Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Keywords: Notch; hematopoietic stem cell; hemogenic endothelium; somite.

Figure 1. Notch3 is required for HSPC specification

A WISH of notch3 viewed dorsally at 13 hpf (left) and laterally in the trunk at 19 (middle) and 24 hpf (right). Black arrowheads denote somitic expression; red arrowheads denote PLM expression at 13 hpf and endothelial expression at 19 and 24 hpf. B, C WISH of the HSPC marker runx1 at 26 hpf (B) and cmyb at 36 hpf (C) on uninjected and notch3 morphants. Arrowheads indicate HSPCs in the DA. D, E Confocal fluorescence microscopy images of transgene reporter expression in cmyb:GFP; kdrl:RFP trunk region at 48 hpf (D) and rag2:GFP at 4 dpf (E) transgenics uninjected or with notch3 morpholino injected. Arrowheads in cmyb:GFP; kdrl:RFP embryos indicate double-positive HSPCs, and dotted lines in rag2:GFP embryos outline the thymic lobes where GFP⁺ lymphoid cells should reside. F Enumeration of cmyb:GFP⁺; kdrl:RFP⁺ cells in the floor of the DA at 48 hpf. Bars represent mean ± SEM of double-positive cells for uninjected (n = 12) and notch3 morphants (n = 20). P = 2.3 × 10⁻¹⁵.

Figure 2. Notch3 is dispensable for DA, but required for sclerotome

A Brightfield image of a 26 hpf zebrafish. B Cartoon cross section of the embryonic trunk marking somites in light blue, sclerotome in purple, venous endothelium in yellow, and aortic endothelium in orange. C–H WISH of uninjected and notch3 morphants at 26 hpf for the endothelial marker kdrl (C), dorsal aorta markers efnb2a (D) and dlc (E), the somite marker myod (F), and sclerotome markers foxc1b (G) and twist1b (H). Magnified panels are shown for somitic and sclerotomal markers in lower left corner. Arrowheads indicate tissue-specific gene expression.

Figure 3. Specific temporal activation of Notch signaling is sufficient to rescue HSPCs in notch3 morphants

A, B WISH for runx1 in 26 hpf hsp70:gal4; UAS:NICD-myc uninjected or notch3 morphant transgenic embryos with heat-shock induction at 14 hpf (A) or 20 hpf (B), with or without enforced NICD expression. Arrowheads indicate the presence or absence of HSPCs at the midline. C Quantitation of results recording percentages of embryos displaying normal or decreased numbers of runx1⁺ HSPCs at 26 hpf in notch3 morphants with heat-shock induction conditions.

Figure 4. Notch signaling is required during two distinct time windows for specification of sclerotome and dorsal aorta

A–D WISH for runx1 (A), foxc1b (B), efnb2a (C), and dlc (D) in 26 hpf embryos treated with DMSO vehicle (left), 4 μM γ-secretase Notch inhibitor DBZ at 6–15 hpf (middle), or 15–26 hpf (right). Arrowheads indicate tissue-specific expression.

Figure 5. Specific spatial activation of Notch signaling is sufficient to rescue HSPCs in notch3 morphants

A, B WISH for runx1 in 26 hpf kdrl:gal4 (A) or phldb1:gal4-mcherry (B) crossed to UAS:NICD-myc transgenic embryos either uninjected or injected with notch3 morpholino, with or without enforced NICD expression. Arrowheads indicate the presence or absence of HSPCs at the midline. C Quantitation of results recording percentages of embryos displaying normal or decreased numbers of runx1⁺ HSPCs at 26 hpf in notch3 morphants with tissue-specific induction conditions.

Figure 6. Notch3 cooperates synergistically with dlc and dld to specify HSPCs

A–F WISH for runx1, twist1b, and foxc1b at 26 hpf in uninjected (A), low-dose knockdown of notch3 (B), heterozygotes for dlc mutant bea (C), low-dose knockdown of dld (D), bea heterozygotes with low-dose knockdown of notch3 (E), and combinatorial low-dose knockdown of dld and notch3 (F). Arrowheads indicate tissue-specific expression.

Figure 7. Wnt16, dlc/dld, and notch3 function in a linear pathway to specify HSPCs

A–E Expression of runx1 at 26 hpf in uninjected (A) or injected with wnt16MO (B), wnt16MO with 50 pg dlc/dld mRNA (C), notch3MO with 50 pg dlc/dld mRNA (D), and wnt16MO/notch3MO with 50 pg dlc/dld mRNA (E). Arrowheads indicate the presence or absence of HSPCs.