Worm Breeder's Gazette 14(2): 90 (February 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.
Department of Molecular Biology and Biochemistry Rutgers University New Brunswick NJ 08855
C. elegans mec-4, mec-10, deg-1 and unc-105 encode members of the degenerin gene family that exhibit sequence similarity to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. The vertebrate Na+ channel includes at least three homologous subunits. The channel expressed in the C. elegans touch receptor neurons, hypothesized to be a mechanosensitive channel, includes at least two related subunits, MEC-4 and MEC-10. Recently four additional family members, housed in cosmids T28D9 (II), F58G6 (IV), R13A1 (IV), and E02H4 (X) have been entered into the C. elegans database by the Genome Consortium. We have begun characterization of these newly identified degenerin genes by assaying the expression of lacZ fusions to the predicted open reading frames. In addition, we fused their putative 5' regulatory regions to the toxic mec-4(d), so that the cells in which the new family member is expressed undergo degeneration. These ablation studies help both in identification of the cells in which degenerinX is expressed and in determination of potential mutant phenotypes (provided that degenerinX is needed for cell function). Here we report on the two new family members most similar to the touch receptor degenerins (60% and 66% similarity to MEC-4 respectively), designated R13A1_4 and E02H4_1. R13A1_4 is expressed in about 35 neurons that have cell bodies situated primarily in the ventral cord and nerve ring. Staining is most intense in L1 and L2 animals, although it is evident at late embryonic stages and persists into adulthood. The neuronal processes of stained cells are clearly detectable, which has facilitated identification of some of the cells that express this potential degenerin family member. PDA, PDB, DD, DB and posibly DA motorneurons express R13A1_4. We have not yet unambiguously identified neurons whose cell bodies are situated in the nerve ring, but it is interesting that included among the candidates are the ASH, FLP and OLQ neurons that have been demonstrated by Kaplan and colleagues to mediate nose touch. We are able to state with certainty that the posterior touch receptor neurons do not express this fusion gene. Animals harboring fusions of the toxic mec-4(d) to R13A1_4 are severely uncoordinated. The most affected are nearly paralyzed; an often seen phenotype is that animals are unable to back up and coil when prodded with a pick. Transgenic animals also appear to be touch abnormal and somewhat hypercontracted at the posterior part of the body. Some mobile animals are insensitive to nose touch, (4 out of 50 mosaic transformants examined). Given this data, we examined the genetic map to identify candidate mutations in R13A1 which maps between genes deb-1 and dif-1 on chromosome IV. One very interesting gene in the general region (although not specifically mapped within the deb-1-dif-1 interval) is unc-8. unc-8 has been proposed to encode a member of the degenerin gene family by Shreffler, Wolinsky and colleagues because dominant gain-of-function mutations cause swelling of ventral cord neurons, a phenotype suppressed by mutations in mec-6 (which also suppress swelling and death induced by toxic alleles of degenerin family members mec-4, mec-10 and deg-1). The phenotype of some mosaics of the R13A1_4-mec-4(d) ablation fusion line very closely resembles the phenotype of the semi-dominant unc-8(n491) allele. Cosmid R13A1, however, does not unambiguously rescue unc-8(n491) or unc-8(e49) (a weaker recessive allele). Rescue by increased dosage of the wild type gene might not be expected since unc-8 alleles are gain-of-function alleles and overexpression of the gene might exacerbate the phenotype (unc-8 loss-of-function mutants exhibit no behavioral abnormalities). We consider R13A1 a strong candidate for unc-8 or a gene whose product could interact with unc-8. E02H4_1 is expressed in about 15 neurons whose cell bodies are also situated in the ventral cord and nerve ring. The cells that express this degenerin-related gene appear to be a subset of those cells in which R13A1_4 is expressed. When the E02H4_1 expressing cells were genetically ablated using a mec-4(d) gene fusion, worms were moderately uncoordinated but they retained the ability to back and did not coil. Hypercontraction or touch abnormalities were not evident. E02H4 maps to the middle of X, between genes col-9 and his-24. We are in the process of characterizing mutants in the region to test whether they might correspond to E02H4_1. One theme that emerges is that members of the degenerin gene family are expressed widely in the C. elegans nervous system. What are they all doing? Possibly, Na+ channels made up of degenerin subunits are generally involved in cell volume regulation or in maintenance of osmotic balance that is critical to neuronal function. All might be mechanically gated and some, such as those expressed in the touch neurons, may play essential roles in the conversion of mechanical stimuli to behavioral responses. Interestingly, it has been suggested that A and B type motorneurons act as stretch receptors that help propagate the sinusoidal body wave in response to contraction of body wall muscles. Perhaps degenerins expressed in these motorneurons function in this capacity.