GATA-6 maintains BMP-4 and Nkx2 expression during cardiomyocyte precursor maturation

Abstract

GATA-6 is expressed in presumptive cardiac mesoderm before gastrulation, but its role in heart development has been unclear. Here we show that Xenopus and zebrafish embryos, injected with antisense morpholino oligonucleotides designed specifically to knock-down translation of GATA-6 protein, are severely compromised for heart development. Injected embryos express greatly reduced levels of contractile machinery genes and, at the same stage, of regulatory genes such as bone morphogenetic protein-4 (BMP-4) and the Nkx2 family. In contrast, initial BMP and Nkx2 expression is normal, suggesting a maintenance role for GATA-6. Endoderm is critical for heart formation in several vertebrates including Xenopus, and separate perturbation of GATA-6 expression in the deep anterior endoderm and in the overlying heart mesoderm shows that GATA-6 is required in both for cardiogenesis. The GATA-6 requirement in cardiac mesoderm was confirmed in zebrafish, an organism in which endoderm is thought not to be necessary for heart formation. We therefore conclude that proper maturation of cardiac mesoderm requires GATA-6, which functions to maintain BMP-4 and Nkx2 expression.

Fig. 1. MOs inhibit translation of long and short isoforms of Xenopus GATA-6. (A) MO sequences and the ATG regions to which they bind. The MYQ MO was changed in one nucleotide, denoted in blue, to reduce the chances of MO hairpins forming. (B) Western blot of Xenopus animal caps injected with HA-tagged mRNAs and MOs.

Fig. 2. Depletion of GATA-6 results in heartless Xenopus embryos. MDL MO-injected embryos at stage 43+. (A) Uninjected embryos possess beating hearts (arrow) anterior to the gut. Some injected embryos have a tissue-free region (arrowhead) where the heart should be, but in others this region is occupied by the intestines (B). The heart can be seen clearly in the uninjected embryos, but not in MDL MO-injected embryos (C). The intestines, which are normally present in more posterior sections, are enlarged. SC = spinal cord, S = somites, N = notochord, G = gills, H = heart, I = intestines.

Fig. 3. Expression of Nkx2.5 and MLC2 is downregulated in Xenopus embryos depleted of GATA-6. (A) Whole-mount in situ hybridization for Nkx2.5 and MLC of stage 27/28 embryos injected with MDL MO into the DLMZ at the 4-cell stage. Embryos were classified into four classes: wild-type (type +), mild downregulation (type -), strong downregulation (type - -) and severe downregulation/no expression (type - - -) of the cardiac markers. All phenotypes were observed, with one exception: Nkx2.5 was never completely absent from the injected embryos. (B) Graphical representation of the proportion of embryos in each class. (C) Quantitative real-time RT–PCR of MLC expression in MO-injected embryos. Expression relative to uninjected embryos was determined in four experiments, which were averaged, and the error bars represent standard deviations. MLC expression in uninjected embryos was arbitrarily set at 100%.

Fig. 4. Depletion of GATA-6 results in Xenopus embryos exhibiting cardia bifida and a downregulation of several cardiac genes. (A) Upper panels: ventral views of the heart regions of stage 28 embryos, either uninjected or injected with MDL MO, stained for expression of Nkx2.5 and MLC by whole-mount in situ hybridization. Lower panels: sections showing two separated populations of cardiomyocytes (arrows). Lateral views of stage 28 embryos: CA expression in the heart is absent (B) although somite expression is unaffected, even though this tissue would have received the MO, as revealed by injection of a control fluorescent MO (C) which itself does not affect MLC expression (D). Expression of Nkx2.3 and Nkx2.10 is downregulated in MDL MO-injected embryos (E and F). GATA-4 expression levels are unchanged by injection of MDL MO (G). (H) Nkx2.5 is expressed normally at stage 16. (I) At stage 16, Nkx2.10 expression remains unchanged on injection of MDL MO (H and I, anterior view; D = dorsal, V = ventral). (J) A slight decrease of Nkx2.5 transcripts is seen at stage 22.

Fig. 5. GATA-6 is not essential for BMP initiation, but is required for BMP-4 maintenance. (A, C and E) Embryos injected with MDL MO were analysed for BMP-2 expression. At stage 16 (A), stage 22 (C) and stage 28 (E), BMP-2 expression remained unchanged in the morphants in the heart (arrows). (B, D and F) BMP-4 was also monitored in MDL-depleted embryos. At stage 16 (B) and stage 22 (D), BMP-4 expression was unaltered in the heart (arrows). However, by stage 28, a decrease in transcripts was observed in the heart (F, arrows) in the injected embryos. (C and D) Upper panel lateral view, lower panel ventral view.

Fig. 6. GATA-6 MO effects on Xenopus embryos can be partially rescued by co-injection of human GATA-6 RNA but not by Xenopus GATA-1 RNA. Embryos were injected with MDL MO alone or co-injected with human GATA-6 mRNA (A) or Xenopus GATA-1 mRNA (B), and the expression of Nkx2.5 and MLC was studied by whole-mount in situ hybridization at stage 28. Embryos were classified into classes as in Figure 3A.

Fig. 7. GATA-6 is required in the mesoderm and endoderm for heart formation in Xenopus embryos. Embryos were injected into the DLMZ at the 4-cell stage with MDL MO. At stage 10, DMZs were dissected, the endoderm was removed and replaced with endoderm from an uninjected DMZ; injected endoderm and uninjected mesoderm were also juxtapposed (A); uninjected endoderm and mesoderm, and injected endoderm and mesoderm were reconstitiuted as controls. Conjugates were collected at stage 28/29 equivalent and MLC levels measured by real-time RT–PCR (B). Relative levels were calculated for four experiments (n = 40) and the mean values plotted, with standard errors. For each experiment, readings were normalized to ODC and compared with injected DMZ conjugates, which were arbitrarily designated as 1.

Fig. 8. MO-induced depletion of GATA-6 in zebrafish embryos inhibits maturation of cardiac precursors. (A) The zfMDL MO binds to 25 nucleotides of GATA-6 sequence around the ATG. (B) Western blot showing that the translation of zfGATA6 (MDL) in Xenopus animal caps is inhibited by zfMDL MO. (C) Downregulation of zfGATA6 results in reduced numbers of cells expressing cardiac genes and cardia bifida at 26 h.p.f. (dorsal views). See Supplementary table 1 for numbers. (D) zfSCL MO has no effect on Vmhc expression (dorsal views). Reduced and patchy GATA-1 expression (dorsal view) in embryos injected with zfSCL MO indicates that the MO was functioning. (E) Nkx2.5 expression initiates normally in zfMDL MO-injected embryos but, by 10 somites, expression is slightly reduced in 10/13 embryos. At 18 somites, 13/19 injected embryos showed a stronger reduction. (F and G) GATA-4 and -5 expression is unaffected by zfMDL MO injection.