All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish

Vauti, Franz; Stegemann, Luisa A.; Vögele, Viktoria; Köster, Reinhard W.

doi:10.1371/journal.pone.0237167

Cited by 19 publications

(13 citation statements)

References 28 publications

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“…The detection of expressed mRNAs through WISH is a robust tool for assessing the spatial distribution of gene transcripts [ 48 ]. The zebrafish is especially suitable for this analysis and there are many described gene expression patterns available online at the Zebrafish Information Network (ZFIN, https://zfin.org/action/expression/search/ ) [ 47 , 49 , 50 ].…”

Section: Discussionmentioning

confidence: 99%

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

et al. 2022

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Background The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. Results Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. Conclusions The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

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Section: Discussionmentioning

confidence: 99%

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

et al. 2022

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“…These targeted genes can be related to the larval stage of zebrafish, a decisive phase in which the development of the main organs and tissues is completed. According to Vauti et al [41], 87% of zebrafish genes are expressed in the first six days of development and only 13% in the second to fourth weeks.…”

Section: Discussionmentioning

confidence: 99%

In Silico Target Prediction of Overexpressed microRNAs from LPS-Challenged Zebrafish (Danio rerio) Treated with the Novel Anti-Inflammatory Peptide TnP

Disner

Falcão

Lima

et al. 2021

IJMS

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miRNAs regulate gene expression post-transcriptionally in various processes, e.g., immunity, development, and diseases. Since their experimental analysis is complex, in silico target prediction is important for directing investigations. TnP is a candidate peptide for anti-inflammatory therapy, first discovered in the venom of Thalassophryne nattereri, which led to miRNAs overexpression in LPS-inflamed zebrafish post-treatment. This work aimed to predict miR-21, miR-122, miR-731, and miR-26 targets using overlapped results of DIANA microT-CDS and TargetScanFish software. This study described 513 miRNAs targets using highly specific thresholds. Using Gene Ontology over-representation analysis, we identified their main roles in regulating gene expression, neurogenesis, DNA-binding, transcription regulation, immune system process, and inflammatory response. miRNAs act in post-transcriptional regulation, but we revealed that their targets are strongly related to expression regulation at the transcriptional level, e.g., transcription factors proteins. A few predicted genes participated concomitantly in many biological processes and molecular functions, such as foxo3a, rbpjb, rxrbb, tyrobp, hes6, zic5, smad1, e2f7, and npas4a. Others were particularly involved in innate immunity regulation: il17a/f2, pik3r3b, and nlrc6. Together, these findings not only provide new insights into the miRNAs mode of action but also raise hope for TnP therapy and may direct future experimental investigations.

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“…Larvae were fixed in 4% PFA (in PBS) at 4°C overnight and progressively dehydrated in ascending concentrations of ethanol (25, 50, 75, and 100%) before being kept in 100% methanol at −20°C until needed. We optimized previously described ISH protocols in zebrafish ( 60 , 61 ) for 5 dpf larvae and for transcripts of interest. Larvae were incubated for 1 h at room temperature in ethanol/xylol solution (1:1 vol/vol) and then progressively rehydrated in descending concentrations of ethanol (90, 75, 50, 25% and then H 2 O).…”

Section: Methodsmentioning

confidence: 99%

Behavioral and Gene Regulatory Responses to Developmental Drug Exposures in Zebrafish

et al. 2022

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Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring—including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD—is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2, were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab, and junbb, which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits.

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All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish

Cited by 19 publications

References 28 publications

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

In Silico Target Prediction of Overexpressed microRNAs from LPS-Challenged Zebrafish (Danio rerio) Treated with the Novel Anti-Inflammatory Peptide TnP

Behavioral and Gene Regulatory Responses to Developmental Drug Exposures in Zebrafish

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