regression of the epibranchial placodes 2 and 3.

Latrophilin2 is involved in neural crest cell migration and placode patterning in Xenopus laevis.

Latrophilin2 (Lphn2) is an adhesion-class of G protein-coupled receptor with an unknown function in development. Here, we show that Xenopus laevis lphn2 (Xlphn2) is involved in the migration and differentiation of neural crest (NC) cells and placode patterning in Xenopus laevis embryos. Although Xlphn2 mRNA was detected throughout embryogenesis, it was expressed more abundantly in the placode region. Morpholino antisense oligonucleotide-mediated knockdown of Xlphn2 caused abnormal migration of NC cells, irregular epibranchial placode segmentation, and defective cartilage formation. Transplantation of fluorescently-labeled NC regions of wild-type embryos into Xlphn2 morpholino-injected embryos reproduced the defective NC cell migration, indicating that Xlphn2 regulates the migration of NC cells in a non-cell autonomous manner. Our results suggest that Xlphn2 is essential for placode patterning and as a guidance molecule for NC cells.

Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals.

ABSTRACTPlacodes are focal thickenings of the surface ectoderm which, together with neural crest, generate the peripheral nervous system of the vertebrate head. Here we examine how, in embryonic mice, apoptosis contributes to the remodelling of the primordial posterior placodal area (PPA) into physically separated otic and epibranchial placodes. Using pharmacological inhibition of apoptosis-associated caspases, we find evidence that apoptosis eliminates hitherto undiscovered rudiments of the lateral line sensory system which, in fish and aquatic amphibia, serves to detect movements, pressure changes or electric fields in the surrounding water. Our results refute the evolutionary theory, valid for more than a century that the whole lateral line was completely lost in amniotes. Instead, those parts of the PPA which, under experimental conditions, escape apoptosis have retained the developmental potential to produce lateral line placodes and the primordia of neuromasts that represent the major functional units of the mechanosensory lateral line system.

Regulation of continuous but complex expression pattern of Six1 during early sensory development

In vertebrates, cranial sensory placodes give rise to neurosensory and endocrine structures, such as the olfactory epithelium, inner ear, and anterior pituitary. We report here the establishment of a transgenic mouse line that expresses Cre recombinase under the control of Six1-21, a major placodal enhancer of the homeobox gene Six1. In the new Cre-expressing line, mSix1-21-NLSCre, the earliest Cre-mediated recombination was induced at embryonic day 8.5 in the region overlapping with the otic-epibranchial progenitor domain (OEPD), a transient, common precursor domain for the otic and epibranchial placodes. Recombination was later observed in the OEPD-derived structures (the entire inner ear and the VIIth-Xth cranial sensory ganglia), olfactory epithelium, anterior pituitary, pharyngeal ectoderm and pouches. Other Six1-positive structures, such as salivary/lacrimal glands and limb buds, were also positive for recombination. Moreover, comparison with another mouse line expressing Cre under the control of the sensory neuron enhancer, Six1-8, indicated that the continuous and complex expression pattern of Six1 during sensory organ formation is pieced together by separate enhancers. mSix1-21-NLSCre has several unique characteristics to make it suitable for analysis of cell lineage and gene function in sensory placodes as well as nonplacodal Six1-positive structures. Developmental Dynamics 247:250-261, 2018. © 2017 Wiley Periodicals, Inc.

An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis.

Craniofacial morphogenesis requires proper development of pharyngeal arches and epibranchial placodes. We show that the epibranchial placodes, in addition to giving rise to cranial sensory neurons, generate a novel lineage-related non-neuronal cell population for mouse pharyngeal arch development. Eya1 is essential for the development of epibranchial placodes and proximal pharyngeal arches. We identify an Eya1-Notch regulatory axis that specifies both the neuronal and non-neuronal commitment of the epibranchial placode, where Notch acts downstream of Eya1 and promotes the non-neuronal cell fate. Notch is regulated by the threonine phosphatase activity of Eya1. Eya1 dephosphorylates p-threonine-2122 of the Notch1 intracellular domain (Notch1 ICD), which increases the stability of Notch1 ICD and maintains Notch signaling activity in the non-neuronal epibranchial placodal cells. Our data unveil a more complex differentiation program in epibranchial placodes and an important role for the Eya1-Notch axis in craniofacial morphogenesis.

Distinct and common expression of receptors for inflammatory mediators in vagal nodose versus jugular capsaicin-sensitive/TRPV1-positive neurons detected by low input RNA sequencing.

Capsaicin-sensitive sensory C-fibers derived from vagal ganglia innervate the visceral organs, and respond to inflammatory mediators and noxious stimuli. These neurons play an important role in maintenance of visceral homeostasis, and contribute to the symptoms of visceral inflammatory diseases. Vagal sensory neurons are located in two ganglia, the jugular ganglia (derived from the neural crest), and the nodose ganglia (from the epibranchial placodes). The functional difference, especially in response to immune mediators, between jugular and nodose neurons is not fully understood. In this study, we microscopically isolated murine nodose and jugular capsaicin-sensitive / Trpv1-expressing C-fiber neurons and performed transcriptome profiling using ultra-low input RNA sequencing. RNAseq detected genes with significantly differential expression in jugular and nodose neurons, which were mostly involved in neural functions. Transcriptional regulators, including Cited1, Hoxb5 and Prdm12 showed distinct expression patterns in the two C-fiber neuronal populations. Common and specific expression of immune receptor proteins was characterized in each neuronal type. The expression of immune receptors that have received little or no attention from vagal sensory biologists is highlighted including receptors for certain chemokines (CXCLs), interleukins (IL-4) and interferons (IFNα, IFNγ). Stimulation of immune receptors with their cognate ligands led to activation of the C-fibers in isolated functional assays.

Annexin A6 controls neuronal membrane dynamics throughout chick cranial sensory gangliogenesis

Cranial sensory ganglia are components of the peripheral nervous system that possess a significant somatosensory role and include neurons within the trigeminal and epibranchial nerve bundles. Although it is well established that these ganglia arise from interactions between neural crest and neurogenic placode cells, the molecular basis of ganglia assembly is still poorly understood. Members of the Annexin protein superfamily play key roles in sensory nervous system development throughout metazoans. Annexin A6 is expressed in chick trigeminal and epibranchial placode cell-derived neuroblasts and neurons, but its function in cranial ganglia formation has not been elucidated. To this end, we interrogated the role of Annexin A6 using gene perturbation studies in the chick embryo. Our data reveal that placode cell-derived neuroblasts with reduced Annexin A6 levels ingress and migrate normally to the ganglionic anlage, where neural crest cell corridors correctly form around them. Strikingly, while Annexin A6-depleted placode cell-derived neurons still express mature neuronal markers, they fail to form two long processes, which are considered morphological features of mature neurons, and no longer innervate their designated targets due to the absence of this bipolar morphology. Moreover, overexpression of Annexin A6 causes some placode cell-derived neurons to form extra protrusions alongside these bipolar processes. These data demonstrate that the molecular program associated with neuronal maturation is distinct from that orchestrating changes in neuronal morphology, and, importantly, reveal Annexin A6 to be a key membrane scaffolding protein during sensory neuron membrane biogenesis. Collectively, our results provide novel insight into mechanisms underscoring morphological changes within placode cell-derived neurons that are essential for cranial gangliogenesis.