Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta
- Weizmann Institute of Science, Israel
- Victor Chang Cardiac Research Institute, Australia
- Technion-Israel Institute of Technology, Israel
- Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- Institute for Stem Cell and Regenerative Medicine, The University of Washington, United States
- Institute for Molecular Bioscience, The University of Queensland, Australia
- The University of Sydney, Australia
- Children’s Medical Research Institute, Australia
- University of New South Wales, Australia
Figures
Figure 1 with 1 supplement
Identification of novel Nkx2-5 and Isl1 cardiac enhancers.
(A) Location of the Nkx2-5 enhancer in the mouse genome. (B–C) Expression patterns of the enhancer-driven GFP/RFP formed in the cardiac crescent at St. 8, compared to corresponding control vectors. …
Figure 1—figure supplement 1
Characterization of the novel cardiac Isl1 enhancer.
(A–D) Expression pattern of the Isl1-en at different developmental stages. (E) The GFP pattern formed in the cardiac crescent at St. eight overlapped with the Isl1 mRNA expression pattern and its …
Figure 2 with 1 supplement
Nkx2-5 and Isl1 enhancers mark distinct cardiac progenitor populations.
(A) Experimental design of the ex ovo electroporation technique. An electric field is used to introduce different plasmids into the embryo at gastrulation (St. 3), followed by EC culture and …
Figure 2—figure supplement 1
Migration of Nkx2-5-en+ cells from outside the cardiac crescent towards the heart tube.
(A–H) Time lapse analysis of a live chick embryo expressing GFP under the control of Nkx2-5-en analyzed at different time points. A single cell (white arrow and circle) migrating from the border of …
Figure 3 with 1 supplement
The Nkx2-5 enhancer is expressed in a hemangiogenic cell population.
(A–A''') A St. 7 embryo co-electroporated with the Nkx2-5-en (RFP) and Hb-en (GFP). The dashed line marks the boundaries of the cardiac crescent. Red arrows represent Nkx2-5-en+ cells in the cardiac …
Figure 3—figure supplement 1
The Hb enhancer is active in endothelial and blood progenitors.
(A) In situ hybridization of Tal1 mRNA at St.8 (B–B") The Hb-en is expressed in the extra-embryonic region with a similar pattern to Tal1 endogenous mRNA. Expression can also be detected in the …
Figure 4 with 1 supplement
Hemangiogenic progenitors migrate to the heart through the inflow tract, and contribute to the endocardium.
(A–G) Time-lapse images taken from an embryo electroporated with Hb-en (GFP). Images span a 24 hr time frame, from St. 6 to St. 11 of the same embryo. The orange and cyan circles distinguish between …
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Figure 4—source data 1
The distribution of Hb-en+ cells in the chick embryo.
Analysis is based on six embryos from two independent experiments. The first column represents the tissues checked for Hb-en+ cells. The second column represents the percentage of embryos in which Hb-en+ cells were detected. The third column represents the percentage of double positive Hb-en+/CD45+ cells out of the total CD45+ cells.
- https://doi.org/10.7554/eLife.20994.012
Figure 4—figure supplement 1
Double positive Nkx2-5-en+/Hb-en+ cells migrate towards the heart and yolk sac.
Time-lapse images taken from an embryo electroporated with Hb-en (GFP) and Nkx2-5-en (RFP). Images span an 18 hr time frame, from St. six to St. 11 of the same embryo. Images were taken in a way …
Figure 5
Hb-en+ and Nkx2-5-en+ cells contribute to the hemogenic endothelium of the dorsal aorta.
(A–A”) A St.17 embryo sectioned at the level of the AGM and stained with novel anti Cdh5 (VE-cadherin) and CD45 antibodies delineating the endothelium and hematopoietic cells, respectively. (B–B''') …
Figure 6
Hb-en+ and Nkx2-5-en+ cells give rise to hemogenic endocardium.
(A) Section through a heart of a St.12–13 embryo expressing the Nkx2-5-en. Staining for Cdh5 delineates the endocardium (white arrow) which expresses the Nkx2-5-en. (B) High magnification of a …
Figure 7 with 1 supplement
Nkx2.5 is expressed in the nascent mesoderm in the chick primitive streak.
(A) The cardiovascular network gradient. In situ hybridization of key genes involved in blood, vascular and cardiac development. (B) Semi qRT-PCR analysis of cardiovascular genes in early St. 3 (red …
Figure 7—figure supplement 1
Key cardiovascular gene expression gradient in a chick embryo.
(A) Super-position of the different cardiovascular factors on a single embryo based on in situ hybridization and semi qRT-PCR analysis. (B) Semi qRT-PCR analysis of cardiovascular genes in a St.8- …
Figure 8 with 2 supplements
Ectopic expression of Nkx2.5 induces angioblast gene expression.
(A–E) In situ hybridization for Tal1, Flk1, Ets1 and Lmo2 and Pax3 in control embryos. (A’–E’) Embryos electroporated at St.3 with a vector over-expressing Nkx2.5 (pCIG-Nkx2.5-GFP). Embryos were …
Figure 8—figure supplement 1
Tal1 over-expression inhibits cardiogenesis.
The coding sequence of chick Tal1 was cloned into an expression vector and electroporated into St.3 embryos. (A,C,E,G) Expression profile in WT control embryos. (B,D,F,H) Expression profile in Tal1 …
Figure 8—figure supplement 2
Tal1 over-expression impairs normal heart development.
(A) Control St.13 WT embryo. (B) Tal1 over-expressing embryo with edema. (C) Tal1 over-expressing embryo with defected cardiac looping and size. (D) Tal1 over-expressing embryo with ectopic …
Figure 9 with 5 supplements
An Nkx2-5+ lineage contributes to hemogenic endothelium of the dorsal aorta in the mouse.
(A–D) Lineage tracing β-gal staining of Nkx2-5irescre;R26R embryos. (A) E7.5 showing β-gal staining beginning in the cardiac crescent (cc) and yolk sac (ys). (B) E8.0 showing β-gal staining in the …
Figure 9—figure supplement 1
Nkx2-5 is transiently expressed in yolk sac mesoderm from cardiac crescent stages.
(A) Lateral view of whole mount β-gal staining in an E7.0 mid-gastrula-stage Nkx2-5+/+;R26R (control) embryo. Weak background is detected sporadically across the yolk sac (ys). (A’) Lateral view of …
Figure 9—figure supplement 2
Nkx2-5+ lineage contributes to hemogenic endothelium of yolk sac vasculature.
Co-immunostaining of E9.0 Nkx2-5irescre;ROSAYFP (A–C’’’) and Nkx2-5irescre;Z/EG (D–E’’’) embryos. (A-B’’’; D–D’’’). Cryosections of yolk sac immunostained for YFP (green) and endothelial marker …
Figure 9—figure supplement 3
Nkx2-5 expression in single cells of mouse E7. 0–7.75 embryos.
(A) t-SNE plots were used to visualize single cells transiting through stages of mouse mesodermal development in vivo, with cell clustering and identification of subtypes based on categorizations …
Figure 9—figure supplement 4
Spatial RNA-seq analysis of Nkx2-5 expression in mouse gastrula-stage embryos.
(A–C) ‘Corn plots’ showing examples of the spatial domains of Nkx2-5 expression in the epiblast, ectoderm and mesoderm of mouse embryo at different stages of gastrulation (taken from published …
Figure 9—figure supplement 5
Nkx2-5 lineage+ cells in the cardio-pharyngeal region.
(A–E”) Co-immunostaining of cryo-sections from E10.5 Nkx2-5irescre; ROSAYFP (A–D) and Nkx2-5irescre;Z/EG (E–E”) embryos. (A) Section showing Nkx2-5 lineage traced cells (YFP+) in the endothelium of …
Figure 10
Nkx2.5 marks hemogenic angioblasts that contribute to the formation of the endocardium and dorsal aorta.
Cardiovascular progenitors begin to form at the onset of gastrulaion. These populations segregate as the cells begin to migrate in a lateral fashion towards the extraembryonic tissue. During early …
Videos
Video 1
Time lapse movie of a developing embryo expressing the Isl1 enhancer.
Relates to Figure 1. An embryo electroporated with the Isl1-en (GFP) plasmid at St. 3. The time-lapse video follows from St.5to St.11 when the heart tube has already formed and the embryo has begun t…
Video 2
Time lapse movie of a developing embryo co-expressing the Nkx2-5 and Isl1 enhancers.
Relates to Figure 2. A St. 3 embryo was co-electroporated with the Isl1-en (RFP) and Nkx2-5-en (GFP) plasmids. The embryo was cultured overnight and time-lapse analysis started at St.6. The embryo …
Video 3
Time lapse movie of a developing embryo expressing the Hb enhancer.
Relates to Figure 4. A St.3 embryo electroporated with the Hb-en plasmid (GFP) and cultured overnight. The movie covers the development of the embryo between St.6 to St. 11–12. The BF and GFP …
Video 4
Time lapse movie of a developing embryo co-expressing the Hb and Nkx2-5 enhancers.
Relates to Figure 4. A St.3 embryo electroporated with both the Hb-en (GFP) and Nkx2-5-en (RFP) plasmids subsequently cultured overnight. The movie covers the development of the embryo between St.6 …
