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First published online 29 March 2006
doi: 10.1242/dev.02357

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Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans

Molecular Neurobiology Program, Department of Pharmacology, Skirball Institute, NYU School of Medicine, New York, NY 10016, USA.

View larger version (47K): [in a new window]   Fig. 1. wnt, frizzled and vps-35 mutants have PLM polarity defects. (A) Wild-type mec-4::gfp, left and right PLM, ventral view. mec-4::gfp is expressed in mechanosensory neurons ALM, PLM, AVM and PVM. (B) lin-17; PLMs with a reversed polarity. The sister of PLM, ALN, can often adopt a PLM-like fate in lin-17 mutants; ALN expresses mec-4::gfp and has an inverted polarity. Asterisks indicate the two PLM and one ALN cell bodies. (C) lin-44; arrows indicate the PLM with a symmetric polarity, the other PLM has a wild-type polarity. (D) lin-44; egl-20; PLMs with a reversed polarity. (E) vps-35; PLMs with a symmetric polarity. (F) ALM and PLM are bilaterally symmetric pairs of neurons located near the midbody and the tail, respectively; only left ALM and PLM are shown. The posterior ALM process is rarely present and is not depicted. Boxed area indicates region shown in fluorescence photomicrographs. Anterior is leftwards and dorsal is towards the top, unless indicated otherwise.

View larger version (93K): [in a new window]   Fig. 2. Synaptic vesicle localization is altered in lin-17 mutants. Synaptic vesicles were visualized in PLM using the mec-7::snb-1-gfp reporter. Arrows indicate synaptic vesicle (SNB-1-GFP) enrichment and triangles indicate PLM bodies. Autofluorescence in intestine is labeled (int.). (A) Wild-type mec-7::snb-1-gfp; synaptic vesicles are enriched in anterior PLM process. (B) lin-17; synaptic vesicles accumulate in long posterior PLM process. (C) lin-17; synaptic vesicles are enriched in both anterior and posterior PLM processes when they are symmetric in length. Anterior is leftwards; dorsal is towards the top. (D) PLM polarity defects in lin-17 mutants and lin-17 mutants harboring mec-4::lin-17a or mec-4::lin-17b extrachromosomal arrays. n=24-162 PLMs.

View larger version (32K): [in a new window]   Fig. 3. wnt and vps-35 mutants have ALM polarity defects. (A) Wild-type mec-4::gfp; ALM has a long anterior process. (B) cwn-1; cwn-2; ALM with a symmetric polarity. Arrows indicate the long anterior and posterior processes. (C) vps-35; ALM with a reversed polarity, PVM migrates anteriorly rather than posteriorly and is found near ALM (see text for details). (D) ALM and PLM cell bodies and process trajectories; boxed area indicates region shown in fluorescence photomicrographs. Anterior is leftwards; dorsal is towards the top.

View larger version (50K): [in a new window]   Fig. 4. wnt mutants have axon guidance and branching defects. (A) Wild type; PVM process enters VNC and turns anteriorly (ventral view). Arrows indicate PVM process, triangles label PLM process. (B) cwn-1; egl-20; PVM process enters the VNC and turns posteriorly. (C) ALM, PLM, AVM and PVM cell bodies and process trajectories. AVM is on the right-hand side; PVM is on the left-hand side. Boxed areas show regions depicted in fluorescence photomicrographs. (D) Wild type; ALM and AVM processes form branches at their distal ends that make contact within the nerve ring. Arrows indicate AVM nerve ring branch and anterior extension past nerve ring. (E) cwn-1; cwn-2; ALM and AVM fail to form nerve ring branches. AVM process also stops short. Arrow labels termination site of AVM process. Anterior is leftwards; dorsal is towards the top, unless indicated. (F) Phenotypes of AVM and PVM processes in wild type and cwn-1; egl-20 double mutants. Because of overlap with AVM process in anterior body, we were often unable to score the precise end point of the anteriorly directed PVM process; nonetheless, the PVM process was typically shorter in cwn-1; egl-20 double mutants than in wild type.

View larger version (8K): [in a new window]   Fig. 5. Molecular analysis of vps-35. (A) Schematic of vps-35 genomic region on chromosome II (LGII). Arrows depict hypothetical gene transcripts and direction of transcription (www.wormbase.org). Cosmid clone F59G1 (2/2 lines) and a 5.6 kb PstI-MscI genomic fragment (3/3 lines) (shown) rescued vps-35 mutants. Based on rescue and expression studies, vps-35 is likely to be the first gene in a multigene operon rather than tsp-18, as indicated on WormBase Release WS150. (B) vps-35 gene structure is based on cDNA and genomic sequences. vps-35 transcript is trans-spliced to SL1. Our results confirm essential features of vps-35 (F59G1.3) (WormBase Release WS150), except that the initiation ATG is at position 5907784 rather than 5907868. The deletion in vps-35(zd163) is marked. Black boxes indicate exons and the white box depicts the 3' UTR.

View larger version (56K): [in a new window]   Fig. 6. VPS-35 expression and localization. (A) Wild-type mab-5::gfp; GFP expression is seen in QL descendants QL.a and QL.p in L1. (B) vps-26; mab-5::gfp expression is not detected in QL descendants. Positions of cells were determined by DIC microscopy (denoted by arrows). (C) vps-35::gfp; GFP expression in embryo. (D) vps-35::gfp; GFP expression in tail epidermis of a young larva. (E) Golgi marker mannosidase-YFP (MANS-YFP) expression in tail epidermis using the lin-44 promoter. (F) VPS-35-mCherry expression in tail epidermis. (G) Merged image of E and F. A-D, anterior is leftwards; E-G, anterior is rightwards; dorsal is towards the top.

View larger version (12K): [in a new window]   Fig. 7. Model of PLM polarity regulation by LIN-44. (A) Schematic of embryo showing positions of PLM neuron (green), lin-44-expressing tail epidermal cells (red), and egl-20-expressing epidermal and muscle cells (blue). Based on their expression patterns, hypothetical gradients of EGL-20 and LIN-44 are presented for wild type and lin-44 mutants. Axonal structure of PLM is shown. A high level of LIN-44 is needed to induce correct PLM polarity. (B) The PLM/ALN precursor divides along the AP body axis to produce the anterior daughter PLM and posterior daughter ALN (a, anterior; p, posterior). The centrosomes of PLM and ALN (red spots) are on opposite poles after cell division. In response to LIN-44 signaling via LIN-17, the PLM centrosome moves to the posterior pole and PLM adopts the correct AP polarity. In the absence of LIN-44 or LIN-17, no centrosome movement occurs and PLM polarity is reversed. Axonal structures of PLM and ALN are shown.

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