LAD-1, the Caenorhabditis elegans L1CAM homologue, participates in embryonic and gonadal morphogenesis and is a substrate for fibroblast growth factor receptor pathway-dependent phosphotyrosine-based signaling

Abstract

This study shows that L1-like adhesion (LAD-1), the sole Caenorhabditis elegans homologue of the L1 family of neuronal adhesion molecules, is required for proper development of the germline and the early embryo and embryonic and gonadal morphogenesis. In addition, the ubiquitously expressed LAD-1, which binds to ankyrin-G, colocalizes with the C. elegans ankyrin, UNC-44, in multiple tissues at sites of cell-cell contact. Finally, we show that LAD-1 is phosphorylated in a fibroblast growth factor receptor (FGFR) pathway-dependent manner on a tyrosine residue in the highly conserved ankyrin-binding motif, FIGQY, which was shown previously to abolish the L1 family of cell adhesion molecule (L1CAM) binding to ankyrin in cultured cells. Immunofluorescence studies revealed that FIGQY-tyrosine-phosphorylated LAD-1 does not colocalize with nonphosphorylated LAD-1 or UNC-44 ankyrin but instead is localized to sites that undergo mechanical stress in polarized epithelia and axon-body wall muscle junctions. These findings suggest a novel ankyrin-independent role for LAD-1 related to FGFR signaling. Taken together, these results indicate that L1CAMs constitute a family of ubiquitous adhesion molecules, which participate in tissue morphogenesis and maintaining tissue integrity in metazoans.

Figure 1.

LAD-1 is the sole C. elegans homologue of the L1CAM family. (A) Intron-exon structure of LAD-1 (based on the cDNAs isolated). Blue boxes indicate exons. The hatched box indicates the unique 5′ coding region for the short cDNA isoform. Distinct 3′ UTRs and poly A tails of the cDNAs are also shown. Note that the shorter cDNA isoform lacks exons 1–4. (B, i) Predicted amino acid sequence for the longer lad-1 cDNA. The italicized NH₂ terminus sequence in maroon indicates a predicted signal sequence peptide (SignalP; http://www.cbs.dtu.dk/services/SignalP). The ORF encodes the canonical L1CAM of six Ig domains (each Ig domain is flanked by red cysteines) followed by five fibronectin type III repeats. (Each repeat is flanked by a dark blue 5′ histidine or phenylalanine and a light blue 3′ tyrosine.) The transmembrane domain is bold and underlined, whereas the ankyrin-binding motif is highlighted in green. The last three amino acids marked in fuchsia are a consensus PDZ-binding motif. (B, ii) The pink italicized letters outline the start of the predicted signal sequence peptide for the second lad-1 isoform. The pink italicized letters in B, i, completes this signal sequence peptide. The resulting cDNA encodes for a LAD-1 isoform, which lacks the first two Ig domains. The schematics for the two LAD-1 isoforms are shown in E. (C) A phylogenetic analysis of the L1CAM family members indicates LAD-1 is not an ortholog of any one vertebrate L1CAM family member. (D) Chromosomal deficiencies stDf7 and stDf8 remove the lad-1 gene located on chromosome IV, whereas deficiencies nDf41, eDf18, and eDf19 do not.

Figure 2.

LAD-1 is localized to the plasma membrane at sites of cell–cell contact in all cells throughout development. (A, i) An immunoblot of total C. elegans lysates reveals four major LAD-1 polypeptides (200, 185, 120, and 65 kD) that were detected by a rabbit polyclonal antibody raised against the LAD-1 cytoplasmic domain (lane 2). The lower bands are not LAD-1 related, since they are also recognized by ¹²⁵ I-labeled protein A (lane 3). (Lane 1) Coomassie blue staining of total C. elegans lysate. Equivalent lysate amounts. (ii) nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1 immunodetection (green) unlike stDf7 homozygous embryos, which lack the lad-1 gene; only control antibody staining of JAM-1 is seen in red (iii). B and C show immunodetection of LAD-1 in embryos. (B) LAD-1 is localized to sites of cell–cell contact, including the partial plasma membrane of a developing two-cell staged embryo (arrow). A one-cell embryo, which lacks contact sites, does not show LAD-1 localization (arrowhead). (C) LAD-1 is found in the developing nervous system (arrow) with robust LAD-1 staining in the nerve ring, the major C. elegans neuropil (arrowhead). D and E show LAD-1 expression in multiple tissues in a larva and adult, respectively. LAD-1 is present in the nerve ring and ventral nerve cord (D, arrowhead) and in the hypodermis (skin cells) (D, arrows), the plasma membrane of body wall muscles (F, arrow), neuronal cell bodies (G, long arrow), dendrites (G, arrowhead), and the pharynx (G, short arrow). LAD-1 is also present in the syncytial germline where its localization at contact sites is maintained. A schematic of the C. elegans germline is shown in H: arrowheads point to oocytes and arrows point to germ nuclei. The cross section of the cylindrical gonad reveals a single layer of germ nuclei around a cytoplasmic core with T-shaped membranes between each nucleus. In a grazing section of the germline shown in I, LAD-1 is localized at oocyte–oocyte contact (arrowhead) and only the vertical part of the T-shaped membrane, which divides two neighboring germline nuclei (arrows). The top of the T does not show LAD-1 localization. Bars: (A) 20 μm; (B–I) 25 μm.

Figure 3.

The LAD-1 cytoplasmic tail binds and recruits mammalian ankyrin to the plasma membrane in cultured cells and similar to LAD-1 UNC-44 ankyrin is localized to the plasma membrane at sites of cell–cell contact in multiple tissues in C. elegans. (A) Cytoplasmic localization of exogenous ankyrin in human embryonic kidney 293 cells transfected with murine GFP-tagged ankyrin_G (Zhang et al., 1998). Upon cotransfection with a neurofascin–LAD-1 cytoplasmic tail chimera, cytoplasmic ankyrin_G (green) is recruited to the plasma membrane, colocalizing with the L1CAM chimera (B, red). Immunofluorescence labeling of UNC-44 in multiple tissues in embryos as shown in C and D. The long arrows in C point to cytoplasmic ankyrin in the intestinal primordium. No unc-44 ankyrin expression is evident in early embryos as indicated by the short arrow, which shows a 12-cell staged embryo. The arrows in D and E point to strong unc-44 ankyrin expression in the nerve ring in the embryo and adult, respectively. In the adult (F), UNC-44 ankyrin is detected in the pharynx (short arrow), body wall muscles (small arrowhead), body wall muscle sarcomeres (large arrowhead), and commissural axons (long arrow). Bars: (A and B) 5 μm; (C and D) 10 μm; (E and F) 50 μm.

Figure 4.

Phosphorylation of LAD-1 at the conserved tyrosine residue of the ankyrin-binding motif, FIGQY, occurs in vivo, and LAD-1P localizes to polarized sites in epithelial tissues. (A) Immunoblots of BSA-coupled LAD-1 peptides containing phosphorylated or nonphosphorylated FIGQY-tyrosine (+P and −P, respectively): i, LAD-1P antibody; ii, LAD-1NP antibody. (B) Immunoblots of total C. elegans lysates blotted with antibodies against the LAD-1 cytoplasmic tail (lane 2), LAD-1P (lane 4), LAD-1NP (lane 5), and ¹²⁵I-labeled protein A (lane 3). Lane 5 shows that the 185 and 65 kD LAD-1 polypeptides contain the FIGQY epitope; only the 185-kD product is phosphorylated (lane 4, arrow). Lane 1 shows a Coomassie blue–stained C. elegans lysate and relative molecular weight markers. (C) Control nDf41 homozygous embryos, which contains the lad-1 gene, show LAD-1P (i) and LAD-1NP (ii) staining (green) unlike embryos homozygous for stDf7, which lacks the lad-1 gene; positive staining of JAM-1 is in red. (D) Preincubation of the LAD-1P antibody with the phosphorylated FIGQY peptide eliminated LAD-1P signal in stained animals; only JAM-1 immunostaining in red was detected (i). LAD-1P signal (green) is not displaced by preincubation of the LAD-1P antibody with the nonphosphorylated FIGQY peptide (ii). (E) Immunodetection of LAD-1P is localized in the pharynx and intestine (i, long and short arrow, respectively) and in the hypodermis (ii, arrow). LAD-1P (green) colocalized with JAM-1 (red) at the adherens junctions of hypodermal and intestinal cells. This colocalization is seen in the overlay panels as a yellow signal. Bar: (C and D) 10 μm; (E) 20 μm.

Figure 5.

LAD-1 FIGQY-tyrosine phosphorylation requires the FGFR pathway. In egl-15(n1454) homozygous-null larvae (B), LAD-1P levels (green) are dramatically reduced compared with similarly arrested let-23(sy17) homozygous-null larvae (A). JAM-1 immunostaining is red. Levels and localization of LAD-1NP are not affected in the egl-15 (n1454) homozygous animals (C). Bar, 10 μm.

Figure 6.

Localization of LAD-1P is complementary to that of LAD-1NP and UNC-44 ankyrin in the pharynx. (A) A schematic of the lateral view and cross-section of the pharynx. The pharynx has an anterior (1) and a posterior bulb (2). The cross-section shows the muscle cells (yellow and marked with “M”) and the marginal cells (pink). The lumen-facing apical surface is outlined in green and indicated by the green arrow. The adherens junctions are in red in the cross-section and indicated by short red arrows. LAD-1P localization (B) is compared with those of UNC-44 ankyrin (C) and LAD-1NP (D) in the pharynx. The long arrow points to the adherens junction and the short arrow (C) points to the apical surface, whereas arrowhead 1 indicates the pharyngeal anterior bulb and arrowhead 2 the posterior bulb. The white dots in B, i–iii, C, iii, and D, iii, outline the entire pharyngeal organ. LAD-1P (green) appears to be localized to the pharyngeal apical surface (short arrows) outlined by the adherens junctions (red and long arrow) revealed by JAM-1 labeling. LAD-1NP and UNC-44 ankyrin does not colocalize with LAD-1P. Bar, 10 μm.

Figure 7.

LAD-1P (A, green) is colocalized with JAM-1 (red) at intestinal adherens junctions but is distinct from UNC-44 ankyrin (B) and LAD-1NP (C) localization. Arrows in A–C point to the intestinal adherens junction. Bar, 20 μm.

Figure 8.

LAD-1P is detected along the sublateral axons and commissural axons crossing the sublateral axons in a regularly spaced punctate pattern. (A) A schematic of the C. elegans nervous system with two major nerve cords (ventral and dorsal) and smaller sublateral axon tracts on either side of the body. Sublateral axons grow along the body wall muscle basement membrane unlike the dorsal and ventral nerve cords. LAD-1P is localized specifically to sublateral axons (B, arrow) and the part of the commissural axons in contact with body wall muscles (B, arrowhead). A magnified portion of the axon tract shown in B highlights the regular punctate localization pattern of LAD-1P. (C and E) LAD-1P (green) is detected only in sublateral axons that overlay the body wall muscles (D, red, and E). The body wall muscle (red) is detected by MH24, a monoclonal antibody that recognizes vinculin. The arrow in C points to a sublateral axon tract, whereas the arrowhead points to the transversing commissural axon. Bars: (A) 50 μm; (B) 5 μm; (C) 20 μm.

Figure 9.

LAD-1 plays a role in embryonic morphogenesis and in germline and early embryo development. (A) Transgenic animals expressing a dominant negative form of LAD-1 display morphogenetic defects: anterior Vabs (i) and posterior Vabs (ii) (a, anterior; p, posterior). Wild-type animals are shown in iii and iv. (B) Apparent cell adhesion defects between oocytes result in mispositioned and misshapen oocytes, (i, arrows, and iii, short arrow). In addition, misplaced germ cells and immature-looking oocytes were detected (iii, long arrows). Apparent cell adhesion defects in early embryos result in misshapen cells and embryos, indicated by arrows in ii, that have yet to be laid through the vulva as designated by the arrowhead. The wild-type germline is displayed in iv, showing normal germline nuclei (long arrows), properly shaped and positioned oocytes (short arrows), and wild-type embryos (mid-sized arrows). Bar, 25 μm.

Figure 10.

LAD-1 is required for the gonad morphogenesis. (A) A cartoon (not to scale) of a wild-type U-shaped gonad in C. elegans. Below each panel (A–F) is a schematic of an animal opened dorsally and spread out, displaying the migration path of the DTCs as seen in the corresponding DIC micrograph. In wild-type animals (A and B), the gonad arms, led by a DTC on each arm, grow in opposite directions along the ventral length of the body. Each DTC makes the appropriate turns to meet the other at the middle of the body. DL, dorsal left; VL, ventral left; VR, ventral right; DR, dorsal right; red bar, vulva. In C, one DTC migrated a little too far to meet the other DTC that did not migrate far enough. D–F show various gonadal morphogenesis defects due to abnormal cell adhesion and/or DTC migration. The green arrows in B–F indicate the DTC, the numbered arrowheads indicate the progression of the DTC migration, and the red arrowhead points to the vulva. Bar, 50 μm.

Figure 11.

A schematic model of L1CAM functions involving both unphosphorylated and phosphorylated states.