Eomes serves as the primary cardiac mesoderm regulator.

The authors' assertion that Eomes occupancy at these T-box sites directly activates the MEsp1 expression region that marks the cardiac progenitors is supported by the knowledge that T-box transcription factor is present at these T-box sites. In all vertebrates, brachyury is required for the development of the posterior body. The results of experiments d and e show that the T-box transcription factor Eomesodermin (Eomes) controls the cardiac mesoderm at the most fundamental level.
The fate-mapping experiments in Figures d and e aim to identify the offspring of Eomes. These are made of two expressing cells that make up the endocardium and the myocardium of the heart, the endoderm and vasculature of the primary gut tube, the head mesenchyme and less frequently regulate other mesoderm tissues formed from lateral and paraxial plate mesoderm. Both the sections in the two figures were counterstained with eosin to highlight non-labelled cells. Black lines indicate the plane of section in figure d and e. On the other hand, the two figures differ considerably such that figure d uses dp- MAPKK blots from the animal cap the lysates incubated for fifteen minutes with and without FGF2 in the presence of increasing concentrations of MEK1 inhibitor and FGFR1 inhibitor. The drugs used removed FGF-induced MAPK activation in a dosage-dependent manner. On figure e the embryo is treated with the ten nil of 2 Mm DMSO, U0126 or SU5402 into the blastocoel thereafter it was cultured with an identical inhibitor as the one used in experiment d at stage 8 or 12.5. The treated embryos at stage eight were fixed at stage 11.5 and 29-30 for in situ hybridization in situ for cardiac actin and Xbra respectively. The use of the two inhibitors ensured gastrulation movements and mesoderm specification when treated at stage eight. Besides, in figure e the embryos treated at stage 12.5 with SU54O2 displayed morphogenetic defects while those treated with U0126 were unaffected.

The authors concluded that the process of vertebrate gastrulation needs coordination of mesoderm specification with morphogenetic movements. Although these processes are initiated under the influence of FGF signaling, it is not fully understood how cell movements and mesoderm specification are coordinated during gastrulation.

The authors have identified Spred and Sprouty families as the regulators of receptor tyrosine kinase signaling. Besides, the authors have discovered two genes for each family in xenopus tropicalis: Xtspred1, Xtsprouty2, Xtspred2, and Xtsprouty1. By demonstrating through loss and gain-of -function experiments the authors show that XtSpred and Xtsprounty proteins regulate various signaling pathways downstream of the FGF receptor (FGFR) and subsequently different developmental processes. Particularly, XtSproutys hinder morphogenesis and PKC and Ca2+ signaling, leaving mesoderm specification and MAPK activation intact. On the other hand, XtSpreds hinders mesoderm specification and MAPK activation but with minimal effect on PKC and Ca2+ signaling. According to the authors, these variations coupled with the developmental expression shows that a mechanism switches FGFR signal interpretation to coordinate cell movement and mesoderm formation during gastrulation.



An alternative explanation involves an investigation on Mesp1/2 activation which usually occurs during cardiovascular lineage commitment. In this experiment P19C16 embryonal carcinoma cell line that effectively differentiates into beating cardiomyocytes in the presence of one percent dimethylsulphoxide (DMSO). At the second day of the experiment transient, Eomes expression is seen but it is downregulated by day six alongside the associated Mesp1 expression levels. Activation of Eomes oestrogen-receptor fusion protein in the presence of tamoxifen induces a maximum Mesp1 expression within twenty-four hours as assayed by qPCR. Eomes occupancy at the Mesp1/2 locus is directly evaluated by carrying out chromatin immunoprecipitation analysis with day four (DMSO)-treated P19Cl6 cells alongside tamoxifen-treated P19Cl6EomesER cells.

The T-box-site containing regions within the Mesp1/2 locus which are bind by Eomes can be seen clearly. The EME which usually is known to regulate the initial expression in nascent mesoderm triggers the strongest signal. In this experiment, the occupancy at other uninduced cells remains undetectable. The entire T-box sites next to the Mesp2 TSS are recognized to be occupied by TBx6 in presomitic mesoderm. Mesp2 and Mesp1 expression are regulated during the later stages of somitogenesis by the minimal 220-bp Mesp1 EME T-box element and cis-acting regulatory element. Besides, the T-box family members are also expressed during the initial gastrulation stage of the embryo.

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