Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors
- Johns Hopkins University, United States
- Oregon Health & Science Universtiy, United States
- Medical University of Graz, Austria
- Yale University, United States
- Victor Chang Cardiac Research Institute, Australia
Figures
Figure 1 with 1 supplement
Numb and Numbl are required for PA2 and heart development.
(A) Schema of cardiac differentiation in ES cell system. (B) Expression profiles of genes indicated during cardiac differentiation of ES cells. Gene expression was analyzed by qPCR. Data are mean ± …
Figure 1—figure supplement 1
Numb is ubiquitously expressed in developing embryos.
(A–C) Mesp1 lineage-traced embryos (Mesp1Cre; Ai9) at E7 (A), E8 (B), or E9 (C). RFP marks Mesp1+ cells and their progeny. (A′, A″, B′, B″, C′, C″) Confocal images of corresponding embryos …
Figure 2 with 3 supplements
Deletion of Numb and Numbl depletes Mesp1+ progenitor-derived Isl1+ Nkx2.5– cells in PA2.
(A) Generation of Numb/Numbl DKO embryos expressing RFP in Mesp1 progeny (Mesp1Cre; Numbflox/flox; Numbl−/−; Ai9). (B) Relative mRNA levels of Numb in RFP+ cells isolated from control and Numb/Numbl …
Figure 2—figure supplement 1
Numb/Numbl DKO embryos are grossly normal at E8.5.
(A, A′, F, F′) Lateral views of Mesp1+ cell-traced control (A and A′) or Numb/Numbl DKO (F and F′) embryos analyzed at E8.5. RFP marks Mesp1 progeny. (B–E′ and G–J′) Representative confocal images …
Figure 2—figure supplement 2
Mef2c expression in PA2 and OT of control and Numb/Numbl DKO embryos.
Scale bars, 25 mm. pa2, second pharyngeal arch; ot, outflow tract.
Figure 2—figure supplement 3
Histogram of RFP + cell induction from Mesp-Cre; Ai9 ES Cells.
RFP+ cells are induced from day 2–4.
Figure 3 with 3 supplements
Mesp1+ progenitor-derived Isl1+ Nkx2.5– cells expand in PA2 and differentiate into Nkx2.5+ heart cells after leaving PA2.
(A) Whole-mount view of EdU (green)-treated embryo at E9.0. (B) Enlargement of the boxed area in (A), showing enrichment or lack of EdU+ cells in the PA2 or heart region, respectively. (C) …
Figure 3—figure supplement 1
Dual Injection of EdU and BrdU.
EdU and BrdU were sequentially injected at E9.5 at a 4-hr interval. The resulting embryos were harvested after 4 hr (8 hr after EdU injection) and analyzed by immunostaining. EdU+ cells were mostly …
Figure 3—figure supplement 2
Time lapse images of 3-D matrigel PA2 culture.
These show growth and migration of Mesp1 progeny (RFP+) in the PA2 (Video 1). Boxed area is shown in Video 2.
Figure 3—figure supplement 3
Explant culture of PA1 and PA2 dissected from Nkx2.5GFP embryo.
pa, pharyngeal arch; ot, outflow tract; rv, right ventricle; ra, right atrium; lv, left ventricle.
Figure 4 with 2 supplements
PA2 cells promote CPC expansion and suppress cardiac differentiation.
(A) FACS-purification of RFP+ Isl1+ Nkx2.5− CPCs induced from ES cell-derived precardiac mesoderm and their culture with no, embryonic PA2, or embryonic heart feeders. Images show RFP+ cells at day …
Figure 4—figure supplement 1
Percentages of Nkx2.5+ or cTnT + cells cultured with No, PA2, or embryonic heart feeders at day 8.
NF, no feeder; PA2, PA2 co-culture; HT, heart cell co-culture. Data are mean + SD; n = 3; *p<0.05. p values were determined using the paired Student t test.
Figure 4—figure supplement 2
PA2 conditioned medium mimics PA2 co-culture.
Con, control; CM, PA2-conditioned medium; PA2, PA2 co-culture. Data are mean + SD; n = 3; *p<0.05. p values were determined using the paired Student t test.
Figure 5 with 1 supplement
Generation of Mesp1 lineage-specific somatic cells lacking Numb and Numbl in vivo.
(A) Scheme for generation of RFP+ Numb/Numbl DKO cells in Mesp1 lineage. In this study, three independent sets of blastocyst injection were carried out and 16 chimeras were obtained from 132 …
Figure 5—figure supplement 1
Numb/Numbl DKO cells are normally specified into OT cells.
(A–C) Confocal images of PA2 sections of E9.0 chimera, immunostained with RFP and Isl1 (A), RFP and Mef2c (B) or RFP, Isl1 and Mef2c (C) antibodies. RFP+ Isl1+ Mef2c+ cells are outlined in white (C).…
Figure 6 with 1 supplement
Numb and Numbl are required for proliferation of Mesp1+ progenitor-derived Isl1+ Nkx2.5– cells in PA2.
(A–G′) Confocal images of PA2 sections of control (A–C‴), Numb/Numbl DKO (D), and chimeric (E–G′) embryos, immunostained with PH3, RFP, Isl1 antibodies. Asterisks indicate PH3+ RFP+ Isl1+ (triple …
Figure 6—figure supplement 1
Knockdown efficiency of Numb siRNA and Numbl siRNA.
Relative mRNA expression levels of Numb and Numbl in CPCs after transfected with scrambled siRNA (Con), Numb siRNA (Numb KD), or Numbl siRNA (Numbl KD), determined by qPCR. Data are mean + SD; n = 3;…
Figure 7
Model for Renewal and Niche of Mesp1+ progenitor-derived CPCs.
https://doi.org/10.7554/eLife.02164.023Videos
Video 1
3D-cultured PA2 in Matrigel.
This video shows expansion and migration of Mesp1 progeny (RFP+) in ex-vivo cultured PA2.
Video 2
High magnification of inset in Video 1.
This video shows RFP+ beating cardiac myocytes at a migrating edge of ex-vivo cultured PA2.
Video 3
2D-cultured PA2.
This video shows a beating monolayer of cardiomyocytes derived from RFP+ Mesp1 Progeny in ex-vivo cultured PA2.
