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Fig. 1 – Carboxy-terminal domain (362–440) of GATA4 is required for cardiogenesis. (A) Schematic representation of GATA4
structure showing transcriptional activation domains (TAD), Zn fingers (Zn), nuclear localisation signal (nls) and the
position of mutations tested in this study. Right – sequence of the C-terminal region of GATA4 (rat, NP_653331; Xenopus
NP_001085355) and GATA5 (rat NP_001019487; Xenopus NP_001081962), showing poor conservation in these cardiogenic
factors. (B) Deletion of C-terminal 78 amino acids abolishes cardiogenic activity of GATA4. Animal caps injected with wt or
1–361 mutant rGATA4 were tested for expression of cardiomyocyte-specific genes myl7 and myh6 (synonyms: Myosin Light
Chain 2 and Myosin Heavy Chain α, respectively) at st.34 and for levels of GATA4 protein at st. 10 (lower panel). Note that
the effect of this deletion is negligible on endothelial marker aplnr (apelin receptor; synonym- msr) and is less pronounced
on hba3 (haemoglobin alpha 3; synonym- αglobin), a blood marker. odc1- loading control (expression of ornithine
decarboxylase 1). (C) Nuclear localisation of 1–361 rGATA4 was determined by immunohistochemistry at st. 7–8. (D) Lack of
activity of 1–361 in vivo. Embryos injected with 1–361 rGATA4 at 8–16 cell-stage in anterior ectodermal precursors were
analysed for myl7 expression by whole mount hybridisation. myl7 expression was only observed in the heart (h) in all
embryos examined (n = 50, from 2 independent experiments). In contrast, cardiogenic GATA4 versions readily induce
ectopic myl7 expression (Figs 3 and 5). (E) Non-cardiogenic 1–361 GATA4 mutant retains gene expression-inducing activity
in animal cap explants. At st. 10 the mutant 1–361 induces endogenous gata4 and (F) at st. 34 pan-endodermal marker
alpha-2 macroglobulin (a2m; synonym – endodermin) is induced (as well as by N-terminal deletions 153–440 and 201–440
non-cardiogenic GATA4 (Gallagher et al., 2012)). |
