Worm Breeder's Gazette 15(3): 32 (June 1, 1998)
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.
| 1 | Laboratory of Molecular Biology, Dept of Ecological, Engineering, Toyohashi, University of Technology, Toyohashi 441, Japan |
| 2 | DNA Library Lab, National Institute of Genetics, Mishima, 411, Japan |
Kinesin is the founding member of a superfamily of ATPases that transportcellular cargo on microtubule tracks. In C. elegans unc-116 gene encodes the kinesin heavy chain (Patel et al., 1993). We plan to characterize all members of the kinesin superfamily, and have recently described idenitification of klp-1 - klp-14 genes encoding different kinesin like proteins in C.elegans (Khan et al., 1997). These include five previously identfied genetic loci, osm-3, unc-104, unc-116, vab-8 and zen-4. Here we report temporal and spatial expression of the UNC-116 kinesin heavy chain (KHC), using RNA insitu hybridization and a newly constructed unc-116::lacZ reporter gene during embryonic and post-embryonic development. Previously we have reported expression of the unc-116::lacZ reporter gene in the set of interneurons tentatively identified as RMG, RIFL, RIS, ADA,ADE,AIM, AIY, AVA, AVB, etc. in the anterior ganglia located in the head, and PVT,PDA, PDB, located in the pre-anal ganglion.(WBG 14, #5, p70, 1997, and The 1997 C. elegans meeting abstracts p682).However this pattern was a bit unusual since the unc-116 mutants are defective in locomotion, coil as young larvae, and display abnormal backward movement during late larval and adult life, thus we expected unc-116 expression in the ventral cord motor neurons. We therefore constructed a larger unc-116 promoter that added about 0.4 kb upstream sequence in the promoter region, and fused it with the pPD95.57 (A. Fire) lacZ expresion vector. Germline transgenics were obtained by injecting this large promoter construct and stained for the lacZ activity. Intense staining was observed in late embryos, all larval stages and in adults of both sexes. In particular the body wall and pharyngeal muscles, stained weakly in young larvae, but staining increases gradually, reaching a maximum in L3-L4 stage and stays that way during adult stage. Staining of the nervous system is strong, and include the set of motor neurons along the ventral nerve cord,nerve ring in the head, neurons in the anterior and posterior ganglia. In the pharynx, cells in the anterior and terminal bulb are strongly stained.Staining of sensory neurons in the head (amphid and labial) and in the tail (phasmid) is weak but noticeable. The ventral and dorsal nerve cords and some lateral processes are also stained. We have also perofrmed in situ RNA hybridizations using a unc-116 cDNA clone, during embryonic (Tabara et al., 1996), and post-embryonic development (Y. Kohara, unpublished), and found that the large promoter expression is consistent with the in situ hybridization patten observed in the larval and adult stages. During early embryonic cleavages, heavy expression of the unc-116 gene is observed, that is somewhat reduced in later stages, but continues during most of the embryonic development. Dissected gonads show strong hybridization signal in developeing oocytes, but meiotic cells in the gonad apparently do not express the signal. We have begun analysis of unc-116::lacZ reporter gene expression in different mutant backgrounds, and find that expression in the muscle and motor neurons is significantly reduced in the unc-104(rh 1016) mutant background, especially during the larval stages. Similarly expression of the reporter gene is eliminated or extremely reduced in the nerve ring and some muscle cells in the vab-8(e1017) mutant background. Since UNC-104 and VAB-8 are kinesin like proteins, these observations suggest interaction of multiple kinesins in muscle and neural development of C. elegans. Experiments are in progress using various mutant backgrounds to test the embryonic and postembryonic interaction of UNC-116 KHC with other kinesin and non-kinesin proteins. We thank D. Thierry-Mieg, E. Hedgecock, A. Otsuka,, D. Hall, R. Holmgren, J. Miwa, H. Tabara and J. white for encouragement; Theresa Stierngale and A. Fire for strains and vectors, and Sanger Centre for genomic sequence information.