A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.

Spinal cord precursors utilize neural crest cell mechanisms to generate hybrid peripheral myelinating glia.

During development, oligodendrocytes and Schwann cells myelinate central and peripheral nervous system axons, respectively, while motor exit point (MEP) glia are neural tube-derived, peripheral glia that myelinate axonal territory between these populations at MEP transition zones. From which specific neural tube precursors MEP glia are specified, and how they exit the neural tube to migrate onto peripheral motor axons, remain largely unknown. Here, using zebrafish, we found that MEP glia arise from lateral floor plate precursors and require foxd3 to delaminate and exit the spinal cord. Additionally, we show that similar to Schwann cells, MEP glial development depends on axonally derived neuregulin1. Finally, our data demonstrate that overexpressing axonal cues is sufficient to generate additional MEP glia in the spinal cord. Overall, these studies provide new insight into how a novel population of hybrid, peripheral myelinating glia are generated from neural tube precursors and migrate into the periphery.

Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos.

Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (Ciona and Phallusia for Phlebobranchia, Molgula and Halocynthia for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in Ciona. All genes, but one in Molgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in Molgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that Molgula is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.

Expression of Toll-like receptors 4 and 7 in murine peripheral nervous system development.

BACKGROUND: Toll-Like Receptors (TLRs) play a critical role in the innate and adaptive immune system. They are the mammalian orthologs of Drosophila melanogaster protein Toll, which has been proved to have an early morphogenetic role in invertebrate embryogenesis that in the adult switches to an immune function. AIM: The aim of this study was to evaluate the expression of TLR4 and TLR7 during dorsal root ganglia (DRG), paravertebral ganglia (PVG), and enteric nervous system (ENS) murine development. METHODS: Mouse embryos from different stages (i.e. E12 to E18) were processed for immunolocalization analysis on formalin-fixed paraffin-embedded sections, and isolated intestine were processed for whole-mount preparations. RESULTS: We observed a differentially regulated expression of TLR4 and TLR7 during embryogenesis and an overall increased expression of both receptors during development. While TLR4 was detectable in neurons of DRG and PVG starting from E14 and only from E18 in the ENS, TLR7 was already expressed in scattered neurons of all the investigated regions at E12. CONCLUSIONS: TLR4 and TRL7 expression temporal patterns suggest a morphogenetic role for these receptors in the development of neural crest derivatives in mammals.

Cell fate decisions during the development of the peripheral nervous system in the vertebrate head.

Sensory placodes and neural crest cells are among the key cell populations that facilitated the emergence and diversification of vertebrates throughout evolution. Together, they generate the sensory nervous system in the head: both form the cranial sensory ganglia, while placodal cells make major contributions to the sense organs-the eye, ear and olfactory epithelium. Both are instrumental for integrating craniofacial organs and have been key to drive the concentration of sensory structures in the vertebrate head allowing the emergence of active and predatory life forms. Whereas the gene regulatory networks that control neural crest cell development have been studied extensively, the signals and downstream transcriptional events that regulate placode formation and diversity are only beginning to be uncovered. Both cell populations are derived from the embryonic ectoderm, which also generates the central nervous system and the epidermis, and recent evidence suggests that their initial specification involves a common molecular mechanism before definitive neural, neural crest and placodal lineages are established. In this review, we will first discuss the transcriptional networks that pattern the embryonic ectoderm and establish these three cell fates with emphasis on sensory placodes. Second, we will focus on how sensory placode precursors diversify using the specification of otic-epibranchial progenitors and their segregation as an example.

Transplantation of Neural Tissue: Quail-Chick Chimeras.

Tissue transplantation is an important approach in developmental neurobiology to determine cell fate, to uncover inductive interactions required for tissue specification and patterning as well as to establish tissue competence and commitment. Combined with state-of-the-art molecular approaches, transplantation assays have been instrumental for the discovery of gene regulatory networks controlling cell fate choices and how such networks change over time. Avian species are among the favorite model systems for these approaches because of their accessibility and relatively large size. Here we describe two culture techniques used to generate quail-chick chimeras at different embryonic stages and methods to distinguish graft and donor tissue.

Neurovascular Interactions in the Nervous System.

Molecular cross talk between the nervous and vascular systems is necessary to maintain the correct coupling of organ structure and function. Molecular pathways shared by both systems are emerging as major players in the communication of the neuronal compartment with the endothelium. Here we review different aspects of this cross talk and how vessels influence the development and homeostasis of the nervous system. Beyond the classical role of the vasculature as a conduit to deliver oxygen and metabolites needed for the energy-demanding neuronal compartment, vessels emerge as powerful signaling systems that control and instruct a variety of cellular processes during the development of neurons and glia, such as migration, differentiation, and structural connectivity. Moreover, a broad spectrum of mild to severe vascular dysfunctions occur in various pathologies of the nervous system, suggesting that mild structural and functional changes at the neurovascular interface may underlie cognitive decline in many of these pathological conditions.

Cell migration and axon guidance at the border between central and peripheral nervous system.

The central and peripheral nervous system (CNS and PNS, respectively) are composed of distinct neuronal and glial cell types with specialized functional properties. However, a small number of select cells traverse the CNS-PNS boundary and connect these two major subdivisions of the nervous system. This pattern of segregation and selective connectivity is established during embryonic development, when neurons and glia migrate to their destinations and axons project to their targets. Here, we provide an overview of the cellular and molecular mechanisms that control cell migration and axon guidance at the vertebrate CNS-PNS border. We highlight recent advances on how cell bodies and axons are instructed to either cross or respect this boundary, and present open questions concerning the development and plasticity of the CNS-PNS interface.

Neuron-Glia Signaling in Synapse Elimination.

Maturation of neuronal circuits requires selective elimination of synaptic connections. Although neuron-intrinsic mechanisms are important in this process, it is increasingly recognized that glial cells also play a critical role. Without proper functioning of these cells, the number, morphology, and function of synaptic contacts are profoundly altered, resulting in abnormal connectivity and behavioral abnormalities. In addition to their role in synaptic refinement, glial cells have also been implicated in pathological synapse loss and dysfunction following injury or nervous system degeneration in adults. Although mechanisms regulating glia-mediated synaptic elimination are still being uncovered, it is clear this complex process involves many cues that promote and inhibit the removal of specific synaptic connections. Gaining a greater understanding of these signals and the contribution of different cell types will not only provide insight into this critical biological event but also be instrumental in advancing knowledge of brain development and neural disease.

Characterization of Drosophila Nidogen/entactin reveals roles in basement membrane stability, barrier function and nervous system patterning.

Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). Recent in vivo studies challenged the initially proposed role of NDG as a major ECM linker molecule by revealing dispensability for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. Although loss of Laminin strongly affected BM localization of NDG, Ndg-null mutants exhibited no overt changes in the distribution of BM components. Although Drosophila Ndg mutants were viable, loss of NDG led to ultrastructural BM defects that compromised barrier function and stability in vivo Moreover, loss of NDG impaired larval crawling behavior and reduced responses to vibrational stimuli. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system, especially in the chordotonal organs and the neuromuscular junction (NMJ). Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.

Mutations in dock1 disrupt early Schwann cell development.

BACKGROUND: In the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood. METHODS: Through a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes. RESULTS: We show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development. CONCLUSIONS: Together, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.

Neurocristopathies: New insights 150 years after the neural crest discovery.

The neural crest (NC) is a transient, multipotent and migratory cell population that generates an astonishingly diverse array of cell types during vertebrate development. These cells, which originate from the ectoderm in a region lateral to the neural plate in the neural fold, give rise to neurons, glia, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others. Neurocristopathies (NCP) are a class of pathologies occurring in vertebrates, especially in humans that result from the abnormal specification, migration, differentiation or death of neural crest cells during embryonic development. Various pigment, skin, thyroid and hearing disorders, craniofacial and heart abnormalities, malfunctions of the digestive tract and tumors can also be considered as neurocristopathies. In this review we revisit the current classification and propose a new way to classify NCP based on the embryonic origin of the affected tissues, on recent findings regarding the molecular mechanisms that drive NC formation, and on the increased complexity of current molecular embryology techniques.

Segmentation of the chick central and peripheral nervous systems.

The chick embryo has provided a prominent model system for the study of segmental patterning in the nervous system. During early development, motor and sensory axon growth cones traverse the anterior/rostral half of each somite, so avoiding the developing vertebral components and ensuring separation of spinal nerves from vertebral bones. A glycoprotein expressed on the surface of posterior half-somite cells confines growth cones to the anterior half-somites by a contact repulsive mechanism. Hindbrain segmentation is also a conspicuous feature of chick brain development. We review how its contemporary analysis was initiated in the chick embryo, and the advantages the chick system continues to provide in its detailed elucidation at both molecular and neural circuit levels.

Retinoic acid signaling and neurogenic niche regulation in the developing peripheral nervous system of the cephalochordate amphioxus.

The retinoic acid (RA) signaling pathway regulates axial patterning and neurogenesis in the developing central nervous system (CNS) of chordates, but little is known about its roles during peripheral nervous system (PNS) formation and about how these roles might have evolved. This study assesses the requirement of RA signaling for establishing a functional PNS in the cephalochordate amphioxus, the best available stand-in for the ancestral chordate condition. Pharmacological manipulation of RA signaling levels during embryogenesis reduces the ability of amphioxus larvae to respond to sensory stimulation and alters the number and distribution of ectodermal sensory neurons (ESNs) in a stage- and context-dependent manner. Using gene expression assays combined with immunohistochemistry, we show that this is because RA signaling specifically acts on a small population of soxb1c-expressing ESN progenitors, which form a neurogenic niche in the trunk ectoderm, to modulate ESN production during elongation of the larval body. Our findings reveal an important role for RA signaling in regulating neurogenic niche activity in the larval amphioxus PNS. Although only few studies have addressed this issue so far, comparable RA signaling functions have been reported for neurogenic niches in the CNS and in certain neurogenic placode derivatives of vertebrates. Accordingly, the here-described mechanism is likely a conserved feature of chordate embryonic and adult neural development.

Commissural neurons transgress the CNS/PNS boundary in absence of ventricular zone-derived netrin 1.

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in Ntn1 hypomorphic embryos and Dcc knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in Ntn1 and Dcc mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a Ntn1 conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.

Conserved gene regulatory module specifies lateral neural borders across bilaterians.

The lateral neural plate border (NPB), the neural part of the vertebrate neural border, is composed of central nervous system (CNS) progenitors and peripheral nervous system (PNS) progenitors. In invertebrates, PNS progenitors are also juxtaposed to the lateral boundary of the CNS. Whether there are conserved molecular mechanisms determining vertebrate and invertebrate lateral neural borders remains unclear. Using single-cell-resolution gene-expression profiling and genetic analysis, we present evidence that orthologs of the NPB specification module specify the invertebrate lateral neural border, which is composed of CNS and PNS progenitors. First, like in vertebrates, the conserved neuroectoderm lateral border specifier Msx/vab-15 specifies lateral neuroblasts in Caenorhabditis elegans Second, orthologs of the vertebrate NPB specification module (Msx/vab-15, Pax3/7/pax-3, and Zic/ref-2) are significantly enriched in worm lateral neuroblasts. In addition, like in other bilaterians, the expression domain of Msx/vab-15 is more lateral than those of Pax3/7/pax-3 and Zic/ref-2 in C. elegans Third, we show that Msx/vab-15 regulates the development of mechanosensory neurons derived from lateral neural progenitors in multiple invertebrate species, including C. elegans, Drosophila melanogaster, and Ciona intestinalis We also identify a novel lateral neural border specifier, ZNF703/tlp-1, which functions synergistically with Msx/vab-15 in both C. elegans and Xenopus laevis These data suggest a common origin of the molecular mechanism specifying lateral neural borders across bilaterians.

In vivo time-lapse imaging reveals extensive neural crest and endothelial cell interactions during neural crest migration and formation of the dorsal root and sympathetic ganglia.

During amniote embryogenesis the nervous and vascular systems interact in a process that significantly affects the respective morphogenesis of each network by forming a "neurovascular" link. The importance of neurovascular cross-talk in the central nervous system has recently come into focus with the growing awareness that these two systems interact extensively both during development, in the stem-cell niche, and in neurodegenerative conditions such as Alzheimer's Disease and Amyotrophic Lateral Sclerosis. With respect to the peripheral nervous system, however, there have been no live, real-time investigations of the potential relationship between these two developing systems. To address this deficit, we used multispectral 4D time-lapse imaging in a transgenic quail model in which endothelial cells (ECs) express a yellow fluorescent marker, while neural crest cells (NCCs) express an electroporated red fluorescent marker. We monitored EC and NCC migration in real-time during formation of the peripheral nervous system. Our time-lapse recordings indicate that NCCs and ECs are physically juxtaposed and dynamically interact at multiple locations along their trajectories. These interactions are stereotypical and occur at precise anatomical locations along the NCC migratory pathway. NCCs migrate alongside the posterior surface of developing intersomitic vessels, but fail to cross these continuous streams of motile ECs. NCCs change their morphology and migration trajectory when they encounter gaps in the developing vasculature. Within the nascent dorsal root ganglion, proximity to ECs causes filopodial retraction which curtails forward persistence of NCC motility. Overall, our time-lapse recordings support the conclusion that primary vascular networks substantially influence the distribution and migratory behavior of NCCs and the patterned formation of dorsal root and sympathetic ganglia.

Functional dissection of the Pax6 paired domain: Roles in neural tube patterning and peripheral nervous system development.

During development of the CNS, stem and progenitor cell proliferation, cell fate designation, and patterning decisions are tightly regulated by interdependent networks of key transcriptional regulators. In a genetic approach we analyzed divergent functionality of the PAI and RED sub-domains of the Pax6 Paired domain (PD) during progenitor zone formation, motor and interneuron development, and peripheral connectivity at distinct levels within the neural tube: within the hindbrain, mutation of the PAI sub-domain severely affected patterning of the p3 and pMN domains and establishment of the corresponding motor neurons. Exit point designation of hypoglossal axons was disturbed in embryos harboring either mutations in the PD sub-domains or containing a functional Pax6 Null allele. At brachial spinal levels, we propose a selective involvement of the PAI sub-domain during patterning of ventral p2 and pMN domains, critically disturbing generation of specific motor neuron subtypes and increasing V2 interneuron numbers. Our findings present a novel aspect of how Pax6 not only utilizes its modular structure to perform distinct functions via its paired and homeodomain. Individual sub-domains can exert distinct functions, generating a new level of complexity for transcriptional regulation by one single transcription factor not only in dorso-ventral, but also rostro-caudal neural tube patterning.

Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems.

BACKGROUND: Rac1 is a critical regulator of cytoskeletal dynamics in multiple cell types. In the nervous system, it has been implicated in the control of cell proliferation, neuronal migration, and axon development. RESULTS: To systematically investigate the role of Rac1 in axon growth and guidance in the developing nervous system, we have examined the phenotypes associated with deleting Rac1 in the embryonic mouse forebrain, in cranial and spinal motor neurons, in cranial sensory and dorsal root ganglion neurons, and in the retina. We observe a widespread requirement for Rac1 in axon growth and guidance and a cell-autonomous defect in axon growth in Rac1 (-/-) motor neurons in culture. Neuronal death, presumably a secondary consequence of the axon growth and/or guidance defects, was observed in multiple locations. Following deletion of Rac1 in the forebrain, thalamocortical axons were misrouted inferiorly, with the majority projecting to the contralateral thalamus and a minority projecting ipsilaterally to the ventral cortex, a pattern of misrouting that is indistinguishable from the pattern previously observed in Frizzled3 (-/-) and Celsr3 (-/-) forebrains. In the limbs, motor-neuron-specific deletion of Rac1 produced a distinctive stalling of axons within the dorsal nerve of the hindlimb but a much milder loss of axons in the ventral hindlimb and forelimb nerves, a pattern that is virtually identical to the one previously observed in Frizzled3 (-/-) limbs. CONCLUSIONS: The similarities in axon growth and guidance phenotypes caused by Rac1, Frizzled3, and Celsr3 loss-of-function mutations suggest a mechanistic connection between tissue polarity/planar cell polarity signaling and Rac1-dependent cytoskeletal regulation.