Developing Zebrafish Brain Research Articles

Structural and mechanistic insights into secretagogin-mediated exocytosis

Secretagogin (SCGN) is a hexa-EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.

Hexb enzyme deficiency leads to lysosomal abnormalities in radial glia and microglia in zebrafish brain development.

Sphingolipidoses are severe, mostly infantile lysosomal storage disorders (LSDs) caused by defective glycosphingolipid degradation. Two of these sphingolipidoses, Tay Sachs and Sandhoff diseases, are caused by β‐Hexosaminidase (HEXB) enzyme deficiency, resulting in ganglioside (GM2) accumulation and neuronal loss. The precise sequence of cellular events preceding, and leading to, neuropathology remains unclear, but likely involves inflammation and lysosomal accumulation of GM2 in multiple cell types. We aimed to determine the consequences of Hexb activity loss for different brain cell types using zebrafish. Hexb deficient zebrafish (hexb−/−) showed lysosomal abnormalities already early in development both in radial glia, which are the neuronal and glial progenitors, and in microglia. Additionally, at 5 days postfertilization, hexb −/− zebrafish showed reduced locomotor activity. Although specific oligosaccharides accumulate in the adult brain, hexb−/−) zebrafish are viable and apparently resistant to Hexb deficiency. In all, we identified cellular consequences of loss of Hexb enzyme activity during embryonic brain development, showing early effects on glia, which possibly underlie the behavioral aberrations. Hereby, we identified clues into the contribution of non‐neuronal lysosomal abnormalities in LSDs affecting the brain and provide a tool to further study what underlies the relative resistance to Hexb deficiency in vivo.

Parentally inherited long non-coding RNA Cyrano is involved in zebrafish neurodevelopment.

Transfer of genetic material from parents to progeny via fusion of gametes is a way to ensure flow of information from one generation to the next. Apart from the genetic material, gametes provide a rich source of other factors such as RNA and proteins which can control traits of the embryo. Non-coding RNAs are not only carriers of regulatory information but can also encode memory of events of parental life. Here, we explore the possibility of parental inheritance of non-coding RNAs, especially long non-coding RNAs. Meta-analysis of RNA-seq data revealed several non-coding RNAs present in zebrafish oocyte, sperm and 2cell-stage. The embryo is transcriptionally silent at this stage, we rationalize that all the RNAs detectable at 2cell-stage are deposited either by sperm or oocyte or both and thus inherited. In the inherited pool, we noticed a conserved lncRNA, Cyrano previously known for zebrafish brain development. Knockdown of inherited Cyrano by miR-7 without changing zygotic Cyrano altered brain morphology at 24 hpf and 48 hpf. This defect could be partially rescued by injecting full length Cyrano lncRNA or a mutant resilient to knock-down by miR-7. In future, there is ample scope to check the possibility of inherited lncRNAs as carriers of memory of parental life events and building blocks that set up an initial platform for development.

Germ cell depletion in zebrafish leads to incomplete masculinization of the brain

Zebrafish sex differentiation is under the control of multiple genes, but also relies on germ cell number for gonadal development. Morpholino and chemical mediated germ cell depletion leads to sterile male development in zebrafish. In this study we produced sterile males, using a dead end gene morpholino, to determine gonadal-brain interactions. Germ cell depletion following dnd inhibition downregulated the germ cell markers, vasa and ziwi, and later the larvae developed as sterile males. Despite lacking proper testis, the gonadal 11-ketotestosterone (11-KT) and estradiol (E2) levels of sterile males were similar to wild type males. Qualitative analysis of sexual behavior of sterile males demonstrated that they behaved like wild type males. Furthermore, we observed that brain 11-KT and E2 levels in sterile males remained the same as in the wild type males. In female brain, 11-KT was lower in comparison to wild type males and sterile males, while E2 was higher when compared to wild type males. qRT-PCR analysis revealed that the liver transcript profile of sterile adult males was similar to wild type males while the brain transcript profile was similar to wild type females. The results demonstrate that proper testis development may not be a prerequisite for male brain development in zebrafish but that it may be needed to fully masculinize the brain.

Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA): An aquatic model for autism

Autism spectrum disorder (ASD) has complex neurodevelopmental impairments and origins that are linked to both genetic and environmental factors. Hence, there is an urgency to establish animal models with ASD-like characteristics to understand the underlying mechanisms of ASD. Prenatal exposure to valproic acid (VPA) produced ASD-like symptoms in humans, rats, and recently zebrafish. The present study investigated the use of VPA exposure to generate an ASD model in zebrafish. Early life stage exposures produced ASD-like phenotypes in the developing brain development and behavioral changes in embryonic and larval zebrafish. Our findings revealed that treating zebrafish embryos with VPA starting at 8h post fertilization (hpf) resulted in significant: increase in the ASD macrocephalic phenotype; hyperactivity of embryo/larvae movement behaviors; and increases of ASD-like larval social behaviors. Further analysis showed increases in cell proliferation, the proportion of mature newborn neurons, and neural stem cell proliferation in the brain region, which may contribute to the brain overgrowth and macrocephaly observed following VPA exposure. Our study demonstrated that VPA exposure generates ASD-like phenotypes and behaviors, indicating that zebrafish is an alternative model to investigate underlying ASD mechanisms.

Temporal and spatial expression of insulin‐like peptide (insl5a and insl5b) paralog genes during the embryogenesis of Danio rerio

Relaxin (RLN) and insulin (INSL)-like peptides are member of the INSL/RLN superfamily, which are encoded by seven genes in humans and can activate the G-protein coupled receptor RXFP 1-4. These peptides evolved from a common ancestor, RLN3-like gene. Two rounds of whole genome duplication (WGD) in early vertebrate evolution, together with an additional WGD in the teleost lineage, caused an expansion of RLN genes set in the genome of Danio rerio. In particular, six RLN genes are present: a single copy of rln and insl3 genes, and two paralogs for the rln3 gene (rln3a and rln3b), and the insl5 gene (insl5a and insl5b). We have already reported the presence of rln3a and rln3b genes in the developing zebrafish brain, as well as the expression of rln gene in the developing zebrafish brain and extraneural territories, such as thyroid gland and pancreas. Here, we report for the first time the expression of the two parologs genes for insl5, insl5a, and insl5b in D. rerio embryonic development. The corresponding transcripts of both the paralogs are present in all embryonic stages analyzed by RT-qPCR. In situ hybridization analyses showed a restricted signal in intestinal cells and the pancreatic region at 72 hpf for insl5a, while at 96 hpf both genes are expressed in specific intestinal cells. Furthermore, in adult zebrafish intestine tissue, in situ hybridation experiments showed that insl5a transcript is specifically localized in the goblet cells, while insl5b transcript is in enteroendocrine cells. These data revealed a high degree of gene expression pattern conservation for such genes in vertebrate evolution.

Comparison of molecular marker expression in early zebrafish brain development following chronic ethanol or morpholino treatment.

This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons.

Maternal cortisol stimulates neurogenesis and affects larval behaviour in zebrafish

Excess glucocorticoid transferred from stressed mother to the embryo affects developing vertebrate offspring, but the underlying programming events are unclear. In this study, we tested the hypothesis that increased zygotic glucocorticoid deposition, mimicking a maternal stress scenario, modifies early brain development and larval behaviour in zebrafish (Danio rerio). Cortisol was microinjected into the yolk at one cell-stage, to mimic maternal transfer, and the larvae [96 hours post-fertilization (hpf)] displayed increased activity in light and a reduction in thigmotaxis, a behavioural model for anxiety, suggesting an increased propensity for boldness. This cortisol-mediated behavioural phenotype corresponded with an increase in primary neurogenesis, as measured by incorporation of EdU at 24 hpf, in a region-specific manner in the preoptic region and the pallium, the teleostean homolog of the hippocampus. Also, cortisol increased the expression of the proneural gene neurod4, a marker of neurogenesis, in a region- and development-specific manner in the embryos. Altogether, excess zygotic cortisol, mimicking maternal stress, affects early brain development and behavioural phenotype in larval zebrafish. We propose a key role for cortisol in altering brain development leading to enhanced boldness, which may be beneficial in preparing the offspring to a stressful environment and enhancing fitness.

Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae.

A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain. We also generated stmn4 maternal-zygotic knockout zebrafish by the CRISPR/Cas9 system. Unexpectedly, only less than 10% of stmn4 mutants showed similar phenotypes observed in that of stmn4 morphants. It might be due to the complementation of the increased stmn1b expression observed in stmn4 mutants. In addition, time-lapse recordings revealed the changes in cellular proliferation and differentiation in stmn4 morphants. Stmn4 morphants displayed a longer G2 phase that could be rescued by Cdc25a. Furthermore, the inhibition of Wnt could reduce stmn4 transcripts. These results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during the neural keel stage.

Trim69 regulates zebrafish brain development by ap-1 pathway.

Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway.

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain

Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA). The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4- or 7-day post-fertilization (dpf) zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B), expressed in the brain, using three different in situ/in vivo strategies: (1) Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols; (2) Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus); and (3) Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay) at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF, or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα). Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP) did not show estrogenic activity in our model.

Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

Immunoimaging with Light Sheet Microscopy: Microglial Dynamics in the Developing Zebrafish Brain

The immune system is integral to the natural development and homeostasis of the brain, and in its response to injury and disease. These processes are highly sensitive to cell position and dynamics, and live imaging is important for quantification of these aspects of the immune response. The zebrafish is well established as a model organism for developmental biology due to its relative transparency and ease of study by microscopy. Recent zebrafish transgenic lines are extending its use to the study of the innate immune system. However, the immune system is characterized in part by the fast, organism-scale motility of its cells, presenting challenges for proper observation of its roles, both in the development of the organism and in immune responses. We apply 1- and 2-photon light sheet microscopy, or selective plane illumination microscopy (SPIM), to achieve rapid, large volume imaging of the developing zebrafish brain and the dynamics of microglia - the main innate immune cell of the brain. The high-speed, low-photodamage imaging by SPIM enables the direct tracking of microglia throughout the entire zebrafish brain in early development. Further, the high spatio-temporal resolution allows observation of the microglial processes during development, as well as of the active state in response to pathological stimuli. In capturing these dynamics, across the full organ, we begin to gain a more complete understanding of neuroimmunology not available with other approaches. This work lays the foundation for the measurement of neuroimmune dynamics and their relationship to developing brain circuitry and vasculature as well as activity and function.

Single neuron morphology in vivo with confined primed conversion.

Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion,"which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.

Identification of Wnt Genes Expressed in Neural Progenitor Zones during Zebrafish Brain Development.

Wnt signaling regulates multiple aspects of vertebrate central nervous system (CNS) development, including neurogenesis. However, vertebrate genomes can contain up to 25 Wnt genes, the functions of which are poorly characterized partly due to redundancy in their expression. To identify candidate Wnt genes as candidate mediators of pathway activity in specific brain progenitor zones, we have performed a comprehensive expression analysis at three different stages during zebrafish development. Antisense RNA probes for 21 Wnt genes were generated from existing and newly synthesized cDNA clones and used for in situ hybridization on whole embryos and dissected brains. As in other species, we found that Wnt expression patterns in the embryonic zebrafish CNS are complex and often redundant. We observed that progenitor zones in the telencephalon, dorsal diencephalon, hypothalamus, midbrain, midbrain-hindbrain boundary, cerebellum and retina all express multiple Wnt genes. Our data identify 12 specific ligands that can now be tested using loss-of-function approaches.

How the optic tectum stacks up

Sticky proteins help create distinctive pattern in visual center of the zebrafish brain.

Mecp2 regulates neural cell differentiation by suppressing the Id1 to Her2 axis in zebrafish.

Rett syndrome (RTT) is a progressive neurological disorder caused by mutations in the X-linked protein methyl-CpG-binding protein 2 (MeCP2). The endogenous function of MeCP2 during neural differentiation is still unclear. Here, we report that mecp2 is required for brain development in zebrafish. Mecp2 was broadly expressed initially in embryos and enriched later in the brain. Either morpholino knockdown or genetic depletion of mecp2 inhibited neuronal differentiation, whereas its overexpression promoted neuronal differentiation, suggesting an essential role of mecp2 in directing neural precursors into differentiated neurons. Mechanistically, her2 (the zebrafish ortholog of mammalian Hes5) was upregulated in mecp2 morphants in an Id1-dependent manner. Moreover, knockdown of either her2 or id1 fully rescued neuronal differentiation in mecp2 morphants. These results suggest that Mecp2 plays an important role in neural cell development by suppressing the Id1-Her2 axis, and provide new evidence that embryonic neural defects contribute to the later motor and cognitive dysfunctions in RTT.

Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain.

In rodents, the Npas4 gene has recently been identified as being an important regulator of synaptic plasticity and memory. Homologs of Npas4 have been found in invertebrate species though their functions appear to be too divergent for them to be studied as a proxy for the mammalian proteins. The aim of this study, therefore, was to ascertain the suitability of the zebrafish as a model organism for investigating the function of Npas4 genes. We show here that the expression and regulation of the zebrafish Npas4 homolog, npas4a, is remarkably similar to that of the rodent Npas4 genes. As in mammals, expression of the zebrafish npas4a gene is restricted to the brain where it is up-regulated in response to neuronal activity. Furthermore, we also show that knockdown of npas4a during embryonic development results in a number of forebrain-specific defects including increased apoptosis and misexpression of the forebrain marker genes dlx1a and shha. Our work demonstrates that the zebrafish is a suitable model organism for investigating the role of the npas4a gene and one that is likely to provide valuable insights into the function of the mammalian homologs. Furthermore, our findings highlight a potential role for npas4a in forebrain development.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.