Worm Breeder's Gazette 16(3): 23 (June 1, 2000)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New York, NY 10032
Neuroanatomical studies in vertebrates long ago revealed that certain forms of insult such as axotomy or activity blockade result in the outgrowth of additional neurites. These observations suggest that postmitotic neurons contain an intrinsic capacity to assess their integrity and to modulate their anatomy accordingly. In C.elegans, several genetic lesions have been described that lead to the outgrowth of additional neurites ("neurite sprouting")(1,2). We have found that a null mutation in the lim-6 LIM homeobox gene causes neurite sprouting in the DVB motor neuron (3). Using this observation as a starting point, we have set out to understand the molecular components of neurite sprouting and to further dissect the role of lim-6 in this process. First, we used a candidate gene approach to test mutations in proteins involved in different aspects of neuron and muscle function. Second, we performed an unbiased genetic screen for mutants that phenocopy the lim-6 sprouting defect in DVB.
Since activity blockade at the neuromuscular junction in vertebrates and flies causes neurite sprouting, we tested whether loss of the neurotransmitter GABA causes sprouting of the GABAergic DVB motorneuron. We indeed find that mutations in unc-25 cause neurite sprouting of DVB. These defects can be enhanced by mutations in unc-31, which presumably abolishes peptinergic neurotransmission. Furthermore, blocking the activity of the enteric muscle targets of DVB with an egl-2(gf) mutation, which activates a K-channel, leads to neurite sprouting of DVB, as does the complete removal of the enteric muscles in hlh-8/twist mutants. We also find that a gain-of-function mutation in CamKII/unc-43, a gene whose role in neurite outgrowth has been described extensively in vertebrates, causes neurite sprouting in DVB.
Using a clonal, fluorescence microscope-based screen of 3300 haploid genomes, we identified nine mutants, ot1 through ot9, that affect neurite sprouting of DVB. These mutants fall into at least 4 complementation groups and are not allelic to any of the genes described above. The mutants do not cause sprouting in other defined sensory or interneurons tested. We also find that the as yet uncloned unc-122 gene causes specific neurite sprouting of DVB. We are in the process of mapping and cloning these genes. So far, we have mapped unc-122 very close (<0.1 map unit) to the unc-54 locus on LGI and are in the stage of cosmid rescue.
Neurite sprouting caused by the genetic lesions described above are strictly temperature dependent and most pronounced at 25C. The temperature dependence of the process may explain why unc-25 defects had previously escaped detection. It is also important to note that the sprouting defects observed in DVB (and for that matter, sprouting defects observed in other neurons with other markers) are somewhat dependent on the GFP reporter gene. We have used transgenic integrants of two distinct unc-47 promoter GFP fusions (oxIs12 and otIs37). While both display <5% sprouting in wildtype animals, the reporters show differences in the penetrance of sprouting in several of the mutant backgrounds described above (e.g. 73% in lim-6(nr2073); oxIs12; 51% in lim-6(nr2073); otIs37). Nevertheless, we are confident that neurite sprouting is not an reporter-gene induced artifact since, as evidenced in lim-6 mutants, sprouting can be observed with antibody staining and in the presence of several independent GFP reporter genes. In conclusion, reporter genes may sensitize the genetic background; while this may be beneficial in the sense that it facilitates the identification of mutants affecting sprouting, the unknown nature of this sensitization effect may add a complicating factor to our understanding of the molecular basis of neurite sprouting.
(1) Peckol et al., 1999, Development 126, 1891
(2) Zhao and Nonet, 2000, Development 127, 1253
(3) Hobert et al., 1999, Development 126, 1547