Correction: scMultiome analysis identifies a single caudal hindbrain compartment in the developing zebrafish nervous system

BackgroundA key step in nervous system development involves the coordinated control of neural progenitor specification and positioning. A long-standing model for the vertebrate CNS postulates that transient anatomical compartments – known as neuromeres – function to position neural progenitors along the embryonic anteroposterior neuraxis. Such neuromeres are apparent in the embryonic hindbrain – that contains six rhombomeres with morphologically apparent boundaries – but other neuromeres lack clear morphological boundaries and have instead been defined by different criteria, such as differences in gene expression patterns and the outcomes of transplantation experiments. Accordingly, the caudal hindbrain (CHB) posterior to rhombomere (r) 6 has been variably proposed to contain from two to five ‘pseudo-rhombomeres’, but the lack of comprehensive molecular data has precluded a detailed definition of such structures.MethodsWe used single-cell Multiome analysis, which allows simultaneous characterization of gene expression and chromatin state of individual cell nuclei, to identify and characterize CHB progenitors in the developing zebrafish CNS.ResultsWe identified CHB progenitors as a transcriptionally distinct population, that also possesses a unique profile of accessible transcription factor binding motifs, relative to both r6 and the spinal cord. This CHB population can be subdivided along its dorsoventral axis based on molecular characteristics, but we do not find any molecular evidence that it contains multiple pseudo-rhombomeres. We further observe that the CHB is closely related to r6 at the earliest embryonic stages, but becomes more divergent over time, and that it is defined by a unique gene regulatory network.ConclusionsWe conclude that the early CHB represents a single neuromere compartment that cannot be molecularly subdivided into pseudo-rhombomeres and that it may share an embryonic origin with r6.

The round-spotted pufferfish (Tetraodon fluviatilis) had the genome size of 380 Mb which is the smallest of any vertebrate. We used this organism for complete determination of the genomic structure and characterization of the promoter region of disease-related genes (ret and Menkes-related genes) and all JAK/STAT genes. All pufferfish JAK kinase genes are composed of 24 exons except JAK1 gene, which has an additional exon. A comparison of the exon-intron organization of these genes reveals that all splice sites of JAK kinase genes are nearly indentical, suggesting that the pufferfish JAK kinase genes may evolve from a common ancestor. The putative promoter regions of four JAK kinase genes were tested either in a carp CF cell line or in zebrafish embryos by using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo. We have also completely determined the genomic structure of seven STAT genes. Currently, we have constructed the constitutively active form of different STAT cDNAs and are analyzing their DNA binding and transactivation activity in various cell lines. At present, we are trying different approaches such as the generation of transgenic zebrafish that carried the constitutively active STAT or microinjection of constitutively active STAT mRNA into one-cell stage zebrafish embryos to test its effect in vivo. We have also cloned and characterized the mnk gene that encodes the copper transporter of the roundspotted pufferfish. The mnk gene is composed of 23 exons spanning about 12.5 kb comparing to ＞150 Kb in human. The intron-exon junctions are all highly conserved between the round-spotted pufferfish and human. By comparing the amino acid sequence, the pufferfish MNK protein shows 62.1% identity to the corresponding human Menkes disease gene product. In addition, we are working on some novel intron-less protein kinases and one brain-specific protein kinase that may involve in the development of zebrafish nervous system. Some techniques, such as wholemount in situ hybridization, transgenic zebrafish, and microinjection of RNAi/double-stranded RNA into zebrafish embryo have been established and used to study the possible function of these protein kinases.

Semaphorin6A (Sema6A) and its PlexinA2 (PlxnA2) receptor canonically function as repulsive axon guidance cues. To understand downstream signaling mechanisms, we performed a microarray screen and identified the "clutch molecule" shootin-1 (shtn-1) as a transcriptionally repressed target. Shtn-1 is a key proponent of cell migration and neuronal polarization and must be regulated during nervous system development. The mechanisms of Shtn-1 regulation and the phenotypic consequences of loss of repression are poorly understood. We demonstrate shtn-1 overexpression results in impaired migration of the optic vesicles, lack of retinal pigmented epithelium, and pathfinding errors of retinotectal projections. We also observed patterning defects in the peripheral nervous system. Importantly, these phenotypes were rescued by overexpressing PlxnA2. We demonstrate a functional role for repression of shtn-1 by PlxnA2 in development of the eyes and peripheral nervous system in zebrafish. These results demonstrate that careful regulation of shtn-1 is critical for development of the nervous system.

Chapter Seven - Effects of Ethanol Exposure on Nervous System Development in Zebrafish

Neurofilament light chain (NEFL), a subunit of neurofilament, has been shown to play an important role in pathogenic neurodegenerative disease and in radial axonal growth. However, information remains largely lacking regarding the function of NEFL in early development to date. In this study, we demonstrated the presence of two nefl genes, nefla and neflb, in zebrafish, generated by fish-specific third round genome duplication. These duplicated nefl genes were predominantly expressed in the nervous system with an overlapping and distinct expression pattern. Both gene knockdown and rescue experiments show that it was neflb rather than nefla that played an indispensable role in nervous system development. It was also found that neflb knockdown resulted in striking apoptosis of the neurons in the brain and spinal cord, leading to morphological defects such as brain structure disorder and trunk bending. Thus, we report a previously uncharacterized role of NEFL that NEFLb impairs the early development of zebrafish nervous system via regulation of the neuron apoptosis in the brain and spinal cord.

Avobenzone and nanoplastics affect the development of zebrafish nervous system and retinal system and inhibit their locomotor behavior

The Slitrk family of leucine-rich repeat (LRR) transmembrane proteins bears structural similarity to the Slits and the Trk receptor families, which exert well-established roles in directing nervous system development. Slitrks are less well understood, although they are highly expressed in the developing vertebrate nervous system. Moreover, slitrk variants are associated with several sensory and neuropsychiatric disorders, including myopia, deafness, obsessive-compulsive disorder (OCD), schizophrenia, and Tourette syndrome. Loss-of-function studies in mice show that Slitrks modulate neurite outgrowth and inhibitory synapse formation, although the molecular mechanisms of Slitrk function remain poorly characterized. As a prelude to examining the functional roles of Slitrks, we identified eight slitrk orthologs in zebrafish and observed that seven of the eight orthologs were actively transcribed in the nervous system at embryonic, larval, and adult stages. Similar to previous findings in mice and humans, zebrafish slitrks exhibited unique but overlapping spatial and temporal expression patterns in the developing brain, retina, and spinal cord. Zebrafish express Slitrks in the developing central nervous system at times and locations important to neuronal morphogenesis and synaptogenesis. Future studies will use zebrafish as a convenient, cost-effective model organism to characterize the functional roles of Slitrks in nervous system development.

Polysialic acid (polySia) is mainly described as a glycan modification of the neural cell adhesion molecule NCAM1. PolySia-NCAM1 has multiple functions during the development of vertebrate nervous systems including axon extension and fasciculation. Phylogenetic analyses reveal the presence of two related gene clusters, NCAM1 and NCAM2, in tetrapods and fishes. Within the ncam1 cluster, teleost fishes express ncam1a (ncam) and ncam1b (pcam) as duplicated paralogs which arose from a second round of ray-finned fish-specific genome duplication. Tetrapods, in contrast, express a single NCAM1 gene. Using the zebrafish model, we identify Ncam1b as a novel major carrier of polySia in the nervous system. PolySia-Ncam1a is expressed predominantly in rostral regions of the developing nervous system, whereas polySia-Ncam1b prevails caudally. We show that ncam1a and ncam1b have different expression domains which only partially overlap. Furthermore, Ncam1a and Ncam1b and their polySia modifications serve different functions in axon guidance. Formation of the posterior commissure at the forebrain/midbrain junction requires polySia-Ncam1a on the axons for proper fasciculation, whereas Ncam1b, expressed by midbrain cell bodies, serves as an instructive guidance cue for the dorso-medially directed growth of axons. Spinal motor axons, on the other hand, depend on axonally expressed Ncam1b for correct growth toward their target region. Collectively, these findings suggest that the genome duplication in the teleost lineage has provided the basis for a functional diversification of polySia carriers in the nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

Fenpropathrin has been a commonly used insecticide to control agricultural and household insects over a few decades. Up to now, fenpropathrin residue in soil and water has been often determined due to its widespread use, which poses serious threat to environment and aquatic organisms. The potential of fenpropathrin to affect aquatic lives is still poorly understood. In this study, we used zebrafish (Danio rerio) embryo as an experimental model system to evaluate the toxicity of fenpropathrin to the development of zebrafish nervous system. Zebrafish embryos were separately exposed to fenpropathrin at the dose of 0.016mg/L, 0.032mg/L, 0.064mg/L, starting at 6h post-fertilizationhpf (hpf) up to 96 hpf. The results showed that fenpropathrin exposure gives rise to physiological, behavioral, and neurodevelopmental impairments in zebrafish embryos, including enhanced acetylcholinesterase (AChE) activity, abnormal swimming behavior, karyopyknosis in brain cells, increased intercellular space, and uneven migration of neuron in brain area. In addition, the expressions of genes concerning neurodevelopment and neurotransmitter system were inhibited following fenpropathrin exposure. We also found that fenpropathrin exposure distinctly induced oxidative stress by increasing reactive oxygen species (ROS) generation and inhibiting the production of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Expectedly, some apoptosis-associated genes were induced and the apoptosis appeared in the brain and heart cells of zebrafish embryos. Moreover, fenpropathrin exposure also inhibited the expressions of genes in Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1) and SOD. In summary, the results of this study indicate that oxidative stress-triggered apoptosis may be an underlying fundamental of fenpropathrin-induced neurotoxicity in zebrafish embryos.

The implications of autophagy-related genes in serious neural degenerative diseases have been well documented. However, the functions and regulation of the family genes in embryonic development remain to be rigorously studied. Here, we report on for the first time the important role of atg5 gene in zebrafish neurogenesis and organogenesis as evidenced by the spatiotemporal expression pattern and functional analysis. Using morpholino oligo knockdown and mRNA overexpression, we demonstrated that zebrafish atg5 is required for normal morphogenesis of brain regionalization and body plan as well as for expression regulation of neural gene markers: gli1, huC, nkx2.2, pink1, β-synuclein, xb51 and zic1. We further demonstrated that ATG5 protein is involved in autophagy by LC3-II/LC3I ratio and rapamycin-induction experiments, and that ATG5 is capable of regulating expression of itself gene in the manner of a feedback inhibition loop. In addition, we found that expression of another autophagy-related gene, atg12, is maintained at a higher constant level like a housekeeping gene. This indicates that the formation of the ATG12–ATG5 conjugate may be dependent on ATG5 protein generation and its splicing, rather than on ATG12 protein in zebrafish. Importantly, in the present study, we provide a mechanistic insight into the regulation and functional roles of atg5 in development of zebrafish nervous system.

Expression of unconventional myosin genes during neuronal development in zebrafish

Influence of Nanoparticle Exposure on Nervous System Development in Zebrafish Studied by Means of Light Sheet Fluorescence Microscopy

Today, less than 1% of compounds that are registered for commercial use with the United States Environmental Protection Agency (EPA) have associated developmental neurotoxicity data. There is a need to identify human developmental neurotoxins and prioritize them for future studies. Two chemicals for which there is little neurotoxicity data are the insecticides carbaryl and malathion, that are commonly used in modern agriculture. Despite their benefits to agricultural yield, overspray and runoff of insecticides from fields may contaminate bodies of water, resulting in serious damage to non‐target aquatic fish. In this study, the toxicity of these two insecticides was measured by examining zebrafish locomotor activity and nervous system development, specifically the retino‐tectal area of the brain. Since both of these insecticides are acetylcholinesterase (AChE) inhibitors, it is expected that carbaryl and malathion will decrease locomotor activity and decrease the retino‐tectal area. Results indicate that carbaryl concentrations decreased swimming speed, but malathion concentrations had no effect on swimming speed. The experimental concentrations of carbaryl and malathion in this experiment can show disruptions of the nervous system by decreasing swim speed, and decreasing the retino‐tectal area. Funding for this research project was provided by the Westminster College Drinko Center.

Chapter 16 - Using Zebrafish to Assess Developmental Neurotoxicity