Zebrafish Development Research Articles

TDMPP activation of estrogen receptor 2a regulates smc2 and p53 signaling to interfere with liver development in zebrafish (Danio rerio)

Tris (2,6-dimethylphenyl) phosphate (TDMPP), a novel organic phosphorus flame retardant (OPFR), has been found to have estrogenic activity. Estrogens are critical in regulating various biological responses during liver development. However, the effects of TDMPP on zebrafish liver development remain largely unexplored. Here, we utilized a chemical genetic screening approach to assess the estrogenic effects of TDMPP on liver development and to elucidate the underlying molecular mechanism. Our findings revealed that zebrafish larvae exposed to environmentally relevant concentrations of TDMPP (0.05 and 0.5 μM) exhibited concentration-dependent liver impairments, including reduced liver size, histopathological changes, and hepatocyte apoptosis. In addition, E2 caused similar adverse effects to TDMPP, but the pharmacological blockade of estrogen synthesis alleviated the effects on liver development. Chemical inhibitors and morpholino knockdown assays indicated that the reduction of esr2a blocked TDMPP-induced liver impairments, which was further confirmed in the esr2a-/- mutant line. Subsequently, transcriptomic analysis showed that the estrogen receptor activated by TDMPP inhibited the expression of smc2, which was linked to the suppression of liver development through p53 activation. Consistently, overexpression of smc2 and inhibition of p53 evidently rescued hepatic damages induced by TDMPP. Taken together, the above findings identified esr2a, downstream smc2, and p53 as important regulators for the estrogenic effects of TDMPP on liver development. Our work fills crucial gaps in the current knowledge of TDMPP's hepatotoxicity, providing new insights into the adverse effects of TDMPP and the molecular mechanisms of action. These findings underscore the need for further ecological risk assessment and regulatory considerations.

In vitro study of ATP1A3 p.Ala275Pro mutant causing alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism.

We previously reported that ATP1A3 c.823G>C (p.Ala275Pro) mutant causes varying phenotypes of alternative hemiplegia of childhood and rapid-onset dystonia-parkinsonism in the same family. This study aims to investigate the function of ATP1A3 c.823G>C (p.Ala275Pro) mutant at the cellular and zebrafish models. ATP1A3 wild-type and mutant Hela cell lines were constructed, and ATP1A3 mRNA expression, ATP1A3 protein expression and localization, and Na+-K+-ATPase activity in each group of cells were detected. Additionally, we also constructed zebrafish models with ATP1A3 wild-type overexpression (WT) and p.Ala275Pro mutant overexpression (MUT). Subsequently, we detected the mRNA expression of dopamine signaling pathway-associated genes, Parkinson's disease-associated genes, and apoptosisassociated genes in each group of zebrafish, and observed the growth, development, and movement behavior of zebrafish. Cells carrying the p.Ala275Pro mutation exhibited lower levels of ATP1A3 mRNA, reduced ATP1A3 protein expression, and decreased Na+-K+-ATPase activity compared to wild-type cells. Immunofluorescence analysis revealed that ATP1A3 was primarily localized in the cytoplasm, but there was no significant difference in ATP1A3 protein localization before and after the mutation. In the zebrafish model, both WT and MUT groups showed lower brain and body length, dopamine neuron fluorescence intensity, escape ability, swimming distance, and average swimming speed compared to the control group. Moreover, overexpression of both wild-type and mutant ATP1A3 led to abnormal mRNA expression of genes associated with the dopamine signaling pathway and Parkinson's disease in zebrafish, and significantly upregulated transcription levels of bad and caspase-3 in the apoptosis signaling pathway, while reducing the transcriptional level of bcl-2 and the bcl-2/bax ratio. This study reveals that the p.Ala275Pro mutant decreases ATP1A3 protein expression and Na+/K+-ATPase activity. Abnormal expression of either wild-type or mutant ATP1A3 genes impairs growth, development, and movement behavior in zebrafish.

Effect of pH on Development of the Zebrafish Inner Ear and Lateral Line: Comparisons between High School and University Settings.

Increasing carbon dioxide levels associated with climate change will likely have a devastating effect on aquatic ecosystems. Aquatic environments sequester carbon dioxide, resulting in acidic conditions that can negatively affect fish development. Increasing climate change impacts in the coming decades will have an outsized effect on younger generations. Therefore, our research had two interconnected goals: 1) understand how aquatic acidification affects the development of zebrafish, and 2) support a high school scientist's ability to address environmental questions of increasing importance to her generation. Working with teachers and other mentors, the first author designed and conducted the research, first in her high school, then in a university research laboratory. Zebrafish embryos were reared in varying pH conditions (6.7-8.2) for up to 7 days. We assessed fish length and development of the inner ear, including the otoliths; structures that depend on calcium carbonate for proper development. Although pH did not affect fish length, fish reared in pH 7.75 had smaller anterior otoliths, showing that pH can impact zebrafish ear development. Furthermore, we demonstrate how zebrafish may be used for high school students to pursue open-ended questions using different levels of available resources.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e.,"cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

MiR-29b-triggered epigenetic regulation of cardiotoxicity following exposure to deltamethrin in zebrafish

Deltamethrin is a classical pyrethroid insecticide that is frequently detected in aquatic environments and organisms. Furthermore, deltamethrin has been detected in samples related to human health and is a potential risk to public health. This study aimed to investigate the mechanism of cardiotoxicity induced by deltamethrin. Zebrafish were exposed to 0.005, 0.05, or 0.5 μg/L deltamethrin for 28 days. The results showed a significant reduction in male reproduction compared to female reproduction. Additionally, the heart rate decreased by 15.75 % in F1 after parental exposure to 0.5 μg/L deltamethrin. To evaluate cardiotoxicity, deltamethrin was administered to the zebrafish embryos. By using miRNA-Seq and bioinformatics analysis, it was discovered that miR-29b functions as a toxic regulator by targeting dnmts. The overexpression of miR-29b and inhibition of dnmts resulted in cardiac abnormalities, such as pericardial edema, bradycardia, and abnormal expression of genes related to the heart. Similar changes in the levels of miR-29b and dnmts were also detected in the gonads of F0 males and F1 embryos, confirming their effects. Overall, the results suggest that deltamethrin may have adverse effects on heart development in early-stage zebrafish and on reproduction in adult zebrafish. Furthermore, epigenetic modifications may threaten the cardiac function of offspring.

Elucidating the reproductive toxicity mechanisms in female zebrafish: A transcriptomic study of lifetime tris(2-chloroethyl) phosphate exposure

Tris(2-chloroethyl) phosphate (TCEP), emerging as a predominant substitute for brominated flame retardants (BFRs), is now increasingly recognized as a prevalent contaminant in aquatic ecosystems. The extent of its reproductive toxicity in aquatic species, particularly in zebrafish (Danio rerio), remains insufficiently characterized. This study subjected zebrafish embryos to various concentrations of TCEP (0, 0.8, 4, 20, and 100 μg/L) over a period of 120 days, extending through sexual maturation, to assess its impact on female reproductive health. Notable reductions in body weight (0.59- and 0.76-fold) and length (0.71- and 0.77-fold) were observed at concentrations of 20 and 100 μg/L, with a concomitant decrease by 0.21- to 0.61-fold in the gonadal somatic index across all treatment groups. The reproductive output, as evidenced by egg production and hatchability, was adversely affected. Histopathological analysis suggested that TCEP exposure impedes ovarian development. Endocrine alterations were also evident, with testosterone and 11-ketotestosterone levels significantly diminished by 0.38- and 0.08-fold at the highest concentration tested, while 17β-estradiol was elevated by 0.09- to 0.14-fold in all exposed groups. Transcriptomic profiling illuminated numerous differentially expressed genes (DEGs) integral to reproductive processes, including hormone regulation, neuroactive ligand-receptor interactions, oocyte meiosis, and progesterone-mediated maturation pathways. Collectively, these findings indicate that lifelong exposure to TCEP disrupts ovarian development and maturation in female zebrafish, alters gene expression within the hypothalamic-pituitary-gonadal axis, and perturbs sex hormone synthesis, culminating in pronounced reproductive toxicity.

Neurodevelopmental toxicity of a ubiquitous disinfection by-product, bromoacetic acid, in Zebrafish (Danio rerio)

Disinfection of public drinking water and swimming pools is crucial for preventing waterborne diseases, but it can produce harmful disinfection by-products (DBPs), increasing the risk of various diseases for those frequently exposed to such environments. Bromoacetic acid (BAA) is a ubiquitous DBP, with toxicity studies primarily focused on its in vitro cytotoxicity, and insufficient research on its neurodevelopmental toxicity. Utilizing zebrafish as a model organism, this study comprehensively explored BAA’s toxic effects and uncovered the molecular mechanisms through neurobehavioral analysis, in vivo two-photon imaging, transcriptomic sequencing, pharmacological intervention and molecular biological detection. Results demonstrated BAA induced significant changes on various indicators in the early development of zebrafish. Furthermore, BAA disrupted behavioral patterns in zebrafish larvae across locomotion activity, light-dark stimulation, and vibration stimulation paradigms. Subsequent investigation focused on larvae revealed BAA inhibited neuronal development, activated neuroinflammatory responses, and altered vascular morphology. Transcriptomic analysis revealed BAA-stressed zebrafish exhibited downregulation of visual transduction-related genes and activation of ferroptosis and cellular apoptosis. Neurobehavioral disorders were recovered by inhibiting ferroptosis and apoptosis. This study elucidates the neurodevelopmental toxicity associated with BAA, which is crucial for understanding health risks of DBPs and for the development of more effective detection methods and regulatory strategies.

Cxcr4a regulates heart progenitor development and cardiac rhythm in zebrafish

Cxcr4a is involved in multiple organ development including coronary vasculature formation and heart left-right (LR) patterning, whether it is involved in heart progenitor determination and cardiac rhythm regulation is not addressed. Here we showed that in cxcr4a mutants, from 2 days post fertilization (dpf) to 4dpf the embryos transiently displayed pericardial edema and increased cardiac rhythm. While from 5dpf, the heart phenotype disappeared. Detailed analysis demonstrated that, at 36hpf and 48hpf, even though there was no distinct difference in the heart size between cxcr4a mutants and controls, the expression of myl7 was decreased. Further data showed that, the heart progenitors were decreased at 18SS(Somite Stage). Mechanically, RNA-seq, RT-qPCR and in situ experiments showed that the retinoic acid (RA) signaling was upregulated, and the up-regulation of RA signaling may mediate the role of cxcr4a in regulating heart progenitor development. In addition, we also identified that low dose of RA treatment accelerated the cardiac rhythm, being similar to that in cxcr4a mutants. Decreasing RA signaling partially restored the rapid cardiac rhythm in cxcr4a mutants, implying the possibility that RA signaling partially mediates the role of cxcr4a in regulating cardiac rhythm. In conclusion, our study identified cxcr4a simultaneously regulates heart progenitor determination and cardiac rhythm.

TRNA expression and modification landscapes, and their dynamics during zebrafish embryo development.

tRNA genes exist in multiple copies in the genome of all organisms across the three domains of life. Besides the sequence differences across tRNA copies, extensive post-transcriptional modification adds a further layer to tRNA diversification. Whilst the crucial role of tRNAs as adapter molecules in protein translation is well established, whether all tRNAs are actually expressed, and whether the differences across isodecoders play any regulatory role is only recently being uncovered. Here we built upon recent developments in the use of NGS-based methods for RNA modification detection and developed tRAM-seq, an experimental protocol and in silico analysis pipeline to investigate tRNA expression and modification. Using tRAM-seq, we analysed the full ensemble of nucleo-cytoplasmic and mitochondrial tRNAs during embryonic development of the model vertebrate zebrafish. We show that the repertoire of tRNAs changes during development, with an apparent major switch in tRNA isodecoder expression and modification profile taking place around the start of gastrulation. Taken together, our findings suggest the existence of a general reprogramming of the expressed tRNA pool, possibly gearing the translational machinery for distinct stages of the delicate and crucial process of embryo development.

Cohesin composition and dosage independently affect early development in zebrafish.

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed 'cohesinopathies' are characterized by germline variants of cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear whether mutations in individual cohesin subunits have independent developmental consequences. Here, we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single-cell RNA sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21, mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

Corticosteroids alter kidney development and increase glomerular filtration rate in larval zebrafish (Danio rerio).

Pharmaceutical drugs and other chemicals can impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants. Corticosteroids are administered to pregnant women at risk of preterm delivery in order to reduce neonatal morbidity and mortality. In addition, high-dose corticosteroid exposure of very preterm infants regularly serves to maintain blood pressure and to prevent and treat bronchopulmonary dysplasia, a form of chronic lung disease in prematurely born infants. Despite clinical benefits, there is increasing evidence of corticosteroid-mediated short- and long-term detrimental developmental effects, especially in the kidney. Here, we performed a detailed morphological and functional analysis of corticosteroid-mediated effects on pronephros development in larval zebrafish. About 24-h postfertilization (hpf) transgenic Tg(wt1b: EGFP) zebrafish larvae were exposed to a set of natural and synthetic corticosteroids (hydrocortisone, dexamethasone, 6α-methylprednisolone, betamethasone, prednisolone, fludrocortisone, 11-deoxycorticosterone) with varying glucocorticoid and mineralocorticoid potency for 24 h at different concentrations. A semiautomated, multiparametric in vivo workflow enabled simultaneous assessment of kidney morphology, renal FITC-inulin clearance, and heart rate within the same larva. All corticosteroids exerted significant morphological and functional effects on pronephros development, including a significant hypertrophy of the pronephric glomeruli as well as dose-dependent increases in FITC-inulin clearance as a marker of glomerular filtration rate. In conclusion, the present study demonstrates a significant impact of corticosteroid exposure on kidney development and function in larval zebrafish. Hence, these studies underline that corticosteroid exposure of the fetus and the preterm neonate should be carefully considered due to potential short- and long-term harm to the kidney.

Synthesis and Structure of Novel Phenothiazine Derivatives, and Compound Prioritization via In Silico Target Search and Screening for Cytotoxic and Cholinesterase Modulatory Activities in Liver Cancer Cells and In Vivo in Zebrafish.

Phenothiazines (PTZ) are antipsychotics known to modulate a variety of neurotransmitter activities that include dopaminergic and cholinergic signaling and have been identified as potential anticancer agents in vitro. However, it is important to also test whether a highly cytotoxic, repurposed, or novel PTZ has low toxicity and neuromodulatory activity in vivo using vertebrate model organisms, such as zebrafish. In this study, we synthesized novel phenothiazines and screened them in vitro in liver cancer and in vivo in zebrafish embryos/larvae. The syntheses of several intermediate PTZ 10-yl acyl chlorides were followed by elemental analysis and determination of 1H NMR and 13C NMR mass (ESI+) spectra of a large number of novel PTZ 10-carboxamides. Cytotoxicities of 28 PTZ derivatives (1-28) screened against Hep3B and SkHep1 liver cancer cell lines revealed five intermediate and five novel leads along with trifluoperazine (TFP), prochlorperazine (PCP), and perphenazine, which are relatively more cytotoxic than the basic PTZ core. Overall, the derivatives were more cytotoxic to Hep3B than SkHep1 cells. Moreover, in silico target screening identified cholinesterases as some of the commonest targets of the screened phenothiazines. Interestingly, molecular docking studies with acetylcholinesterase (AChE) and butyrylcholinesterase proteins showed that the most cytotoxic compounds 1, 3, PCP, and TFP behaved similar to Huprin W in their amino acid interactions with the AChE protein. The highly cytotoxic intermediate PTZ derivative 1 exhibited a relatively lower toxicity profile than those of 2 and 3 during the zebrafish development. It also modulated in vivo the cholinesterase activity in a dose-dependent manner while significantly increasing the total cholinesterase activity and/or ACHE mRNA levels, independent of the liver cancer cell type. Our screen also identified novel phenothiazines, i.e., 8 and 10, with significant cytotoxic and cholinesterase modulatory effects in liver cancer cells; yet both compounds had low levels of toxicity in zebrafish. Moreover, they modulated the cholinesterase activity or expression of ACHE in a cancer cell line-specific manner, and compound 10 significantly inhibited the cholinesterase activity in zebrafish. Accordingly, using a successful combination of in silico, in vitro, and in vivo approaches, we identified several lead anticancer and cholinesterase modulatory PTZ derivatives for future research.

Male germ cell-associated kinase is required for axoneme formation during ciliogenesis in zebrafish photoreceptors.

Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.

Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

Comparative Study of Condensed and Hydrolysable Tannins during the Early Stages of Zebrafish Development.

In this study, we present data on the effects of condensed tannins (CTs) and hydrolysable tannins (HTs), polyphenols extracted from plants, at different concentrations on zebrafish development to identify the range of concentrations with toxic effects. Zebrafish embryos were exposed to CTs and HTs at two different concentration ranges (5.0-20.0 μgL-1 and 5.0-20.0 mgL-1) for 72 h. The toxicity parameters were observed up to 72 h of treatment. The uptake of CTs and HTs by the zebrafish larvae was assessed via HPLC analysis. A qRT-PCR analysis was performed to evaluate the expressions of genes cd63, zhe1, and klf4, involved in the hatching process of zebrafish. CTs and HTs at 5.0, 10.0, and 20.0 μgL-1 were not toxic. On the contrary, at 5.0, 10.0, and 20.0 mgL-1, HTs induced a delay in hatching starting from 48 h of treatment, while CTs showed a delay in hatching mainly at 48 h. The analysis of gene expression showed a downregulation in the group exposed to HTs, confirming the hatching data. We believe that this study is important for defining the optimal doses of CTs and HTs to be employed in different application fields such as the chemical industry, the animal feed industry, and medical science.

Rohon-beard neurons do not succumb to programmed cell death during zebrafish development

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1+ (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1+ RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement.

Gene expression patterns and DNA methylation of neuron and pancreatic β-cell developments in zebrafish embryos treated with bisphenol F and AF

Bisphenol F (BPF) and bisphenol AF (BPAF) are structural analogues of bisphenol A (BPA) that are used in the manufacture of a myriad of BPA-free products; however, there is a paucity of information regarding their developmental effects. The present study investigates the effects of BPF and BPAF on neurodevelopment and pancreatic β-cell differentiation via altering DNA methylation and gene expression patterns using the zebrafish model. BPF and BPAF induced behavioral perturbations: increased average speed, increased maximum acceleration, increased mania time and decreased static time, in 0.3 and 1.0 μM groups in zebrafish embryos. Glucose level was significantly increased in 1.0 μM BPF (28 %); while a monotonic increase of 29 %, 55 %, and 74 % were observed in 0.1, 0.3, and 1.0 μM BPAF, respectively. Consistent with a decreased insulin mRNA level, the expression of two critical transcription factors (pdx-1 and foxa2) essential for the development and functioning of beta-cells decreased following the bisphenols exposure. In addition, embryonic exposure to BPF and BPAF upregulated the transcription of developmental genes (vegfa, wnt8a, and mstn1) and neuron-related genes (mbp, elavl3, gap43, gfap). Also, the expressions of DNA methyltransferases (dnmt1, dnmt3, dnmt4, dnmt5, dnmt6, dnmt7, and dnmt8) were significantly aberrant compared with the control group. The Bisulfite PCR results indicate increased DNA methylation at promoter regions of pdx-1 in BPF (8.2 %) and BPAF (7.6 %); α1-tubulin in BPF (5.3 %) and in BPAF (4.1 %), congruous with the increased dnmt1 and dnmt3 transcription, at early stage of zebrafish development. The present study indicates that zebrafish embryonic exposure to BPF and BPAF elicits islet dysfunction and neuron perturbations resulting in increased DNA methylation levels.

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and invivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.