Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

• MeSH
• Zebrafish MeSH
• Erythropoietin * MeSH
• Erythroid Cells MeSH
• Ligands MeSH
• Zebrafish Proteins genetics   MeSH
• Stem Cell Factor genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Erythropoietin * MeSH
• kitlga protein, zebrafish MeSH Browser
• Ligands MeSH
• Zebrafish Proteins MeSH
• Stem Cell Factor MeSH

Hematopoiesis is a precisely orchestrated process regulated by the activity of hematopoietic cytokines and their respective receptors. Due to an extra round of whole genome duplication during vertebrate evolution in teleost fish, zebrafish have two paralogs of many important genes, including genes involved in hematopoiesis. Importantly, these duplication events brought increased level of complexity in such cases, where both ligands and receptors have been duplicated in parallel. Therefore, precise understanding of binding specificities between duplicated ligand-receptor signalosomes as well as understanding of their differential expression provide an important basis for future studies to better understand the role of duplication of these genes. However, although many recent studies in the field have partly addressed functional redundancy or sub-specialization of some of those duplicated paralogs, this information remains to be scattered over many publications and unpublished data. Therefore, the focus of this review is to provide an overview of recent findings in the zebrafish hematopoietic field regarding activity, role and specificity of some of the hematopoietic cytokines with emphasis on crucial regulators of the erythro-myeloid lineages.

• Keywords
• cytokine, erythropoiesis, genome duplication, hematopoiesis, myelopoiesis, zebrafish,
• Publication type
• Journal Article MeSH
• Review MeSH

This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

• MeSH
• Cell Culture Techniques MeSH
• Cytokines genetics   MeSH
• Zebrafish blood   MeSH
• Hematopoietic Stem Cells cytology   MeSH
• Hematopoiesis * MeSH
• Carps blood   MeSH
• Culture Media chemistry   MeSH
• Molecular Biology methods   MeSH
• Zebrafish Proteins genetics   MeSH
• Recombinant Proteins genetics   MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cytokines MeSH
• Culture Media MeSH
• Zebrafish Proteins MeSH
• Recombinant Proteins MeSH

Vertebrate erythrocytes and thrombocytes arise from the common bipotent thrombocytic-erythroid progenitors (TEPs). Even though nonmammalian erythrocytes and thrombocytes are phenotypically very similar to each other, mammalian species have developed some key evolutionary improvements in the process of erythroid and thrombocytic differentiation, such as erythroid enucleation, megakaryocyte endoreduplication, and platelet formation. This brings up a few questions that we try to address in this review. Specifically, we describe the ontology of erythro-thrombopoiesis during adult hematopoiesis with focus on the phylogenetic origin of mammalian erythrocytes and thrombocytes (also termed platelets). Although the evolutionary relationship between mammalian and nonmammalian erythroid cells is clear, the appearance of mammalian megakaryocytes is less so. Here, we discuss recent data indicating that nonmammalian thrombocytes and megakaryocytes are homologs. Finally, we hypothesize that erythroid and thrombocytic differentiation evolved from a single ancestral lineage, which would explain the striking similarities between these cells.

• MeSH
• Cell Differentiation physiology   MeSH
• Erythrocytes cytology   MeSH
• Humans MeSH
• Megakaryocytes cytology   MeSH
• Vertebrates physiology   MeSH
• Blood Platelets cytology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH