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First published online 19 April 2006
doi: 10.1242/dev.02365

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Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.

View larger version (62K): [in a new window]   Fig. 1. In vitro differentiation potential of cranial paraxial mesoderm and splanchnic mesoderm in chick embryos. (A) Dissected cranial paraxial mesoderm (CPM, purple) and splanchnic mesoderm (SpM, orange) of a stage 10 chick embryo: (A1) Ventral view; (A2) following the removal of the linear heart tube; (A3) transverse section of the embryo (dotted line in A2). (B,C) CPM and SpM explants were dissected from stage 10 embryos. RNA was harvested from the explants either immediately (0) or after 3 days in culture. RT-PCR analysis was performed for the indicated cardiac markers and signaling molecules. Although cranial paraxial mesoderm explants underwent myogenesis in culture (n=17/20), splanchnic mesoderm explants underwent cardiogenesis (n=20/20). T, total chick RNA; da, dorsal aorta; nc, notochord; nt, neural tube; oft, outflow tract; ph, pharynx.

View larger version (109K): [in a new window]   Fig. 2. Gene expression analysis of candidate regulatory molecules that specify the head mesoderm. (A-J) Whole-mount in situ hybridization for the indicated genes in stage 16 chick embryos. (A1-J4) Transverse sections at four levels (indicated by the dotted lines in A). (A1-J1) First branchial arch. (A2-J2) Outflow tract/second branchial arch. (A3-J3) Epithelial splanchnic mesoderm/third branchial arch. (A4-J4) Inflow tract. ba1, branchial arch 1; ba2, branchial arch 2; da, dorsal aorta; dm, dorsal mesocardium; end, endoderm; V, trigerminal ganglion; ift, inflow tract; mn, motoneuron; nt, neural tube; oft, outflow tract; ov, otic vesicle; ph, pharynx; spm, splanchnic mesoderm.

View larger version (71K): [in a new window]   Fig. 3. Bmp4 signaling promotes cardiogenesis and blocks myogenesis in vitro. (A) RT-PCR analysis of CPM and SpM explants. Explants cultured in the absence (-) or presence (+) of BMP4-conditioned medium. RT-PCR analysis was performed after 3 days. (B) RT-PCR analysis of explants cultured for 3 days in the absence (-) or presence (+) of Noggin protein. BMP4 induced cardiogenesis and blocked skeletal muscle differentiation in CPM explants after 2 and 3 days in culture (n=15/15), while Noggin induced myogenesis in splanchnic mesoderm explants after 3 days in culture (n=7/8).

View larger version (58K): [in a new window]   Fig. 4. BMP4 induces cardiac gene expression and blocks skeletal muscle differentiation in vivo. (A) Dorsal view of a stage 9 embryo, implanted with cells expressing HA-BMP4 (dashed circle). (B) Western blot analysis of HA-tagged BMP4 in the conditioned medium of the transfected cells. (C-H) In situ hybridization analyses for the indicated genes. Whole-mount and transverse sections of the embryos are shown (left panel, control; right panel, Bmp4 treated). Induction (white arrowhead) or reduction (open arrowhead) of the expression of cardiac and skeletal muscle markers following BMP4 application is indicated. Ectopically-applied Bmp4 induced expression of the cardiac markers Nkx2.5 (n=7/7), Gata5 (n=7/14) and Capsulin (n=5/6). By contrast, BMP4 cells blocked the expression of the skeletal markers Myf5 (n=4/4) and Tbx1 (n=3/4). A dual effect was observed for Isl1: although Bmp4 abolished the expression of Isl1 in the trigeminal ganglion (V), an elevated expression of this gene was found in the mesenchyme and ectoderm of the first BA (n=7/7). as, aortic sac; ba1, ba2, first, second branchial arches; lr, lateral rectus; nt, neural tube.

View larger version (107K): [in a new window]   Fig. 5. Cranial paraxial mesoderm cells contribute to the myocardium and endocardium of the cardiac outflow tract in vivo. (A-P) Tracking of CPM cells by Dil labelling. DiI injections into the left (A) or right (E,I,M) CPM of stage 10 (or stage 8, I) chick embryos. Lateral (B,F,J) or transverse (C,G,K) views of embryos at stage 17-18 are shown as an overlay of bright field and fluorescence images. (D,H,L) Higher magnification of dissected hearts. Cells labeled on the left side of the CPM contribute to the inner curvature of the cardiac OFT (D), whereas cells from the right side contribute to the outer curvature (H,L). (O,P) The CPM cells contribute to both the myocardium and endocardium: cross section (O) and transverse section (P) of the OFT. (M,N) Embryo dissected shortly after the DiI injection (M) to verify the location of the dye in the paraxial mesoderm (N). ba1, first branchial arch; ba2, second branchial arch; cc, conus cordis; da, dorsal aorta; end, endocardium; ic, inner curvature; myo, myocardium; nc, notochord; nt, neural tube; oc, outer curvature; oft, outflow tract; ov, otic vesicle; ph, pharynx; rv, right ventricle; ta, truncus arteriosus.

View larger version (51K): [in a new window]   Fig. 6. Cellular and molecular analyses of cranial paraxial mesoderm en route to the heart. (A) Diagram of a stage 16 embryo (ventral view), following removal of the heart. Dotted lines represent the three section levels displayed in the lower panels. (B) Diagram of quail-chick chimera assay. (C-I) Transverse sections at the first branchial arch (ba) level (upper dotted line in A). (C1-I1) Transverse sections at the aortic sac level (middle line). (C2-I2) Transverse sections at the outflow tract (OFT) level (lower line). (C-C2) Sections of CM-DiI-labeled cells at the aforementioned levels. (D-D2) Transverse sections through the quail-chick chimera, followed by immunostaining with the quail-specific antibody (QCPN, x10 bright field and fluorescence images) (D'-D2') x20 fluorescence images of the marked area shown in the x10 images (D-D2). (E-E2) Immunostaining for Myosin heavy chain using MF20 antibody. (F-I2) Sections through embryos subjected to whole-mount in situ hybridization with the indicated markers. aaa1, aortic arch artery 1; aaa2, aortic arch artery 2; as, aortic sac; ba1, ba2, first, second branchial arches; end, endocardium; myo, myocardium; nt, neural tube; ov, otic vesicle; ph, pharynx.

View larger version (32K): [in a new window]   Fig. 7. A model for cranial paraxial mesoderm specification in the chick embryo. Our model proposes that cells within the CPM that migrate through the first branchial arch (marked by orange line), first adopt a myogenic lineage (Myf5, Capsulin and Tbx1; highlighted in the upper box). Those cells migrating further towards the aortic sac, which connects the branchial arches to the OFT, initiate cardiogenesis (note the expression of cardiac markers Gata5, Gata6, Capsulin, Isl1 and Nkx2.5; middle box). A smaller portion of these cells may reach the myocardium and endocardium of the OFT, where different cardiac markers are expressed (e.g. Gata4 and cMHC; lower box). The gradual shift from a skeletal muscle to a cardiac cell fate is correlated with the spatiotemporal expression of Bmp4. Moreover, ectopic application of Bmp4 both in vitro and in vivo promotes cardiogenesis in the cranial paraxial mesoderm, and blocks the skeletal muscle differentiation programs.