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First published online 9 July 2008
doi: 10.1242/dev.015289

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The C. elegans F-spondin family protein SPON-1 maintains cell adhesion in neural and non-neural tissues

Wei-Meng Woo^1,*,, Emily C. Berry^1,2,*, Martin L. Hudson¹, Ryann E. Swale¹, Alexandr Goncharov^2,3 and Andrew D. Chisholm^2,

¹ Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.
² Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.
³ Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA 92093, USA.

View larger version (75K): [in this window] [in a new window]   Fig. 1. spon-1 mutants are defective in epidermal morphogenesis and muscle attachment. (A) The wild-type L1 larva has a smooth epidermal shape. (B,C) spon-1(e2623) L1 larvae and adults display variable defects in epidermal morphology. The bent body phenotype is more obvious during early larval stages (B), whereas some adults display `pinched' head morphology (arrow, C); other e2623 phenotypes include egg laying defects and a slightly shorter body length. (D) A wild-type embryo at 3-fold stage. (E) spon-1(ju402) mutant embryo, displaying elongation arrest and uneven epidermal morphology (arrow). (F) spon-1(ju430) mutant hatchling showing elongation arrest with lumpy 2-fold morphology and detached body wall muscles (arrow). Scale bar: 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 2. spon-1 encodes a member of the F-spondin family. (A) Genetic map position, gene organization, and minimal spon-1 rescuing DNA fragment (pCZ695). The nc30 mutation affects the last base pair of exon 3 (CAGgtaa to CAAgtaaa; codon 104), which is likely to force the use of an alternative cryptic splice site, resulting in a frameshift and a premature stop in the reelin domain. (B) Domain organization of SPON-1, and percentage identity to Drosophila fat-spondin and rat F-spondin. (C) Phylogenetic tree of spondin domains of C. elegans SPON-1, Drosophila fat-spondin (CG6953), CG17739, CG30203 and CG30046, rat (Rn) F-spondin, zebrafish (Br) spondin 1a and 1b (F-spondin 1 and 2), Xenopus (Xl) spon-1A and Amphioxus Amphi-F-spondin (Bf, Branchiostoma floridae), made with ClustalW and displayed using njplot (Perrière and Gouy, 1996). (D) Alignment of the thrombospondin type I repeats from SPON-1; missense alterations are shown in red.

View larger version (100K): [in this window] [in a new window]   Fig. 3. SPON-1 is synthesized in muscle and localizes to basement membranes. (A) Ventral view of an adult midbody showing that Pspon-1-GFP (juEx593) is expressed in body wall muscles (arrows, two muscle quadrants). (B) DIC optics showing the muscle quadrants (arrows) on the ventral side near the vulva. (C) Embryonic expression of Pspon-1-GFP from the comma stage onwards. (D-H) SPON-1::GFP localization visualized by anti-GFP immunostaining. (D) In 1.5-fold stage embryos SPON-1::GFP is localized to muscle quadrants (arrowhead). (E) In juEx1111 larvae, SPON-1::GFP localizes at muscle dense bodies (inset, large arrowhead) and at M lines (inset, small arrowhead). (F) Colocalization of SPON-1::GFP (anti-GFP) and the dense body component vinculin (MH24 immunostaining) in juEx734 adult muscles. (G,H) SPON-1 was detected on the pharynx surface (overexpressing line juEx698, arrow, G) and within coelomocytes (SPON-1::GFP, arrowheads, H) in juEx734 adults. (I) Functional SPON-1::GFP expressed under the control of the pharynx-specific myo-2 gene (juEx1302) localizes to muscle BMs (arrowhead) in 3-fold stage embryos (anti-GFP immunostaining). (J,K) Immunostaining with anti-SPON-1 antibodies of animals of genotype spon-1(ju402); juEx698[spon-1(+)]. (J) Dorsal view of a 1.5-fold stage embryo stained with anti-SPON-1 (red), and anti-Myotactin (MH46) and anti-AJM-1 (MH27) (both green), which mark muscle-epidermal attachments and epidermal adherens junctions, respectively. SPON-1 is in muscle cells, beneath Myotactin staining. (K) Larva stained with anti-SPON-1 (red) and anti-MHC (green) to show body muscles. SPON-1 expression was detected in or adjacent to body muscles (arrow) and the excretory canal (arrowhead). Scale bars: 10 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 4. SPON-1 is required for late elongation and interacts with INA-1 -integrin. (A-P) Frames from time-lapse analysis of wild type (A-D), spon-1(ju402) mutants (E-H), putative ina-1(gm144); spon-1(ju430)/+ mutants (I-L) and spon-1 ina-1 double mutants (M-P). Most spon-1(ju402) embryos developed normally up to either 2- or 3-fold stage (F,G), but then retracted to 2.5-fold (H) by the time the wild-type embryo has developed to the 3-fold stage (D). spon-1(ju402) embryos displayed normal muscle twitching movements from 1.75- to 3-fold stage before elongation arrest (see Movie 1 in the supplementary material). Body constrictions became apparent when muscle movements slowed down and later stopped. At the restrictive temperature, spon-1(ju430ts) mutants resemble ju402 in phenotype. More spon-1(ju402) embryos retracted to a 2-fold stage (12/19, 63%) than did spon-1(ju430ts) embryos (7/33, 21%). Sixty-three percent of spon-1(ju402) and 79% of spon-1(ju430ts) embryos reached the 3-fold stage before retraction. spon-1(ju348) embryos were indistinguishable from ju402 in embryonic phenotype. ina-1(gm144) mutants displayed normal elongation (not shown). (I-L) From strains of genotype ina-1(gm144); spon-1(ju430)/mIn1 mIs14 mutants, 41% (18/44) GFP-positive embryos (either ina-1;spon-1/mIn1 mIs14 or ina-1; mIn1 mIs14) displayed late-onset elongation defects. No ina-1 mIn1 mIs14 embryos (n=11) showed elongation defects. We infer that 60% of spon-1/+ heterozygotes have elongation defects in the gm144 background. Six out of seven ju430; gm144 homozygotes (GFP-negative embryos) displayed 1.5-fold arrest (M-P). (Q) Early elongation rates (mean±s.e.m.). spon-1 mutants elongate normally from comma to 2-fold stage, emb-9 mutants were slightly slower, pat-3 mutants were significantly slower (**P<0.05, ANOVA). (R) The spon-1(ju430ts) temperature-sensitive period begins at the 2-fold stage.

View larger version (160K): [in this window] [in a new window]   Fig. 5. SPON-1 maintains muscle-epidermal attachment and muscle organization. (A-D) Muscle quadrants in 2-fold (A,B) and 3-fold (C,D) embryos; anti-MHC-A staining is counterstained with anti-AJM-1 in A and B. 3-fold stage spon-1(ju402) mutants display gaps in MHC staining due to muscle detachment (arrowhead, D). (E,F) Anti-PAT-3/β-integrin (MH25) staining in wild type and in arrested spon-1(ju402) embryos. (G,H) Perlecan (MH3) in wild-type and spon-1(ju402) embryos. Epidermal cell outlines are shown by anti-AJM-1 staining. Similar to MHC-A, both β-integrin and perlecan staining were discontinuous because of muscle detachment (arrowhead in F,H), but were normal in other regions; the gaps were variable in size and position from embryo to embryo. (I,J) Epidermal circumferential F-actin bundles (CFBs), detected by phalloidin labeling in wild-type and arrested spon-1(ju402) embryos. In the wild type, CFBs in lateral epidermal cells were circumferentially oriented (I, arrowhead). In arrested spon-1(ju402) embryos, CFBs in lateral epidermal cells were misoriented (J, arrow), becoming perpendicular to the circumference. (K,L) Myotactin patterns (MH46 antibody staining) in the wild type (K) and in arrested spon-1(ju402) embryos (L). Myotactin patterns are discontinuous along the body; however, normal circumferential double band patterns were observed in the remaining Myotactin. Scale bar in A-L: 10 µm. (M,N) Post-embryonic muscle surfaces visualized using trIs10; note muscle detachment in ju430 (arrowhead, N). (O) Percentage of animals displaying muscle-muscle detachment (n>40 for each condition; Fisher exact test, *P<0.05, ***P<0.001). (P-R) Ultrastructure of larval body wall muscle and BM. (P) Wild-type body muscle, showing dense bodies (db) and regular myofilament lattice; cuticle is at top. (Q) Muscle in spon-1(ju430) larvae grown at 22.5°C; note absence of dense bodies (db), disorganized myofilament lattice and invagination of basal lamina (arrowhead). (R) Higher magnification of basal lamina invagination into muscle (arrowhead). Scale bars in P-R: 500 nm.

View larger version (41K): [in this window] [in a new window]   Fig. 6. SPON-1 is required for motoneuron fasciculation and dorsoventral guidance. (A-E) Motoneuron processes (juIs76) in wild-type (A,C) and spon-1(ju430) mutant (B,D,E) L4s. (B) Defasciculation of D neuron ventral processes in ju430 mutants (arrowhead). (C) Motoneuron commissures in the wild type extend from ventral to dorsal on the right side. (D,E) Motor commissures in spon-1(ju430) mutants turn laterally (arrowhead) upon contacting the dorsal muscle quadrant (m, dotted line) and either extend and terminate subdorsally (D) or turn back to the dorsal midline (arrowhead, E). (F,G) Commissural guidance defects occur independently of muscle detachment (trIs10); commissure guidance can be normal (arrowhead) in regions of body wall muscle detachment (F) and can be aberrant in regions of normal attachment (arrowhead, G). (H-J) D neuron outgrowth in wild type (juIs76, H), in a ju430 juIs76 embryo (I, progeny of animal shifted to 25°C as L4) and in a spon-1(ju402) arrested hatchling (J, ynIs37 marker); all six D neuron commissures (DD1-6) extend dorsally. Scale bars: 10 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 7. SPON-1 maintains process position at the ventral midline. (A) PVQ axons in wild type (oyIs14). (B) In spon-1(ju430), PVQL crosses over to the right-hand VNC (open arrowhead), as in the wild type, and undergoes two ectopic crossovers (white arrowheads). (C) Midline crossing is more penetrant in spon-1 adults than in L1s, is enhanced by shifting L1s from 15 to 25°C, and is suppressed by growth in 1 mM levamisole (**P<0.01, ***P<0.001). (D) Suppression of spon-1(ju430) midline crossing by zig-4 and egl-15 (at 22.5°C; the number of animals is shown in the column) and enhancement by dig-1 (at 20°C; **P<0.01). (E) Model for the interaction of SPON-1 and ZIG-4/EGL-15A pathways; a cross-section of ventral nerve cord is shown. SPON-1 in the BM (green) mediates adhesion of PVQ axons (blue) to their normal environment; ZIG-4 and EGL-15 (red) locally inhibit axon-BM adhesion at the midline. (F-J) spon-1(ju430ts) oyIs14 animals were shifted from 15°C to 25°C in the L1 stage and PVQ scored in young adults. The adults were sectioned in the region of PVQ crossing over posterior to the vulva; approximate levels of sections are shown in F. (G) Anterior to PVQR crossover (0 µm), the left-hand ventral nerve cord contains its normal three processes, the PVQL, PVPR and AVKR (arrowheads and colored blue in G-J). (H,I) Between 90 and 150 µm a fourth process, presumably PVQR, has crossed over and fasciculated with the left-hand bundle. (J) At 160 µm posterior, the left-hand bundle again contains three processes. The ventral hypodermal ridge (HYP, green) and the right-hand ventral nerve cord (RVC, blue outline) appear normal in both crossover regions. Similar results were obtained for a second animal (not shown). Scale bars: in A,10 µm; in G-J, 0.2 µm.

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