The nematode Caenorhabditis elegans is an organism well suited for genetic analyses of nervous system development and function. First, C. elegans is easily maintained in the laboratory and has a rapid generation time of only 3 days. Second, most proteins in the C.
elegans genome are highly conserved. Third, functional redundancy has proven to be less of a problem in C. elegans than in the vertebrate systems. The genome of C. elegans frequently has single copies of genes that are found in multiple copies in vertebrates. Fourth, the greatest benefit of studying a biological process in C. elegans is that gene disruptions can be easily generated and even mutations that severely disrupt nervous system function are likely to be viable, and recoverable in a genetic screen.
Three types of approaches were developed to study these processes: forward genetics, reverse genetics and a combination of these two techniques. The aim of forward genetic technique is to identify mutations that produce a certain phenotype. In classical forward genetic screening, individuals are treated with mutagens to induce DNA lesions and mutants with a phenotype of interest are sought. Once mutants have been isolated, the mutated gene can be molecularly identified. Detailed studies of the mutant phenotype coupled with molecular analyses of the gene allow elucidation of its function. While forward genetic screens are productive, a more straightforward approach is to study the phenotype which results from mutating a given gene. This is called reverse genetics, which involves generation of knockout mutants through the application of RNA interference or the creation of transgenic organisms which do not express the gene of interest. The most productive analyse is to use both of these techniques: first, screens for mutants with a given phenotype to identify new proteins which function specifically in the process of interest; second, gene-targeting techniques to elucidate the roles of genes which play modulatory or subtle roles.
3.4.1 FORWARD GENETIC SCREENS
The main strength of C. elegans is its use in forward genetic screens. Forward genetics approaches, namely screens for visible phenotypes and/or suppressors of mutant phenotypes, have been responsible for understanding many biological processes. For example, many of the key components involved in signal transduction were identified by using the forward genetic approach. Genes that control neuronal specification have been identified through forward genetic screens for mutants in which individual neuron classes with easily notable functions or morphologies (for example motorneurons or sensory neurons) either do not differentiate or function appropriately, resulting in characteristic defects. Also, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons (Harris et al., 2001). Using behavioural and pharmacological selection criteria, genetic screens have identified many key proteins required for synaptic transmission. Genetic screens using a synaptic vesicle-associated GFP marker have identified key players in synaptic target recognition and organization of the presynaptic terminals.
Despite the success of forward genetic screens in C. elegans, certain classes of genes will not be isolated in these screens. In particular, essential genes that mutate to lethality are lost in most forward screens. Furthermore, genes which play a modulatory role are likely to lead to extremely subtle phenotypes that are easily overlooked in a genetic screen. To address these problems, two reverse genetic approaches can be used: RNA interference of gene function and the generation of targeted deletions.
3.4.2 REVERSE GENETIC TECHNIQUES
In reverse genetics, the functional study of a gene starts with the gene sequence. Using various techniques, a gene function is altered and the effect on the development or behaviour of the organism is analysed. Reverse genetics techniques complement the forward genetics ones.
The function of a gene found to be involved in a process of interest in another organism, but for which no forward genetic mutants have yet been identified, can be studied using reverse genetics. The availability of complete genome sequences combined with reverse genetics offer the possibility of every gene to be studied.
Two main methods of perturbing gene function are commonly used in C. elegans: RNA interference and the creation of deletion mutants. These techniques can also be combined, using RNAi for rapid screening of loss of function phenotypes and then obtaining deletion mutants to study genes of particular interest. RNAi can also be carried out on a global scale, where knockdown of almost every gene is tested for inducing a given phenotype. In this case,
the reverse genetics technique of RNAi can be considered as a forward genetic screening tool (Ahringer, 2006).
Although RNAi is an effective method (Fire et al., 1998) for disrupting genes required early during worm development, it has certain limitations. The method is less effective for proteins required post-embryonically, and is considered ineffective against proteins expressed in neurons (Fraser et al., 2000). One way to overcome these problems is to use RNAi supersensitive strains, like rrf-3 (Simmer et al., 2002) eri-1 (Kennedy et al., 2004), eri-1; lin-15B (Wang et al., 2005) or lin-35 (Lehner et al., 2006).
In this work, we used a candidate gene approach, which investigates the role of specific genes based on previous observations, and we have focused on the investigation of the C.
elegans lnp-1 involvement in the nervous system.
3.4.3 LUNAPARK(LNP) A NOVEL MOUSE GENE THAT SHARES THE GLOBAL CONTROL REGION (GCR)
WITH EVX2 AND HOXD GENES
To map the “digit” enhancer sequence, Prof. Duboule’s laboratory developed a large-scale transgenic approach using mouse and human BACs containing part of the Hoxd complex and upstream sequences (1Mb and 700 kb respectively). While performing sequence analysis, they found an open reading frame of 429 amino acids coding for an unknown protein, which was named Lunapark (Lnp), within both human and mouse BACs (Spitz et al., 2003) (Figure 12). A consensus sequence was identified in genomic clones from different organisms such as yeast, Arabidopsis, C. elegans, Drosophila, zebrafish, Xenopus, etc., suggesting the existence of Lnp orthologs. several enhancers, defines segments of the genome wherein genes are under the same the Lnp-Evx2-Hoxd locus). The underlying mechanisms may involve both global and local enhancer-promoter interactions (A), perhaps as a result of the formation of a functional or/and structural configuration facilitating long-range gene regulation from the GCR (B) In this view, the GCR would have concentrated, in the course of evolution, several important enhancers, due to an intrinsic property to work at a distance (from Spitz et al., 2003).
Databases analyses suggested that in all species examined, a single gene encodes lnp. The initial search for motifs related to well established functional domains did not yield definitive results. However, we detected an interesting consensus sequence conserved amongst all organisms analyzed. This motif contains 4 cysteine residues with a configuration which may be related to a zinc finger motif. A more detailed bioinformatical analysis of the LNP sequence will be presented in the Results, Chapter 4.3.
Lnp expression was first observed in 10.5 days old mouse foetuses in the posterior distal bud, highly similar to Evx2 and posterior Hoxd genes expression. Lnp was found expressed at basal level in most tissues, but strongly expressed in distal limb buds, genital bud and developing central nervous system. In the CNS, LNP is expressed in the ventral neural tube, extending to the upper part of the rombencephalon, in the presumptive cerebellum, in the dorsal neurons and in the columns of the ventral interneurons, as well as in some regions of the midbrain and forebrain (Spitz et al., 2003).
The function of Lnp in limb formation and neural development is unknown. Its expression pattern, similar to those of Evx-2 and Hox genes, suggests that Lnp may be a component of a complex required for morphogenesis. Therefore, elucidation of the role of Lnp in limb and nervous system development and investigation of the molecular events underlying its biological actions may provide novel insights into differentiation and morphogenesis during development.
Evx2 and Lnp coexpression in the nervous system
At the moment, there is no evidence that functionally links Evx2 and Lnp. Genetic studies in C. elegans, Drosophila and mouse have shown that the Evx homologues distinguish alternative fates in the motorneuron circuit (Esmaeili et al., 2002; Landgraf et al., 1999; Moran-Rivard et al., 2001). Eve, the Evx ortholog in Drosophila is necessary to form motor axons projections along the dorsal intersegmental nerve that innervates the dorsal muscles (Landgraf et al., 1999). In Eve mutants, the dorsal projections of motor axons are always abnormal, arrested in the ventral or dorsolateral region of the muscle field. In C. elegans, a homolog of the Drosophila eve gene, vab-7, is also expressed in a set of motorneurons that go to dorsal targets, and is required for correct pathfinding (Esmaeili et al., 2002).
Lnp-1, like vab-7, is expressed in the ventral nerve cord, in a subset of motor, sensory and inter neurons in C. elegans transgenic worms expressing a putative Lnp-1 promoter:GFP construct (see Results, Chapter 4.1).
Hoxd complex, Evx2 and Lnp Coexpression in Limb
The functional significance of the coexpression of the Hox complex, Evx2 and Lnp during limb development is not clear. According to Spitz and colleagues, Evx2 and Lnp expression in the developing limb would be explained by the fact that these genes are located near the Hoxd genes, and consequently fall under the influence of the digit enhancer. Even though Evx2 and Lnp genes appear not to have a critical function in digit formation (Spitz et al., 2003), they may participate in this process through the presence of their promoters, which will impinge upon the contact between the digit enhancer and the Hoxd promoters through a titration mechanism (Kmita et al., 2002; Monge et al., 2003).
Linkage of Hox with Evx2 and Lnp during evolution
In contrast to the situation in vertebrates, Hox, Evx and Lnp orthologs in flies and C. elegans are not clustered in the same genomic region (see Figure 13). In C. elegans, the Hox-like genes are located in the chromosome III; vab-7, the Evx ortholog, while located in the chromosome III is not tightly linked to the Hox-like genes. Lnp is located on the chromosome X. In Drosophila, Evx and Lnp orthologs are located in the chromosome 2R (but not in the same region) and Hox orthologs in the chromosome 3R.
Hox clustering during evolution has been well documented (reviewed in (Aboobaker and Blaxter, 2003a; Ferrier and Holland, 2001). Comparison of the Hox cluster of flies, worms and humans showed that the different genes of the cluster are most tightly linked in vertebrates, less in Drosophila and even less linkage in C. elegans. Moreover, C. elegans has a reduced number
Figure 13. Comparisons of the Hox clusters of flies, worms and humans. Each cluster is mapped at the same scale, with the coloured boxes representing each gene drawn proportionally to the length of the gene on the chromosome. The C. elegans and D. melanogaster clusters are each broken between antennapedia-like genes and more posterior genes, with the two portions separated by ~4 Mb and ~10 Mb, respectively; the cluster fragments of both species are still on the same chromosome. H. sapiens, like other vertebrates, has
of Hox genes that have strongly diverged at the sequence level compared with other phyla. The highly derived Hox gene set of C. elegans was first interpreted as being representative of ancestral bilaterian features. However, recent evidence suggests that nematodes have evolved from an ancestor that possessed a complete set of Hox genes (Aboobaker and Blaxter, 2003b).
Strong evolutionary changes either at the sequence level or of overall genomic organization of the Hox cluster may have lead to evolutionary change in developmental processes, such as the switch from the regulative processes observed in other phyla to the lineage-mode development (Houthoofd et al., 2003; Lahl et al., 2003; Schierenberg, 2001).
The separation of the Evx gene from the Hox gene cluster in flies and in C. elegans is probably also a derived condition, as the Evx-like gene is closely linked to Hox-like genes in Cnidaria (Miller and Miles, 1993). However it does not necessarily mean that these genes are not part of a same synexpression group. It is interesting to speculate as to whether the loss of linkage between Lnp, Evx and the Hox genes is functionally significant in these species. Indeed the function of Evx genes during gastrulation, dorsal-ventral patterning and neurogenesis is probably conserved throughout all the bilaterian animals. In vertebrates, there are two Evx genes, Evx1 and Evx2, the latter one is located in the immediate vicinity of the Hoxd gene cluster, next to the Hoxd13 gene. The Evx1 gene is physically linked to the Hoxa complex, but more distant from the corresponding Hoxa13 gene. One striking difference between Evx2 and the other Evx-like genes, including Evx1, is the lack of expression of Evx2 during gastrulation (Dush and Martin, 1992). It has been proposed (Dolle et al., 1994) that the disappearance of some expression traits common to Evx genes is due to the close physical linkage of Evx2 to the Hoxd complex. Moreover, in Drosophila as well as C. elegans, the lack of linkage between these genes is associated with early gastrulation expression of Evx (Ahringer, 1996; Macdonald et al., 1986), suggesting that separation between Evx and Hoxd cluster during evolution may have allowed the recruitment of Evx during gastrulation. To determine whether this new role may be implicated in the appearance of the basal triploblast, it would be necessary to know whether the Evx-like gene, which is linked to the Hox-like cluster, is expressed during gastrulation in the Cnidaria.
Even though Lnp does not appear to be functionally related to the Evx and Hox genes, the fact that its expression pattern is coincident with these genes may suggest that Lnp is implicated in a common pathway. We know that Lnp is not located near these genes in Drosophila and in C. elegans, in contrast to the situation in vertebrates. Our observations in GFP transgenic C. elegans animals showed that Lnp is expressed early during gastrulation similarly to vab-7 gene (Evx-like).