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First published online 21 September 2005
doi: 10.1242/dev.02042

Development 132, 4621-4633 (2005)
Published by The Company of Biologists 2005
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Syndecan regulates cell migration and axon guidance in C. elegans

Christa Rhiner1,2, Stephan Gysi1, Erika Fröhli3, Michael O. Hengartner1,2,* and Alex Hajnal3

1 Institute of Molecular Biology, University of Zurich, 8057, Switzerland
2 Neuroscience Center, 8057, Zurich, Switzerland
3 Institute of Zoology, University of Zurich, 8057, Switzerland



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  		Fig. 1. Structure of cell surface HSPGs. (A) Predicted structure of the C. elegans syndecan SDN-1 and glypican GPN-1 proteins. Syndecan is a single-pass transmembrane protein with a short conserved cytoplasmic domain containing a PDZ-binding motif. HS side chains are attached to the extracellular domain of the core protein at conserved serine residues. The yellow box represents a typical tetrasaccharide linker region, which connects the HS chain to the serine. Glypican is attached to the cell surface by a GPI anchor. (B) Genomic structure of the sdn-1 locus on chromosome X. Boxes represent exons (blue, coding; black, non-coding) and red triangles indicate putative HS attachment sites. Two isoforms of the sdn-1 mRNA have been predicted to code for identical proteins. The null allele sdn-1(zh20) affects exons 1-5. The allele ok449 was previously shown to produce a truncated syndecan form lacking the two major HS attachment sites (Minniti et al., 2004Go). (C) Northern blot analysis of sdn-1. A single message of approximately 0.9 kb is detected in wild-type worms, whereas no message can be found in sdn-1(zh20) mutants. 18S and 28S ribosomal RNAs, as loading controls, are shown to the right.

 


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  		Fig. 2. SDN-1 is required for the migration of neurons and coelomocytes. (A) Schematic depicting the migration of HSN neurons during embryogenesis. (B) In wild-type animals both HSN cell bodies (arrowheads) locate close to the vulva. (C,D) In sdn-1 mutants, HSN neurons fail to migrate to the vulva from their original position in the tail (C) or stall prematurely anterior to the vulva (D). Axons of mispositioned HSN bodies (arrowheads) that initially grow out incorrectly in a posterior direction eventually turn (arrows) and reach the nerve ring in the head. (E) HSN migration (adult stages) demonstrated by the genetic interaction of sdn-1 nulls with HS-modifying enzyme mutants. `% animal defective' represents the fraction of animals with at least one defective HSN neuron. Numbers are shown with the standard error of proportion. (F) Schematic of the six coelomocytes in wild-type and sdn-1(zh20) hermaphrodites. (G) In the wild type, coelomocytes born in the head migrate caudally during embryonic development, past the intestinal valve that separates the pharynx from the intestine (arrowhead). (H) In sdn-1 null mutants, coelomocytes are found in the head region, anterior and posterior of the intestinal valve (arrowhead). In order to define anatomical locations, some pictures were taken with DIC optics in the fluorescent channel (C,G,H). N, number of animals scored. Scale bars: 10 µm.

 


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  		Fig. 3. sdn-1 mutants show midline crossover defects of VNC interneurons. (A) Schematic of PVQL/R interneurons. (B) PVQL/R interneurons run parallel to the head in the wild type. (C) PVQ axons inappropriately cross the midline in sdn-1 mutants, leading to collapsed axon tracks (arrowheads). (D) Schematic of the AVKL/R interneurons. (E) Wild-type AVKL/R run next to each other, whereas midline crossing of AVKL/R axons can be observed in sdn-1(zh20) worms (arrowheads) (F). (G) Schematic of HSNL/R motoneurons. (H) HSNL/R stay ipsilateral in the wild type, whereas HSNL/R axons frequently cross to the contralateral side in sdn-1(zh20) null mutants (arrowheads) (I). All schematics and photos are ventral views, anterior is to the left. N, number of animals scored. Scale bars: 10 µm.

 


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  		Fig. 4. Loss of SDN-1 affects the guidance and left/right choices of commissural motoneurons. (A) Schematic of DD and VD motoneurons. (B,E) DD/VD commissures in young adults and (C,D) embryonic DD commissures in the L1 larva. (B) Ventral nerve cord (VNC) view of an sdn-1(zh20) animal. D-type motoneurons inappropriately grow out on the left side of the animal (arrowhead) or stop prematurely before reaching the dorsal nerve cord (DNC) (arrows). L, left side; R, right side. (C) Six DD commissures form a continuous dorsal nerve cord in the wild-type L1 larva. (D) In sdn-1 null mutants, DD commissures branch prematurely and fail to reach the DNC (arrow). (E) D-type motoneuron development analyzed for: (1) circumferential outgrowth of DD/VD commissures to the DNC; (2) left/right choice to exit the VNC. (1) N represents the total number of commissures scored. Right and left denote the percentage of commissures that reach the DNC when growing out on the correct (right side) and incorrect side (left side), respectively. Note that guidance is considerably more affected if axons grow out on the wrong side of the animal. (2) N represents the number of animals analyzed. Each column gives the percentage of animals that show the indicated amount (1-8) of commissures inappropriately exiting the VNC on the left side. Scale bars: 10 µm.

 


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  		Fig. 5. SDN-1 expression in neural and hypodermal tissue. (A-F) Expression pattern of the translational Psdn-1::sdn-1::gfp reporter. (A') SDN-1::GFP is broadly expressed in the three-fold embryo, and is particularly strong in the pharynx (pha) and the motoneurons (mn) of the VNC. (B) In L1 larvae, expression is seen in the VNC (arrow) and around the seam cells (arrowhead). SDN-1::GFP expression is also visible in embryonic DD commissures (out of focal plane in B). (C) During later larval stages, SDN-1::GFP is predominantly found in the nervous system. The nerve ring (nr), and the VNC motoneurons and commissures (com) show the highest expression levels (arrowheads; B,F), but the reporter is also present in touch neurons (arrow; E) and other sensory neurons in the head (arrow; F). SDN-1::GFP can also be detected at a lower level in the body wall hypodermis (arrowhead; D); expression is stronger in hypodermal tissue in the tail (arrow; C).

 


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  		Fig. 6. Double mutant analysis of SDN-1 and HS-modifying enzymes. (A) Circumferential guidance of DD commissures (L1 larvae), as shown by the genetic interaction of sdn-1 nulls with HS-modifying enzyme mutants. Data represent the number of commissures that reach the level of the DNC per L1 larva. Error bars indicate s.e.m. (B,B') Analysis of DD commissures in L1 larvae. (B') In hse-5; sdn-1 double-null mutants, most DD commissures either fail to grow out dorsally or branch prematurely (arrowhead). (C,C') VNC view of L1 larvae. (C') The VNC of hse-5; sdn-1 double-null mutants is highly defasciculated (arrowhead). (D,D') DD/VD commissures of L4 larvae. (D') Circumferential guidance of DD/VD motoneurons is disrupted in hse-5; sdn-1 double mutants (arrowheads). D and D' show confocal z-stack images to visualize all misguided commissures in different focal planes. (E) The PVQ crossover phenotype demonstrated by the genetic interactions of sdn-1 null worms with HS-modifying enzyme mutants and slt-1(eh15). Error bars indicate the standard error of proportion. Asterisks denote statistical significance as follows: ***P<0.0001; n.s., not significant; n, number of animals scored. Scale bars: 10 µm in B-C'; 25 µm in D,D'.

 


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  		Fig. 7. Model for syndecan function in axon guidance signaling. Syndecan (SDN-1) on the neural growth cone exposes HS chains with `neuron-specific' sugar modifications, here simplified by 6Osulfations. The HS side chains of syndecan bind specific guidance cues (e.g. SLT-1) and modulate signaling by cognate high-affinity receptors, such as the SAX-3/Robo receptor (co-receptor function). Genetic analysis suggests that parallel signaling to syndecan relies on distinct sugar motifs on additional HSPGs in a context-dependent manner. In PVQ interneurons, this parallel pathway involves HS with epimerization (green), whereas in motoneuron development it involves both epimerization and 6O-sulfation. The HS modification patterns may thus define the specificity of different ligand/receptor interactions; A and B represent different ligands.

 

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© The Company of Biologists Ltd 2005