Worm Breeder's Gazette 14(4): 68 (October 1, 1996)
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||Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114|
|2||Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japan|
Worms have the capacity to sense and store temperature information such that wild-type animals move to approximately their cultivation temperature when placed on a temperature gradient. ttx-3 mutant animals were isolated based on their defective thermotactic behaviour; they move independently of the cultivation temperature to colder temperatures.They do not exhibit defects in chemo- or odortaxis. We mapped the ttx-3 mutation to the lin-14, sma-5 interval on the X chromosome. Doing GENEFINDER and BLAST searches on the corresponding physical map we noticed the presence of a LIM homeobox (LHX) gene in this region (on cosmid C40H5). We injected that cosmid and a subclone bearing only the LHX gene including 3.1 kB upstream region into ttx-3 and found both arrays to rescue the thermotactic defect. Sequencing the ttx-3(ks5) allele revealed a G to A mutation in a conserved splice donor site between exon 5 and exon 6, both of which encode the second LIM domain. Because of the presence of a stop codon after the aberrant splicing site, this mutation is predicted to delete half of the protein including its homeodomain. Such a deletion is predicted to cause a null phenotype which is confirmed by the observation that a ttx-3(ks5) / chromosomal deficiency heterozygote has the same phenotype as a ttx-3(ks5) homozygote. We cloned the cDNA of ttx-3 and by aligning its predicted protein product to other LHX proteins we found it to be the ortholog of Drosophila APTEROUS and vertebrate LH2/LHX2 proteins, both of which have been shown to be expressed and function in nervous system development (Lundgren et al., 1995, Xu et al., 1993). Next, we constructed a ttx-3 promoter fusion to GFP to monitor ttx-3 expression in wildtype and ttx-3 mutant animals. In wildtype, we found ttx-3-GFP to be expressed exclusively in the AIY interneurons of adult animals. In embryos we found expression in additional, as yet unidentified cells. Laser ablation of AIY phenocopies the cryophilic behaviour of the ttx-3 mutation, consistent with a ttx-3 function in the AIY neurons. Using ttx-3-GFP to reveal AIY process morphology, we analysed the neuroanatomy of AIY in ttx-3 mutant animals. We found AIY to be generated; however,we observed multiple axon morphology defects in adult animals. The outgrowth of additional axons represents the most penetrant phenotype but prematurely terminated processes and misguided axons were also apparent. We conclude that ttx-3 is not necessary for the generation of AIY but required for its functional specification. The TTX-3 LIM homeodomain protein might regulate the expression of downstream target genes which mediate neural connectivity, signaling or axonal outgrowth in a single interneuron of the thermotaxis sensation and memory pathway.