New tools for ‘ZEBRA-FISHING’

Briefings in Functional Genomics, 20(6), 2021, 407–419 https://doi.org/10.1093/bfgp/elab001 Advance Access Publication Date: 19 February 2021 Review Paper New tools for ‘ZEBRA-FISHING’ Veronica Bergo and Eirini Trompouki Abstract Zebrafish has been established as a classical model for developmental studies, yet in the past years, with the explosion of novel technological methods, the use of zebrafish as a model has expanded. One of the prominent fields that took advantage of zebrafish as a model organism early on is hematopoiesis, the process of blood cell generation from hematopoietic stem and progenitor cells (HSPCs). In zebrafish, HSPCs are born early during development in the aorta–gonad–mesonephros region and then translocate to the caudal hematopoietic tissue, where they expand and finally take residence in the kidney marrow. This journey is tightly regulated at multiple levels from extracellular signals to chromatin. In order to delineate the mechanistic underpinnings of this process, next-generation sequencing techniques could be an important ally. Here, we describe genome-wide approaches that have been undertaken to delineate zebrafish hematopoiesis. Key words: zebrafish; hematopoiesis; genomics; epigenetics; genome-wide techniques; multi-omics Introduction Delineating the molecular mechanisms that govern developmental processes has been instrumental for understanding cell fate decisions. Traditional tools like genetic manipulation, lineage tracing and imaging have been extensively and successfully used to understand the complex paths of development. However, the complexity of these processes cannot be fully captured without novel multi-omics tools. Indeed, these tools permit a deeper exploration of the transcriptional output of different cell types and even single cells, or the epigenetic landscape and the role of various transcription factors that govern developmental fates. Lineage tracing methods also received a lift-over, since computational analysis and clustered regularly interspaced short palindromic repeats (CRISPR) mutagenesis or barcoding permitted the reevaluation of old questions with novel tools. During the last decade or more, multi-omics methods have been established as a standard strategy for investigating the molecular mechanisms of development at a much higher resolution than before. One of the best-established animal models, which is ideal for studying developmental questions, is zebrafish (Danio rerio). Zebrafish have been used for studying various biological processes over the past four decades, especially hematopoiesis, during both physiological and pathological conditions [1–5]. The hematopoietic process, from development until adulthood, is highly conserved among vertebrates, even though some differences do exist. Despite the fact that hematopoietic niches are different in zebrafish and mammals, the genetic programs regulating hematopoietic stem and progenitor cell (HSPC) development and their interaction with the niche are largely conserved [6]. This conservation of molecular mechanisms has made it possible in the past to transfer the obtained knowledge and discoveries from zebrafish to mammals, and facilitated drug discovery [7–11]. Embryonic hematopoiesis is a multi-step process that occurs in different spatial locations in three distinct waves [3, 5, 6, 12–15]. In zebrafish, erythroid and myeloid cells are produced during the primitive wave from the medial and anterior lateral mesoderm, respectively. Erythroid–myeloid progenitors also arise from the posterior blood island (PBI) during a transient intermediate wave. During the definitive wave, hematopoietic stem cells (HSCs) emerge from the aorta–gonad–mesonephros Veronica Bergo is a IMPRS-IEM PhD student at the Max Planck Institute of Immunobiology and Epigenetics (Freiburg, Germany). Her passion is to dissect the molecular mechanisms that govern developmental, adult and pathological hematopoiesis. Eirini Trompouki is a group leader at the Department of Cellular and Molecular Immunology at the Max Planck Institute of Immunobiology and Epigenetics (Freiburg, Germany). She is associated to the Centre for Integrative Biological Signaling Studies (CIBSS) of the University of Freiburg, Germany. Her laboratory generates and integrates multi-omics data to understand physiological and pathological hematopoiesis. © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: 407 Corresponding author: Eirini Trompouki, Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, 79108, Germany. E-mail: 408 Bergo and Trompouki and permitting the progression towards the hematopoietic fate [53]. In addition, the identification of Gpr183 as a crucial repressor of Notch signaling before EHT and HSPC emergence was also possible due to extensive expression analysis [54]. Expression profiling has also been used to explore malignant hematopoiesis. In a zebrafish model of acute lymphoblastic leukemia, RNA-seq of a side cell population enriched for leukemia propagating cells identified the Wnt pathway as a candidate genetic driver [55]. Furthermore, RNA-seq revealed the metabolic function of the transcription factor HLX in normal and malignant hematopoiesis [56]. Taken together, bulk RNA-seq has been and will continue to be an excellent choice, not only for identifying molecular mechanisms, but also for providing new insights on druggable targets. Expression analysis refines hematopoietic cell isolation Transcriptomics Expression analysis has been used to refine hematopoietic cell characterization and isolation in zebrafish. Compared with human and mouse, the purification of HSPCs from adult zebrafish kidney has been more difficult due to the absence of well-defined cell surface markers. Indeed, the main approach used for isolation of distinct hematopoietic populations is based on the expression of fluorescent transgenic markers. Recently, the application of transcriptomic analyses combined with functional transplantation assays on a transgenic line expressing a combination of two HSPC-related transgenes, Tg(gata2a:GFP)la3 [57] and Tg(Mmu.Runx1:NLS-mCherry)cz2010 [16], has improved HSPC isolation from zebrafish kidney, providing new insights into the molecular hallmarks of the HSPC population [58]. Expression of coding and noncoding elements in hematopoiesis in bulk Gene expression in hematopoietic niches Over the past decade, the application of RNA sequencing (RNAseq) has shaped our understanding of many different biological aspects. Bulk RNA-seq assumes that all the sequenced cells exhibit the same behavior and thus represent a homogeneous population (Figure 1). However, it has the advantage of most likely representing the dominant cell population and can lead to identification of druggable or genetically manipulable targets that can reshape or rescue phenotypic differences. Bul (...truncated)