C. elegans Bristol (N2) strain nematodes were cultured using standard techniques [21]. let-756 rescuing activity was assayed by injecting tester DNA at 50 ng per µl into gravid s2887 strain hermaphrodites, establishing transformed lines and scoring the Dpy progeny for a viable phenotype. The s2887 allele used in these experiments was generated on a dpy-17(e164) unc-32(e189) chromosome after UV irradiation [22]; the sDp3, a portion of chromosome III, balances the lethal phenotype of the s2887 strain. The rescue activity was tested for the presence in the progeny of live Dpy animals. Dpy animals were genotyped for the presence of the inversion in the let-756 locus characteristic of the s2887 strain by PCR on single worm, as described in [23]. The strains dpy-17(e164) unc-32 (e189) and unc-32 (e189) used as control, were obtained from the Caenorhabditis Genetics Center (CGC).

The hypomorph FF628 let-756(s2613) unc-32(e189) strain was isolated by D. Thierry-Mieg from BC4253 and outcrossed an additional 7 times. Homozygous animals are viable and fertile, but slow growing, transparent and small. These animals were coinjected with P_let-756::let-756 and the pPD93.97 plasmid which contains P_myo3::GFP. Fluorescent cells contain arrays bearing both plasmids and mosaic animals were analyzed.

The promoters used in muscle-specific expression of let-756::gfp were myo-2 (pharyngeal muscles), myo-3 (body muscles) and egl-17 (sex muscles) [24, 8]. The constructs driving the coding region of let-756::gfp and let-756^R318::gfp under the control of P_let-756 promoter have already been reported [25]. The P_myo-3::let-756::gfp expression construct was made by ligating the PCR product containing the let-756 cDNA in the XbaI-BamHI digested pPD93.97. The XbaI-EcoRI fragment containing the let-756::gfp fusion was then excised and inserted in the NheI-BglII digested pPD96.48 vector to obtain P_myo-2::let-756::gfp expression construct. The full length let-756 genomic DNA under the control of egl-17 promoter (NH#469) was a gift from M. Stern (Yale, USA).

The method used for immunofluorescence experiment was essentially as described by Ruvkun and Giusto [26]. Animals were fixed in a mixture of 1% paraformaldehyde, 20% methanol, permeabilized in 1% triton. Fixed animals were then incubated overnight with a 1:400 dilution of anti-GFP antiserum (Roche), washed extensively and then incubated for another two hours in a 1:500 dilution of the anti- mouse antibody conjugated to Texas Red (Molecular Probes). After extensive washing, the animals were mounted in DAPI-containing glycerol solution. In some experiments, DiI (Molecular Probes) dye filling was performed on living preparations of worms as previously described [27] to identify chemosensory neurons [28].

To analyze phenotypes, worms were collected on agar pads, anesthetized with levamisol and observed on a Zeiss LSM 510 laser scanning microscope equipped with Normasky optics. The number of s2887 transformed strains analyzed was: 2 for the P_let-756:: let-756::gfp construct, 3 for P_let-756:: let-756^R318::gfp, 3 for P _myo-3::let-756::gfp, 4 for P_myo-2::let-756::gfp and 2 for P_egl-17::let-756. Three s2613 strains transformed by P_let-756::let-756::gfp were analyzed. In the wild type context, 3 5 [rol] strains were compared to 2 strains transformed with P_let-756::let-756::gfp and two strains transformed with P_let-756:: let-756^R318::gfp.

To assess body size in transgenic animals, cultures were synchronized by bleaching gravid animals. Eggs were allowed to grow for 90 hours in the adult stage. Selected plates containing adult rescued [dpy] animals were washed off in M9 medium and fixed in a mixture of 1% paraformaldehyde, 20% methanol. Body sizes (length and width at the vulva) were measured on 100 individual animals using LSM 510 software. Non-parametric Mann-Whitney u test was used. Differences were considered as significant when p values were < 0.01.

To determine where let-756 is required to insure worm viability, we examined the cells that express a GFP reporter from ~ 4 kb of promoter and enhancer sequences of let-756 (P_let-756::gfp). Animals carrying this transgene express GFP in many cells throughout development and in the adult, as reported by us and others [7, 11, 14].

The major site of let-756 expression was found in descendant cells of mesodermic precursors (muscle cells). However, some cephalic neurons, CAN cells, and the two glandular cells G1 and G2 (Fig. 1A) were also positive.

Fig. (1) LET-756 expression in wild type and let-756R318 transformed worms. (A) In this mosaic worm, GFP driven from the let-756 promoter is expressed in body muscles (bm), pharynx procorpus (ph), cephalic neurons (cn), CAN cells (can), and G1 and G2 gland cells (gc). (B – E) worms were transformed by Plet-756::let-756::gfp, fixed and immunostained with anti-GFP antiserum. Accumulation of fluorescence is observed in a punctuate pattern at the junctions between body muscle cells (C) and around the pharynx (D). CAN cells are stained (E). (F) worms were transformed by Plet-756::let-756R318::gfp; the construct was mainly expressed in neurons, which were identified as the ASE chemosensorial neurons since they do not fill with the lipophilic DiI dye and exhibit typical endings (magnified in the right insert).

Because LET-756 is secreted [23], we expected to find the FGF at some distance from its site of synthesis. We compared the sites of expression to those of LET-756 action as revealed by the localization of a LET-756::GFP fusion. We used a construct where identical let-756 upstream sequence drives the LET-756::GFP fusion protein. We found accumulation of LET-756::GFP mainly in body wall muscle cells and incidentally in vulva, sphincter and pharyngeal muscle cells. However, under microscope observation only few animals were positive for LET-756::GFP despite the fact that they were perfectly rescued; in addition, the fluorescence was often lost with time in rescued strains although the rescuing ability was not.

To correlate protein level expression with rescue capacity we performed immunofluorescence staining of the rescued animals with anti-GFP antibody. Immunodetection of GFP is more sensitive than the observation of its direct fluorescence when exposed to UV light and thus more suitable to detect small amounts of protein. Immunostaining was seen in all rescued individuals, even in the absence of the GFP direct fluorescence, indicating that only animals containing a small number of arrays were able to survive and that these arrays did not contain enough copies of GFP to allow visualization by direct fluorescence.

After GFP immunostaining, animals displayed accumulation of fluorescence in a punctuate pattern. It was not possible to discriminate between a localization of LET-756 at the cell membrane on the cytoplasm side or, more likely, extracellularly on the basement membrane of body wall muscle cells (Fig. 1B,C) and around the pharynx (Fig. 1D), in regions where collagen [29] and UNC-52/perlecan [30] accumulate.

The fusion was observed in the nucleus and not in the nucleolus of CAN cells (Fig. 1E), in agreement with the results obtained with Cos-1 transfected mammalian cells [25]. A construct that mimicked the partial loss-of-function allele, which we have previously denominated LET-756^R318, fused to GFP and under the control of the same upstream sequence, was expressed in muscle cells. Surprisingly, it was also expressed in some neurons exhibiting peculiar endings unable to be filled by the DiI lipophilic dye (Fig. 1F), which we tentatively identified as the chemosensorial ASE neurons. We never observed this type of fluorescent cells when using the let-756 promoter driving GFP. One possibility is that this staining reflects the specific translocation of the mutated protein in this cell type, but we do not have any definite explanation for this observation. Unlike cells with wt FGF, the nucleolus was also stained in these cells.

let-756 is expressed mainly in various muscle cells. To determine which of these locations are essential for viability, we created transgenic animals expressing let-756 in various muscles. Constructs were made with tissue-specific promoters and the let-756::gfp C-terminal fusion. For body wall muscle expression we used P_myo-3::let-756 [24] and for pharyngeal expression, we used P_myo-2::let-756 [24]. For sex myoblast expression, we used P_egl-17::let-756 [9], which drives expression specifically in primary fate vulval precursors and their descendants. These constructs were introduced in animals carrying the let-756(s2887) null allele [dpy->9)] and animals were examined for both rescue activity and phenotype. In addition, we compared the rescuing activity of the wt LET-756 molecule with its truncated form LET-756^R318, which mimics the partial loss-of-function allele (s2613).

All types of tested ectopic muscle expression rescued the lethal phenotype: 7, 6 and 3 strains were obtained using myo-3, myo-2 and egl-17 promoters respectively, indicating that muscle expression is sufficient to insure viability.

Variations in body length and proportions were observed in the various rescued animals described above. To identify the action of spatially-restricted gene expression on body length and proportions we analyzed transgenic animals with wt let-756 expression driven by both endogenous and heterologous promoters in the null allele (s2887) background. Three types of spatial expression patterns were studied: two spatially-restricted expression patterns (anterior and central regions of the worm body, corresponding to the expression of the transgene under the control of the heterologous promoters P_myo-2 and P_egl-17, respectively) [24, 9] and a widespread expression pattern corresponding to the expression of the transgene under the control of the endogenous and P_myo-3 promoters [24].

Body sizes of animals were measured - without assessing the size of individual cells - in these different conditions. Individual total body length, mouth-to-vulva length, and width at the vulva are shown in Table 1.

Table 1.

Morphometric Analysis of the let-756 Rescued s2887 Allele

Expression of wt let-756 from the myo-3 promoter produced animals with the same characteristics as those rescued by using the let-756 promoter (Fig. 2B,2E). MYO-3 is expressed in body wall muscles, which are distributed all along the body from head to tail. Therefore, LET-756 expression in body wall muscles is sufficient for full rescue of the lethal phenotype and insures the same body length development as expression driven by the proper promoter. The respective proportions of anterior and posterior body parts in strains rescued with P_let-756and P_myo-3 promoters being similar (Fig. 3), the contribution of pharyngeal LET-756 expression seems to be minor.

Fig. (2) Worms transformed with let-756 under the control of tissue specific promoters were phenotypically different. Worms transformed by let-756 (B) or let-756R318 (C) under the control of Plet-756 promoter are not different from the control [Dpy Unc] strain (A) although the former are bigger (see Fig. (3) for morphometric analysis). Worms transformed with let-756 under the control of the Pmyo-2 promoter show reduction in the size of the posterior part of the body (D). Animals with let-756 under the control of Pmyo-3 develop harmoniously (E). Animals with let-756 under the Pegl-17 promoter show enlargement of the body width at the vulva (F). Arrows point to the vulva.

Fig. (3) Morphometric analysis. Anterior (shaded area) and posterior (unshaded) sizes of the different strains are represented (in µm) as well as their relative contribution in the total body length.

Expression of LET-756 from the P_myo-2 promoter, which drives expression exclusively in the pharynx musculature, led to shorter animals than the one described above. In addition, they exhibited a misplaced vulva due to a better development of the anterior (defined as the length from mouth to vulva) than the posterior part of the body (Fig. 2D). These animals exhibited a ratio “anterior length over total” larger than control individuals or individuals with LET-756 or LET-756^R318 expression driven by P_let-756 and P_myo-3 (Table 1).

Expression of LET-756 from the egl-17 promoter led to animals with larger width size than control animals (Fig. 2F). EGL-17 is expressed in sex muscles [9]. It is thus coherent that LET-756 expression driven by the egl-17 promoter in the middle of the animal increased locally the size of the animals. In addition, the posterior length was also increased compared to control strain and animals rescued with the let-756^R318 transgene. Sex muscles arise from a pair of bilaterally symmetrical sex myoblasts; these cells are born in the posterior body. During larval development of the transgenic animals, enough LET-756 vehiculated by migrating myoblasts may allow the growth of the posterior part of the animals.

We have previously reported that no major morphological and/or behavior differences was observed between lines rescued with P_let-756::let-756 and P_let-756::let-756^R318 constructs [25], which mimic wt and partial-loss-of-function alleles, respectively. However, in light of our results on body size we analyzed the length and width of these transgenic animals.

As compared to [dpy-17(e164) unc-32(e189)] animals, which have only two copies of the wt let-756 gene, animals from the s2887 strain (null allele) rescued by let-756 expressed under the control of its own promoter had enlarged body length mostly due to a better development of the anterior part of the animal (Table 1, Fig. 2A,B). In contrast, the body length of animals rescued by let-756^R318 was smaller than the length of those rescued by the wt molecule, and was in the range of the body length of the control (dpy-17 (e164) unc-32(e189)) animals (Figs. 2C,3). As already reported [29], we found a four-fold increase in the number of transgene copies in let-756^R318 strains compared to strains rescued with the wt let-756. This increase in array copy number could be explained by different activities of the proteins encoded by transgenes on the tyrosine kinase FGFR/EGL-15, since both FGFs are secreted (23) or by the different subcellular localizations of the two molecules in the expressing cells [25].

Thus, in all circumstances, LET-756 increased the animal body size in areas where it was expressed and body muscle cell expression was sufficient for ensuring normal body length. Quantification indicated that, as compared to [dpy-17(e164) unc-3(e189)] animals, the total length of all transgenic animals (except for P_myo-2::let-756) was significantly increased (p < 0.01) in a hierarchy P_let-756::let-756 > P_myo-3::let-756 > P_let-756::let756^R318 = P_egl-17::let-756> no transgene > P_myo-2::let-756. In contrast, the anterior part of animals transformed with P_myo-2::let-756 was significantly (p <10^-5) larger than untransformed worms or worms transformed with P_egl-17::let-756 or P_let-756::let756^R318 transgene. It was not different from worms transformed with P_myo-3::let-756 and smaller than those transformed with P_let-756::let-756. The posterior part of P_myo-2::let-756 was shorter than all the other transgenic or wild-type strains (p <10^-7). Finally, the width of P_myo-2::let-756 and P_egl-17::let-756 transgenic strains was increased (p <10^-4) as compared to all other animals (Fig. 3).

Some genes coding for cuticle collagens modify worm body length [20]. We were thus concerned by a possible effect of dpy-17 (20), which was used as a balancer in the establishement of homozygote strain for let-756 loss-of-function allele, and rol-6 [31], a largely used coinjection marker of transformation, both encoding cuticle collagens.

Due to early lethality of the let-756 loss-of-function allele, we could not compare the size of rescued vs unrescued animals. We thus compared the ratio between body length and width in the various rescued strains, as a hallmark of the Dpy phenotype. Dpy mutants are defined as small, fatty worms and thus display a larger width to length size ratio than wt animals. The body dimensions were increased in the rescued strains expressing the wt let-756 transgene under the control of the P_let-756 or P_myo-3 compared to the dpy-1((e164) unc-3(e189) reference strain, but, importantly, the width to length size ratio was not modified (Table 1). Thus, overexpression of let-756 cannot suppress the Dpy phenotype of the dpy-17 mutant allele.

To eliminate unambiguously any effect of the dpy-17 background on the LET-756-induced body length, we compared the consequence of let-756 transgene expression under the control of its own promoter in the [let-756(s2613) unc-32(e189)] hypomorph as well in wt N2 animals. In the hypomorph, the myo-3 marker was used as an indicator of which area of the body was transformed, and in wt N2 animals, the rol-6 marker was used to identify transformed animals. Expression of LET-756 increased both length and width in the [let-">9)] hypomorph in which the dpy-17 mutation had being outcrossed (Table 2). As compared to the hypomorph (Fig. 4A), the rescued animals appeared larger and developed a normal number of eggs (Fig. 4B). They did not look different from the unc-32 (e189) strain (Fig. 4C), which contains two wt let-756 alleles. However, in mosaic animals where LET-756 was expressed only in the anterior part of the animal, the posterior part looked scrawny and thinner than normal (Fig. 4D). Reciprocally, animals where FGF expression occurred only in the posterior part of the animals exhibited a thinner anterior part (Fig. 4E,F). Thus, the same phenotypes as those described in the worms carrying the loss-of-function allele s2887 were observed in the animals carrying the partial loss-of-function allele s2613, showing that spatial expression of LET-756 increases body size independently of the genetic background.

Table 2.

Morphometric Analysis of the let-756 Rescued s2613 Allele

Fig. (4) Rescue of the hypomorph by let-756 expressed under the control of its own promoter. let-756(s2613) unc-32(e189) worms (A) were transformed by the Plet-756::let-756 construct. (B) shows the phenotype of animals rescued by let-756 expressed in all body muscles. These animals resemble the unc-32 strain (C). Animals shown in (D) exhibit LET-756 expression in anterior muscles: development of the posterior part of the animal is impaired and reciprocally, animals shown in (E) and (F) exhibit LET-756 expression in posterior muscles and their anterior part is thinner than normal. The autofluorescence of the gut granules is visible in (A) and (C).

Finally, we examined the effect of let-756 over-expression in a wt context. We found that N2 animals injected with with P_let-756::let-756 or P_let-756::let756^R318 were not different in size (Table 3) than those injected with P_let-756::gfp although smaller than untransformed worms, due to the presence of rol-6 (su1006) allele, the transformation marker gene. Thus, overexpression of LET-756 or LET-756^R318 in a wt context did not result in an overgrowth phenotype. A threshold ligand amount expressed by endogenous alleles of the N2 worms and sufficient to saturate the EGL-15 receptors may explain size conservation.

Table 3.

Morphometric Analysis of the let-756 Surexpression in Wild-Type Animals