|
Fig. 1. GATA4 induces the expression of ventricular and proepicardial markers in animal cap explants. (A) Ventricular gene expression is induced in animal cap explants injected with GATA4 (75%, n = 50) using WMISH analysis for the expression of myl3. (B, C) Close-up of animal cap explants shown in (A); black arrowheads are indicating myl3 expression. (D, E) Lateral and ventral views of a myl3 expression in control embryo (stage 39). (F–H) Animal caps injected with GATA4 showed incompletely overlapping expression of pan-myocardial marker tnni3 and ventricular marker myl3 (36% (n = 55), 13% showed tnni3 expression only). Similarly to the heart of control embryos (I, J), GATA4 injected AC explants show a region of overlapping expression (black arrowheads) and region expressing tnni3 alone (white arrowhead). (K) RT-PCR analysis in AC explants at stage 40 injected with GATA4 or co-injected with GATA4 and Dkk-1 mRNA showed the expression of the ventricular (myl3 and irx4) and proepicardial (tbx18) markers as in sibling control embryos at the same stage. A representative experiment out of 3 repeats is shown. a, atrium; ih, interhyoid muscle of the jaw; v, ventricle. The scale bar corresponds to 100 μm.
|
|
Fig. 2. Cardiac cell diversification is lost in dissociated and reaggregated animal cap explants but can be restored by blocking BMP signalling. (A) Restricted cardiac gene expression is lost in GATA4 injected explants dissociated and reaggregated. Inhibition of BMP signalling through dominant-negative BMP type I receptor (tBr) restores ventricular and proepicardial gene expression in GATA4 co-injected reaggregated explants. (B). Ventricular gene expression (myl3) is partially restored in explants also co-injected with GATA4 and Dkk-1 mRNA. (C) The second proepicardial marker, WT1, shows the same pattern of expression as Tbx18 in intact (Fig. 1K) and reaggregated animal cap explants expressing GATA4, GATA4 and Dkk-1 or GATA4 and tBr. AC, Animal Caps; G4, GATA4; R, dissociated and reaggregated animal caps. RT-PCR was performed at stage 40 according to control embryos. A representative experiment is shown out of 3 (A, B) or 2 (C).
|
|
Fig. 3. Ventricular and proepicardial gene expression is restored in reaggregated animal cap explants expressing GATA4 upon transplantation into host embryos. (A–F) Dissociated and reaggregated GATA4 injected explants (CAG-GFP) were transplanted at stage 20 into host (CAG-GFP) at the same stage. GFP expression was recorded at stage 39. (C, F) lineage tracing (rhodamine dextran) of transplants in D and E. (G, H) Transplants were analysed for the expression of myl7 and myl3. 48% of transplants (n = 45) showed patterned gene expression with regions of overlapping expression between myl7 and myl3 corresponding to the ventricle and regions expressing myl7 alone. 30% of transplants expressed only myl7, and in 17% there was a complete overlap between myl7 and myl3. (G′, H′) Close-up of transplants shown in G and H. Black head arrow shows overlapping gene expression and white head arrow shows region of myl7 expression alone. (I, J) 65% of transplants (n = 30) express both Tbx18 and myl7. (I′,J′) Close-up of transplants shown in I and J. h, heart; ih, interhyoid muscle of the jaw; p, proepicardium; v, ventricle. The scale bar corresponds to 100 μm.
|
|
Fig. 4. Secondary heart formation is lost in UV-ventralised hosts. (A, B) GATA4 injected reaggreagates (CAG-GFP) were transplanted at stage 20 in control (A, A′) or UV-treated hosts (B, B′) at the same stage. Transplants were viable (A′, B′) and were also positive for GFP expression in both control and UV-transplants analysed at stage 39. (C, D) Double-WMISH analysis for the expression of myl7 and myl3 indicated that whilst control transplants showed patterned expression of both myl7 and myl3 markers (C), transplants in UV-hosts (D) only expressed myl7 expression (white arrowhead). (C′, D′). Higher magnification of transplants shown in C and D. Region of overlap between myl3 and myl7 (purple) is indicated by black arrowhead. h, heart; ih, interhyoid muscle of the jaw; t, transplant; v, ventricle. The scale bar corresponds to 100 μm.
|
|
Fig. 5. Host shows broad spatial competence for the development of the secondary heart. (A) GATA4 injected reaggregates (CAG-GFP) were transplanted at stage 20 into host embryos (CAG-GFP) at the same stage in a number of different regions, as indicated by the number. (B, B′) 51% of transplants in the head showed GFP expression but did not beat (n = 20). (C–E′) Transplants in the lateral (position 5) and the caudal side (position 4) of host embryos developed beating SH as control ventral transplants. Transplant were all analysed at stage 39. (B, E′) lineage tracing (rhodamine dextran) of transplants whose cardiac GFP activity is shown in B′ and E, respectively. h, heart.
|
|
Fig. 6. Dorsal GATA4-expressing transplants show a Dkk-1 sensitive delay of cardiogenesis. (A, A′) Control transplants of GATA4-injected reaggregates (CAG-GFP) showed cardiac differentiation by GFP expression at stage 37 as indicated by the arrows. (B, B′) In contrast, transplants placed in the dorsal side of the host (position 6 in Fig. 5A) were negative for GFP expression at stage 37 (100%; n = 20), but 50% were positive after stage 39 (C, C′). No beating activity was detected in these reaggregates. (D, D′) Double-WMISH showed that 20% (n = 94) of dorsal transplants express myl7 alone (white arrowhead) and only a small group of cells express both myl7 and myl3 (black arrowhead). (E–F′) Both control and dorsal transplants of reaggregates (CAG-GFP) co-injected with GATA4 and Dkk-1 showed evidence of cardiac differentiation (GFP activity) at stage 37. (G, G′) Cardiac expression is maintained in dorsal transplants at stage 40. Beating activity was never detected in dorsal transplants. (H, H′) Double WMISH shows overlapping expression between myl7 and myl3 (black arrowhead) and regions of myl7 expression alone (white arrowhead) in 35% of dorsal transplants (n = 84; the remaining transplants were negative for cardiac gene expression). a, atrium; ih, interhyoid muscle of the jaw; t, transplant; v, ventricle. The scale bar corresponds to 100 μm.
|
|
Fig. 7. Competence to generate a secondary heart is maintained until the onset of cardiac differentiation. GATA4-injected animal cap reaggregates (CAG-GFP) were transplanted at stage 13 (A, A′), 24 (B, B′), 26 (C, C′) and 28 (D, D′) in the ventral side of host embryos at stage 20 (CAG-GFP) and were analysed at stage 39–40 for cardiac GFP expression and for lineage tracing (rhodamine-dextran). (E–G′) Patterned expression of myl7 and myl3 was found in reaggregates transplanted at stage 20 and 24. Overlapping expression is indicated by black arrowheads and myl7-only expression by white arrowheads. (H) Summary of frequency of GFP expression and beating activity. The capacity to generate a beating structure declines with the increasing age of the transplants. (I) Data summarised the number of embryos positive to the gene expression of myl7 and myl3 in double WMISH. h, heart; t, transplant. The scale bar corresponds to 100 μm.
|
|
Fig. 8. Growth of secondary hearts is dependent on the host embryo. Transplants in samples analysed by double-WMISH for myl7 and myl3 at stage 39 were visualised by lineage tracing (pink-light red colour). (A, B) Examples of control transplants (R20H20; GATA-4 injected reaggregates transplanted at stage 20 into host embryos at the same stage) show that cardiac tissue occupies a substantial part of the transplant. In contrast, cardiac tissue in (C) R28H20 and (D) R20H20UV (UV-ventralised hosts) transplants occupies a much smaller area (arrows). (D) Inside view of a dissected sample is presented to better show cardiac tissue (myl7, pointed by an arrow). Lineage trace that is visible from the outside is outlined. The scale bar corresponds to 100 μm. All images except the insert in (C) (showing the heart dissected from control embryo at stage 39) are at the same magnification. At least 5 samples were analysed for each treatment, with comparable results.
|
|
Figure S1. Morphogenesis of Second Hearts. Confocal microscopy was performed on SH (n=41; 73% beating) which were visualised by immunohistochemistry using anti-tropomyosin antibody CH1. (A) 35% of the SH showed morphology similar to a linear heart tube whilst (B) remaining SH were unstructured. (B) shows skeletal muscle staining in addition to SH.
|
|
Figure S2. Cardiac cell fate diversification requires intact heart fields. (A) Diagram of the experiment. Heart Fields (HF) were explanted at stage 20 and were analysed at stage 40. (B) Dissociated and reaggregated HFs do not express ventricular and proepicardial markers, which are expressed by intact HF explants as well as by HFs from which endoderm was removed. RT-PCR analysis was performed for indicated markers. Double-WMISH analysis showing regions of overlapping expression between myl7 and myl3 in (C, D) control sibling embryos at stage 39 (C- lateral view, D- ventral view) and in HF explants (E,F). (E) All HF analysed express myl7 and myl3. (F) close up of 3 HF explants showing areas corresponding to the ventricle (myl7+/myl3+; black arrowheads) and myl7+/myl3- (white arrowheads). a- atria, ih- interhyoid muscles of the jaw, v- ventricle, HF R; heart field dissociated and reaggregated, WE; whole embryos.
|
|
Figure S3. UV-treated host does not support the growth of the reaggregate. Representative examples of (A) control transplants (n=40) and (B) UV-treated transplants (n=40) that were analysed at stage 39 using the fluorescent microscope. Transplants (CAG-GFP), marked with white rectangular, were positive in all transplant samples for GFP expression. The 2D analysis showed that 80% (n=32) of transplants placed in UV-treated host embryos have smaller size when compared to control transplants. (Aâ, Bâ) Higher magnification of transplants into control and UV-treated hosts. The bar corresponds to 100μm.
|
|
Figure S4. Secondary heart develops without obvious cellular contribution from the host. GATA4-injected reaggregates (CAG-GFP) were transplanted at stage 20 into host embryos at the same stage. For this analysis GATA-4 was injected without lineage tracer (rhodamine dextran) and host embryos were injected with lineage tracer to facilitate visualisation of labelled host cells localisation. An area of a representative embryo carrying a transplant in the caudal area (arrows) shown to reveal (A) lineage tracer or (B) GFP-expressing secondary beating heart in the caudal region of the host. This low-resolution analysis offers no evidence of extensive cellular contribution of the host to the transplant; given the limitations of the method, a small number of host cell contribution to the transplant cannot be excluded. 20 beating transplants were examined with no evidence of host contribution in any.
|
