Extended Data Figure 10: Meox1 acts as a repressor and directly regulates the expression of cxcl12b in zebrafish.

a–d′, Reduction in cxcl12 signalling rescues the meox1^−/− phenotype. Addition of AMD3100 (20 μM b, b′) or injection of with a morpholino against the cxcl12b gene (c, c′) prevents the expansion of HSCs evident in meox1 mutants (a, a′). Expression of cxcl12b from the endotome specific pax3a promoter induces clustering of HSC. d, d′, Clustering of cmyb positive HSCs is induced upon expression of a cxcl12b protein fused to mCherry from the endotome specific pax3a promoter. e, cxcl12b expressed on endotome-derived endothelial cells rescues the HSC deficit in embryos lacking global Cxcl12b activity. unject, uninjected siblings embryos. morp: Embryos injected with a cxcl12b morpholino, designed against the 5′UTR of cxcl12b gene. This morpholino knocks down global endogenous Cxcl12b activity in the developing embryo in a similar manner to the original ATG targeted morpholino, but does not target the open reading frame of cxc12b present within the pax3a:cxcl12b mCherry construct. morp. pax3a–mCherry: Embryos injected with the 5′UTR morpholino and pax3a mCherry vector only control that does not contain the cxcl12b open reading frame. morp. pax3a;cxcl12bmcherry: Embryos injected with the 5′UTR morpholino and a construct expressing cxcl12b fused to mCherry from the pax3a promoter. In these injected animals, endotome-derived endothelial cells expressing cxcl12b are able to rescue the deficit of global knock down of cxcl12b. Counts are total cmyb positive (cmyb+ve) HSCs in the DA, six somites anterior to the end of the yolk extension. Data are mean+ s.e.m., significance **P < 0.005 from an unpaired t-test. f, Human aortic endothelial cells (HAECs) were transfected with an expression construct in which Meox1 is fused to GFP. Cells were cultured for 24 h and sorted by FACs for GFP expression. Real time PCR was used to determine the relative level of expression of cxcl12 to ribosomal protein, large, P0 (RPLP0). Fold expression changes between GFP-positive and GFP-negative cells were calculated from three separate transfection experiments. Data are mean ± s.e.m. transfections n = 3, PCR on each transfection performed in triplicate. g, UCSC genome browser showing alignment using the Phastcons algorithim of cxcl12b across 5 fish species revealing specific conserved non-coding elements (CNEs). All alignment algorithms tested (Phastcons, Phylof, Multiz) reveal the same seven (labelled 1–7) CNEs within introns and approximately 6.5 kb upstream of the ATG start site. Possum software was consequently used to locate Meox1 binding sites within these regions using the Meox1 positional weight matrix (PH0103.1) obtained from the Jaspar database⁵⁴. Of the 7 selected regions only 4 (red boxes) had matching Meox1 binding sites. h, Chromatin immunoprecipitation (ChIP)-qPCR analysis of the 4 selected regions was conducted with 3 non-overlapping primer sequences spanning the conserved region, and showed Meox1 occupancy is enriched in regions 1, 2 and 6 but is not found at region 7. Data are mean+ s.e.m. i, A model for endotome formation and its contribution to definitive HSC induction. A number of distinct phases contribute to the induction of haematopoietic stem cells by somite-derived endothelial cells. (i), The somite is initially separated into a primary posterior myogenic domain (red) and a non myogenic anterior compartment (pink). (ii), The anterior somitic compartment is further partitioned into either endotome (orange) or dermomyotome (yellow) by the activity of the meox1 gene. Endotome cells expressing the chemokine cxcl12b migrate from the somite. (iii), The dorsal aorta (DA) is colonized by endotome cells, which contribute to endothelial cells and a second set of cells termed vascular associated cells (VACs). A potential fate of VACs could be mural cells, which give rise to vascular associated pericytes and smooth muscle cells of the vasculature. This is unlikely, however, as DA associated expression of smooth muscle actin, which specifically marks both these cell types, does not occur until 3dpf, a phase of development well after the processes we observe here⁵⁵. (iv), HSC (green) induction requires cxcl12b activity (arrowheads) secreted from endotome-derived endothelial cells. NT, neural tube; NC, notochord; DA, dorsal aorta; PCV, posterior cardinal vein.