Gpr182-mediated calcium mobilization is required for definitive hematopoiesis - Abstract
Hematopoietic stem cells (HSCs) are self-renewing cells that produce all major blood cell lineages during an
individual’s lifetime. During embryonic specification of HSCs, axial vasculature must be appropriately patterned;
vascular endothelial cells of the embryonic aorta can be broadly categorized into hemogenic endothelium (HE)
and non-hemogenic endothelium. HSCs transdifferentiate specifically from HE and enter circulation. It has long
been understood that proper tissue patterning and transcriptional regulation are required for adequate
specification and production of HSCs. More recently, it has become clear that cell populations surrounding the
sites of HSC development—known as the HSC microenvironment—provide inductive signaling cues that are
required for HSC specification. Signaling molecules established in HSC specification, such as Wingless (Wnt)
and Hedgehog (Hh), activate G protein-coupled receptor (GPCR)-driven signaling pathways in endothelial cells.
Our lab has demonstrated that Gpr182, an endothelial GPCR without a currently known ligand, regulates
intracellular Ca2+ mobilization during the time of HSC specification. Transient loss of Gpr182 leads to a reduction
in HSC precursors. These data suggest that a Gpr182-induced increase in calcium levels may be required for
HSC specification. Our lab has also shown that pharmacological inhibition of the calcium sensor and effector
proteins calmodulin and calcineurin disrupts HSC specification. The nuclear factor of activated T cells (NFAT)
family of transcription factors act downstream of calcineurin. Several NFAT family members are expressed at
the right time and place to potentially influence HSC specification in the zebrafish model. However, it is unknown
whether or how these transcription factors HSC specification. While in vitro data have shown a connection
between Gpr182 expression and NFAT response element activity, no connection between these two molecules
has been made in vivo. I hypothesize that Gpr182-mediated activation of the Ca2+-calcineurin-NFAT
signaling axis is required for HSC specification. To test this hypothesis, I will first evaluate the role of Gpr182
activity in Ca2+ mobilization and HSC specification by defining that Gpr182 is required for HSC specification,
evaluating the effects of gpr182 gene knockout on Ca2+ mobilization, assessing the contribution of Gpr182
candidate co-receptor Ramp2 to endothelial Ca2+ mobilization, and determining if Gpr182 functions cell-
autonomously in HSC precursors. Second, I will determine if NFAT transcription factors are required for HSC
specification through performing loss-of-function genetic studies with morpholino oligonucleotides, generating
stable mutant knockout zebrafish, and assessing the potential for constitutively active NFAT to rescue
hematopoietic defects caused by Gpr182 loss-of-function. These studies will advance the field of developmental
hematopoiesis by establishing a previously unknown role for Ca2+ mobilization and NFAT activity in HSC
specification. These findings will better inform efforts of reprogramming HSCs from induced pluripotent stem
cells (iPSCs), which has been a longstanding goal of regenerative medicine.
