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14 pages, 8 figures, supplementary information https://doi.org/10.1038/s41588-022-01089-w.-- Data availability: Raw and aligned sequencing data are available at https://danio-code.zfin.org/dataExport/. The raw sequencing data produced for this study are available on the European Bioinformatics Institute (EBI) European Nucleotide Archive (ENA) under study numbers PRJNA824720, PRJNA821001, PRJNA821088, PRJNA821148 and PRJNA821034. Annotation tracks are available at http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer10&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer10) and http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer11&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer11). Code availability: The processing pipelines for the individual assays are available at https://gitlab.com/danio-code. The code used for the analysis is available at https://github.com/DANIO-CODE/DANIO-CODE_Data_analysis (https://doi.org/10.5281/zenodo.6424702). The script to generate the TrackHub is available at https://gitlab.com/danio-code/TrackHub, Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals, We thank our main funders, the Horizon 2020 MSCA-ITN project ZENCODE-ITN by the European Commission to F.M., B.L., C.O.D., J.M.V. and B.P. (GA no: 643062), BBSRC support (DanioPeaks, P61715) to B.L., F.M. and F.C.W., Wellcome Trust (Joint-Investigator award 106955/Z/15/Z) to F.M. and B.L. and AQUA-FAANG (Horizon 2020, GA 817923) to B.L., D.B. and F.M. and BBSRC (BB/R015457/1) to F.vE and Key Special Project for Introduced Talents Team to Z.C. (GML2019ZD0401) and PrecisionTox project by the European Commission (GA no: 965406), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Background Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. Results We identified two novel tetrapod‐teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue‐specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. Conclusion This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression., Key Findings Identified a novel enhancer near zebrafish zic3/zic6 locus.The novel enhancer drives tissue‐specific expression in the habenula.Zebrafish transgenic line generated in this study can be a useful resource for studying development of habenula.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.

Glutamate–cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate–cysteine ligase catalytic ( Ace -GCLC) and modifier subunits ( Ace -GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace - gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51 kDa, respectively. The Ace -GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa , Onchocerca volvulus , Brugia malayi , and Ascaris suum , whereas the Ace -GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace -GCLC and Ace -GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35 dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate–cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace -GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace -GCLC or Ace -GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.

Hookworms, a group to which Ancylostoma ceylanicum belongs, are gastrointestinal nematodes that infect more than 700 million people around the world. They are a leading cause of anemia in developing countries. In order to effectively prevent hookworm infections research is conducted to develop an effective vaccine using recombinant antigens of the parasite. The aim of this study was to examine the influence of the hosts' on protection against ancylostomiasis and the shaping of the humoral immune response among Syrian hamsters after immunization with a cocktail of five A. ceylanicum recombinant antigens. Ace-ASP-3, Ace-ASP-4, Ace-APR-1, Ace-MEP-6 and Ace-MEP-7 were obtained in the pET expression system. Immunization with a vaccine cocktail resulted in a 33.5% worm burden reduction. The immunogenicity of the recombinant proteins were determined using ELISA. Statistical analysis showed that vaccinated hamsters developed stronger humoral responses to four of five recombinant antigens (the exception being Ace-ASP-3) compared to hamsters from the control group.