Hecate/Grip2a Acts to Reorganize the Cytoskeleton in the Symmetry-Breaking Event of Embryonic Axis Induction

Abstract

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis.

Author Summary

One of the earliest and most crucial events in animal development is the establishment of the embryonic dorsal axis. In amphibians and fish, this event depends on the transport of so-called “dorsal determinants” from one region of the egg, at the pole opposite from the site where the oocyte nucleus lies, towards the site of axis induction. There, the dorsal determinant activates the Wnt signaling pathway, which in turn triggers dorsal gene expression. Dorsal determinant transport is mediated by the reorganization of a cellular network composed of microtubules. We determine that hecate, a zebrafish gene active during egg formation that is essential for embryonic axis induction, is required for an early step in this microtubule reorganization. We find that hecate corresponds to glutamate receptor interacting protein 2a, which participates in other animal systems in Wnt-based pathways. We also show that the microtubule reorganization dependent on hecate results in a subtle symmetry-breaking event that subsequently becomes amplified by a more general transport process independent of hecate function. Our data reveal new links between glutamate receptor interacting protein 2a, Wnt signaling and axis induction, and highlights basic mechanisms by which small changes early in development translate into global changes in the embryo.