Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

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 Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design.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.

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos.

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology.

A molecular roadmap of the AGM region reveals BMPER as a novel regulator of HSC maturation.

In the developing embryo, hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros (AGM) region, but the molecular regulation of this process is poorly understood. Recently, the progression from E9.5 to E10.5 and polarity along the dorso-ventral axis have been identified as clear demarcations of the supportive HSC niche. To identify novel secreted regulators of HSC maturation, we performed RNA sequencing over these spatiotemporal transitions in the AGM region and supportive OP9 cell line. Screening several proteins through an ex vivo reaggregate culture system, we identify BMPER as a novel positive regulator of HSC development. We demonstrate that BMPER is associated with BMP signaling inhibition, but is transcriptionally induced by BMP4, suggesting that BMPER contributes to the precise control of BMP activity within the AGM region, enabling the maturation of HSCs within a BMP-negative environment. These findings and the availability of our transcriptional data through an accessible interface should provide insight into the maintenance and potential derivation of HSCs in culture.

Developing HSCs become Notch independent by the end of maturation in the AGM region.

The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.