Worm Breeder's Gazette 16(4): 30 (October 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.
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032
We have become increasingly interested in uncovering the molecular events underlying the so-called neurite "sprouting" defect observed in several Lim homeodomain mutants such as lim-6 [1]. In the course of testing more than a dozen known unc mutants for neurite sprouting in the DVB motorneuron, we discovered that a previously identified but uncharacterized gene, unc-122, causes a 25-40% penetrant DVB sprouting defect. The sprouting defect caused by unc-122 has thus far proven to be specific for DVB as we have not detected ectopic neurites in the head sensory neurons, the interneuron AIY, or the A, B, or D-type motorneurons. unc-122 does not display an obvious defecation phenotype although more subtle defects such as changes in the relative timing of individual muscle contractions are currently being assessed.
Previous deficiency mapping data (J.Hodgkin) had placed the unc-122 gene within the sur-2 unc-54 interval on LGI. Our initial attempts to rescue the mutant phenotype of unc-122 with DNA spanning this region were unsuccessful. We used additional 3-factor mapping and tests of several more deficiencies within this region to refine the original mapping and unambiguously place unc-122 immediately to the right of unc-54. Cosmid rescue followed by single gene rescue demonstrated that F11C3.2 corresponds to the unc-122 locus. One incomplete Kohara cDNA exists for unc-122 and 5’ RACE was used to confirm the 5’ end of the gene and construct a full-length cDNA.
The protein encoded by unc-122 is predicted to be a type II transmembrane protein, with a 19 amino acid N-terminal intracellular domain. The extracellular region of the protein contains a collagen-like repeat followed by a highly conserved domain, the OLF domain. While the function of the OLF domain is entirely unknown, it is found in several other invertebrate and vertebrate transmembrane and extracellular proteins. The OLF domain is named for olfactomedin, an extracellular protein highly enriched in the olfactory epithelium and proposed to be involved in the development of the apical olfactory dendrite [2]. Latrophilin, another OLF domain protein, is a 7-TM, G-protein coupled receptor for a-latrotoxin and has been implicated in calcium-independent synaptic vesicle exocytosis [3]. While these homologies do not point to a clear function of UNC-122, the neuronal linkage is especially intriguing. DNA sequencing revealed molecular lesions in two independent unc-122 alleles. Each allele represents a G->A transition leading to a premature stop codon at the beginning of the OLF domain (e2520) or in the middle of the OLF domain (n2916). In light of the fact that e2520 behaves as a genetic null, the molecular change in e2520 is consistent with a crucial role of the OLF domain in the function of unc-122.
In addition to the DVB phenotype, unc-122 mutants are strong ventral coilers suggesting a defect in motorneurons or the muscles they innervate. We have thus far been unable to detect gross morphological defects in the A, B, or D-type motorneurons as assessed by GFP reporters (unc129::GFP, del-1::GFP, unc-47::GFP, unc-25::VAMP-GFP). Double mutants of unc-30 with unc-122 show both shrinker and coiler phenotypes indicating that the GABAergic D-type motorneurons are not involved in the coiler phenotype. We find that unc-122 does show hypersensitivity to both aldicarb and levamisole indicating a defect in cholinergic synaptic transmission. These drug studies suggest that one function of unc-122 is to negatively regulate acetylcholine signaling. Furthermore, we find that unc-122 enhances the DVB sprouting defect of unc-25(0) and thus likely acts in parallel to GABAergic signaling in DVB. Interestingly, similar enhancement of unc-25 sprouting is observed in unc-31 mutants where peptinergic neurotransmission is presumably disabled.
To localize the expression of unc-122, we generated GFP reporter constructs containing the unc-122 promoter alone as well as a C-terminal translational fusion containing the unc-122 genomic region fused to GFP followed by the unc-54 5’UTR. Transgenic lines derived from the promoter-GFP fusion show extremely strong fluorescence in coloemocytes only. As we cannot find a coloemocyte phenotype nor can we reconcile the coiler phenotype with expression of unc-122 in coloemocytes alone, we believe that this limited expression pattern does not reflect the true expression pattern of unc-122. The translational fusion reporter neither rescues the unc-122 coiler phenotype nor shows any GFP fluorescence, even in coloemocytes. New GFP reporters are currently being tested. We are also generating peptide antibodies to UNC-122 as well as performing tissue specific rescue experiments to define the location of UNC-122 action.