Eomesodermin is functionally conserved between zebrafish and mouse in spite of different mutant phenotypic severities, and controls left/right organiser formation via interlocking feedforward loops

Abstract

The T-box family transcription factor Eomesodermin (Eomes) is present in all vertebrates, with many key roles in the developing mammalian embryo and immune system. Homozygous Eomes mutant mouse embryos exhibit early lethality due to defects in both the embryonic mesendoderm and the extraembryonic trophoblast cell lineage. In contrast, zebrafish lacking the predominant Eomes homologue A (Eomesa) do not suffer complete lethality and can be maintained. This suggests fundamental differences in either the molecular function of Eomes orthologues or the molecular configuration of processes in which they participate. To explore these hypotheses we initially analysed the expression of distinct Eomes isoforms in various cell mouse types. Next we compared the functional capabilities of these murine isoforms compared to zebrafish Eomesa. These experiments provided no evidence for functional divergence. Next we examined the functions of zebrafish Eomesa and other T-box family members expressed in early development, as well as its paralogue Eomesb. Though Eomes is a member of the Tbr1 subfamily we found strong evidence for functional redundancy without complete functional equivalence with the Tbx6 subfamily member Tbx16, known to be absent from eutherians and other mammals. Finally, we analysed the ability of Eomesa to induce zebrafish left-right organiser progenitors (known as dorsal forerunner cells) known to be positively regulated by vgll4l, a gene we had previously shown to be repressed by Eomesa. Here we demonstrate that Eomesa indirectly upregulates vgll4l expression via interlocking feedforward loops, suggesting a role in establishment of left/right asymmetry. Overall these findings demonstrate conservation of Eomes molecular function and participation in similar processes, but differential requirements across evolution due to the expanded complement of T-box factors in teleosts. Our analyses also provide insights into the role of Eomesa in left-right organiser formation in zebrafish. Author summaryRecent studies provide evidence for both shared and unique molecular pathways controlling early development in different vertebrate organisms. The transcription factor Eomesodermin plays essential roles during mammalian development and has potent functional capabilities in zebrafish embryos yet is seemingly dispensable for viability. Here we compared functional contributions of the predominant zebrafish Eomesodermin homologue - Eomesa - with multiple isoforms of mouse Eomesodermin. We found them to be functionally indistinguishable in the early embryo. Surprisingly, a distant relative of Eomesodermin in the zebrafish embryo - Tbx16 - which is absent in mammals shows a degree of functional overlap with Eomesodermin, underscoring the different evolutionary requirements for Eomesodermin in teleosts and mammals. Finally, we demonstrate that Eomesodermin differentially regulates the transcriptional cofactor vgll4l, which plays key roles in formation of the zebrafish left/right organiser, at different stages of development. Thus, our work demonstrates the molecular function of Eomesodermin is likely to be conserved throughout vertebrate evolution despite differences in mutant phenotypes, and reveals regulatory mechanisms controlling left/right asymmetric patterning.