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PubMed2025

The snakehead retrovirus promoter functions independently of the 3'ORF protein and its products are maternally inherited in transgenic zebrafish.

The exogenous snakehead retrovirus (SnRV) is an unclassified member of the Orthoretrovirinae subfamily, discovered in cell lines derived from several fish species. SnRV resembles complex lentiviruses and potentially encodes accessory proteins, including the product of the 3' open reading frame (3'ORF). The 3'ORF protein was suggested to function as a transactivator of transcription (Tat). Here, we constructed an infectious molecular clone for SnRV and tested the effects of 3'ORF mutations on SnRV transcription. Although replacing 3'ORF with foreign sequences strongly reduced virus expression and production, an out-of-frame point mutation in 3'ORF had only a minimal effect on SnRV replication. This latter result suggests that the 3'ORF protein does not function as Tat and that SnRV transcription is largely independent of the product of this ORF. We also show that in vitro, the SnRV promoter is versatile and robustly functioning in both fish and mammalian cultured cells. Finally, the SnRV promoter was transiently active in injected zebrafish embryos as early as the blastula stage. In transgenic zebrafish, this promoter drives enhanced expression in sensory organs and gonads, and its generated products are maternally inherited. Considering these characteristics, the SnRV promoter emerges as a promising candidate for developing versatile expression vectors applicable to research and biotechnological applications.

Animals↗

PubMed1992

Axonogenesis and morphogenesis in the embryonic zebrafish brain.

We have examined early neuronal differentiation and axonogenesis in the fore- and midbrain of zebrafish embryos to address general issues of early vertebrate brain development. AChE expression and HNK-1 antibody immunoreactivity were used as markers for differentiated neurons and axons, respectively. The pattern of neuronal differentiation followed a stereotyped sequence. AChE-positive cells first appeared between 14 and 16 hr in three small, isolated, bilaterally symmetrical clusters on the surface of the brain. The three clusters--the dorsorostral, ventrorostral, and ventrocaudal clusters--proved to be the progenitors of the telencephalon, ventral diencephalon, and mesencephalic tegmentum, respectively. With further development, more cells were added to these three clusters, and new clusters appeared in the anlage of the epiphysis (18 hr) and in the pituitary and dorsal mesencephalon (by 24 hr). Subsequently, as more neurons differentiated, the gaps of unlabeled cells were reduced; by 48 hr, the cluster boundaries were indistinguishable. Axonogenesis also followed a stereotyped sequence. The first HNK-1-labeled processes arose from the first three clusters of AChE-positive cells and connected the clusters. The earliest axonal growth cones appeared at 16 hr, directed caudally from two to three neurons of the ventrocaudal cluster and pioneering the ventral longitudinal tract. By 18 hr, the tract of the postoptic commissure was initiated by growth cones directed caudally from the ventrorostral cluster toward the ventrocaudal cluster. By 20 hr, axons from the dorsorostral cluster projected ventrally to form the supraoptic tract. The other dorsoventral tracts (the dorsoventral diencephalic tract and the tract of the posterior commissure) became evident between 20 and 24 hr. These observations provide a continuous record of the topological distortions involved in the conversion of the tubular embryonic brain into the contorted adult form. The telencephalon, ventral diencephalon, and hypothalamus originate from the same rostrocaudal level of the neural tube. The pattern of differentiation demonstrated that the early development of the rostral neural tube occurs simultaneously in several independent centers, similar to the overtly segmental development of the hindbrain.

Acetylcholinesterase↗

PubMed2025

H3K4me2 distinguishes a distinct class of enhancers during the maternal-to-zygotic transition.

After egg fertilization, an initially silent embryonic genome is transcriptionally activated during the maternal-to-zygotic transition. In zebrafish, maternal vertebrate pluripotency factors Nanog, Pou5f3 (OCT4 homolog), and Sox19b (SOX2 homolog) (NPS) play essential roles in orchestrating embryonic genome activation, acting as "pioneers" that open condensed chromatin and mediate acquisition of activating histone modifications. However, some embryonic gene transcription still occurs in the absence of these factors, suggesting the existence of other mechanisms regulating genome activation. To identify chromatin signatures of these unknown pathways, we profiled the histone modification landscape of zebrafish embryos using CUT&RUN. Our regulatory map revealed two subclasses of enhancers distinguished by presence or absence of H3K4me2. Enhancers lacking H3K4me2 tend to require NPS factors for de novo activation, while enhancers bearing H3K4me2 are epigenetically bookmarked by DNA hypomethylation to recapitulate gamete activity in the embryo, independent of NPS pioneering. Thus, parallel enhancer activation pathways combine to induce transcriptional reprogramming to pluripotency in the early embryo.

Animals↗

PubMed2027

Genomic Profiling of Chromatin State Using CUT&Tag.

Alterations in chromatin state, mediated through histone modifications and the incorporation of histone variants, are fundamental to establishing transcriptional networks and cell identity. Recent advances in low-input epigenome profiling methods, such as CUT&Tag and CUT&RUN, have enabled the study of chromatin states from very limited starting materials. In this chapter, we describe procedures for generating CUT&Tag libraries to profile histone modifications and histone variants in early-developing zebrafish embryos.

Animals↗

PubMed1992

Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture.

Previous culture studies have demonstrated that muscle cells autonomously express and cluster ACh receptors (AChRs) and that contact by neurites induces a reorganization of these clusters. We studied these phenomena in zebrafish embryos where the same cells could be examined in vivo and in vitro, and where contacts between cells could be viewed repeatedly. Receptor clusters first appeared when the pioneer growth cones emerged from the spinal cord, were always associated with labeled branches, and developed normally in the presence of neuromuscular transmission blockers. When motoneurons were removed, the muscles failed to cluster receptors. In contrast, muscle cells grown in cell culture uncontacted by nerves clustered AChRs. Our results suggest that clustering of AChRs in living embryos is induced by the presence of neurites and does not occur in the absence of neuronal contact. We suggest that conditions in cell culture, which differ from those in the intact embryo, induce clusters on isolated muscle cells. Moreover, our results demonstrate that receptors cluster without binding transmitter and in the absence of neuronal activity.

Animals↗

PubMed2026

Neurodevelopmental toxicity of 2-(Methylthio)benzothiazole (MTBT) in zebrafish: Insights into PTGS2- associated dysregulation of the neuroactive ligand-receptor interaction pathway.

2-(Methylthio)benzothiazole (MTBT), an important derivative of benzothiazoles, has extensive applications in industrial processes, pharmaceuticals, and environmental monitoring. It can enter aquatic environments through surface runoff and has been detected at relatively high concentrations in various environmental systems. However, studies investigating the aquatic toxicity of MTBT remain limited. In this study, zebrafish embryos were exposed to MTBT at concentrations of 0, 10, 100, and 1000 μg/L for 144 h to evaluate its developmental and neurotoxic effects. MTBT exposure significantly reduced the survival rate, hatching rate, spontaneous movement, and body length of zebrafish larvae. MTBT also impaired locomotor behavior, reduced fluorescence of Tg(huc:eGFP) larvae in the central nervous system and inhibited motor neuron axonal development. Protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses indicated that MTBT-induced neurotoxicity may be associated with disruption of the neuroactive ligand-receptor interaction pathway. Further validation experiments revealed that MTBT induced oxidative stress, inflammation, and apoptosis, suggesting that these adverse effects may underlie its neurodevelopmental toxicity. Collectively, these findings provide biological evidence that MTBT induces neurodevelopmental toxicity in zebrafish larvae and suggest that dysregulation of the PTGS2-related neuroactive ligand-receptor interaction pathway may be involved in this process.

2-(Methylthio)benzothiazole (MTBT)↗

PubMed1992

Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea.

Recessive lethal mutations and mutations at the gol-1 locus were induced in the zebrafish by exposure of mature sperm to the alkylating agent ethyl nitrosourea (ENU). Embryonic lethal phenotypes were recognized among the parthenogenetic progeny of mutagenized animals or among the progeny of daughters of mutagenized animals. Novel specific locus mutations were identified by the failure of mutagenized chromosomes to complement pre-existing mutant alleles at the gol-1 locus. Each mutagenized individual harboured approximately 10 embryonic lethal mutations in its germ line and about 1 in 500 mutagenized animals harboured a new mutation at the gol-1 locus. Three lines of evidence indicate that the majority of mutations that were recovered following treatment of mature sperm with ENU were probably point mutations. First, the soma and germ lines of mutagenized animals were mosaic, as expected following simple alkylation of sperm DNA. Second, mutations induced by ENU at the gol-1 locus affected pigmentation but not viability, unlike the majority of mutations induced at this locus with gamma-irradiation. Third, the ratio of specific locus:recessive lethal mutations induced by ENU was approximately 50-fold lower than the ratio observed following mutagenesis with gamma-rays. Comparison of the incidence with which embryonic recessive lethal mutations were induced with the incidence with which specific locus mutations arose indicates that there are greater than 5000 genes essential to the development and viability of the zebrafish embryo.

Alkylation↗

PubMed1991

Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis.

The paired box-containing (pax) gene family encodes a group of putative transcription factors differentially expressed during embryonic development. In this study, we describe the cloning and expression of a zebrafish gene pax[zf-b], which most probably is a direct homologue to the mouse Pax2 gene. The putative protein encoded by pax[zf-b] contains a paired box, an octapeptide, but no homeobox. However, a region of homology to the N-terminal half of paired-type homeoboxes is detected C-terminal to the pax[zf-b] paired domain. In zebrafish embryos, pax[zf-b] transcripts are first seen during the formation of the neural keel. At 9-10 h of development, two laterally located transverse stripes of cells expressing the gene appear in the rostral 1/3 of the embryo. The two areas subsequently move towards the midline and form the posterior portion of the midbrain. In the following stages of development, at 10-12 h, transcripts are detected in the otic placode, the Wolffian duct including the nephritic primodium and in the optic stalk. At a later time point, beginning at 14-15 h, single cells along the spinal cord, presumably interneurons, start to express the gene. The characteristic expression pattern of pax[zf-b] in the neural tube suggests an involvement of this gene in the regionalization of the midbrain as well as in the specification of neuronal cell fates at early embryonic stages.

Amino Acid Sequence↗

PubMed1992

Growth cone guidance in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have allowed researchers to make a detailed characterization of pathfinding by identifiable growth cones. The growth cones follow precise cell-specific pathways to their targets. Analyses of pathfinding in mutant and experimentally manipulated wild type embryos have shown that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant.

Animals↗

PubMed1992

Coordinate embryonic expression of three zebrafish engrailed genes.

We have identified three genes, expressed in zebrafish embryos, that are members of the engrailed gene family. On the basis of sequence comparisons and analyses of their expression patterns, we suggest that two of these genes, eng2 and eng3, are closely related to the En-2 gene of other vertebrates. The third gene, eng1, is probably the zebrafish homolog of En-1. Subsets of cells at the developing junction between the midbrain and hindbrain express three different combinations of these genes, revealing a previously unknown complexity of this region of the CNS. Other cells, for example, jaw and myotomal muscle precursors, express two of the three genes in combinations which, in the myotomal muscles, change during development. Cells in the developing hindbrain and fins express only a single engrailed gene. We propose that the fates and patterning of these cells may be regulated by the coordinate expression of particular combinations of these closely related homeoproteins.

Amino Acid Sequence↗

PubMed1992

Cell movements and cell fate during zebrafish gastrulation.

The early lineages of the zebrafish are indeterminate and a single cell labeled before the late blastula period will contribute progeny to a variety of tissues. Therefore, early cell lineages in the zebrafish do not establish future cell fates and early blastomeres must necessarily remain pluripotent. Eventually, after a period of random cell mixing, individual cells do become tissue restricted according to their later position within the blastoderm. The elucidation of a fate map for the zebrafish gastrula (Kimmel et al., 1990), has made it possible to study the processes by which cellular identity is conferred and maintained in the zebrafish. In this chapter, I describe single cell transplantation experiments designed to test for the irreversible restriction or 'commitment' of embryonic blastomeres in the zebrafish embryo. These experiments support the hypothesis that cell fate in the vertebrate embryo is determined by cell position. Work on the spadetail mutation will also be reviewed; this mutation causes a subset of mesodermal precursors to mismigrate during gastrulation thereby leading to a change in their eventual cell identity.

Animals↗

PubMed2026

Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN.

The transition of neural crest cells (NCCs) from a multipotent state to lineage-restricted derivatives, including melanocytes, is governed by tightly regulated epigenetic mechanisms that orchestrate cell type specific gene expression programs. Histone post-translational modifications (PTMs), in particular, play an important role in modulating chromatin accessibility, enhancer activation, and transcription factor occupancy, thereby facilitating dynamic chromatin and transcriptional reprogramming required during development. However, profiling such chromatin states in rare and transient Neural Crest Cell (NCC) populations in vivo remains technically challenging. To address this, we present an optimized low-input Cleavage Under Targets and Release Using Nuclease (CUT&RUN) workflow tailored for fluorescence-activated cell sorting (FACS) isolated NCCs from zebrafish embryos. This approach enables high-resolution and low-background mapping of key histone modifications, including H3K27ac, H3K4me3, and H3K27me3, from limited cell numbers. Collectively, these methodologies provide a robust framework for dissecting chromatin state dynamics in developmental systems and can also offer insights into epigenetic dysregulation associated with disease.

Animals↗

PubMedDate not supplied

Molecular analysis of the ependymin gene and functional test of its promoter region by transient expression in Brachydanio rerio.

Ependymins are secretory products of meningeal cells and represent the predominant glycoproteins in the cerebrospinal fluid from various orders of teleost fish. In the zebrafish, their expression starts between 48 and 72 h post-fertilization. Generally, they share characteristics with proteins involved in cell-contact phenomena. Here, we characterize the ependymin gene from Brachydanio rerio and its flanking regions. The sequence was obtained from clones generated using the polymerase chain reaction (PCR), including a variation of an "anchored" PCR. Also, clones from a conventional phage library were analyzed. We found that the transcribed portion is arranged in six exons. Transient expression of an ependymin-promoter-lacZ gene fusion in zebrafish embryos revealed that the 2.0-kb upstream regulatory region used is sufficient to direct the ependymin-specific correct temporal and spatial expression pattern of the lacZ reporter gene.

Amino Acid Sequence↗

PubMed2026

Pseudomonas aeruginosa mgtC gene is under the control of PhoP and CbrAB regulators, and its expression can be visualized in macrophages.

The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa. In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro. Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation.IMPORTANCEThe adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC, a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa. An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC. The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas-specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

Pseudomonas aeruginosa↗

PubMed1992

Retinoic acid-induced duplication of the zebrafish retina.

Exogenous treatment of zebrafish embryos with retinoic acid induces a duplication of the retinas during development. These effects occur only when retinoic acid is applied within a 2-hr period prior to and during the initial formation of the optic primordia, and they are concentration-dependent. Light microscopic examination reveals that the second retina derives from cells in the ventral region of the developing eyecup that normally become pigment epithelial cells. Two distinct ganglion cell fields are usually observed in eyes with duplicated retinas. Bundles of axons from each ganglion cell field join as they leave the eye and innervate the contralateral tectum.

Animals↗

PubMed2024

Cilia defects upon loss of WDR4 are linked to proteasomal hyperactivity and ubiquitin shortage.

The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.

Animals↗

PubMed2026

Stem Cell Differentiation Disperses Transcriptional Clusters via a Conserved Surface-Condensate Trajectory.

Stem cells exhibit exceptionally prominent transcriptional clusters, which dissolve with progressing differentiation. Although these clusters are assigned central roles in embryonic gene regulation, their formation and loss during differentiation remain poorly understood. This study reveals that these prominent clusters disperse along a conserved trajectory in mouse embryonic stem cells, fruit fly testes, and zebrafish embryos. Imaging and lattice simulations show that these clusters form via surface condensation on H3K27ac-marked super-enhancer regions, which act as genomic scaffolds. Upon differentiation, partial loss of these active epigenetic marks and transcription-driven unfolding lead to dispersal of the prominent clusters. The block copolymer-based lattice simulations explain this process as a conserved trajectory through a three-dimensional state space, governed by surface condensation principles that extend beyond canonical liquid-liquid phase separation. This work marks surface condensation as a biophysical mechanism for the dynamic organization of stem cell-specific transcriptional hubs and demonstrates evolutionary conservation in several organisms. By uncovering a conserved biophysical mechanism for transcriptional organization in development, our work illustrates how polymer properties can contribute to the control of cell identity and fate.

Animals↗

PubMed1992

Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons.

Individually identified primary motoneurons of the zebrafish embryo pioneer cell-specific peripheral motor nerves. Later, the growth cones of secondary motoneurons extend along pathways pioneered by primary motor axons. To learn whether primary motor axons are required for pathway navigation by secondary motoneurons, we ablated primary motoneurons and examined subsequent pathfinding by the growth cones of secondary motoneurons. We found that ablation of the primary motoneuron that pioneers the ventral nerve delayed ventral nerve formation, but a normal-appearing nerve eventually formed. Therefore, the secondary motoneurons that extend axons in the ventral nerve were able to pioneer that pathway in the absence of the pathway-specific primary motoneuron. In contrast, in the absence of the primary motoneuron that normally pioneers the dorsal nerve, secondary motoneurons did not pioneer a nerve in the normal location, instead they formed dorsal nerves in an atypical position. This difference in the ability of these two groups of motoneurons to pioneer their normal pathways suggests that the guidance rules followed by their growth cones may be very different. Furthermore, the observation that the atypical dorsal nerves formed in a consistent incorrect location suggests that the growth cones of the secondary motoneurons that extend dorsally make hierarchical pathway choices.

Animals↗