Abstract
BackgroundDevelopment of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo.ResultsWe used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes in vivo, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation.ConclusionOur ability to image the dynamics of F-actin distribution during neuronal morphogenesis in vivo is unprecedented, and our experiments provide insight into the regulation of cell motility as neurons develop in the intact embryo. We identify specific motile cell behaviors affected by LIM-HD transcription factor activity and reveal how transcription factors differentially control the formation and growth of two axons from the same neuron.
Highlights
Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching
We found that disruption of LIM homeodomain (LIM-HD) activity did not affect filamentous actin (F-actin)-based protrusive activity or the ability of F-actin to accumulate at peripheral axon initiation sites, indicating that F-actin dynamics were unperturbed by dominant negative CLIM (DN-CLIM)
Proportion of RB neurons (12%) in DN-CLIM-expressing embryos extended a peripheral axon that exited the spinal cord and grew along the correct pathway to the skin. These axons had aberrant morphology (Figure 5D, compare to Figure 2D; Additional file 5), and displayed significantly slower rates of extension (Figure 5E) and fewer branches (Figure 5F) than wild-type peripheral axons, indicating that the dynamics of peripheral outgrowth and branching are disrupted by DN-CLIM. Together these results suggest that DN-CLIM primarily affects peripheral axon initiation, and influences other cell motility processes required for peripheral axon extension and the ability to form secondary branches in the periphery
Summary
Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo. Development of proper neuronal structure requires precise regulation of multiple cellular processes, including axon formation, outgrowth and branching [1,2,3,4]. Axon formation creates a distinct molecular and suppress protrusions These processes are critical for neuronal development, yet the mechanisms that regulate axon formation, consolidation and branching in the complex in vivo environment remain poorly understood. Our understanding of the mechanisms controlling the process of initial axon emergence from the cell body and the underlying actin rearrangements in vivo is far from complete
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