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.
Highlights
Dorsoventral axis formation is a fundamental process in early vertebrate embryogenesis
A gynogenesis-based forward genetic screen in the zebrafish led to the isolation of a mutation in hec, which results in axis induction defects [17,20,23]
We find that hec functions to reorganize and align microtubule bundles that are involved in the symmetry-breaking transport of factors localized to the vegetal cortex, which is essential for axis induction
Summary
Dorsoventral axis formation is a fundamental process in early vertebrate embryogenesis In many vertebrates such as amphibians and teleosts, evidence indicates that maternally-supplied dorsal determinants trigger the formation of the future dorsal organizer. Embryological manipulations have indicated that the dorsal determinants are initially localized to the vegetal pole and subsequently translocate animally to the prospective dorsal side in a microtubule-dependent process in both amphibians (reviewed in [1]) and teleosts [2,3,4]. Translocation of the signal from the vegetal pole to the dorsal side is initiated by cortical rotation, the microtubule-dependent movement of the egg cortex with respect to its core that is triggered by fertilization and is implemented by transport along microtubule tracks (reviewed in [1]). Studies showed that fluorescent polystyrene beads injected at the vegetal pole were transported animally along microtubule-based cortical tracks in a microtubule dependent manner [2], and that this movement had temporal dynamics and functional requirements similar to that of the movement of putative dorsal determinants as Author Summary
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