Axonal Branching





Axonal outgrowth is generally thought to be controlled by direct interaction of the lead growth cone with guidance cues, and, in trailing axons, by fasciculation with pioneer fibers. Responses of axons and growth cones are examined as cultured retinal ganglion cell (RGC) axons encounter repellent cues. Contact with either cells expressing ligands for Eph receptor tyrosine kinase receptors or mechanical probing increases the probability of lead growth cone retraction. Lateral extension of filopodia and lamellipodia hundreds of microns behind the lead growth cone can be correlated with this collapse. Transmission electron microscopy shows that some of the lateral extensions originate from the pioneer axon, whereas others represent growth cones of defasciculating trailing axons. We are examining the cellular and molecular signals that determine characteristics of the axonal response following growth cone collapse.




A temporal RGC axon is retracting after contact with a posterior tectal neuron. Lamellipodia and filopodia appear along the axonal shaft shortly after collapse of the growth cone.

EM Study



Phase-contrast time-lapse images were recorded as a mouse temporal RGC growth cone contacted and retracted from a caudal superior colliculus cell . Immediately after the appearance of lateral extensions, the culture was fixed and prepared for EM. Camera lucida drawings outline the lateral extension and indicate the position of the micrograph (10000x). A single axon was detected along the extent of axon shown at the top. The newly formed lamellipodia is not fully visible in the micrograph due to its relative thinness. The micrograph reveals the kinked axon and its associated distorted microtubules and accumulation of membrane-bound vesicles, organelles and microfilaments . The most exaggerated distortions in microtubules appeared as in this case in single axons that underwent collapse and displayed kinks in the retinal fiber that were visible with light microscopy. Scale bars: 10 µm and 1 µm



Intracellular Signaling



We are currently examining cellular signals that relay between growth cone collapse and axonal branching. Such signaling may be widespread across the nervous system and affect neuronal outgrowth in many regions. We have detected that Rho-GTPases are involved in the response of the growth cone to repellent molecules and are examining their role in the formation of axonal branches.