EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

Abstract

SummaryThe mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit β-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.HighlightsAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.Fzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.WNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.EGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.

Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells

“…We confirmed secretion of F7-Ab, F7, L6, and F7L6 from CHO cells by SDS-PAGE and Coomassie Blue staining and immunoblotting ( Figure 1B). Using a dot blot with lysates of HEK293T cells carrying loss of function mutations in FZD1, 2 and 7 (F127-KO) (Voloshanenko et al, 2017) or in LRP6 (LRP6-KO) (Grainger et al, 2019) confirmed binding specificity of each of these molecules. FZD7-specific binders (F7 and F7L6) only reacted with lysates upon FZD7 overexpression (note that endogenous FZD7 expression in LRP6 KO is below the level of detection), whereas LRP6 specific binders (L6 and F7L6) only reacted with lysates of cells expressing LRP6 ( Figure 1C).…”

“…For example, Reck, a glycosylphosphatidylinositol-anchored cell surface protein, acts together with Gpr124, a 7transmembrane protein, to promote Wnt7a-Fzd-Lrp5/6 signaling to promote angiogenesis in the developing central nervous system (Cho et al, 2017;Cho et al, 2019;Eubelen et al, 2018;Vallon et al, 2018). Furthermore, we recently identified a novel function for Egfr in mediating Wnt9a-Fzd9-Lrp5/6 signaling during hematopoietic development in zebrafish (Grainger et al, 2019). The use of engineered proteins capable of engaging specific WNT receptors, such as F7L6, may overcome the need for the recruitment of specificity-conferring co-receptors, such as Reck, Gpr124 and Egfr, and thus replace the need for purification of biologically active WNT proteins.…”

“…Hematopoietic stem and progenitor cells can be derived from hPS cells using defined culture conditions (Ng et al, 2008;Ng et al, 2005) and can be identified by dual expression of the cell surface markers CD34 and CD45 ( Figure 8A). We previously showed that a specific WNT signal involving WNT9A and FZD9 increased the efficiency of this differentiation protocol, as monitored by an increase in the yield of CD34/CD45 double positive cells (Grainger et al, 2019;Richter et al, 2018). Given this highly selective requirement for WNT9A/FZD9 signaling, we reasoned that activation of FZD7 signaling with F7L6 would likely interfere with the differentiation of hPS cells towards the hematopoietic lineage.…”

“…For example, the cell surface proteins Reck and Gpr124 promote Wnt7 signaling through Fzd (Cho et al, 2017;Eubelen et al, 2018). Furthermore, Egfr promotes a specific interaction of Wnt9a with Fzd9 and Lrp5/6 (Grainger et al, 2019). Likewise, Wnt3a may recruit additional, and currently unknown, co-receptors to activate signaling in hPS cells.…”

“…All cell lines used in these studies were directly obtained from either ATCC (CHO, HEK293, L) Addgene Plasmid #12456, RRID:Addgene_12456) were previously described (Bauer et al, 2013). HEK293T cells carrying knockout mutations in LRP6 were previously described (Grainger et al, 2019). Analysis System (Sartorius) and cultured without media changes for 5 days at 37°C.…”