Polarization and orientation of retinal ganglion cells in vivo

Abstract

In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.

Analysis of the Retina in the Zebrafish Model

“…The H2A-GFP transgene, for example, allows one not only to visualize cell nuclei but also to distinguish when cells undergo mitosis, and even to determine the orientation of mitotic spindles in the retinal neuroepithelium (Cui et al ., 2007; Pauls et al ., 2001). Similarly, GFP-centrin can be used to monitor the position of the centrosome in differentiating ganglion cells (Zolessi et al ., 2006), and GFP fused to a mitochondrial localization sequence can be applied to observe the distribution of mitochondria (Kim et al ., 2008). GFP fused to the 44 C-terminal amino acids of rod opsin is targeted to the photoreceptor outer segment and can be used as a specific marker of this structure (Perkins et al ., 2002).…”

“…GFP fused to the 44 C-terminal amino acids of rod opsin is targeted to the photoreceptor outer segment and can be used as a specific marker of this structure (Perkins et al ., 2002). FPs can also be applied to mark specific cell membrane domains: PAR-3/EGFP fusion, for example, labels the apical surface of retinal neuroepithelial cells (Zolessi et al ., 2006). …”

“…In the simplest case, the donor-derived cells are labeled with a fluorescent tracer or a transgene and directly analyzed in whole embryos using conventional or confocal miscroscopy (Zolessi et al ., 2006). Such analysis is sufficient to provide information about the position and sometimes the morphology of donor-derived cells.…”

Neuron's little helper: The role of primary cilia in neurogenesis

“…3B). 11,34,40 Remarkably, these structures are always located away from the site of axon outgrowth and close to the base of the forming dendrites. Prolonged erratic movements of the centrosome have been observed in these cells upon Laminin a1 knockdown, a treatment that also caused defects in neuronal polarization.…”

“…In the zebrafish retina, where detaching retinal ganglion cells drag apical marker proteins at the tip or their apical processes, the same proportion of subapical cilia was observed before and after the initiation of neurogenesis. 11,34 Alternatively, an apical abscission mechanism has been described in chick neural tube. 35 The authors observed that detaching neurons actively left behind a small region of the apical process containing several apical complex proteins as well as the primary cilium (Fig.…”