BIOLOGICAL OVERVIEW

The results from an enhancer trap approach have identified loco (locomotion defects) as a gene whose expression in glial cells depends on the activity of Pointed. loco is expressed in most lateral CNS glial cells throughout development. Embryos lacking loco function have a normal overall morphology, but fail to hatch. Ultrastructural analysis of homozygous loco null mutant embryos reveals a severe glial cell differentiation defect. Mutant glial cells fail to properly ensheath longitudinal axon tracts and do not form the normal glial-glial cell contacts, resulting in a disruption of the blood-brain barrier (Granderath, 1999).

loco encodes two variants of the first known Drosophila member of the family of Regulators of G-protein signaling (RGS) proteins, known to interact with alpha subunits of G-proteins. RGS proteins act as GTPase-activating proteins (GAPs) toward the alpha subunit of heterotrimeric G proteins. RGS proteins were first described in yeast and C. elegans (De Vries, 1995; Druey, 1996; Koelle and Horvitz, 1996). RGS proteins stimulate the GTPase activity of different Galpha subunits as much as 100-fold (Watson, 1996), accelerating the transition from the GTP-bound active to the inactive GDP-bound form and thereby terminating trimeric G-protein signaling. Different RGS proteins vary in their specificities for the Galphai and Galphao subunits. The RGS domain itself is sufficient for both binding Galpha and GTPase activation (De Vries, 1995). Loco specifically interacts with the Drosophila Galphai-subunit. This interaction and the coexpression of Loco and Galphai suggests a function for G-protein signaling in glial cell development (Granderath, 1999).

The strongest loco allele is represented by the embryonic lethal mutation loco^delta13. No abnormal CNS axon pattern phenotype is detected using the mAb BP102, which labels the overall axon pattern. mAb 1D4 recognizes the Fasciclin II protein, which is expressed on a subset of longitudinal fascicles. In mutant loco^delta13 but not in mutant loco^delta293 embryos, a slight defasciculation of axons can be found. In addition, an occasional crossing of Fasciclin II-positive axons within the longitudinal connective is observed. To analyse the different glial cells in mutant loco embryos, anti-Repo antibodies were used; these antibodies label most lateral glial cells. No gross defects are detected in the number and position of these cells. This indicates that birth and migration of the lateral glial cells do not depend on loco function. To analyse terminal differentiation of glial cells, the M84 enhancer trap marker was used. In wild-type stage 16/17 embryos, a regular pattern of evenly spaced glial cells can be detected. In loco^delta293 as well as in loco ^L1 mutant embryos defects in the positioning of some of the M84-positive cells are observed. In particular, beta-galactosidase expression is reduced specifically in the A and B glial cells compared to more laterally positioned glial cells. A similar phenotype can be seen for the longitudinal glial cells. The nuclei are found at relatively normal positions, but no glial cell processes can be detected within the connectives. In addition, the intimate glial-glial cell contact, observed in wild-type embryos, is severely disrupted in loco mutant embryos. Often axons are found on the dorsal surface of longitudinal glial cells, apparently in direct contact with the hemolymph. In summary, the loco mutant phenotype can be described as a late glial cell differentiation defect, where the formation of glial cell processes enwrapping neuronal cell bodies and axons does not occur (Granderath, 1999).

Although glial cells are an important component in any complex nervous system, not much is known about the molecular mechanisms underlying glial development. In Drosophila, a number of gene functions and mechanisms required during glial development are emerging. Following lineage specification, terminal differentiation of glial cells is mediated by transcription factors encoded by repo and pointed. The identification of genes activated by pointed in glial cells should provide new insights in the molecular mechanisms underlying glial differentiation. loco might represent such a pointed target gene. Analysis of the loco promotor region reveals the presence of GCM- and ETS-binding sites suggesting that loco might be a direct target of gcm as well. loco promotor-lacZ fusion constructs reveal a small promotor fragment that is capable of directing lacZ expression in almost all loco-expressing glial cells. This promotor fragment is indeed dependent on pointed function and ectopic pointed expression as well as ectopic gcm expression result in a corresponding ectopic lacZ expression. Sequence analysis and in vitro mutagenesis reveal both Gcm- and Pointed-binding sites within this element. These data, as well as the phenotypes observed in loco and pointed mutant embryos, suggest that loco is indeed a target of pointed. However, it is important to emphasize that loco expression in the tracheal system does not appear to depend on pointed function (Granderath, 1999).

In loco mutants the blood-brain barrier is not established. Due to the high potassium concentration in the hemolymph, this is likely to result in a disruption of axonal conductance. The adult, paralytic phenotype of the weak EMS-induced loco alleles might be a consequence of such a defect as well. Similar phenotypes were found in neurexin or gliotactin mutants (Auld, 1995 and Baumgartner, 1996). Here too, the formation of the blood-brain barrier is defective and the animals are paralysed. Gliotactin is a transmembrane protein expressed by a subset of glial cells; Neurexin is a transmembrane protein that is required for the formation of septate junctions. In contrast to the above mentioned proteins, Loco is likely to be localized within the cell (Granderath, 1999).

What causes the mutant phenotype found in loco mutant embryos? Loco physically interacts with Galphai. Strikingly, the interaction with Galphai is not confined to the RGS domain but can also be mediated by C-terminal sequences, possibly by a stretch of 51 amino acids that is conserved between Loco and RGS12. It is interesting to note that rat RGS12 also interacts with Gai (Snow, 1998a). Several G-proteins have been identified in Drosophila. Beside their role in phototransduction and learning, only a few functions, to date, have been associated with G-proteins. Interestingly Galphai RNA (but not Galphas and Galphao) is expressed in dorsal CNS cells at a position typical of glial cells (Wolfgang, 1991). This, as well as the interaction data presented, suggests that loco function is required to regulate Galphai signaling in glial cells. Taken together, the data argue for an important role of G-protein-mediated signaling in terminal glial cell differentiation. G-protein signaling is thought to be triggered by binding of a ligand to a seven transmembrane domain receptor. To date, no such receptor has been reported to be expressed in the Drosophila glial cells. Recently cross talk between receptor-tyrosine-kinases and G-proteins has been described. Interestingly, the CNS expression of heartless, the Drosophila FGF-receptor2 gene, is restricted to glial cells (Beiman, 1996; Gisselbrecht, 1996; Shishido, 1997). heartless mutant embryos show a defect in lateral glial development. Based on immunostaining using anti-Heartless antibodies, mutant glial cells appear rounded and are incapable of increasing their surface area (Shishido, 1997). This is reminiscent of the phenotype of mutant loco embryos described here. It is thus tempting to speculate that Heartless and G-protein signaling involving Loco act in concert to trigger glial cell shape changes in response to extracellular signals (Granderath, 1999).

GENE STRUCTURE

loco-c2 exons, the non-coding II-0 and the coding exon II-1, are located upstream of the first exon (I-1) of loco-c1. I-1, the glia-specific exon of loco-c1, encodes only the initiator methionine, such that the remaining cDNA sequences are shared with the loco-c2 transcript. A second, in-frame ATG is found 46 codons 3' of the c1 initiator. The introns between exons 2/3 and 3/4 (the common exons for the two transcripts) are 67bp and 72 bp (Granderath, 1999).

Exons - Three common exons, and two upstream exons for transcript c2, and one upstream exon for transcript c1

PROTEIN STRUCTURE

Amino Acids - 828aa (Loco C1); 1175aa (Loco C2)

Structural Domains

The deduced protein sequences of both transcripts were compared with those in the EMBL database using the FASTA program. A conserved domain of 125 amino acids, encoded by exon 2, identifies Loco as a member of the RGS (Regulators of G-Protein signaling) protein family. To date about 16 different members of the family have been described. loco represents the first Drosophila RGS family member. Within the RGS domain, LOCO is most similar (>40% homology) to rat RGS12 and rat RGS14 (Snow, 1997). Interestingly, homology to rRGS12 and rRGS14 extends beyond the RGS domain to the C terminus of the deduced LOCO sequences. Three additional regions of homology were designated as B (48 of 160 aa are identical in Loco and rRGS12, which corresponds to 30% identity), C (14/59 identical, 26% identity), and D (20/51 identical, 39% identity). No homologies are found in the Loco-c2-specific domain (Granderath, 1999).

date revised: 28 March 99

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