Wnt16 Is Required for Specification of Vertebrate Hematopoietic Stem Cells through Notch.
Authors: Clements Wilson K. , Ong Karen G. , Kim Albert D. , Traver David 
Published: October 2019

Abstract
Abstract 2616
Hematopoietic stem cells (HSCs) are self-renewing progenitor cells that provide all adult blood lineages over the lifetime of an individual. Understanding the signaling events that regulate specification of these cells during embryonic development is a key precondition to recapitulating those processes in vitro for the purposes of regenerative medicine. Here we report that non-canonical Wnt signaling by the conserved vertebrate ligand, Wnt16 is required for specification of HSCs in zebrafish. WNT16 was originally identified as a gene aberrantly expressed in pre-B acute lymphoblastic leukemia (ALL) cells containing the t(1;19) chromosomal translocation, leading to expression of E2A-PBX1. Wnt16 is expressed in mammals at times when hemogenic endothelial cells first appear in the dorsal aorta. In zebrafish, wnt16 is expressed in somites, which lie adjacent to the dorsal aorta. Knock down of Wnt16 function by injection of antisense morpholino oligonucleotides leads to loss of HSCs and definitive blood lineages, such as T-cells, during development. Non-blood tissues including vasculature appear largely unaffected. Thus, wnt16 is required for HSC specification.
To better understand the Wnt16 signal transduction pathway, we examined its ability to activate transcription of target genes through β-catenin/Tcf-dependent “canonical” signaling. Although Wnt16 overexpression causes morphological abnormality, it does not yield ectopic expression of endogenous or transgenic canonical reporter genes at time points relevant to blood specification. Thus, Wnt16 signals independently of β-catenin/Tcf through a “non-canonical” Wnt pathway.
Notch signaling is required for specification of HSCs across phyla. To determine whether Notch signaling is disrupted in Wnt16 morphants, we examined the expression of all Notch ligands and receptors in these animals and found that expression of two ligands, deltaC (dlc) and deltaD (dld) are decreased. Zebrafish mutants, homozygous for a null allele of dlc, and embryos injected with a dld morpholino each display decreased numbers of HSCs during development, but recover. Dlc mutants injected with dld morpholino show complete loss of HSCs. To determine whether defects in Notch signaling are responsible for loss of HSCs in Wnt16 morphants, we performed a rescue experiment. Transgenic animals carrying an inducible dominant activator of Notch target genes were injected with Wnt16 morpholino, and Notch activity was either induced or not. Wnt16 morphants with enforced Notch activity recovered HSC marker expression. Taken together, our results indicate that Wnt16 regulates expression of the Notch ligands dlc and dld, and these are redundantly required for HSC specification during development.
Chimera experiments in mouse using wild type and Notch1-deficient cells indicate that there is a cell-autonomous requirement for Notch signaling in specification of HSCs. To determine when the first Notch-responsive cells that contribute to the adult hematopoietic system appear, we used animals carrying a transgene encoding a photconvertible, green-to-red Kaede protein under the control of a Notch-responsive promoter. By photoconverting all embryonic cells at various times during development, we determined that cells destined to become HSCs first experience a Notch signal at approximately the time when HSCs first appear, just before 24 hours post fertilization (hpf). At earlier time points, Notch responsive cells were present, but did not contribute to blood, although they did contribute to tissues in or near the dorsal aorta, which contains the hemogenic endothelium that gives rise to HSCs. Surprisingly, HSC rescue in Wnt16 morphants by Notch activation could only be achieved earlier than 16 hpf, long before a cell-autonomous Notch signal is received in pre-HSCs. Moreover, loss of the critical ligands, dlc and dld, in Wnt16 morphant animals was confined to somitic tissue prior to 20 hpf, while ligand expression in or near the dorsal aorta at later times was relatively normal. Together, our results strongly suggest a previously unappreciated, non-cell-autonomous requirement for Notch signaling in the somites. We hypothesize that somitic Notch signaling regulates a morphological process or expression of a relay signal required for HSC specification during development.

Disclosures:
No relevant conflicts of interest to declare.