Septins are a family of GTP binding proteins involved in cytokinesis in diverse organisms. They are also expressed in post-mitotic cells, suggestive of other cellular functions. C. elegans have two septins, encoded by the
unc-59 and
unc-61 genes (1). We have recently described a novel role for the worm septins in ventral cord motor neuron axonal migration (2). We hypothesize that the septins are required for establishment and/or maintenance of cortical domains that are necessary for process outgrowth. To further define the role of septins in axonal migration, we are studying process outgrowth in primary cultures derived from wild-type and septin mutant worm embryos expressing the pan-neuronal
unc-119::GFP reporter using the culture method of Christensen et al. (3) for isolation of primary embryonic cells. Extension of processes in
unc-119::GFP expressing cells derived from wild type embryos begins soon after plating, with approximately 40% of neurons displaying processes after one day in culture. Processes continue to extend and elaborate over the first week in culture, at the end of which approximately 70% of wild type
unc-119::GFP expressing cells display processes. In contrast, fewer than 20% of
unc-119::GFP expressing cells derived from null mutant
unc-61(
e228) embryos or from
unc-59(
n391) embryos extend processes initially, and this remains essentially unchanged after a week in culture. Those processes that are seen in the
unc-61(
e228) cells and in
unc-59 (
n391) cells are shorter and less elaborate than those cultured from wild-type strains. During the first week in culture approximately 70% of cells cultured from all three strains express
unc-119::GFP, and essentially all cells are viable as revealed by trypan blue exclusion. Phalloidin staining of the cultured cells reveals a loss of F-actin in the septin mutant neurons, suggesting that the septins may regulate cytoskeletal assembly. These results are consistent with our previous in vivo findings, and suggest that the primary cultured neurons will be a useful system for further dissection of septin function in axonal migration. 1.Nguyen, T. Q., Sawa, H., Okama, H. & White, J. G. (2000) J. Cell Sci. 113, 3825-3837. 2.Finger, F. P., Kopish, K. R. & White, J. G. (2003) Dev. Biol. 261, 220-234. 3.Christensen, M., Estevez, A., Yin, X., Fox, R., Morrison, R., McDonnell, M., Gleason, C., Miller, D. M., 3rd & Strange, K. (2002) Neuron 33, 503-14.