3224 – NFAT TRANSCRIPTION FACTORS ARE REQUIRED FOR DEFINITIVE HEMATOPOIESIS

Abstract

Hematopoietic stem cells (HSCs) are a population of tissue-specific stem cells that give rise to all mature blood lineages throughout adult life. During embryonic development, HSCs arise from specialized endothelium in the ventral wall of the dorsal aorta (DA), known as the hemogenic endothelium (HE). Key transcription factors encoded by gata2b, runx1, and cmyb mark HSCs during their earliest specification and emergence from the HE and are functionally required across vertebrate phyla. Here we show that members of the nuclear factor of activated T-cells (Nfat) family of transcription factors are also required for proper HSC specification. Pharmacological inhibition of upstream activators of Nfat—calmodulin and calcineurin—reduces the number of HSC precursors in the developing zebrafish embryo. Although five nfat family members are expressed in the DA at 24 hours post fertilization (hpf), knockdown of only two—nfatc1 and nfatc3b—is sufficient to diminish HSC specification as indicated by reduction of precursors expressing runx1 and cmyb, as well as a loss of early definitive T lymphocytes expressing rag1. We show that despite definitive hematopoietic defects in Nfatc1/c3b morphants, earlier vascular patterning (cdh5) and arterial identity (notch1b and efnb2a) are intact, and that competent hemogenic endothelium (gata2b) is available. We are currently genetically verifying findings by generating nfatc1 and nfatc3b knockout zebrafish lines. We are also testing if endothelial-specific expression of nfatc1 and nfatc3b is sufficient to drive HSC generation. These results uncover a previously unknown role of two Nfat family transcription factors in combinatorially regulating specification of HSCs in the developing embryo. Hematopoietic stem cells (HSCs) are a population of tissue-specific stem cells that give rise to all mature blood lineages throughout adult life. During embryonic development, HSCs arise from specialized endothelium in the ventral wall of the dorsal aorta (DA), known as the hemogenic endothelium (HE). Key transcription factors encoded by gata2b, runx1, and cmyb mark HSCs during their earliest specification and emergence from the HE and are functionally required across vertebrate phyla. Here we show that members of the nuclear factor of activated T-cells (Nfat) family of transcription factors are also required for proper HSC specification. Pharmacological inhibition of upstream activators of Nfat—calmodulin and calcineurin—reduces the number of HSC precursors in the developing zebrafish embryo. Although five nfat family members are expressed in the DA at 24 hours post fertilization (hpf), knockdown of only two—nfatc1 and nfatc3b—is sufficient to diminish HSC specification as indicated by reduction of precursors expressing runx1 and cmyb, as well as a loss of early definitive T lymphocytes expressing rag1. We show that despite definitive hematopoietic defects in Nfatc1/c3b morphants, earlier vascular patterning (cdh5) and arterial identity (notch1b and efnb2a) are intact, and that competent hemogenic endothelium (gata2b) is available. We are currently genetically verifying findings by generating nfatc1 and nfatc3b knockout zebrafish lines. We are also testing if endothelial-specific expression of nfatc1 and nfatc3b is sufficient to drive HSC generation. These results uncover a previously unknown role of two Nfat family transcription factors in combinatorially regulating specification of HSCs in the developing embryo.