XB-ART-40529PLoS One October 28, 2009; 4 (10): e7650.
Early activation of FGF and nodal pathways mediates cardiac specification independently of Wnt/beta-catenin signaling.
BACKGROUND: Cardiac induction, the first step in heart development in vertebrate embryos, is thought to be initiated by anterior endoderm during gastrulation, but what the signals are and how they act is unknown. Several signaling pathways, including FGF, Nodal, BMP and Wnt have been implicated in cardiac specification, in both gain- and loss-of-function experiments. However, as these pathways regulate germ layer formation and patterning, their specific roles in cardiac induction have been difficult to define. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanisms of cardiac induction directly we devised an assay based on conjugates of anterior endoderm from early gastrula stage Xenopus embryos as the inducing tissue and pluripotent ectodermal explants as the responding tissue. We show that the anterior endoderm produces a specific signal, as skeletal muscle is not induced. Cardiac inducing signal needs up to two hours of interaction with the responding tissue to produce an effect. While we found that the BMP pathway was not necessary, our results demonstrate that the FGF and Nodal pathways are essential for cardiogenesis. They were required only during the first hour of cardiogenesis, while sustained activation of ERK was required for at least four hours. Our results also show that transient early activation of the Wnt/beta-catenin pathway has no effect on cardiogenesis, while later activation of the pathway antagonizes cardiac differentiation. CONCLUSIONS/SIGNIFICANCE: We have described an assay for investigating the mechanisms of cardiac induction by anterior endoderm. The assay was used to provide evidence for a direct, early and transient requirement of FGF and Nodal pathways. In addition, we demonstrate that Wnt/beta-catenin pathway plays no direct role in vertebrate cardiac specification, but needs to be suppressed just prior to differentiation.
PubMed ID: 19862329
PMC ID: PMC2763344
Article link: PLoS One
Genes referenced: a2m acta2 actl6a acvr1 aplnr cer1 chrd.1 dkk1 eomes fgfr1 gsc hhex irx4 mapk1 mespb mixer mlc1 myf5 myl2 myl3 myod1 nkx2-5 nodal nodal1 nodal3.1 nodal3.2 post sia1 sox17a sox17b.2 tal1 tbx18 tbx5 tbxt tcf3 tnni1 tnni3 tubb2b ventx2.2 wnt8a xpo1
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|Figure 1. Anterior endoderm is sufficient to induce cardiogenesis in animal cap cells.Gene expression in explants were analyzed either by RT-PCR (A,B,E) or by in situ hybridization (C). (A) AC/AE conjugates express Nkx2.5 at st. 16, but animal caps and anterior endoderm alone do not. Cardiomyocyte-specific markers MLC2, MHCα and cardiac TnI, as well as cardiogenic transcription factor Tbx5 are all expressed in AC/AE conjugates at st. 34. (B) Posterior Endoderm (PE) is incapable of inducing cardiogenesis in animal caps. (C) Cardiac tissue (MLC2 expression; C1) is induced by anterior endoderm in animal cap·. Animal caps injected with biotinylated dextran were used to make conjugates with anterior endoderm, and after in situ hybridization for cardiac TnI (C2), lineage tracer was revealed by Fast Red staining on sections (C3,4). (D) A drawing of NF st. 10 embryo describing anterior endoderm explants and their subdivision into anterior (A) and posterior (P) parts. Source of drawing: www.xenbase.org. (E) Anterior half of anterior endoderm explants is greatly enriched in Hex expression (St. 10.5) and is a more efficient inducer of cardiogenesis in animal caps at st. 34. (F) Cardiac-inducing activity resides in the anterior (Ant) part of anterior endoderm explants. Double anterior endoderm/animal cap conjugates show two foci of cardiac TnI expression (F1), whereas double posterior endoderm/animal cap conjugates contain only few positive cells (F2). Dissociated and reaggregated anterior endoderm (Diss; F3) induces multiple smaller foci of cardiac gene expression in animal cap (in all 5 positive conjugates; n = 12). F4: control conjugates most frequently have a single cluster of cardiomyocytes (8/8 in this experiment, and 88% of all expressing explants overall, n = 74).|
|Figure 2. Specificity of anterior endoderm as inducer of cardiac fate.(A) anterior endoderm induces markers of cardiovascular cell types: blood (Scl, Globin), smooth muscle (SM actin), macrophages (Lurp-1) and endothelium (Msr), but not skeletal muscle (MLC1) or neural tissue (N-tubulin). (B) Quantification of RT-PCR shown in A. (C) Late tadpole-stage AC/AE explants express markers of ventricular myocardium Irx4 and MLC1v and of the proepicardium, Tbx18. (D) animal cap/vegetal pole conjugates (Nieuwkoop sandwiches) express skeletal muscle and neural, but not cardiac markers, in contrast to AC/AE conjugates.|
|Figure 3. Two hours of contact between animal cap and anterior endoderm is sufficient for cardiogenesis.(A) Conjugates were made between st. 10.25 anterior endoderm explants and animal caps of aged animal caps isolated at st. 8.5, and MHCα expression was analyzed a day and a half later, at st. 34. (B) Conjugates were made between st. 8.5 animal caps and aged anterior endoderm explants isolated at st. 10, as indicated. MHCα expression was analyzed at st. 34. (C) Strategy for a peeled animal cap assay. Rhodamine-dextran injected anterior endoderm is conjugated with animal caps, and the caps are removed after 1 or 2 hours. Anterior endoderm-free (rhodamine-negative) animal caps are cultured until st. 34 or for a total of 2.5 hours after the initial contact, corresponding to st. 10.5 (D) Only animal caps that were in contact with anterior endoderm for 2 hours express MHCα, but other markers tested – Msr, Globin, Edd – are also induced after 1 hour of contact. (E) Early markers of endoderm (Sox17, Hex, Cerberus, Mixer), Organizer (Goosecoid (Gsc), Chordin) and mesoderm (Xbra, Eomesodermin) are expressed after 1 hour contact with anterior endoderm. However Mesp1, a marker of migrating cardiac precursors , was not induced. (F) Myogenic regulatory factors MyoD and Myf5 as well as posterior markers Xpo and Vent2 are not induced by AE.|
|Figure 4. FGF and Nodal signaling are required during the first hour of cardiac induction.(A) AC/AE conjugates were made with animal cap expressing δFGFR1 or with uninjected animal cap that were continuously incubated with SU5402 (50 µM) or U0126 (35 µM) from the time of conjugation, or were treated with DMSO (Control). MLC2 expression was analyzed at st. 34. (B) CerS (injected in animal cap) or continuous incubation with SB-431542 (75 µM) or A-83-01 (75 µM) block induction of MHCα. (C) FGF and Nodal signaling are only required the first hour of contact between AC and AE. (D) In situ hybridisation analysis of cardiac TnI expression in AC/AE conjugates. D1,2- control embryo and AC/AE explant. 83% (n = 23) explants are cTnI+. D3- expression of δFGFR1 in animal caps leads to a greatly reduced expression of cTnI in 89% (n = 19) explants. D4- 0/15 of CerS explants express cTnI. D5- mosaic expression of δActRI (red) in animal caps prevents expression of cardiac TnI (0/11 cTnI+ explants in δActRI-injected cells), but not in neighboring tissue with intact Nodal signaling (5/11 cTnI+ explants; arrow in D5). D6- constitutively active Alk4* receptor induces cardiac expression cell-autonomously in animal caps (4/4 cTnI+, n = 12).|
|Figure 5. ERK is activated during the first 4 hrs and is required for cardiogenesis over the same period.(A) ERK is activated for at least 4 hours after conjugation of animal cap and anterior endoderm. Animal cap, anterior endoderm or AC/AE explants were collected at indicated times after conjugation and were subjected to Western analysis for doubly-phosphorylated ERK (dpERK) and total ERK. The latest time-point (St. 14) corresponds to 8–10 hours after conjugation. (B) U0126 treatment for the first 2 or 4 hours after conjugation effectively blocks ERK activation. (C) ERK activity is required during at least first four hours after conjugation for efficient cardiogenesis.|
|Figure 6. BMP signaling is not required for cardiogenesis in AC/AE model.(A) Animal caps expressing truncated type I BMP receptor (tBR) or Cerberus (1 ng of mRNA each), were conjugated with anterior endoderm and conjugates were analyzed for expression of MHCα and N-tubulin at st. 34 by RT-PCR. (B) Quantification of RT-PCR in A. Both anti-BMP reagents used were active, as shown by induction of N-tubulin expression, but had no effect on cardiogenesis. (C) Dose-dependent inhibition of cardiogenesis in AC/AE explants by Cerberus. Animal caps from embryos injected with 1, 2 or 4 ng of Cerberus mRNA were conjugated with anterior endoderm and conjugates were analyzed for MHCα expression when sibling control embryos reached st. 34. The effect of Cerberus was quantified and normalized to control conjugates (% C AC/AE). (D) Phenotypic controls for Cerberus mRNA, showing dose-dependent increase in cement gland tissue.|
|Figure 7. Dkk-1 enhances cardiogenesis.(A) animal cap injected with 0.5 ng of ΔTcf3 or 1 ng of Dkk-1, or uninjected control animal cap, were conjugated with anterior endoderm and analyzed for MHCα expression. The effect of Dkk-1 was quantified and shown below the image. In two additional experiments, Dkk-1 stimulated cardiogenesis 5- and 9-fold. (B) Dkk-1 cannot act in Nodal-independent manner, as it is blocked by CerS. The effectiveness of ΔTcf3 was shown by posteriorization of embryos (data not shown).|
|Figure 8. Elevated Wnt/β-catenin signaling opposes cardiogenesis after specification.(A) Animal cap expressing either CSKA-Wnt8 or LEF-β-catenin-GR were conjugated and dexamethasone was added as indicated. All treatments block cardiogenesis. (B) LEF-β-GR activated at st. 9 and st. 13 has no effect on Nkx2.5 and Tbx5 expression at st. 18. (C) The effect of various agonists on the Wnt/β-catenin pathway: LiCl and BIO cause strong transient activation, Wnt8 causes weak and sustained activation, and LEF-β-catenin-GR causes strong and prolonged effect. Siamois and Xnr3 expression was analysed 2 and 6 hours after excision of animal caps/Treatment with LiCl, BIO and Dex was immediately after the excision. (D) The expression of the LEF-β-catenin-GR fusion protein was analyzed by Western blotting for HA tag. The protein was expressed efficiently at all stages at which dexamethasone was added and it persists for many hours. (E, F) LiCl or BIO treatment at st. 9 has no effect on cardiac differentiation but induces skeletal muscle. Later treatment at st. 21 or 24 blocks cardiac differentiation, and treatment at st. 27 has no effect.|
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