BIOLOGICAL OVERVIEW

The Drosophila lim1 cDNA has been isolated by polymerase chain reaction (PCR) using degenerate primers designed from the conserved sequences within the Xenopus Xlim-1 and murine Lim-1 (Lhx1) homeoboxes. The sequence and expression of dLim1 is highly related to its vertebrate homologs. Within the Drosophila embryo, Lim1 is expressed in the head primordia, the brain lobes, and in distinct sets of motorneurons and interneurons within the ventral nerve cord. For comparison, in vertebrates the lim homeodomain proteins [Lim-1 along with Lim-3 (Lhx3), Gsh-4 (Lhx4), Isl-1 and Isl-2] are expressed in developing motorneurons along the spinal column, where their overlapping expression suggests a role lim proteins in the establishment of specific motorneuron subtypes. Drosophila Lim1 is absent from all cells expressing Islet, Lim3, and Apterous (Ap), an indication that these proteins function independently within the Drosophila embryo. Drosophila lim1 is an essential gene that when mutated results in lethality near the larval-pupal boundary. Mice mutant for Lim-1 fail to form anterior structures, resulting in the complete absence of a fore- and mid-brain in mutant embryos (Shawlot, 1995). In contrast to this, Drosophila lim1 has no apparent role in anterior patterning of the Drosophila embryo. Thus Drosophila lim1 has been evolutionarily conserved, however the Drosophila lim1 gene exhibits unique properties that distinguish it from its vertebrate homologs (Lilly, 1999).

While the vertebrate Lim-1 gene is expressed in the head folds of the young embryo, Drosophila Lim1 is expressed as a stripe within the head primordia just after cellularization. At later stages both genes are found in subsets of motorneurons and interneurons within their respective nerve cords. The similarities in sequence and expression between the vertebrate and Drosophila orthologs strongly suggest that their function has also been conserved. Consistent with this, the molecular capabilities of the Drosophila Lim1 protein have been compared with those of vertebrates. Just as the vertebrate proteins, Xlim-1 and Xldb-1 (NLI, CLIM-2) interact in vitro, Drosophila Lim1 and Chip exhibit a comparable association. Thus, within each species these factors have retained common characteristics that are important to the function of these proteins. However, within each organism the Lim-1 proteins have adapted species-specific functions that reflect divergent specialization (Lilly, 1999).

Lim1 is expressed in many cells along the ventral nerve cord of the Drosophila embryo. Lim1 is found in neural cells but not glial cells within the ventral nerve cord. Expression is observed in interneurons and motorneurons, specifically RP2, aCC and the U motorneurons, all of which innervate the dorsal muscles of the embryo. Because of the combinatorial expression observed with LIM homeodomain members in the chick spinal column and the Drosophila ventral nerve cord, it was of interest to assess the relative expression of the LIM homeodomain proteins in Drosophila. In the vertebrate spinal column the overlapping expression of Lim-1, Lim-3, Isl-1 and Isl-2 demarcates regions of motorneurons with common targets. In Drosophila, the LIM homeodomain genes (Lim1, Lim3, Isl, and Ap) are expressed in select subtypes of neurons within the ventral nerve cord. Drosophila Lim1 is expressed in motoneurons that innervate dorsal muscles, while Isl-expressing motoneurons innervate ventral muscles. Additionally, the pools of interneurons in which Lim1 and Isl are expressed do not overlap. Moreover, the results show that Lim1 and Lim3 have mutually exclusive expression patterns. The significance of this exclusive expression has yet to be determined, however based on the vertebrate model and the combinatorial relationship observed for isl and lim3 in Drosophila, it seems feasible that Lim1 may provide pathfinding identity to subclasses of neurons. As in the chick spinal column, the overlapping or exclusive expression of these proteins provides instructional information to subsets of neurons. This is supported by the pathfinding defects observed in the ap, lim3 and isl mutants and the combinatorial relationship observed with isl and lim3. The subtlety of the pathfinding defects seen with ap, isl, and lim3, and the apparent lack of any defects in Lim1 mutants, supports the idea that there are multiple factors involved in regulating this process (Lilly, 1999).

In an effort to elucidate the role of Lim1 in Drosophila, loss-of-function alleles were generated in the Lim1 locus. As a function of the screening strategy, all Lim1 mutations recovered were lethal. These mutants survive to the larval and pupal stages, but never eclose to produce viable adult flies. Morphologically, the Lim1 mutants appear normal, however at the third instar stage, larvae began to exhibit coordination defects and are unable to crawl in a wild-type fashion. Several nervous system markers were examined to assess the cause of lethality. The cells are specified correctly and they appear to extend their axon projections normally. The general behavior of the larvae suggest that these mutants have motor-coordination defects. What these defects are at a morphological and molecular level remains to be determined. What is clear is that the Drosophila Lim1 mutants die during their development, demonstrating that Lim1 is an essential gene. Clonal analysis and characterization of the Lim1 locus may unveil the specific nature of the Lim1 phenotype (Lilly, 1999).

A BLAST search of the genomic clones sequenced by the Drosophila Genome Project using available cDNAs produced hits to two non-overlapping BAC clones. The BAC clone AC012882 contains the first three exons and the BAC clone AC014204 contains the remaining four. Although the genomic sequence of the locus is not complete, all the coding regions are represented, and only part of the sequence of one internal intron is missing. The genomic region known to date spans at least 45 kb and contains three large introns, while two putative binding sites for GAGA transcription factors are found 900 bp upstream of the start of transcription. Comparison of cDNA and genomic sequences reveals a genomic organisation that closely resembles vertebrate Lhx genes. The genomic structure of vertebrate Lhx genes is very conserved and consists of five exons. The Lim domains are contained in each of the first two exons, while most of the homeodomain is in the third exon and it ends in the fourth exon; the fifth exon contains the translation stop codon. Except for the presence of an extra 5' exon, which contains the 5'UTR, the start methionine and the first 13 amino acids, and an internal exon that contains the bridge region between the Lim and Hox domains, the vertebrate intron/exon boundary structure of the regions coding for the Lim and Hox domains (exons II and III, and exons V and VI) is conserved in Drosophila lim1 (Pueyo, 2000).

cDNA clone length - 4145

Bases in 3' UTR - 2435

PROTEIN STRUCTURE

Amino Acids - 505

Structural Domains

The Drosophila Lim1 sequence is most homologous to the Xenopus and mouse Lim-1 proteins. At the amino acid level the Drosophila Lim1 homeodomain is 88% identical, and the LIM domains are 78% identical to the vertebrate Lim-1 proteins. This striking similarity with the vertebrate Lim-1 proteins in the LIM and homeo-domains suggests that the isolated sequence represents the Drosophila ortholog of vertebrate Lim-1 proteins. Reduced stringency screens and Southern hybridization of Drosophila genomic DNA with a Lim1 cDNA probe suggest that there is single Lim1 gene in the Drosophila genome. Outside the LIM and homeodomains there is little overall homology. The size of the Drosophila protein is approximately 100 amino acids larger than the vertebrate proteins. This difference lies within the unconserved regions, including the linker region that separates the LIM domains from the homeodomain. However, as in vertebrates, Drosophila Lim1 has a carboxyl terminus that is rich in proline (16%). Recent analysis of the functional domains of Xlim-1 has revealed a transactivation domain within this region (Breen, 1998). The carboxyl end of Drosophila Lim1 contains of both prolines and glutamines; this is indicative of a strong transactivator. The characteristic domains for which Drosophila Lim1 encodes, suggest that this protein binds to target DNA through its homeodomain, and modulates transcription through positive or negative interactions of its protein-binding LIM domains (Lilly, 1999).

Date revised: 12 January 2000

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