Mesendoderm Patterning by Vg1/Nodal Heterodimers

Abstract

One of the key processes during development is the induction of the mesoderm and endoderm (mesendoderm), which generates the precursors of the heart, liver, gut, pancreas, kidney and other internal organs. Nodal, a member of the TGF-β family of secreted ligands, is considered the key inducer of mesendoderm in vertebrate embryos and embryonic stem cells. Another TGF-β family member, Vg1, has been implicated in mesoderm induction but its role has been unclear and controversial. My graduate work addressed the role of Vg1 during embryogenesis. To interrogate Vg1’s developmental role, we investigated strategies for interfering with mRNA function. First, we developed an antisense oligonucleotide (ASO) approach in zebrafish that allows RNase H-mediated degradation of target RNAs. Second, after the emergence of CRISPR/Cas9 genome editing, we developed a web tool for the selection and design of CRISPR/Cas9 targets called CHOPCHOP. Subsequent developments in the CRISPR/Cas9 technology, combined with large-scale studies of gRNA efficiency, illuminated additional rules for gRNA design that we incorporated into a second version of the web tool. Finally, using CRISPR/Cas9, we knocked out vg1 in zebrafish and uncovered a critical role in mesendoderm formation. vg1 mutants fail to form mesendoderm, closely resembling Nodal loss-of-function mutants. We found that Nodal is processed and secreted without Vg1, but it requires Vg1 for its endogenous activity. Conversely, Vg1 is unprocessed and resides in the endoplasmic reticulum without Nodal, and is only secreted, processed and active in the presence of Nodal. Co-expression of Nodal and Vg1 results in heterodimer formation and mesendoderm induction. Thus, we present a new model of mesendoderm induction that relies on the combination of two TGF-β-related signals: maternal and ubiquitous Vg1, and zygotic and localized Nodal. Modeling reveals that the pool of maternal Vg1 enables rapid signaling at low concentrations of zygotic Nodal.