|
|
Blood, Vol. 110, Issue 13, 4188-4197, December 15, 2007
Runx1-mediated hematopoietic stem-cell emergence is controlled by a Gata/Ets/SCL-regulated enhancer
Blood Nottingham et al.
110: 4188
Supplemental materials for: Nottingham et al
Files in this Data Supplement:
- Table S1. P1, P2 and +23 CNEs and DHS in 270kb mouse Runx1 locus (PDF, 10.3 KB) -
Coordinates are for 270kb Runx1 locus (reverse complement of NCBIM36 Chr16: 92473100-92743100). Species used for MLAGAN alignment/Gumby were mouse, human, dog, opossum, chicken and frog.
- Table S2. Primers used in the generation of +23 and +23-deletion fragments (PDF, 33 KB) -
XhoI and SalI restriction sites added at 5′ end of primers are underlined.
- Table S3. Site-directed mutagenesis of conserved motifs in the +23 enhancer (PDF, 43.7 KB) -
Mutated base pairs are underlined.
- Table S4. Real-time PCR primers and Taqman probes used in ChIP analysis of the +23 element (PDF, 35 KB)
- Figure S1. Sporadic LacZ expression in hematopoietic territories of F0 embryos transgenic for the enhancerless hsp68LacZ promoter-reporter construct is distinct from endogenous Runx1 expression (JPG, 43.5 KB)
-
(A) Widespread mesenchymal and neural LacZ expression, extending into the area of the dorsal aorta, was observed in one E10 hsp68LacZ transgenic embryo (out of 10 embryos generated, 4 were analyzed at E8 and 6 at E10, 2 and 3 of these, respectively, expressed LacZ at random locations, which in one E10 embryo extended into the territories of the dorsal aorta, VU, FL, YS and PB). Comparison with the endogenous Runx1 expression in the dorsal aorta (B), assayed in a Runx1-LacZ KI embryo (North et al. 1999), clearly shows the difference between the two expression patterns. Thus, we concluded that position effects of hsp68LacZ construct integration (Kothary et al. 1989; Rossant et al. 1991) that result in hematopoietic expression, happen sufficiently infrequent (1/10 F0 transgenic embryos) and are sufficiently distinct from the expression pattern of the endogenous Runx1 gene, to allow the use of the hsp68 promoter for the analysis of candidate hematopoietic Runx1 enhancer elements. DA: dorsal aorta. Original magnification ×200. Dorsal = up.
- Figure S2. Activity of the +23 enhancer in the dorsal aorta and placenta of E10 hsp68lacZ+23 F0 transgenic embryos (JPG, 45.9 KB)
-
(A): Activity in a hematopoietic cell cluster attached to the dorso-lateral wall of the aorta is indicated (arrowhead; dorsal = up). In addition, a small subset of cells in the endothelial cell layer and the mesenchyme of the aorta express LacZ. (B): The +23 enhancer is active in putative hematopoietic cells (arrowheads) in the placent (Pl). Original magnification ×200.
- Figure S3. Effects of Runx, Gata and Ets mutations on +23 enhancer activity in (A) E8 and (B) E10 embryos (JPG, 72.1 KB)
-
Ai-iii: sections through the YS BI of E8 hsp68LacZ+23Runx, hsp68LacZ+23Gata and hsp68LacZ+23Ets F0 transgenic embryos. Arrowhead in Ai indicates LacZ expressing blood cells, arrowhead in Aii a LacZ expressing cell in the YS endoderm, and arrowheads in Aiii indicate cells in the YS-BI that show punctuate rather than wider cytoplasmic Xgal staining. Panels Bi,ii: sections through the dorsal aorta and vitelline artery of a E10 F0 hsp68LacZ+23Runx transgenic embryo. LacZ expression was observed in hematopoietic clusters (arrows) and in peripheral blood cells (arrowhead). Expression in aortic clusters often appeared fainter than in VU clusters. Panel Biii: section through the dorsal aorta of an hsp68LacZ+23Gata F0 transgenic embryo. Arrows point at faint LacZ expressing cells in the endothelial cell layer. Panel Biv: section through the dorsal aorta of an E10 hsp68LacZ+23Ets F0 transgenic embryo. Arrow points at a LacZ expressing hematopoietic cluster.
- Figure S4. Generation of hsp68LacZ+23 transgenic mouse lines (JPG, 75.1 KB)
-
(A) Analysis of founders. Out of 20 F0 founders, four independent hsp68LacZ+23 transgenic mouse lines were obtained. All four these lines showed LacZ expression in hematopoietic territories in a pattern similar to what was observed for the F0 transgenic embryos. (B) Activity of the +23 enhancer in E10 embryos of the four hsp68LacZ+23 transgenic lines. Panels a-d: wholemount E10 embryos; panels e-h: sections through the dorsal aorta in the AGM region (dorsal = up); panels i-l: sections through the VU; panels m-p: sections through the YS; panels q-t: sections through the FL. Original magnifications ×200. C) Copy number blot. Genomic DNA isolated from spleens of the transgenic mice was digested with Bgl1. Southern blots were probed with a probe for LacZ to detect the transgene (2.1 kb fragment), and a single copy probe for position 31861-32449 in the 270kb Runx1 locus (5.8kb fragment). Copy numbers were assessed by comparison of a DNA standard that was spiked with a known amount of the transgene. The +23-Line1 and +23-Line12 transgenic lines carry less than 5 copies of the hsp68LacZ+23 transgene. In +23-Line 12, the transgene had integrated on the X chromosome, resulting in mosaic LacZ expression in the female embryos. Thus, +23-Line1 was selected for our functional analyses.
REFERENCES
Kothary, R., S. Clapoff, S. Darling, M.D. Perry, L.A. Moran, and J. Rossant. 1989. Inducible expression of an hsp68-lacZ hybrid gene in transgenic mice. Development 105: 707-14.
North, T., T.L. Gu, T. Stacy, Q. Wang, L. Howard, M. Binder, M. Marin-Padilla, and N.A. Speck. 1999. Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. Development 126: 2563-75.
Rossant, J., R. Zirngibl, D. Cado, M. Shago, and V. Giguere. 1991. Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis. Genes Dev 5: 1333-44.
|
|
