Vertebrate Neural Development Research Articles

Salinization and low-dose levels of pesticides alter brain shape of larval amphibians

Wetland communities are increasingly threatened by multiple stressors simultaneously, such as pesticides and salinization. We examined the effects of ecologically-relevant exposures to broad-spectrum insecticides and salinization on amphibian neurodevelopment, which is strongly linked to how organisms respond behaviorally to environmental change. Prior research showed that exposure to trace concentrations of an organophosphate pesticide (chlorpyrifos) altered the brain shape and behavior of larval and metamorphic amphibians. It is unknown whether brain shape is altered by additional pesticides and road salt. Using outdoor mesocosms, we tested whether salt (NaCl) and representatives from three pesticide families (organophosphates, pyrethroids, and neonicotinoids) altered tadpole (Lithobates pipiens) brain shape. Of the two organophosphates, chlorpyrifos induced relatively longer telencephalon lengths relative to body mass, consistent with previous studies, but malathion had no effect on brain shape. Of the two pyrethroids, permethrin, but not cypermethrin, increased telencephalon length. For the neonicotinoids, there were marginally significant effects of imidacloprid and thiamethoxam on telencephalon length. Thus, the impacts of pesticides on brain shape was not dictated by pesticide family. Exposure to relatively high concentrations of salt resulted in brains that were less wide but had longer optic tecta. Although we failed to find strong interactive effect of salt with pesticides, there was some weak, nonsignificant, evidence that exposure to salt masked responses to pesticides. Together, our results indicate that environmentally realistic levels of pesticides and salinization can alter larval brain shape. Our study highlights the importance of studying the impacts of naturally-occurring levels of pesticides and salinization on vertebrate neural development.

Exploring the Importance of Environmental Complexity for Newly Hatched Zebrafish.

The effects of an early impoverished social or physical environment on vertebrate neural development and cognition has been known for decades. While existing studies have focused on the long-term effects, measuring adult cognitive phenotypes, studies on the effects of environmental complexity on the early stages of development are lacking. Zebrafish (Danio rerio) hatchlings are assumed to have minimal interaction with their environment and are routinely reared in small, bare containers. To investigate the effects of being raised under such conditions on development of behaviour and cognition, hatchlings housed for 10 days in either an enriched or a standard environment underwent two cognitive tasks. The results were mixed. Subjects of the two treatments did not differ in performance when required to discriminate two areas. Conversely, we found a significant effect in a number discrimination task, with subjects from impoverished condition performing significantly worse. In both experiments, larvae reared in impoverished environment showed a reduced locomotor activity. Given the effects that enrichment appears to exert on larvae, a third experiment explored whether hatchlings exhibit a spontaneous preference for more complex environments. When offered a choice between a bare setting and one with objects of different shapes and colors, larvae spent over 70% of time in the enriched sector. Deepening these effects of an early impoverished environment on cognitive development is crucial for the welfare of captive zebrafish populations and for enhancing the quality and reliability of studies on larval zebrafish.

SynCAMs in Normal Vertebrate Neural Development and Neuropsychiatric Disorders: from the Perspective of the OCAs.

Neuronal synaptic junctions connect neurons to enable neuronal signal transmission in the nervous system. The proper establishment of synaptic connections required many adhesion molecules. Malfunctions of these adhesion molecules can result in neural development disorders and neuropsychiatric disorders. How specific synapses are established by various adhesion molecules for proper neural circuitry is a fundamental question of neuroscience. SynCAMs, also named CADMs, Necl, etc., are among the many adhesion proteins found in synapses. Here, we review the current understanding of the physical properties of SynCAMs and their roles in axon pathfinding, myelination, synaptogenesis, and synaptic plasticity. In addition, we discuss the involvement of SynCAMs in neuropsychiatric disorders. Finally, we propose that SynCAM functions can be better viewed and understood from the perspective of orientational cell adhesions (OCAs). In particular, we discuss the possibilities of how SynCAMs can be regulated at the cell-type specific expression, transcription variants, posttranslational modification, and subcellular localization to modulate the diversity of SynCAMs as OCA molecules. Being major components of the synapses, SynCAMs continue to be an important research topic of neuroscience, and many outstanding questions are waiting to be answered.

Ash2l, an obligatory component of H3K4 methylation complexes, regulates neural crest development

The vertebrate nervous system develops from embryonic neural plate and neural crest. Although genetic mechanisms governing vertebrate neural development have been investigated in depth, epigenetic regulation of this process remains less understood. Redundancy of epigenetic factors and early lethality of animals deficient in critical epigenetic components pose major challenges in characterization of epigenetic factors in vertebrate neural development. In this study, we use the amphibian model Xenopus laevis to investigate the roles of non-redundant, obligatory components of all histone H3K4 activating methylation complexes (COMPASS, also known as SET1/MLL complexes) in early neural development. The two genes that we focus on, Ash2l and Dpy30, regulate mesendodermal differentiation in mouse embryonic stem cells and cause early embryonic lethality when removed from mouse embryos. Using targeted knockdown of the genes in dorsal ectoderm of Xenopus that gives rise to future nervous system, we show here that ash2l and dpy30 are required for neural and neural crest marker expression in Xenopus late neurula embryos but are dispensable for early neural and neural plate border gene expression. Co-immunoprecipitation assays reveal that Dpy30 and Ash2L associate with the neural plate border transcription factors, such as Msx1 and Tfap2a. Chromatin immunoprecipitation (ChIP) assay further demonstrates that Ash2L and the H3K4me3 active histone mark accumulate at the promoter regions of the neural crest gene sox10 in a Tfap2a-dependent manner. Collectively, our data suggest that Ash2l and Dpy30 interact with specific transcription factors to recruit COMPASS complexes to the regulatory regions of neural crest specification genes to control their expression and influence development of the nervous system during vertebrate embryogenesis.

Wnt/\u03b2-catenin signaling promotes neurogenesis in the diencephalospinal dopaminergic system of embryonic zebrafish

Wnt/β-catenin signaling contributes to patterning, proliferation, and differentiation throughout vertebrate neural development. Wnt/β-catenin signaling is important for mammalian midbrain dopaminergic neurogenesis, while little is known about its role in ventral forebrain dopaminergic development. Here, we focus on the A11-like, Otp-dependent diencephalospinal dopaminergic system in zebrafish. We show that Wnt ligands, receptors and extracellular antagonist genes are expressed in the vicinity of developing Otp-dependent dopaminergic neurons. Using transgenic Wnt/β-catenin-reporters, we found that Wnt/β-catenin signaling activity is absent from these dopaminergic neurons, but detected Wnt/β-catenin activity in cells adjacent to the caudal DC5/6 clusters of Otp-dependent dopaminergic neurons. Pharmacological manipulations of Wnt/β-catenin signaling activity, as well as heat-shock driven overexpression of Wnt agonists and antagonists, interfere with the development of DC5/6 dopaminergic neurons, such that Wnt/β-catenin activity positively correlates with their number. Wnt/β-catenin activity promoted dopaminergic development specifically at stages when DC5/6 dopaminergic progenitors are in a proliferative state. Our data suggest that Wnt/β-catenin signaling acts in a spatially and temporally restricted manner on proliferative dopaminergic progenitors in the hypothalamus to positively regulate the size of the dopaminergic neuron groups DC5 and DC6.

Sequential stabilization of RNF220 by RLIM and ZC4H2 during cerebellum development and Shh-group medulloblastoma progression.

ABSTRACTSonic hedgehog (Shh) signaling is essential for the proliferation of cerebellar granule neuron progenitors (CGNPs), and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma (MB). During vertebrate neural development, RNF220, a ubiquitin E3 ligase, is involved in spinal cord patterning by modulating the subcellular location of glioma-associated oncogene homologs (Glis) through ubiquitination. RNF220 is also required for full activation of Shh signaling during cerebellum development in an epigenetic manner through targeting embryonic ectoderm development. ZC4H2 was reported to be involved in spinal cord patterning by acting as an RNF220 stabilizer. Here, we provided evidence to show that ZC4H2 is also required for full activation of Shh signaling in CGNP and MB progression by stabilizing RNF220. In addition, we found that the ubiquitin E3 ligase RING finger LIM domain-binding protein (RLIM) is responsible for ZC4H2 stabilization via direct ubiquitination, through which RNF220 is also thus stabilized. RLIM is a direct target of Shh signaling and is also required for full activation of Shh signaling in CGNP and MB cell proliferation. We further provided clinical evidence to show that the RLIM‒ZC4H2‒RNF220 cascade is involved in Shh-group MB progression. Disease-causative human RLIM and ZC4H2 mutations affect their interaction and regulation. Therefore, our study sheds light on the regulation of Shh signaling during cerebellar development and MB progression and provides insights into neural disorders caused by RLIM or ZC4H2 mutations.

Multiple Functions of the Dmrt Genes in the Development of the Central Nervous System.

The Dmrt genes encode the transcription factor containing the DM (doublesex and mab-3) domain, an intertwined zinc finger-like DNA binding module. While Dmrt genes are mainly involved in the sexual development of various species, recent studies have revealed that Dmrt genes, which belong to the DmrtA subfamily, are differentially expressed in the embryonic brain and spinal cord and are essential for the development of the central nervous system. Herein, we summarize recent studies that reveal the multiple functions of the Dmrt genes in various aspects of vertebrate neural development, including brain patterning, neurogenesis, and the specification of neurons.

The many faces of the E3 ubiquitin ligase, RNF220, in neural development and beyond.

Ubiquitin modification plays important roles in many cellular processes that are fundamental for vertebrate embryo development, such as cell division, differentiation, and migration. Aberrant function or deregulation of ubiquitination enzymes can cause developmental disorders, cancer progression, and neurodegenerative diseases in humans. RING finger protein 220 (RNF220) is an evolutionarily conserved RING-type ubiquitin E3 ligase. Recent studies have revealed the roles and mechanisms of RNF220 and its partner protein, zinc finger C4H2-type containing protein (ZC4H2), in embryonic development and human diseases. Using mouse and zebrafish models, it has been shown that RNF220 regulates sonic hedgehog (Shh) signaling via Gli and embryonic ectoderm development (EED), a polycomb repressive complex 2 (PRC2) component, during ventral neural patterning and cerebellum development. In addition, RNF220 also regulates the development and functions of central noradrenergic and motor neurons in mice. By stabilizing β-catenin and signal transducer and activator of transcription 1 (STAT1), RNF220 is also involved in Wnt and interferon (IFN)-STAT1 signaling and thus the regulation of tumorigenesis and immune response, respectively. In humans, both RNF220 and ZC4H2 mutations have been reported to be associated with diseases accompanied by complicated neural defects. In this review, we summarize the current knowledge of RNF220 with special emphasis on its roles and mechanisms of action in signal transduction, vertebrate neural development, and related human disorders.

Essential function and targets of BMP signaling during midbrain neural crest delamination

BMP signaling plays iterative roles during vertebrate neural crest development from induction through craniofacial morphogenesis. However, far less is known about the role of BMP activity in cranial neural crest epithelial-to-mesenchymal transition and delamination. By measuring canonical BMP signaling activity as a function of time from specification through early migration of avian midbrain neural crest cells, we found elevated BMP signaling during delamination stages. Moreover, inhibition of canonical BMP activity via a dominant negative mutant Type I BMP receptor showed that BMP signaling is required for neural crest migration from the midbrain, independent from an effect on EMT and delamination. Transcriptome profiling on control compared to BMP-inhibited cranial neural crest cells identified novel BMP targets during neural crest delamination and early migration including targets of the Notch pathway that are upregulated following BMP inhibition. These results suggest potential crosstalk between the BMP and Notch pathways in early migrating cranial neural crest and provide novel insight into mechanisms regulated by BMP signaling during early craniofacial development.

Aberrant Gcm1 expression mediates Wnt/\u03b2-catenin pathway activation in folate deficiency involved in neural tube defects

Wnt signaling plays a major role in early neural development. An aberrant activation in Wnt/β-catenin pathway causes defective anteroposterior patterning, which results in neural tube closure defects (NTDs). Changes in folate metabolism may participate in early embryo fate determination. We have identified that folate deficiency activated Wnt/β-catenin pathway by upregulating a chorion-specific transcription factor Gcm1. Specifically, folate deficiency promoted formation of the Gcm1/β-catenin/T-cell factor (TCF4) complex formation to regulate the Wnt targeted gene transactivation through Wnt-responsive elements. Moreover, the transcription factor Nanog upregulated Gcm1 transcription in mESCs under folate deficiency. Lastly, in NTDs mouse models and low-folate NTDs human brain samples, Gcm1 and Wnt/β-catenin targeted genes related to neural tube closure are specifically overexpressed. These results indicated that low-folate level promoted Wnt/β-catenin signaling via activating Gcm1, and thus leaded into aberrant vertebrate neural development.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner.

Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.

Adult islet1 Expression Outlines Ventralized Derivatives Along Zebrafish Neuraxis.

Signals issued by dorsal roof and ventral floor plates, respectively, underlie the major patterning process of dorsalization and ventralization during vertebrate neural tube development. The ventrally produced morphogen Sonic hedgehog (SHH) is crucial for vertebrate hindbrain and spinal motor neuron development. One diagnostic gene for motor neurons is the LIM/homeodomain gene islet1, which has additional ventral expression domains extending into mid- and forebrain. In order to corroborate motor neuron development and, in particular, to improve on the identification of poorly documented zebrafish forebrain islet1 populations, we studied adult brains of transgenic islet1-GFP zebrafish (3 and 6 months). This molecular neuroanatomical analysis was supported by immunostaining these brains for tyrosine hydroxylase (TH) or choline acetyltransferase (ChAT), respectively, revealing zebrafish catecholaminergic and cholinergic neurons. The present analysis of ChAT and islet1-GFP label confirms ongoing adult expression of islet1 in zebrafish (basal plate) midbrain, hindbrain, and spinal motor neurons. In contrast, non-motor cholinergic systems lack islet1 expression. Additional presumed basal plate islet1 positive systems are described in detail, aided by TH staining which is particularly informative in the diencephalon. Finally, alar plate zebrafish forebrain systems with islet1 expression are described (i.e., thalamus, preoptic region, and subpallium). We conclude that adult zebrafish continue to express islet1 in the same brain systems as in the larva. Further, pending functional confirmation we hypothesize that the larval expression of sonic hedgehog (shh) might causally underlie much of adult islet1 expression because it explains findings beyond ventrally located systems, for example regarding shh expression in the zona limitans intrathalamica and correlated islet1-GFP expression in the thalamus.

Positive and negative regulation of Shh signalling in vertebrate retinal development.

Cell-to-cell communication is fundamental for embryo development and subsequent tissue homeostasis. This communication is often mediated by a small number of signaling pathways in which a secreted ligand binds to the surface of a target cell, thereby activating signal transduction. In vertebrate neural development, these signaling mechanisms are repeatedly used to obtain different and context-dependent outcomes. Part of the versatility of these communication mechanisms depends on their finely tuned regulation that controls timing, spatial localization, and duration of the signaling. The existence of secreted antagonists, which prevent ligand–receptor interaction, is an efficient mechanism to regulate some of these pathways. The Hedgehog family of signaling proteins, however, activates a pathway that is controlled largely by the positive or negative activity of membrane-bound proteins such as Cdon, Boc, Gas1, or Megalin/LRP2. In this review, we will use the development of the vertebrate retina, from its early specification to neurogenesis, to discuss whether there is an advantage to the use of such regulators, pointing to unresolved or controversial issues.

Radial Glia in Echinoderms.

Radial glial cells are crucial in vertebrate neural development and regeneration. It has been recently proposed that this neurogenic cell type might be older than the chordate lineage itself and might have been present in the last common deuterostome ancestor. Here, we summarize the results of recent studies on radial glia in echinoderms, a highly regenerative phylum of marine invertebrates with shared ancestry to chordates. We discuss the involvement of these cells in both homeostatic neurogenesis and post-traumatic neural regeneration, compare the features of radial glia in echinoderms and chordates to each other, and review the molecular mechanisms that control differentiation and plasticity of the echinoderm radial glia. Overall, studies on echinoderm radial glia provide a unique opportunity to understand the fundamental biology of this cell type from evolutionary and comparative perspectives.

Development of an Embryonic Zebrafish Oligodendrocyte-Neuron Mixed Coculture System.

During vertebrate neural development, oligodendrocytes insulate nerve axons with myelin sheaths. Zebrafish (Danio rerio) has emerged as a useful model organism for studying oligodendrocyte development. However, the absence of an in vitro culture system necessitates in vivo manipulations and analyses, which, in some instances, limits the questions that can be addressed. To fill this gap we developed a mixed coculture system for embryonic zebrafish neurons and oligodendrocyte-lineage cells. Cultures harvested from embryos ≥30 hours postfertilization (hpf) yielded oligodendrocyte progenitor cells (OPCs) positive for olig2 and sox10 transgenic reporters. Cultured OPCs exhibited dynamic, exploratory membrane processes, and cell morphologies resembled those established in vivo. Cells harvested from advanced stage embryos possessed more arborized processes than those from early stage embryos. Advanced stage (>60 hpf) embryo culture produced differentiated, mbp+ oligodendrocytes. Genetically tractable neuron subtypes extended neurites when harvested from embryos ≥19 hpf. Coculture produced juxtaposed oligodendrocytes and neurons, demonstrating the practical usefulness of this technique for future studies examining axon-oligodendrocyte interactions under defined conditions. We expect that zebrafish oligodendrocyte culture will complement existing in vivo strengths and may facilitate future studies elucidating the mechanisms of oligodendrocyte specification, proliferation, differentiation, motility, and axon-oligodendrocyte interactions that shape adult myelination patterns.

Developmental signature, synaptic connectivity and neurotransmission are conserved between vertebrate hair cells and tunicate coronal cells.

In tunicates, the coronal organ represents a sentinel checking particle entrance into the pharynx. The organ differentiates from an anterior embryonic area considered a proto-placode. For their embryonic origin, morphological features and function, coronal sensory cells have been hypothesized to be homologues to vertebrate hair cells. However, vertebrate hair cells derive from a posterior placode. This contradicts one of the principle historical criteria for homology, similarity of position, which could be taken as evidence against coronal cells/hair cells homology. In the tunicates Ciona intestinalis and C. robusta, we found that the coronal organ expresses genes (Atoh, Notch, Delta-like, Hairy-b, and Musashi) characterizing vertebrate neural and hair cell development. Moreover, coronal cells exhibit a complex synaptic connectivity pattern, and express neurotransmitters (Glu, ACh, GABA, 5-HT, and catecholamines), or enzymes for their synthetic machinery, involved in hair cell activity. Lastly, coronal cells express the Trpa gene, which encodes an ion channel expressed in hair cells. These data lead us to hypothesize a model in which competence to make secondary mechanoreceptors was initially broadly distributed through placode territories, but has become confined to different placodes during the evolution of the vertebrate and tunicate lineages.

Identification and functional analysis of SOX10 phosphorylation sites in melanoma.

The transcription factor SOX10 plays an important role in vertebrate neural crest development, including the establishment and maintenance of the melanocyte lineage. SOX10 is also highly expressed in melanoma tumors, and SOX10 expression increases with tumor progression. The suppression of SOX10 in melanoma cells activates TGF-β signaling and can promote resistance to BRAF and MEK inhibitors. Since resistance to BRAF/MEK inhibitors is seen in the majority of melanoma patients, there is an immediate need to assess the underlying biology that mediates resistance and to identify new targets for combinatorial therapeutic approaches. Previously, we demonstrated that SOX10 protein is required for tumor initiation, maintenance and survival. Here, we present data that support phosphorylation as a mechanism employed by melanoma cells to tightly regulate SOX10 expression. Mass spectrometry identified eight phosphorylation sites contained within SOX10, three of which (S24, S45 and T240) were selected for further analysis based on their location within predicted MAPK/CDK binding motifs. SOX10 mutations were generated at these phosphorylation sites to assess their impact on SOX10 protein function in melanoma cells, including transcriptional activation on target promoters, subcellular localization, and stability. These data further our understanding of SOX10 protein regulation and provide critical information for identification of molecular pathways that modulate SOX10 protein levels in melanoma, with the ultimate goal of discovering novel targets for more effective combinatorial therapeutic approaches for melanoma patients.

Sonic hedgehog in vertebrate neural tube development.

The formation and wiring of the vertebrate nervous system involves the spatially and temporally ordered production of diverse neuronal and glial subtypes that are molecularly and functionally distinct. The chick embryo has been the experimental model of choice for many of the studies that have led to our current understanding of this process, and has presaged and informed a wide range of complementary genetic studies, in particular in the mouse. The versatility and tractability of chick embryos means that it remains an important model system for many investigators in the field. Here we will focus on the role of Sonic hedgehog (Shh) signaling in coordinating the diversification, patterning, growth and differentiation of the vertebrate nervous system. We highlight how studies in chick led to the identification of the role Shh plays in the developing neural tube and how subsequent work, including studies in the chick and the mouse revealed details of the cell intrinsic programs controlling cell fate determination. We compare these mechanisms at different rostral-caudal positions along the neuraxis and discuss the particular experimental attributes of the chick that facilitated this work.

Wnt/β-catenin signaling during early vertebrate neural development.

The vertebrate central nervous system (CNS) is comprised of vast number of distinct cell types arranged in a highly organized manner. This high degree of complexity is achieved by cellular communication, including direct cell-cell contact, cell-matrix interactions, and cell-growth factor signaling. Among the several developmental signals controlling the development of the CNS, Wnt proteins have emerged as particularly critical and, hence, have captivated the attention of many researchers. With Wnts' evolutionarily conserved function as primordial symmetry breaking signals, these proteins and their downstream effects are responsible for simultaneously establishing cellular diversity and tissue organization. With their expansive repertoire of secreted agonists and antagonists, cell surface receptors, signaling cascades and downstream biological effects, Wnts are ideally suited to control the complex processes underlying vertebrate neural development. In this review, we will describe the mechanisms by which Wnts exert their potent effects on cells and tissues and highlight the many roles of Wnt signaling during neural development, starting from the initial induction of the neural plate, the subsequent patterning along the embryonic axes, to the intricately organized structure of the CNS. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1239-1259, 2017.