Plexin A3 and Turnout Regulate Motor Axonal Branch Morphogenesis in Zebrafish

Abstract

During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch morphogenesis by destabilizing membrane protrusions before the growth cone completes navigation into the synaptic target field.

Livin’ On The Edge: glia shape nervous system transition zones

“…To identify the molecular mechanisms that mediate this selective permeability at TZs, studies have targeted essential axon pathfinding pathways, including Netrin, Semaphorins and Plexins (Table 1) [29,31–35]. …”

It takes a village to raise a branch: Cellular mechanisms of the initiation of axon collateral branches

“…(1) The growth cone can split and give rise two Y or T shaped axon branches. For example, live imaging of individual zebrafish motor axons reveals that the first axonal branches are generated via bifurcation of the growth cone (Sainath and Granato, 2013). Although growth cone bifurcation can contribute to the formation of branches in specific instances, this is not the major mechanism that contributes to axon branching (discussed in Gallo, 2011).…”

Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis

“…We focused our analysis on ventrally projecting CaP motoneurons [12]. In 26 hpf wildtype embryos, CaP motoneurons (n = 16/16) displayed their typical axonal morphology [10, 12, 28, 29]. In contrast, in mypt1 mutants CaP axons were excessively branched and/or displayed zigzag-like projections (n = 9/15; p = 0.002), consistent with the idea that mypt1 and myosin phosphatase activity regulate motor axon branching (Fig 3C and 3D).…”

“…Before onset of axon initiation at ~16 hpf, CaP motoneurons migrate in response to semaphorin-neuropilin signaling, moving towards the future segmental spinal cord exit point, where motor axons exit from the spinal cord [5, 36]. At 26 hpf CaP cell bodies have reached their position directly above the segmental exit point (Fig 4A) [10, 28, 29, 30, 37]. In contrast, in mypt1 mutants, 33% of CaP cell bodies were shifted rostrally relative to the axon exit point (n = 5/15, p = 0.0177), supporting the idea that mypt1 regulates CaP migration and/or positioning (Fig 4B and 4C).…”