Development and regeneration of the vagus nerve.

The vagus nerve, with its myriad constituent axon branches and innervation targets, has long been a model of anatomical complexity in the nervous system. The branched architecture of the vagus nerve is now appreciated to be highly organized around the topographic and/or molecular identities of the neurons that innervate each target tissue. However, we are only just beginning to understand the developmental mechanisms by which heterogeneous vagus neuron identity is specified, patterned, and used to guide the axons of particular neurons to particular targets. Here, we summarize our current understanding of the complex topographic and molecular organization of the vagus nerve, the developmental basis of neuron specification and patterned axon guidance that supports this organization, and the regenerative mechanisms that promote, or inhibit, the restoration of vagus nerve organization after nerve damage. Finally, we highlight key unanswered questions in these areas and discuss potential strategies to address these questions.

Schwann cell functions in peripheral nerve development and repair.

The glial cell of the peripheral nervous system (PNS), the Schwann cell (SC), counts among the most multifaceted cells of the body. During development, SCs secure neuronal survival and participate in axonal path finding. Simultaneously, they orchestrate the architectural set up of the developing nerves, including the blood vessels and the endo-, peri- and epineurial layers. Perinatally, in rodents, SCs radially sort and subsequently myelinate individual axons larger than 1 µm in diameter, while small calibre axons become organised in non-myelinating Remak bundles. SCs have a vital role in maintaining axonal health throughout life and several specialized SC types perform essential functions at specific locations, such as terminal SC at the neuromuscular junction (NMJ) or SC within cutaneous sensory end organs. In addition, neural crest derived satellite glia maintain a tight communication with the soma of sensory, sympathetic, and parasympathetic neurons and neural crest derivatives are furthermore an indispensable part of the enteric nervous system. The remarkable plasticity of SCs becomes evident in the context of a nerve injury, where SC transdifferentiate into intriguing repair cells, which orchestrate a regenerative response that promotes nerve repair. Indeed, the multiple adaptations of SCs are captivating, but remain often ill-resolved on the molecular level. Here, we summarize and discuss the knowns and unknowns of the vast array of functions that this single cell type can cover in peripheral nervous system development, maintenance, and repair.

Structure-based developmental toxicity and ASD-phenotypes of bisphenol A analogues in embryonic zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that has become more prevalent in recent years. Environmental endocrine disruptor bisphenol A (BPA) has been linked to ASD. BPA analogues (BPs) are structure-modified substitutes widely used as safer alternatives in consumer products, yet few studies have explored the developmental neurotoxicity (DNT) of BPA analogues. In the present study, we used the larval zebrafish model to assess the DNT effects of BPA and its analogues. Our results showed that many BPA analogues are more toxic than BPA in the embryonic zebrafish assay regarding teratogenic effect and mortality, which may partially due to differences in lipophilicity and/or different substitutes of structural function groups such as CF3, benzene, or cyclohexane. At sublethal concentrations, zebrafish embryos exposed to BPA or BPs also displayed reduced prosocial behavior in later larval development, evidenced by increased nearest neighbor distance (NND) and the interindividual distance (IID) in shoaling, which appears to be structurally independent. An in-depth analysis of BPA, bisphenol F (BPF), and bisphenol S (BPS) revealed macrocephaly and ASD-like behavioral deficits resulting from exposures to sublethal concentrations of these chemicals. The ASD-like behavioral deficits were characterized by hyperactivity, increased anxiety-like behavior, and decreased social contact. Mechanistically, accelerated neurogenesis that manifested by increased cell proliferation, the proportion of newborn mature neurons, and the number of neural stem cells in proliferation, as well as upregulated genes related to the K+ channels, may have contributed to the observed ASD-like morphological and behavioral alterations. Our findings indicate that BPF and BPS may also pose significant risks to ASD development in humans and highlight the importance of a comprehensive assessment of DNT effects for all BPA analogues in the future.

De novo transcriptome analysis to search for sex-differentiation genes in the Siberian sturgeon.

The sturgeon family includes many species that are lucrative for commercial caviar production, some of which face critical conservation problems. The purpose of this study was to identify genes involved in gonadal sex differentiation in sturgeons, contributing to our understanding of the biological cycle of this valuable species. A high-quality de novo Siberian sturgeon gonadal transcriptome was built for this study using gonadal samples from undifferentiated fish at 3, 5, and 6 months of age; recently sex-differentiated fish at 9 months of age; and immature males and females at 14-17 months of age. Undifferentiated fish were sexed after validation of forkhead box L2 (foxl2) and cytochrome P450, family 19, subfamily A, and polypeptide 1a (cyp19a1a) as sex markers, and the transcriptomes of the 3-month-old undifferentiated fish, 5-6-month-old future females, and 5-6-month-old putative males were compared. The ovarian program was associated with strong activation of genes involved in estrogen synthesis (cyp19a1, foxl2, and estradiol 17-beta-dehydrogenase 1), stem-cell niche building and regulation, and sex-specific nerve cell development. The genes related to the stem-cell niche were: (1) the family of iroquois-class homeodomain proteins 3, 4, and 5 (irx3, irx4, irx5-1, irx5-2, and irx5-3), which are essential for somatic-germ cell interaction; (2) extracellular matrix remodeling genes, such as collagen type XXVIII alpha 1 chain and collagen type II alpha 1 chain, matrix metalloproteinases 24-1 and 24-2, and NADPH oxidase organizer 1, which, along with the somatic cells, provide architectural support for the stem-cell niche; and (3) mitogenic factors, such as lim homeobox 2, amphiregulin, G2/M phase-specific E3 ubiquitin-protein ligase, and connector enhancer of kinase suppressor of ras 2, which are up regulated in conjunction with the anti-apoptotic gene G2/M phase-specific E3 ubiquitin-protein ligase suggesting a potential involvement in regulating the number of germ cells. Genes related to sex-specific nerve cell developments were: the neurofilament medium polypeptides, the gene coding for serotonin receptor 7, 5-hydroxytryptamine receptor 7; neurotensin, isoform CRA-a, the neuron-specific transmembrane protein Delta/Notch-like epidermal growth factor-related receptor; and insulinoma-associated protein 1. The putative testicular program was poorly characterized by elements of the immune response. The classic markers of maleness were not specifically activated, indicating that testicular differentiation occurs at a later stage. In sum, the ovarian program, but not the testicular program, is in place by 5-6 months of age in the Siberian sturgeon. The female program is characterized by estrogen-related genes with well-established roles in gonadal differentiation, but also by several genes with no previously-described function in the ovarian development of fish. These newly-reported genes are involved in stem-cell niche building and regulation as well as sex-specific nerve development.

Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development.

An unusual variant of the abducens nerve duplication with two nerve trunks merging within the orbit: a case report with comments on developmental background.

This study reports the first case of abducens nerve duplication along its entire intracranial course, ending within the orbit. A distinct abducens nerve duplication reaching the common tendinous ring (annulus of Zinn), as well as another split within the intraconal segment of the nerve have been revealed. Additionally, two groups (superior and inferior) of abducens nerve sub-branches to the lateral rectus muscle were visualised using Sihler's stain. The analysed anatomical variation has never been reported before and it seems to be in the middle of the spectrum between the cases of duplication occurring only within the intracranial segments of the abducens nerve found in the literature and those continuing throughout the whole course of the nerve. Abducens nerve duplication may be treated as a relic of early stages of ontogenesis. Such a variant might result from alternative developmental pathways in which axons of the abducens nerve, specific for a given segment of the lateral rectus muscle, run separately at some stage, instead of forming a single stem.

Gut microbiota depletion delays somatic peripheral nerve development and impairs neuromuscular junction maturation.

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 (Egr2), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel 'Gut Microbiota-Peripheral Nervous System-axis.'

Wide-ranging migration and destination of early olfactory placode-derived neurons in chick embryos.

Cell migration from the olfactory placode (OP) is a well-known phenomenon wherein various cell types, such as gonadotropin-releasing hormone (GnRH)-producing neurons, migrate toward the telencephalon (TEL) during early embryonic development. However, the spatial relationship between early migratory cells and the forebrain is unclear. We examined the early development of whole-mount chick embryos to observe the three-dimensional spatial relationship among OP-derived migratory neurons, olfactory nerve (ON), and TEL. Migratory neurons that express highly polysialylated neural cell adhesion molecule (PSA-NCAM) emerge from the OP and spread over a relatively wide TEL area at the Hamburger and Hamilton (HH) stage 17. Most migratory neurons form a cellular cord between the olfactory pit and rostral TEL within HH18-20. The earliest axons from the olfactory epithelium (OE) were detected along this neuronal cord using DiI-labeling at HH21. Furthermore, a few PSA-NCAM-positive neurons were dispersed around the cellular cord and over the lateral TEL at HH18. A long cellular cord branch extending to the lateral TEL was transiently observed within HH18-24. These results suggest a novel migratory route of OP-derived neurons during the early developmental stages. Following GFP vector introduction into the OP of HH13-15 embryos, labeled neurons were detected around and within the lateral TEL at HH23 and HH27. At HH36, labeled cells were observed in the rostral-lateral TEL, including the olfactory bulb (OB) region. GFP-labeled and calretinin-positive neurons were detected in the OB, suggesting that early OP-derived neurons enter the forebrain and function as interneurons in the OB.

Myelinating Schwann cells and Netrin-1 control intra-nervous vascularization of the developing mouse sciatic nerve.

Peripheral nerves are vascularized by a dense network of blood vessels to guarantee their complex function. Despite the crucial role of vascularization to ensure nerve homeostasis and regeneration, the mechanisms governing nerve invasion by blood vessels remain poorly understood. We found, in mice, that the sciatic nerve invasion by blood vessels begins around embryonic day 16 and continues until birth. Interestingly, intra-nervous blood vessel density significantly decreases during post-natal period, starting from P10. We show that, while the axon guidance molecule Netrin-1 promotes nerve invasion by blood vessels via the endothelial receptor UNC5B during embryogenesis, myelinated Schwann cells negatively control intra-nervous vascularization during post-natal period.

A genetic compensatory mechanism regulated by Jun and Mef2d modulates the expression of distinct class IIa Hdacs to ensure peripheral nerve myelination and repair.

The class IIa histone deacetylases (HDACs) have pivotal roles in the development of different tissues. Of this family, Schwann cells express Hdac4, 5, and 7 but not Hdac9. Here, we show that a transcription factor regulated genetic compensatory mechanism within this family of proteins, blocks negative regulators of myelination ensuring peripheral nerve developmental myelination and remyelination after injury. Thus, when Hdac4 and 5 are knocked-out from Schwann cells in mice, a JUN-dependent mechanism induces the compensatory overexpression of Hdac7 permitting, although with a delay, the formation of the myelin sheath. When Hdac4, 5, and 7 are simultaneously removed, the myocyte-specific enhancer-factor d (MEF2D) binds to the promoter and induces the de novo expression of Hdac9, and although several melanocytic lineage genes are misexpressed and Remak bundle structure is disrupted, myelination proceeds after a long delay. Thus, our data unveil a finely tuned compensatory mechanism within the class IIa Hdac family, coordinated by distinct transcription factors, that guarantees the ability of Schwann cells to myelinate during development and remyelinate after nerve injury.

[Clinical study of age-related sensory innervation of the anterior hard palate].

OBJECTIVES: The present study aimed to explore the innervation of the anterior hard palatine and its relationship with individual development stage. Specifically, the effects of anesthesia on patients of different ages were observed, and neurodevelopment in the maxillofacial region was invesitgated. References that are helpful in selecting local anesthesia were provided. METHODS: A total of 182 patients with mixed dentition were randomly divided into the nasopalatine nerve block and greater palatine nerve block groups. Then, 219 patients with permanent dentition were divided into an adolescent group (13-18 years old) and adult group (over 19 years old), all of whom underwent bilateral greater palatine nerve block. Palatal mucosal pain sensation was tested pre- and post-anesthesia with Von Frey hairs. RESULTS: Among the children with mixed dentition, bilateral greater palatine nerve block tended to result in better anesthetic effects than nasopalatine nerve block (P<0.05), except in the incisive papilla. No difference in anesthetic effect was observed between adolescents and adults (P>0.05). The bilateral greater palatine nerve block was more effective in inducing an anesthestic effect in the anterior hard palatine in mixed dentition than in permanent dentition (over 13 years old; P<0.05). CONCLUSIONS: The sensation of the anterior hard palatine seems mainly dominated by the greater palatine nerve until mixed dentition and gradually shifted to the nasopalatine nerve in conjunction with maxillary development and tooth replacement. Hence, the innervation of the anterior hard palatine induce a secondary development during the development of the maxilla.

Cranial Pair II: The Optic Nerves.

The optic nerves (ONs), one of the 12 pairs of cranial nerves (Pair II), together with the olfactory and the cochlear nerves, are devoted to transmit sensory inputs. In particular, ONs convey visual information from the retina to the brain. In mammals, the ONs are bilateral structures that extend from the optic disc to the optic chiasm containing glial cells and retinal ganglion cells (RGCs) axons. RGCs are the only retinal neurons able to collect visual information and transmit it to the visual centers in the brain for its processing and integration with the rest of sensory inputs. During embryonic development, RGCs born in the retina extend their axons to exit the eye and follow a stereotypic path outlined by the transient expression of a wide set of guidance molecules. As the rest of central nervous system structures, the ONs are covered with myelin produced by oligodendrocytes and wrapped by the meninges. ON injuries or RGCs degenerative conditions may provoke partial or complete blindness because they are incapable of spontaneous regeneration. Here, we first review major advances on the current knowledge about the mechanisms underlying the formation of the ONs in mammals. Then, we discuss some of the human disorders and pathologies affecting the development and function of the ONs and finally we comment on the existing view about ON regeneration possibilities. Anat Rec, 302:428-445, 2019. © 2018 Wiley Periodicals, Inc.

A new season for experimental neuroembryology: The mysterious history of Marian Lydia Shorey.

At the turn of the nineteenth and twentieth centuries, the landscape of emerging experimental embryology in the United States was dominated by the Canadian Frank Rattray Lillie, who combined his qualities as scientist and director with those of teacher at the University of Chicago. In the context of his research on chick development, he encouraged the young Marian Lydia Shorey to investigate the interactions between the central nervous system and the peripheral structures. The results were published in two papers which marked the beginning of a new branch of embryology, namely neuroembryology. These papers inspired ground-breaking enquiry by Viktor Hamburger which opened a new area of the research by Rita Levi-Montalcini, in turn leading to the discovery of the nerve growth factor, NGF.

Schwann Cell Precursors; Multipotent Glial Cells in Embryonic Nerves.

The cells of the neural crest, often referred to as neural crest stem cells, give rise to a number of sub-lineages, one of which is Schwann cells, the glial cells of peripheral nerves. Crest cells transform to adult Schwann cells through the generation of two well defined intermediate stages, the Schwann cell precursors (SCP) in early embryonic nerves, and immature Schwann cells (iSch) in late embryonic and perinatal nerves. SCP are formed when neural crest cells enter nascent nerves and form intimate relationships with axons, a diagnostic feature of glial cells. This involves large-scale changes in gene expression, including the activation of established glial cell markers. Like early glia in the CNS, radial glia, SCP retain developmental multipotency and contribute to other crest-derived lineages during embryonic development. SCP, as well as closely related cells termed boundary cap cells, and later stages of the Schwann cell lineage have all been implicated as the tumor initiating cell in NF1 associated neurofibromas. iSch are formed from SCP in a process that involves the appearance of additional differentiation markers, autocrine survival circuits, cellular elongation, a formation of endoneurial connective tissue and basal lamina. Finally, in peri- and post-natal nerves, iSch are reversibly induced by axon-associated signals to form the myelin and non-myelin Schwann cells of adult nerves. This review article discusses early Schwann cell development in detail and describes a large number of molecular signaling systems that control glial development in embryonic nerves.

Isolation of Schwann Cell Precursors from Rodents.

Schwann cell precursors are the first defined stage in the generation of Schwann cells from the neural crest and represent the glial cell of embryonic nerves. Highly pure cultures of these cells can be obtained by enzymatic dissociation of nerves dissected from the limbs of 14- or 12-day-old rat and mouse embryos, respectively. Since Schwann cell precursors, unlike Schwann cells, are acutely dependent on axonal signals for survival, they require addition of trophic factors, typically ß-neuregulin-1, for maintenance in cell culture. Under these conditions they convert to Schwann cells on schedule, within about 4 days.

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

Genetic Manipulation of Neurofilament Protein Phosphorylation.

Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

Dysphagia and disrupted cranial nerve development in a mouse model of DiGeorge (22q11) deletion syndrome.

We assessed feeding-related developmental anomalies in the LgDel mouse model of chromosome 22q11 deletion syndrome (22q11DS), a common developmental disorder that frequently includes perinatal dysphagia--debilitating feeding, swallowing and nutrition difficulties from birth onward--within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA) signaling, prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V), glossopharyngeal (IX) or vagus (X) cranial nerves (CNs) that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to those in wild-type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and CN development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS.

In vivo evaluation of optic nerve development in non-human primates by using diffusion tensor imaging.

Developmental abnormalities of optic nerve are the leading cause of child blindness. The goal of this study was to use diffusion tensor imaging (DTI) to characterize the optic nerve development of non-human primates during the normal maturation from birth to adulthood. Forty healthy rhesus monkeys aged from 2 weeks to 6 years old were scanned with a clinical 3T scanner. It was demonstrated that the DTI parameters followed an exponential pattern during optic nerve maturation. The time constants of mean diffusivity (MD), fractional anisotropy (FA), axial diffusivity (λ∥) and radial diffusivity (λ⊥) were 16, 14, 18 and 15 months in rhesus monkeys, respectively. Significant decrease in RD was observed firstly at 12 months after birth (p<0.05). No significant differences were observed between the left and right optic nerves in any age group. The in vivo imaging results reveal the normal evolution patterns of DTI parameters during optic nerve maturation in primates. The data might be used as a reference in the examination of optic nerve developmental abnormalities or injury in children or preclinical studies.