The Atypical Rho GTPase CHW-1 Works with SAX-3/Robo To Mediate Axon Guidance in Caenorhabditis elegans

Abstract

During development, neuronal cells extend an axon toward their target destination in response to a cue to form a properly functioning nervous system. Rho proteins, Ras-related small GTPases that regulate cytoskeletal organization and dynamics, cell adhesion, and motility, are known to regulate axon guidance. Despite extensive knowledge about canonical Rho proteins (RhoA/Rac1/Cdc42), little is known about the Caenorhabditis elegans (C. elegans) atypical Cdc42-like family members CHW-1 and CRP-1 in regards to axon pathfinding and neuronal migration. chw-1(Chp/Wrch) encodes a protein that resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and crp-1 encodes for a protein that resembles TC10 and TCL. Here, we show that chw-1 works redundantly with crp-1 and cdc-42 in axon guidance. Furthermore, proper levels of chw-1 expression and activity are required for proper axon guidance. When examining CHW-1 GTPase mutants, we found that the native CHW-1 protein is likely partially activated, and mutations at a conserved residue (position 12 using Ras numbering, position 18 in CHW-1) alter axon guidance and neural migration. Additionally, we showed that chw-1 genetically interacts with the guidance receptor sax-3 in PDE neurons. Finally, in VD/DD motor neurons, chw-1 works downstream of sax-3 to control axon guidance. In summary, this is the first study implicating the atypical Rho GTPases chw-1 and crp-1 in axon guidance. Furthermore, this is the first evidence of genetic interaction between chw-1 and the guidance receptor sax-3 These data suggest that chw-1 is likely acting downstream and/or in parallel to sax-3 in axon guidance.

Keywords: CDC-42; CHW-1; CRP-1; SAX-3/Robo; axon pathfinding.

Figure 1

CHW-1 is similar to Wrch-1/RhoU and Chp/Wrch-2/RhoV. Sequence alignment of CHW-1, human Wrch-1, and human Chp done in biology workbench. Identical residues are marked with an asterisk (*). B) CHW-1 lacks the N-terminal extension found in Wrch-1 and Chp (light gray box). CHW-1 also contains an atypical residue (alanine instead of glycine) at position 18 (analogous to position 12 in Cdc42 and most Rho and Ras family members).

Figure 2

chw-1 works redundantly with cdc-42 and crp-1 in axon guidance. Quantitation of PDE defects. lqIs2 is the osm-6::gfp control transgene. At least 100 neurons were scored for each genotype. P < 0.0001 as determined by Fisher Exact Analysis (Graphpad). The error bars represent the standard proportion of the mean.

Figure 3

Proper expression and activity levels of CHW-1 are required for axon guidance. A) Quantitation of PDE defects. lqIs2 is the osm-6::gfp control transgene. At least 100 neurons were scored for each genotype. P < 0.0001 as determined by Fisher Exact Analysis (Graphpad). The error bars represent the standard proportion of the mean. B) A micrograph of a PDE neuron of a WT adult animal (left panel) and a PDE neuron of an adult animal expressing CHW-1(A18G) (right panel). The white lines indicate the outline of the worm. The scale bar represents 5 μm.

Figure 4

Expression of CHW-1(A18V) results in the formation of ectopic lamellipodia in PDE neurons. A) Quantitation of PDE defects. osm-6::gfp is the control transgene. At least 100 neurons were scored for each genotype. P < 0.0001 as determined by Fisher Exact Analysis (Graphpad). The error bars represent the standard proportion of the mean. B) A micrograph of a PDE neuron of a WT adult animal (left panel) and a PDE neuron of an adult animal expressing CHW-1(A18V) (right panel). The white arrow is pointing to the lamellipodia protrusion. The scale bar represents 5 μm.

Figure 5

Expression of CHW-1(A18G) results in amphid neuron defects. A) Quantitation of amphid neuron defects. lqIs2 is the osm-6::gfp control transgene. The Y-axis denotes the genotype and the X-axis represents the percentage of amphid neuron defects. At least 100 neurons were scored for each genotype. P < 0.0001 as determined by Fisher Exact Analysis (Graphpad). The error bars represent the standard proportion of the mean. B) A micrograph of the amphid neurons of a WT adult animal (left panel), and the amphid neurons of adult animals expressing CHW-1(A18G) (right two panels). The wild-type position of the amphid neuron cell bodies is indicated by the white arrow and the mutant positions are indicated by the yellow arrows. The scale bar represents 10 μm.

Figure 6

Loss of both chw-1 and sax-3, but not other axon guidance molecules, synergistically increases amphid neuron defects. Quantitation of amphid neuron defects. lqIs2 is the osm-6::gfp control transgene. The Y-axis denotes the genotype and the X-axis represents the percentage of amphid neuron defects. At least 100 neurons were scored for each genotype. P < 0.0001 as determined by Fisher Exact Analysis (Graphpad). The error bars represent the standard proportion of the mean.

Figure 7

Loss of both chw-1 and sax-3 increases PDE axon pathfinding defects. Quantitation of PDE defects. lqIs2 is the osm-6::gfp control transgene. The Y-axis denotes the genotype and the X-axis represents the percentage of ectopic lamellipodia formation. At least 100 neurons were scored for each genotype. *P < 0.0001 as determined by Fisher Exact Analysis. The error bars represent the standard proportion of the mean.

Figure 8

Loss of chw-1 but not crp-1 attenuates the defects in the VD/DD motor neurons driven by activated SAX-3. A) A representation of the MYR-SAX-3 construct. B) A micrograph of the VD/DD neurons of a WT adult animal (top panel), and the VD/DD neurons of an adult animal expressing MYR-SAX-3 (bottom panel). C) Quantitation of VD/DD defects. unc-25::myr-sax-3 is the activated SAX-3 receptor driven by the unc-25 promoter, which drives expression in the VD/DD motor neurons. The Y-axis denotes the genotype and the X-axis represents the percentage of ectopic lamellipodia formation. The number of axons scored was greater than 100. *P < 0.0004 as determined by Fisher Exact Analysis. The error bars represent the standard proportion of the mean. The scale bar represents 20 μm.