A Wnt-Frz/Ror-Dsh pathway regulates neurite outgrowth in Caenorhabditis elegans

Abstract

One of the challenges to understand the organization of the nervous system has been to determine how axon guidance molecules govern axon outgrowth. Through an unbiased genetic screen, we identified a conserved Wnt pathway which is crucial for anterior-posterior (A/P) outgrowth of neurites from RME head motor neurons in Caenorhabditis elegans. The pathway is composed of the Wnt ligand CWN-2, the Frizzled receptors CFZ-2 and MIG-1, the co-receptor CAM-1/Ror, and the downstream component Dishevelled/DSH-1. Among these, CWN-2 acts as a local attractive cue for neurite outgrowth, and its activity can be partially substituted with other Wnts, suggesting that spatial distribution plays a role in the functional specificity of Wnts. As a co-receptor, CAM-1 functions cell-autonomously in neurons and, together with CFZ-2 and MIG-1, transmits the Wnt signal to downstream effectors. Yeast two-hybrid screening identified DSH-1 as a binding partner for CAM-1, indicating that CAM-1 could facilitate CWN-2/Wnt signaling by its physical association with DSH-1. Our study reveals an important role of a Wnt-Frz/Ror-Dsh pathway in regulating neurite A/P outgrowth.

Figure 1. GABAergic RME neurons.

(A) A GFP fluorescence image of an unc-30(ju54);juIs76 animal. juIs76 (Punc-25::GFP) highlights the RME GABAergic neurons in unc-30(ju54) mutant animals. The insert shows a schematic drawing of all four RME neurons. Anterior is to the left and dorsal is up. (B) RME neurons at different developmental stages, including a: comma stage; b: two-fold stage; c: three-fold stage, viewed with the juIs76 marker. a, b, and c are DIC images; a′, b′, and c′ are fluorescence images. RMED/V neurons start sending out their posterior processes during the late embryonic stage (c and c′), and continue to grow posteriorly during larval stages (d and e). (C) The relative length of RMED and REMV posterior neurites at different developmental stages. Relative neurite length is defined as the ratio of neurite length to body length. Error bars represent the standard error of the mean (SEM).

Figure 2. Mutants with RMED/V neurite outgrowth defect isolated from the genetic screen.

(A) RME neuron fluorescence images of animals with different genetic backgrounds. unc-30(ju54);juIs76 is treated as wild type (WT). RMED/V posterior neurite outgrowth is unaffected in the D/V guidance cue mutants unc-6(ev400) and slt-1(eh15) and the vulvaless mutant lin-11(n389). (B) Quantification analysis of mutant phenotypes shown in (A). The average relative neurite length in wild type is set as 100. Error bars represent SEM. (C) Phenotypes and list of the three classes of mutants isolated from the genetic screen. Most Class I mutant animals lack both RMED and RMEV processes. Class II mutants display both shortened and normal length of neurites. In Class III mutants, both RMED and RMEV processes are short. Asterisk indicates the end of the process.

Figure 3. cwn-2 regulates RMED/V neurite A/P outgrowth.

(A) xd1 is a C138Y missense mutation in the cwn-2 locus. Black boxes are exons and grey boxes are UTRs. (B) Phenotypic quantification of RMED neurite A/P outgrowth defect in Wnt pathway receptor and ligand mutants. The average relative neurite length in wild type (unc-30;juIs76) is set as 100. Error bars represent SEM. Note that mig-1(e1787);cfz-2(ok1201) double mutants mimic cwn-2(xd1). (C) Pcwn-2::mCherry expression pattern (Red) in different developmental stages. Green is RMED/V neurons highlighted by juIs76 marker. Top panels: fluorescence and bright field images of embryos with Pcwn-2::mCherry. In a 2-fold stage embryo, cwn-2 is mainly expressed in the intestine (arrow). The highest level of mCherry signal is observed in the posterior pharyngeal bulb and the pharyngeal-intestine valve before hatching (arrowhead). After L1 stage, Pcwn-2::mCherry is expressed in the pharynx, body wall muscles and some ventral cord neurons. Asterisks point to the tips of RMED/V neurites. (D) Quantification analysis of the rescue activity of the cwn-2 genomic fragment in cwn-2(xd1). The length of both RMED and RMEV were compared to wild-type controls (unc-30;juIs76). The extent of RMED and RMEV extension was classified into three categories: “WT” stands for wild-type length in both RMED and RMEV neurites; “no D, V” stands for absence of both RMED and RMEV neurites (Class I phenotype); and “short” indicates an intermediate phenotype between “WT” and “No D, V” (including shorter processes and absence of either RMED or RMEV process). Results from two independent transgenic lines are presented.

Figure 4. CWN-2 functions as an attractive cue.

(A) Quantification analysis showing that other Wnts when under control of the cwn-2 promoter can partially rescue cwn-2(xd1) mutant phenotype. Results from two independent transgenic lines are presented. The DNA injection concentration is 20 ng/µl. (B) CWN-2 expression, driven by different promoters, causes different levels of rescuing activity. Posterior extension and anterior growth of RMED and RMEV were evaluated separately. Schematic diagrams of the different posterior and anterior neurite phenotypes are shown at the top. The classification of the posterior phenotypes is the same as for the rescue assays in Figure 3. The color coding in the schematic drawing of the head (left) represents the expression patterns of different promoters. The expression level is relatively higher in the brown regions than in the red regions based on transcriptional mCherry assay. For the lin-11 and egl-17 promoters, the distance from the vulva (indicated by a red oval) to the head is not proportional. Expression of CWN-2 under control of the egl-17 promoter is able to trigger RMED anterior neurite extension (*). (C) Phenotype of anterior extension (arrows) in wild type, cwn-2(xd1) mutants and Pegl-17::CWN-2-rescued cwn-2(xd1) mutants.

Figure 5. cam-1 regulates RMED/V neurite A/P guidance.

(A) RME neuron fluorescence images of animals with different genetic backgrounds. The cam-1 partial loss-of-function mutant xd13 shows a variable phenotype and the null mutant gm122 has a cwn-2 like phenotype. (B) Molecular lesions in the cam-1 mutants xd22, gm122, gm105, ks52 and xd13. The color-coded domain structure of cam-1 is shown. Ig, immunoglobulin domain; CRD, cysteine rich domain; Kr, kringle domain; TM, transmembrane domain; Kinase, kinase domain; S/T, serine and theronine-rich domain. Three cam-1 isoforms are indicated. (C) Quantification of the rescuing activity of cam-1 transgenes. (D) Quantification of the RMEV neurite A/P outgrowth defect in different mutants. cam-1 functions synergistically with cfz-2 and mig-1. The average relative neurite length in wild type is set as 100. Error bars represent SEM. (E) cam-1 is expressed in RME neurons (arrow).

Figure 6. DSH-1 and a conserved Wnt pathway regulate RMED/V neurite outgrowth.

(A) Fluorescence images of RMED/V neurons in various Wnt mutant animals. cwn-2(xd1), dsh-1(xd5) and mig-1(e1787);cfz-2(ok1201) double mutant exhibit the same phenotype. (B) Molecular lesion in the dsh-1 mutant xd5. The domain structure of dsh-1 is shown. DAX: domain present in Dishevelled and axin; PDZ: PSD-95, Dlg, and ZO-1/2 domain; DEP: Dishevelled, Egl-10, and Pleckstrin domain. Two DSH-1 isoforms are indicated. xd5 causes a small deletion. (C) Quantification of the rescuing activity of dsh-1 transgenes. Expression of dsh-1 in RMED/V neurons can rescue dsh-1(xd5) mutant phenotype. (D) Quantification of the RMED neurite A/P outgrowth defect in different mutants, including components of the three different downstream pathways of Wnt and factors affecting the actin-microtubulin cytoskeleton.

Figure 7. The CAM-1 intracellular domain interacts with DSH-1.

(A) The PDZ and DEP domains are important for the binding of DSH-1 to CAM-1 intracellular domain (ICD). Empty vector (Vector) was used as a control. (B) The kinase domain and the region before the kinase domain are crucial for DSH-1 interacting. (C) MIG-1 ICD and CFZ-2 ICD do not interact with CAM-1 or DSH-1.