In vertebrates, Fibroblast Growth Factor (FGFs) are involved in various developmental and pathological processes. In the worm, two FGFs have been described, EGL-17 by M. Sterns group and LET-756 by our group. We have characterised both a loss and a partiel loss of function mutants which have allowed to conclude that LET-756 is a protein essential for worm development (Roubin et al., 1999). Structurally, LET-756 (a 425 amino acid protein) is characterised by a conserved core region common to all FGFs, three putative nuclear localisation signals (NLS), a region of similarity with lamin and the absence of a recognisable signal sequence. To better understand the focus of lethality of
let-756 mutants as well as to make a structure/function analysis of the molecule, we engineered a series of constructs bearing various mutations in the gene coupled to gfp and we injected in wild type and the
let-756 s2887 null allele. Because LET-756 is a ligand which might bound to receptors some distance away from where it is synthesised, we made several constructs, with or without the FGF coding region to study synthesis and action sites of FGF. The construct in which the promoter is fused to GFP in the first exon,
let-756::gfp, is expressed in muscle cells (pharynx and body wall), in some neurons (M2, CAN and yet unidentified peripharyngeal neurons) and in G1 and G2 glandular cells. The rescuing genomic construct, LET-756::GFP, autofluoresced mainly in peripharyngeal neurons. However, after immunostaining with anti-GFP antibodies, we found pharyngeal and body wall muscles stained in the periphery of the cells, and various peripharyngeal neurons as well as the CAN stained intracellularly. The cDNA fused to GFP was capable of rescuing the
let-756 null allele and showed essentially the same pattern of expression as the promoter coupled to GFP. These results indicate 1) that intronic sequences repress the level of FGF expression, 2) that a motif in the coding region of
let-756 is capable of excreting the GFP fused molecule. We made truncations in the LET-756::GFP construct. One corresponded to the partial loss-of-function mutant (
s2613), i.e. we truncated both the genomic and the cDNA rescuing fragments coupled to GFP from the last fourth of the molecule (which interrupts a NLS and eliminate the lamin homology portion of the molecule). GFP expression was the same as in the entire rescuing fragment indicating that this portion of the molecule is not implicated in the addressing of the protein. Rescuing with the truncated form of
let-756 was, as expected, difficult to achieve since overexpression in the null allele allowed the recovery of only one strain. The second contained only the first half of the genomic construct coupled to GFP. When injected in wild type animals, a faint fluorescence distributed all over the body was observed as if this portion of the molecule contained an export signal sequence but no receptor binding site sequence. The studies of the expression patterns and the rescuing activities of other constructs in which we introduced point mutations in either the high affinity receptor binding site or a putative export sequence are in progress. Roubin R., Naert K., Popovici C., Vatcher G., Coulier F., Thiery-Mieg J., Pontarotti P., Birnbaum D., Bailly D., Thiery-Mieg D. (1999) -
let-756, a C. elegansfgf essential for worm development. Oncogene 18:6741-6747