Notch coordinates self-organization of germ layers and axial polarity in cnidarian gastruloids

Dissociation and reaggregation experiments in several animal systems have revealed the stunning capacity of self-organization. Reaggregated early gastrula cells (here called gastruloids) of the sea anemone Nematostella vectensis are able to regenerate with only little delay whole organisms that are virtually indistinguishable from normal developing polyps. However, the molecular control underlying the restoration of body axis and germ layers remains largely unknown. To address this, we established a standardized protocol, which reproducibly generates gastruloids developing into polys with a single body axis. Here, we show that committed mesodermal and endodermal cells are sorted out to the surface of the aggregate, where mesodermal cells form clusters of about 30 cells. At a critical time point, one mesodermal cluster immigrates, along with peripherally attached endodermal cells. Thereby, the inner germ layer and the oral-aboral axis is established in one and the same process. Functional studies demonstrated that this polarization of the endodermal cells requires a feedback loop of Notch and Wnt signaling. The dissociation of the early embryo disrupts Notch signaling in the endodermal cells, which leads to transient adoption of an endomesodermal profile, marked by the expression of the mesodermal cadherin1 until the boundaries between the germ layer identities are re-established. Our results highlight distinct morphogenetic behaviors of mesodermal and endodermal cells and the hitherto unknown role of Notch signaling in germ layer boundary formation in self-organizing gastruloids. Conservation of Notch-mediated boundary formation between endoderm and mesoderm mirrors bilaterian mechanisms, demonstrating how adoption of ancestral regulatory networks governing morphogenesis likely enabled the diversification of metazoan body plans. (1) DMSO treated library: Embryos were raised until 24hpf and then treated from 24hpf to 48hpf with 50uM DMSO as control and then samples were collected for sequencing; (2) LY treated library: Embryos were raised until 24hpf and then treated from 24hpf to 48hpf with 50uM LY411575 as treatment and then samples were collected for sequencing; (3) Gastrula.ACME library: Embryos were raised until 24hpf and then samples were collected for sequencing; (4) 4, 12, 24 and 48 hpa Embryos were raised until 24h and then dissociated, reaggregated and allowed to develop until the specified time when samples were collected.