Hermaphrodite and male C. elegans possess four pairs of sensory neurons that contain the neurotransmitter dopamine, the CEPs, ADEs and PDEs. In addition, the male has three pairs of sex-specific dopaminergic neurons, namely the A neurons of rays 5, 7 and 9 (1). Behavioral analyses of males lacking these ray neurons and of
cat-2 mutants, in which dopamine synthesis is significantly reduced, suggest that dopamine facilitates the execution of one or more steps in male mating behavior (2, 3). The existence of many male behavioral and developmental mutants provides an opportunity to explore the genetic basis for the expression of dopaminergic cell fate and function in the rays. To this end we are examining existing mutants for defects in the specification of the dopaminergic neurons. In order to follow the fate of dopaminergic neurons in mutant backgrounds, we have developed a gfp expression marker that specifically defines terminally differentiated dopaminergic neurons. This marker consists of a translational fusion between gfp and a putative C. elegans tyrosine hydroxylase (TH) gene. Tyrosine hydroxylase is the first enzyme in the dopamine biosynthetic pathway and is frequently used as a marker for dopaminergic cell fate in other systems. We identified this putative C. elegans TH gene in the genome sequencing project database on the basis of its sequence similarity to the rat and Drosophila tyrosine hydroxylase genes. C. elegans TH shares approximately 50% amino acid identity with the rat and Drosophila genes and is located on cosmid C09D7 on linkage Group II. The TH/gfp fusion is specifically expressed in all neurons previously defined as dopaminergic by Sulston et al. (1975), including the neurons of rays 5, 7 and 9. Interestingly, the physical map position of this gene corresponds closely to the genetic map position of
cat-2, a mutation which specifically disrupts dopamine synthesis (1). We are currently investigating whether this putative tyrosine hydroxylase gene can rescue the
cat-2 defect. In our first use of this TH/gfp reporter to screen for mutants defective in dopaminergic cell specification we examined reporter gene expression in TGF-beta pathway mutant backgrounds. Mutations in TGF-beta pathway components
daf-4 and
sma-2, -3, and -4 have been shown to alter the identity of rays 5, 7 and 9 - the rays that contain the dopaminergic neurons. In these mutant backgrounds, the structural cells of rays 5, 7, and 9 take on the identity of a neighbouring ray (4). Thus, TGF-beta pathway activity in rays 5, 7 and 9 is necessary for the expression of their structural cell identity. We were interested in determining whether this pathway is also necessary for expression of the neuronal cell identity of these rays - specifically we asked whether this pathway is required for expression of the dopaminergic fate of the A neuron. To address this question, we introduced our TH/gfp reporter into each of the following backgrounds:
daf-4,
sma-2, -3, and -4 and examined the affects of these mutations on expression of TH/gfp. We find that in all these mutant backgrounds expression of TH/gfp in dopaminergic neurons common to both sexes, namely the CEPs, ADEs and PDEs, is unaffected. In contrast, TGF-beta pathway defects reduce or eliminate expression of TH/gfp in rays 5, 7 and 9. 1. Sulston, J. E., Dew, M., & Brenner, S. (1975) J. Comp. Neurol. 163: 215-226. 2. Loer, C. M. & Kenyon, C. J. (1993) J. Neurosci. 13: 5407-5417. 3. Liu, K. S. & Sternberg, P. W. (1995) Neuron 14: 79-89. 4. Savage, C., Das, P., Finelli, A. L., Townsend, S. R., Sun, C-Y., Baird, S. E. & Padgett, R. W. (1996) Proc. Natl. Acad. Sci. USA 93: 790-794.