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Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter.

Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA - Nature (2009)

Bottom Line: It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs.In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs.Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus-the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

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Runx1 is not required in Vav+ cells for CFU-C or HSC emergencea. Flow cytometric assay of AGM region, vitelline and umbilical arteries (AGM+V+U) and fetal livers from 11.5 dpc R26R-YFP;Vav-Cre conceptuses demonstrating deletion in blood but not in endothelium. Scatter plot shows 7AAD− Ter119− cells gated and analyzed for CD45 and VEC expression. Histograms to the right represent the percent of YFP positive cells (± SEM) in each of the gated populations. The CD45+VEC+ population from the AGM+V+U was too small to analyze for YFP expression. Graph on far right in top row shows mean ± SEM for AGM+V+U. Activation of the R26R-YFP allele in both fetal liver endothelium (CD45− VEC+) and blood (CD45+ VEC− and CD45+ VEC+) with VEC-Cre is shown at the bottom for comparison.b. Dorsal aorta from the AGM region of a 10.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrow points to an intra-aortic cluster, all of which are β-gal−.c. Transverse section through the dorsal aorta of an 11.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrows indicate two β-gal+ cells in the subaortic mesenchyme.d. Detail of β-gal+ cell in the subaortic mesenchyme from boxed region in c.e. β-gal+ cells in the fetal liver (11.5 dpc).f. CFU-C assays from 15.5 dpc fetal livers. Asterisks indicate significant differences from f/+ or f/f animals (P = 0.05, ANOVA and Dunnett’s multiple comparison test). Data are compiled from 3–8 conceptuses of each genotype. Error bars represent 95% confidence intervals.g. PCR genotyping of colonies from CFU-C assays. The gel is from 11.5 dpc AGM+V+U colonies. Horizontal lines above lanes are Runx1f/f;Vav-Cre colonies, and Runx1f/+;Vav-Cre colonies are labeled f/+. Asterisks indicate colonies in which both Runx1f alleles (or in the case of Runx1f/+;Vav-Cre colonies, one Runx1f allele) were completely deleted. Numbers below the gel represent total colonies from 11.5 dpc tissues (AGM+V+U, yolk sac, placenta, fetal liver).h. Engraftment of 11.5 dpc tissues (1 ee) as assessed by FACS on peripheral blood to detect donor derived (Ly5.1−/5.2+) cells. Numbers within bars indicate successfully reconstituted recipients/number transplanted.
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Figure 4: Runx1 is not required in Vav+ cells for CFU-C or HSC emergencea. Flow cytometric assay of AGM region, vitelline and umbilical arteries (AGM+V+U) and fetal livers from 11.5 dpc R26R-YFP;Vav-Cre conceptuses demonstrating deletion in blood but not in endothelium. Scatter plot shows 7AAD− Ter119− cells gated and analyzed for CD45 and VEC expression. Histograms to the right represent the percent of YFP positive cells (± SEM) in each of the gated populations. The CD45+VEC+ population from the AGM+V+U was too small to analyze for YFP expression. Graph on far right in top row shows mean ± SEM for AGM+V+U. Activation of the R26R-YFP allele in both fetal liver endothelium (CD45− VEC+) and blood (CD45+ VEC− and CD45+ VEC+) with VEC-Cre is shown at the bottom for comparison.b. Dorsal aorta from the AGM region of a 10.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrow points to an intra-aortic cluster, all of which are β-gal−.c. Transverse section through the dorsal aorta of an 11.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrows indicate two β-gal+ cells in the subaortic mesenchyme.d. Detail of β-gal+ cell in the subaortic mesenchyme from boxed region in c.e. β-gal+ cells in the fetal liver (11.5 dpc).f. CFU-C assays from 15.5 dpc fetal livers. Asterisks indicate significant differences from f/+ or f/f animals (P = 0.05, ANOVA and Dunnett’s multiple comparison test). Data are compiled from 3–8 conceptuses of each genotype. Error bars represent 95% confidence intervals.g. PCR genotyping of colonies from CFU-C assays. The gel is from 11.5 dpc AGM+V+U colonies. Horizontal lines above lanes are Runx1f/f;Vav-Cre colonies, and Runx1f/+;Vav-Cre colonies are labeled f/+. Asterisks indicate colonies in which both Runx1f alleles (or in the case of Runx1f/+;Vav-Cre colonies, one Runx1f allele) were completely deleted. Numbers below the gel represent total colonies from 11.5 dpc tissues (AGM+V+U, yolk sac, placenta, fetal liver).h. Engraftment of 11.5 dpc tissues (1 ee) as assessed by FACS on peripheral blood to detect donor derived (Ly5.1−/5.2+) cells. Numbers within bars indicate successfully reconstituted recipients/number transplanted.

Mentions: To confirm this and to determine whether Runx1 continues to be required once the endothelial to HSC transition is complete, we examined the outcome of deleting Runx1 with Vav-Cre. Vav1 is a GDP/GTP nucleotide exchange factor for Rho/Rac, whose expression is restricted to hematopoietic cells 20, 21. Vav1 was also implicated as a Runx1 target, as its transcript was not detected in the AGM region or fetal liver of Runx1 deficient embryos 22. Excision of the R26R-YFP allele by Vav-Cre occurred in 68% (±16%) of CD45+VEC− hematopoietic cells from the AGM region, vitelline and umbilical arteries and not in CD45−VEC+ endothelium (Fig. 4a). Excised (β-gal+) cells were not found in intra-arterial clusters at either 10.5 or 11.5 dpc, and were instead located within the sub-aortic mesenchyme and the circulation (Fig. 4b–d). Therefore Vav-Cre marked cells that either originated in the yolk sac, or had upregulated Vav-Cre subsequent to their release from the intra-arterial clusters. β-gal+ cells were detectable in the fetal liver at 10.5 dpc (not shown), and by 11.5 dpc 63% (±13%) of CD45+VEC− cells in the fetal liver were YFP+ (Fig. 4a) and were easily seen by histology (Fig. 4e). Fetal liver HSCs transiently express cell surface VEC 23, 24. Sixty three (±15) percent of the CD45+VEC+ HSC-containing population in the 11.5 dpc fetal liver was YFP+ (Fig. 4a), and by 15.5 dpc 90% of lineage negative Sca1+ Mac1+ fetal liver cells were YFP+ (not shown), similar to the excision frequencies reported previously 21. Thus with Vav-Cre we could execute an excision in CD45+ cells while avoiding deletion in the intra-arterial clusters or endothelium.


Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter.

Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA - Nature (2009)

Runx1 is not required in Vav+ cells for CFU-C or HSC emergencea. Flow cytometric assay of AGM region, vitelline and umbilical arteries (AGM+V+U) and fetal livers from 11.5 dpc R26R-YFP;Vav-Cre conceptuses demonstrating deletion in blood but not in endothelium. Scatter plot shows 7AAD− Ter119− cells gated and analyzed for CD45 and VEC expression. Histograms to the right represent the percent of YFP positive cells (± SEM) in each of the gated populations. The CD45+VEC+ population from the AGM+V+U was too small to analyze for YFP expression. Graph on far right in top row shows mean ± SEM for AGM+V+U. Activation of the R26R-YFP allele in both fetal liver endothelium (CD45− VEC+) and blood (CD45+ VEC− and CD45+ VEC+) with VEC-Cre is shown at the bottom for comparison.b. Dorsal aorta from the AGM region of a 10.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrow points to an intra-aortic cluster, all of which are β-gal−.c. Transverse section through the dorsal aorta of an 11.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrows indicate two β-gal+ cells in the subaortic mesenchyme.d. Detail of β-gal+ cell in the subaortic mesenchyme from boxed region in c.e. β-gal+ cells in the fetal liver (11.5 dpc).f. CFU-C assays from 15.5 dpc fetal livers. Asterisks indicate significant differences from f/+ or f/f animals (P = 0.05, ANOVA and Dunnett’s multiple comparison test). Data are compiled from 3–8 conceptuses of each genotype. Error bars represent 95% confidence intervals.g. PCR genotyping of colonies from CFU-C assays. The gel is from 11.5 dpc AGM+V+U colonies. Horizontal lines above lanes are Runx1f/f;Vav-Cre colonies, and Runx1f/+;Vav-Cre colonies are labeled f/+. Asterisks indicate colonies in which both Runx1f alleles (or in the case of Runx1f/+;Vav-Cre colonies, one Runx1f allele) were completely deleted. Numbers below the gel represent total colonies from 11.5 dpc tissues (AGM+V+U, yolk sac, placenta, fetal liver).h. Engraftment of 11.5 dpc tissues (1 ee) as assessed by FACS on peripheral blood to detect donor derived (Ly5.1−/5.2+) cells. Numbers within bars indicate successfully reconstituted recipients/number transplanted.
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Related In: Results  -  Collection

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Figure 4: Runx1 is not required in Vav+ cells for CFU-C or HSC emergencea. Flow cytometric assay of AGM region, vitelline and umbilical arteries (AGM+V+U) and fetal livers from 11.5 dpc R26R-YFP;Vav-Cre conceptuses demonstrating deletion in blood but not in endothelium. Scatter plot shows 7AAD− Ter119− cells gated and analyzed for CD45 and VEC expression. Histograms to the right represent the percent of YFP positive cells (± SEM) in each of the gated populations. The CD45+VEC+ population from the AGM+V+U was too small to analyze for YFP expression. Graph on far right in top row shows mean ± SEM for AGM+V+U. Activation of the R26R-YFP allele in both fetal liver endothelium (CD45− VEC+) and blood (CD45+ VEC− and CD45+ VEC+) with VEC-Cre is shown at the bottom for comparison.b. Dorsal aorta from the AGM region of a 10.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrow points to an intra-aortic cluster, all of which are β-gal−.c. Transverse section through the dorsal aorta of an 11.5 dpc R26R-lacZ;Vav-Cre conceptus. Arrows indicate two β-gal+ cells in the subaortic mesenchyme.d. Detail of β-gal+ cell in the subaortic mesenchyme from boxed region in c.e. β-gal+ cells in the fetal liver (11.5 dpc).f. CFU-C assays from 15.5 dpc fetal livers. Asterisks indicate significant differences from f/+ or f/f animals (P = 0.05, ANOVA and Dunnett’s multiple comparison test). Data are compiled from 3–8 conceptuses of each genotype. Error bars represent 95% confidence intervals.g. PCR genotyping of colonies from CFU-C assays. The gel is from 11.5 dpc AGM+V+U colonies. Horizontal lines above lanes are Runx1f/f;Vav-Cre colonies, and Runx1f/+;Vav-Cre colonies are labeled f/+. Asterisks indicate colonies in which both Runx1f alleles (or in the case of Runx1f/+;Vav-Cre colonies, one Runx1f allele) were completely deleted. Numbers below the gel represent total colonies from 11.5 dpc tissues (AGM+V+U, yolk sac, placenta, fetal liver).h. Engraftment of 11.5 dpc tissues (1 ee) as assessed by FACS on peripheral blood to detect donor derived (Ly5.1−/5.2+) cells. Numbers within bars indicate successfully reconstituted recipients/number transplanted.
Mentions: To confirm this and to determine whether Runx1 continues to be required once the endothelial to HSC transition is complete, we examined the outcome of deleting Runx1 with Vav-Cre. Vav1 is a GDP/GTP nucleotide exchange factor for Rho/Rac, whose expression is restricted to hematopoietic cells 20, 21. Vav1 was also implicated as a Runx1 target, as its transcript was not detected in the AGM region or fetal liver of Runx1 deficient embryos 22. Excision of the R26R-YFP allele by Vav-Cre occurred in 68% (±16%) of CD45+VEC− hematopoietic cells from the AGM region, vitelline and umbilical arteries and not in CD45−VEC+ endothelium (Fig. 4a). Excised (β-gal+) cells were not found in intra-arterial clusters at either 10.5 or 11.5 dpc, and were instead located within the sub-aortic mesenchyme and the circulation (Fig. 4b–d). Therefore Vav-Cre marked cells that either originated in the yolk sac, or had upregulated Vav-Cre subsequent to their release from the intra-arterial clusters. β-gal+ cells were detectable in the fetal liver at 10.5 dpc (not shown), and by 11.5 dpc 63% (±13%) of CD45+VEC− cells in the fetal liver were YFP+ (Fig. 4a) and were easily seen by histology (Fig. 4e). Fetal liver HSCs transiently express cell surface VEC 23, 24. Sixty three (±15) percent of the CD45+VEC+ HSC-containing population in the 11.5 dpc fetal liver was YFP+ (Fig. 4a), and by 15.5 dpc 90% of lineage negative Sca1+ Mac1+ fetal liver cells were YFP+ (not shown), similar to the excision frequencies reported previously 21. Thus with Vav-Cre we could execute an excision in CD45+ cells while avoiding deletion in the intra-arterial clusters or endothelium.

Bottom Line: It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs.In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs.Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.

ABSTRACT
Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus-the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

Show MeSH
Related in: MedlinePlus