Glutamate is an essential neurotransmitter in the nervous system whose dysfunction can lead to a range of neurodegenerative diseases. Previous molecular studies have characterized some of the components of glutamatergic transmission in C.elegans although this information is not complete yet. The developmental mechanisms that result in terminal differentiation of glutamatergic neurons are incompletely understood both at the level of cis-regulatory elements and trans-acting factors. Extensive work, at the functional and molecular level, has been done to show that
eat-4 is a vesicular glutamate transporter homologue to the mammalian VGluT. Still the expression pattern information is incomplete. We have addressed this gap by using two types of reporters, a fosmid based one and a PCR product containing the whole genomic locus (10 kb aprox). We created a fusion cassette in two pieces such as only when they recombine in vivo the mCherry gene will be transcribed. In both cases we saw a large number of cells expressing
eat-4, either in the head of the worm and in the tail compared to the previous reporter (lee et al, 1999). We have identified most of these cells. Two other genes related to VGluT by sequence homology are K10G9.1 (
vglu-2) and T07A5.3 (
vglu-3). 2 kb promoter fusions of
vglu-2 and
vglu-3 genes showed patterns of expression more restricted than
eat-4 but that may account for "orphan" neurons not yet associated with specific neurotransmitters. Fosmid-based reporters for these genes are being analyzed. On a functional level, we find that
vglu-3 mutants (
tm3990) are defective in motor tasks and impaired in a reversal assay. More functional assays will confirm that these two genes are indeed glutamate transporters. The existence of more than one vesicular glutamate transporter correlates with the three VGluTs described in mammals. Eat-4 expression is regulated in a piece-meal fashion. By dissecting the
eat-4 promoter into individual pieces we have been able to identify independent modules of expression. For instance, in the first 300 bp upstream to the start codon resides the minimal information for
eat-4 expression in the mechanosensory neurons and some head neurons (AUA, ADA and ASK). Moreover
eat-4 is lost in the mechanosensory neurons in
mec-3 mutants and in
unc-86 mutants, indicating that the mechanosensory terminal selector genes,
mec-3/unc-86, might be regulating the glutamatergic fate of this neuronal type. Further studies are being done in order to address the regulatory logic underlying the expression of
eat-4 in this neuronal type. Other components of the glutamate metabolism are being explored as well as its potential regulation by the mechanosensory terminal selector genes.