A surprising insight into the impressive architecture of developing embryos is revealed in a new paper in Development by researchers in CSB.
Building an embryo
The Bruce lab studies the highly regulated process of epiboly, whereby blastoderm tissue of a growing fish embryo slides down over the egg yolk. This is like a ski mask (the embryo) being pulled down over a head (the yolk). The team discovered that a previously elusive signalling pathway coordinates two distinct structural processes essential for epiboly.
During epiboly, thousands of cells must move together while staying perfectly coordinated as the embryo develops and spreads over the yolk.
Actin and myosin proteins in the yolk cell flow toward the spreading blastoderm margin, where they assemble into a contractile actomyosin ring that generates the force driving embryonic movement.
At the same time, yolk membrane is removed, allowing the embryo to continue spreading over the yolk. The removed yolk membrane is recycled in endosomes through a process called macropinocytosis.
An unexpected result in the yolk
Structures in the embryo move and are held together by a cytoskeleton made of flexible actin and rigid microtubules, so it was thought that actin and microtubules in the embryo and yolk might coordinate these movements.
The Camsap2a protein is known to be a microtubule stabilizing protein produced in the yolk that acts even when cells are not dividing. Haoyu Wan, a PhD student in the Bruce lab, deleted the Camsap2a protein for further study of epiboly using gene editing.
As expected, the mutants were delayed in epiboly. However, Wan was shocked to find that microtubules in the yolk were unaffected by losing Camsap2a as measured by abundance, organization and dynamics.
Following the evidence to Rab5ab
The researchers set out to find what was causing epiboly delay with embryonic studies using live cell microscopy.
Actomyosin flow in the embryo, required to form the contractile ring, was slower and misoriented in mutant embryos when viewed with fluorescent tagging.
Tracking yolk membrane removal by fluorescent labelling of endosomes revealed that macropinocytosis was impaired.
They then compared the Camsap mutant phenotypes they observed with other embryonic regulators. Data from human cells and worms revealed that mutants in the Rab5ab protein displayed defects in the same processes. Rab5ab is a small GTPase implicated in signalling cascades to activate downstream processes.
Coordinating two processes
In Camsap2a mutants, Rab5ab is present, but is unable to prevent the defects seen in the mutants during epiboly. Sifa Quibria, as MSc student in the lab conducted rigorous experiments to show that when a constitutively active form of Rab5ab was expressed in the yolk cell, epiboly defects were suppressed, showing that Rab5ab is part of a signal cascade downstream of Camsap2a.
Fitting these pieces of information together revealed a previously unknown signalling axis in epiboly, linking microtubule-binding Camsap2a to Rab5ab for coordinating actomyosin flow to the embryonic contractile ring and for promoting localized membrane removal in the yolk.
It is likely that there are redundant microtubule-regulating proteins in the yolk, which would explain why Camsap2 mutants lack a microtubule defect. This redundancy makes Camsap2’s unique signalling role to actin-based processes in the embryo a fascinating surprise.
This research is available from the journal Development as “Camsap2a regulates actomyosin flow and Rab5ab-mediated macropinocytosis in the yolk cell during zebrafish epiboly”.
Bruce’s lab is excited to pursue the implications of this novel axis further: How is Rab5ab activated during macropinosome formation? How and where do Camsap2a and Rab5ab physically interact? What other proteins are involved in the signalling cascade?
We look forward to more surprising discoveries from this impressive research team.