Mesoderm induction in Ambystoma mexicanum, a urodele amphibian

Understanding how the germ layers are formed is one of the key questions of developmental biology. Abundant studies in the anuran amphibian Xenopus laevis have described that maternal and vegetally localised mRNAs for VegT and Vg1 contribute greatly to the formation of mesoderm and endoderm in the developing embryo. Within Xenopus mesendoderm gene-regulatory network (GRN), Wnt/β-catenin as well as Nodal and Mix family members have been shown to play important roles. The involvement of several members of the Nodal and Mix gene families with redundant functions makes the mesendoderm GRN surprisingly complex and difficult to study in Xenopus laevis. By contrast, mouse and humans have only single copies of Nodal and Mix. Since urodeles have an embryology that is basal to amphibians and that has most likely also been conserved during the evolution of amniotes, including mammals, we have investigated the Mix and Nodal genes in the urodele Axolotl in the hope that their gene families contained fewer members. We cloned one Mix and two Nodal orthologs from the axolotl and showed by Southern blot analysis that there are likely no further copies in the axolotl genome. Morpholino and rescue experiments furthermore showed that AxNodal-1, Mix and Brachyury play essential roles in mesoderm specification in axolotl embryos, suggesting that the urodele Axolotl has a more simplified mesendoderm GRN. In this context, we demonstrate that Mix acts to induce Brachyury expression during mesoderm induction. Mixl1 shRNA knowdown in mouse embryonic stem cells (mESCs) shows that Mixl1 is involved in the production of mesoderm in mESCs too. Analysis of the localisation of the VegT and Vg1 mRNAs in oocytes revealed that they are neither vegetally localised in the Axolotl, nor in the basal fish species lungfish and sturgeon. Furthermore, gain and loss of function assays examining the roles of maternal VegT and β-catenin demonstrated that VegT is not required for mesoderm induction, whereas β-catenin is necessary and sufficient for mesoderm induction by activating AxNodal-1 expression in the axolotl. As these results reveal additional similarities to the GRN in mammals they further support our hypothesis that the regulatory network in the axolotl is more closely related to that in amniotes rather than anuran amphibians