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Regulation of avian cardiogenesis by Fgf8 signaling

Molecular Medicine Unit, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

View larger version (29K): [in a new window]   Fig. 1. Bmp signaling and the regulation of cardiogenesis in the early chick embryo (stage 5-6). The embryo has been divided into three zones, from medial to lateral. Bmp ligands (blue) are expressed in zones II and III, in both anterior and posterior regions of the embryo (Schultheiss et al., 1997). Heart precursors are found in a subportion of the Bmp-expressing zone (zone II) in the anterior part of the embryo. More medial regions of the embryo (zone I) do not express Bmp2 or Bmp4, and do not express cardiac genes. More lateral regions (zone III) are exposed to Bmp signals yet do not express cardiac genes.

View larger version (109K): [in a new window]   Fig. 2. Endoderm is required for the induction and maintenance of Nkx2.5 and Mef2c but not Gata4. Endoderm adjacent to the heart region was removed (as shown by the boxed outline) from embryos at stage 5-6 (A). The unoperated side served as a control. Embryos were incubated until stage 8-9 (B) and processed for in situ hybridization for Nkx2.5 (C,D,G), Mef2c (E,H) or Gata4 (F,I). As seen in C-F, both Nkx2.5 and Mef2c expression were downregulated on the experimental side, while Gata4 expression persisted. On sections, it could be determined where the endoderm had been removed, and it could be seen that endoderm removal led to loss of Nkx2.5 and Mef2c expression in the adjacent cardiac mesoderm (G,H), while mesodermal Gata4 expression was not affected (I). The edges of the removed endoderm are marked in G-I. ecto, ectoderm; edge, edge of removed endoderm; endo, endoderm; hn, Hensen’s node; hp, head process; meso, mesoderm; np, neural plate; ps, primitive streak.

View larger version (101K): [in a new window]   Fig. 3. Expression of Fgf8 during cardiac specification. At stage 6, Fgf8 expression is found in cells of the primitive streak and in a crescent pattern in the anterior half of the embryo (A). The anterior crescent roughly overlaps the anterior expression patterns of Bmp2 (B) and Nkx2.5 (C). Sections reveal that the anterior Fgf8 expression is localized to the endoderm underlying the heart field (E). Fgf8 expression persists in the lateral endoderm of Stage 9 embryos (D), and is expanded medially in the posterior-most part of this domain (arrow in D). endo, endoderm; np, neural plate; ps, primitive streak.

View larger version (129K): [in a new window]   Fig. 4. Fgf8, but not Bmp2, can rescue cardiac gene expression in embryos lacking endoderm. Endoderm was removed from embryos at stage 5-6 and either Bmp2-soaked beads (b) or Fgf8-soaked beads (f) or control beads (c) were placed on top of the now accessible mesoderm. The embryos were cultured until stage 8-9 and processed for in situ hybridization for Nkx2.5 (A-D). Bmp2-soaked beads had little effect in rescuing Nkx2.5 expression (A,B). By contrast, Fgf8 beads rescued expression of Nkx2.5 in the embryos that lack endoderm (C,D). An asterisk (*) marks experimental beads in sections. ecto, ectoderm; edge, edge of endoderm; endo, endoderm; meso, mesoderm; np, neural plate.

View larger version (102K): [in a new window]   Fig. 5. Fgf8 can induce ectopic Nkx2.5 and Mef2c expression lateral to the heart field. Heparin-acrylamide beads soaked in Fgf8 protein (f) or control beads (c) were placed in stage 4-6 embryos (A) and incubated until stage 8-9. Whole-mount in situ hybridization was then performed for Nkx2.5 (B,C,E,G), Mef2c (D,F) and Sox3 (H). As seen in B-D, the Fgf8-soaked bead induced a lateral expansion of both Nkx2.5 and Mef2c expression. While Nkx2.5 expansion was seen in all three germ layers (E), Mef2c lateral expansion was observed only in the mesoderm (F). Fgf8-soaked beads placed medial to the heart field did not induce ectopic Nkx2.5 expression (G). In addition to expanding the heart field, Fgf8 beads also induced the expression of a neural marker, Sox3, in the ectoderm (H). An asterisk (*) marks experimental beads in sections. aip, anterior intestinal portal; ecto, ectoderm; endo, endoderm; hn, Hensen’s node; hp, head process; meso, mesoderm; np, neural plate; ps, primitive streak.

View larger version (33K): [in a new window]   Fig. 6. A pair-wise comparison of Nkx2.5 mesodermal expression areas in Fgf8-treated embryos. Each sample represented on the x-axis is taken from one embryo, with the Fgf8-treated side shaded in blue and the contralateral control side shaded in red. The y-axis is a measurement of the mesodermal area expressing Nkx2.5 from a representative section (see Materials and Methods). In all embryos, the Nkx2.5-expressing area was greater on the Fgf8-treated side, with an average increase of 1.5-fold. Statistical analysis using Student’s two tailed t-test gave a P value of 0.008 (df=12).

View larger version (115K): [in a new window]   Fig. 7. Regulation of Fgf8 expression by Bmp signaling. Bmp2 beads placed in zone I, induced ectopic expression of Fgf8 (compare arrows in treated embryo in B with control in A), and induced ectopic Nkx2.5 expression (E). Note that while Nkx2.5 was induced only immediately surrounding the Bmp2 bead (E), Fgf8 was induced throughout zone I (B). Bmp2-soaked beads placed in zone III, lateral to the heart field, induced neither Fgf8 (C) nor Nkx-2.5 (F) in zone III, but, interestingly, did induce low levels of Fgf8 expression in zone I, some distance away (arrow in C). In contrast to the results in B, beads that were soaked in higher levels of Bmp2 (30 ng/µl), repressed Fgf8 expression (D). aip, anterior intestinal portal; b, Bmp2 bead, 5 ng/µl; bhi, Bmp2 bead, 30 ng/µl; c, control bead.

View larger version (29K): [in a new window]   Fig. 8. Models of the regulation of cardiac gene expression. (A) Cardiogenesis takes place in regions where Bmp and Fgf signaling overlap. Members of the Bmp family are expressed in lateral regions of the embryo (blue). Fgf8 is expressed in the gridded areas. Within the anterior part of the embryo, cardiac gene expression occurs where Bmp and Fgf signaling overlap. Note, however, that the signals also overlap in the posterior primitive streak, although cardiogenesis does not occur in that region, indicating the presence of other regulators of cardiogenesis. (B) Molecular regulation of cardiogenesis. Nkx2.5 appears to be regulated by both Bmp and Fgf signaling, whereas Gata4 is regulated by Bmp but not by Fgf signaling. Mef2c is also regulated by both Bmp and Fgf signaling. The regulation of Mef2c by Bmp and Fgf signaling may be direct or, as Mef2c is expressed several hours after Nkx2.5 and Gata4, work indirectly through Nkx and Gata genes. See text for discussion. (C) Integration of signals that regulate cardiogenesis. Cardiac mesoderm is induced by Bmp signals from the endoderm and ectoderm (blue), and by Fgf8 signals from the endoderm (yellow). Fgf8 is itself regulated by Bmp signaling, and appears to be expressed at intermediate levels of Bmp activity. In addition, cardiac inhibitory signals are present in the embryo (red dashes), emanating from the neural plate, ectoderm or other sources. See text for discussion.

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