XB-IMG-84154
Xenbase Image ID: 84154
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Figure 1. Identification of a Role for miRNAs in Specification of the HSC Lineage
(A) Schematic diagram showing the stepwise differentiation of the blood stem cell lineage in Xenopus, from DLP hemangioblast precusors through to HSCs via hemogenic endothelium. The DLP hemangioblasts give rise to the DA, including the hemogenic endothelium in the ventral wall from which stem cells emerge as part of hematopoietic clusters. The hematopoietic clusters in the DA at stages 42–44 of the Xenopus embryo are analogous to the clusters found in the E11.5 AGM of the mouse.
(B) miRNAs are required for HSC lineage specification. dgcr8 morphants do not produce hemogenic endothelium because of failure to correctly specify hemangioblasts in the DLP. Top: WISH showing Runx1 expression in the DA (arrowheads) at stage 39 is dependent on Dgcr8. Bottom: WISH showing loss of Flk1 expression in the DLP (arrows) of dgcr8 MOs at stage 24. Numbers of embryos represented by each panel, out of the number analyzed, are indicated in the top right corner. The anterior of the embryo is to the left and dorsal to the top in all images.
(C) Heatmap showing hierarchical clustering of miRNA expression profile of sorted cell populations from E11.5 mouse AGM. E: Ter119 CD31+ c-Kit CD45 endothelial cells (including hemogenic endothelium), presumptive pre-HSCs: Ter119 CD31+ c-Kit+ CD45 HSC precursors, and HSPC: Ter119 CD31+ c-Kit+ CD45+ hematopoietic stem and progenitors. Red indicates high expression and green indicates low expression as represented by the Z score. The ‘‘HSPC-miRs’’ cluster is expanded to show miRNA names. All miRNA qRT-PCR array data are available in Table S1. See Figure S1D for gating strategy of the sort and Figure S1E for schematic of AGM populations.
(D) Ranking of miRNAs by scoring peaks within 5 kb up- and downstream of the genomic location of the pre-miRNA in ChIP-seq data for ten hematopoietic TFs from hematopietic progenitor cells (Wilson et al., 2010). Graph showing rankings for the ‘‘HSPC-miRs’’ cluster; only miRNAs with a score greater than zero are shown.
(E and F) Relative expression of the 50 and 30 strands of mir-142, miR-142-5p, and miR-142-3p. (E) qRT-PCR expression analysis on sorted mouse BM adult HSPC (Flt3-KLS) and mouse AGM E, pre-HSC, and HSPC populations. (F) qRT-PCR expression analysis on dissected stage 26 DLP and, for comparison, stage 14 whole embryos. DCt values relative to U6 are shown on a log scale. (Error bars represent mean ± SD.)
See also Figure S1 and Table S1. Image published in: Nimmo R et al. (2013) Copyright © 2013. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |