A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans

Abstract

Introduction: UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance. Methods: Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on unc-5 in growth cone polarity and protrusion. Results: Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion. Discussion: These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.

Keywords: UNC-6/Netrin; axon guidance; growth cone; polarity/protrusion; unc-5 netrin receptor B (UNC5B).

FIGURE 1

Gene structure of unc-5 with isoforms and mutant alleles. (A) The VD and DD motor neurons. Anterior is to the left, and dorsal is up. Black circles and lines indicate DD neurons and axons, and green circles and lines indicate VD neurons and axons. The DD axons grow during embryogenesis. The VD axons extend in the early L2 larva (depicted here) in an anterior-to-posterior temporal gradient (VD1 extends before VD2, and so on). Commissural growth cones of VD6, VD7, and VD8 are shown. unc-6 expression is indicated by the red bar. (B) Gene structure of unc-5 (Wormbase 287) showing five isoforms, A, C, D, F are long isoforms and B is short isoform. The positions of the strong alleles affecting all isoforms (e53, e791, and e553) and the hypomorphic alleles affecting only the long isoforms (ev480, op468, and e152) are indicated on the expanded figure of the 3′ end on unc-5. E152 affects all isoforms but behaves as a hypomorph. The asterisk (*) indicates unc-5(lq190) a precise deletion of intron 7 predicted to abolish the unc-5B short isoform. (C) The sequence of the exon7-intron 7-exon eight region. The C-terminus of the unc-5B short isoform is indicted in intron 7. (D) RNA seq reads in the exon7-intron 7-exon eight region from mixed stage wild-type animals is shown (from the Integrated Genome Browser). 5′ to 3′ is right to left. Reads representing the unc-5B short isoform are indicated with asterisks.

FIGURE 2

Domain structure of UNC-5A and UNC-5B. (A) The modular organization of UNC-5A and UNC-5B isoforms is shown. Domains include extracellular immunoglobulin domains (Ig) and thrombospondin domains (TSP), a transmembrane region (TM), and cytoplasmic ZU5, UPA, and DEATH domains. The position of Y482 in the cytoplasmic juxtamembrane region is indicated. A depiction of the myristoylated (MYR) version of the UNC-5B short cytoplasmic domain is shown. Positions of unc-5 mutations are shown. (B) Graphic summaries of protein alignments from BLAST are shown for UNC-5B from Caenorhabditis elegans and UNC-5A X4 from H. sapiens. The truncated ZU5 domain at the C terminus of each is indicated.

FIGURE 3

VD/DD axon guidance defects of mutants. Fluorescent micrographs of the Punc-25::gfp transgene juIs76 expressed in the VD/DD neurons of L4 animals in different genotypes are shown. Dorsal is up and anterior left. The approximate lateral midline is indicated with a dotted white line, and the dorsal nerve cord by a dashed white line. White arrows indicate axon guidance defects in each genotype. (A) wild-type. (B) unc-5(e791). (C) unc-5(ev4980). (D) unc-5(e152). (E) unc-5A(+) transgenic expression. (F) unc-5B(+) transgenic expression. (G) unc-5(ev480) with unc-5A(+) transgenic expression. (H) unc-5(ev480) with unc-5B(+) transgenic expression. (I) unc-5(e791) with unc-5A(+) transgenic expression. (J) unc-5(e791) with unc-5B(+) transgenic expression. (K) unc-5(lq190). (L) unc-40(n324). (M) unc-5(lq190); unc-40(n324). (N) unc-5(ev480); unc-40(n324). Scale bar in (A) represents 10 μm. Genotypes are indicated in each figure panel.

FIGURE 4

VD growth cone morphology in unc-5 mutants. At least 50 growth cones were scored for each genotype. In the graphs, each point represents a measurement of a single growth cone or filopodium. (A,B) Quantification of VD growth cone area and filopodial length (see Materials and Methods). (A) Area of growth cone, in μm². (B) Filopodial length, in μm. Error bars indicate standard error of the mean. Two-sided t-tests with unequal variance were used to determine the significance between wild-type and mutants. Single asterisks (*) indicate significance at p < 0.05. Double asterisks (**) indicate significance at p < 0.001. (C) A graph showing the percent of dorsally directed filopodial protrusions in VD growth cones of different genotypes (see Materials and Methods). The X-axis is set at 50%, such that bars extending above the X-axis represents above 50%, and bars that extends below represents below 50%. In wild-type, a majority of filopodia (78%) extended from the dorsal half of the growth cone. Significance between wild-type and mutants was determined by Fisher’s exact test. Error bar represents 2x standard error of proportion. 50 growth cones were scored. Single asterisks (*) indicates the significant p < 0.05 double asterisks (**) indicate significant p < 0.001. (D–G) Fluorescence micrographs of wild-type and mutant VD growth cones expressing Punc-25::gfp. Arrows point to filopodial protrusions. Dorsal is up; anterior is left. The scale bar in (D) represents 5 μm. Below each figure is a depiction of the growth cone with the dorsal and ventral regions separated by a dotted line.

FIGURE 5

F-actin dorsal accumulation and microtubule + end distribution in VD growth cones. In all images, dorsal is up and anterior is left. Dashed lines indicate the growth cone perimeter. Scale bars represent 5 μm. (A) mCherry growth cone volume marker of a wild-type VD growth cone. (A’) VAB-10ABD::GFP accumulation at the dorsal edge of a wild-type VD growth cone (arrows) (see Materials and Methods). (A’’) Merge. ((B), B’, B’’) As in A, for an unc-5(ev480) growth cone. ((C), C’, C’’) As in A, for an unc-5(lq190) growth cone. (D) A graph of the dorsal/ventral ratio of GFP/mCherry from multiple growth cones (≥15) in wild-type and mutant animals expressing VAB- 10ABD::GFP and mCherry (a volumetric marker) as described previously (Norris and Lundquist, 2011) (see Materials and Methods). Error bars represent the standard error of the mean of the ratios from different growth cones. Growth cones were divided into dorsal and ventral halves, and the average intensity ratio of VAB-10ABD::GFP/mCherry was determined for each half and represented. Asterisks (*) indicate the significance of difference between wild-type and the mutant phenotype (*p < 0.05) (two-tailed t-test with unequal variance between the ratios of multiple growth cones of each genotype). (E) Box-and-whiskers plot of the number of EBP-2::GFP puncta in the growth cones of different genotypes (≥25 growth cones for each genotype). The boxes represent the upper and lower quartiles, and error bars represent the upper and lower extreme values. Asterisks (*) indicate the significant difference between wild-type and the mutant phenotype (*p < 0.05, **p < 0.001) determined by two-sided t-test with unequal variance. n.s., not significant. (F–H) Fluorescence micrographs of EBP-2 distribution in the VD growth cones of indicated genotypes. Dashed lines indicate the growth cone perimeter. Dorsal is up and anterior is left. Scale bar: 5 μm.

FIGURE 6

Growth cone protrusion and polarity in the short-isoform-specific unc- 5(lq190) mutant. (A–C) Quantification of growth cone area, filopodial length, and dorsal polarity of filopodial protrusion as described in Figure 4. (D–F) Representative images of growth cones as described in Figure 4. Arrows point to filopodia.

FIGURE 7

MYR::UNC-5B does not affect VD growth cone morphology. (A–C) Quantification of growth cone area, filopodial length, and dorsal polarity of filopodial protrusion as described in Figure 4. (D,E) Representative images of growth cones as described in Figure 4. Arrows point to filopodia.

FIGURE 8

VD growth cone morphology in unc-5 hypomorphic mutants and interactions with unc-40 and unc-6. (A–C) Quantification of growth cone area, filopodial length, and dorsal polarity of filopodial protrusion as described in Figure 4. (D–G) Representative images of growth cones as described in Figure 4. Arrows point to filopodia.

FIGURE 9

Effects of transgenic expression of UNC-5A and UNC-5B on VD growth cones. (A–C) Quantification of growth cone area, filopodial length, and dorsal polarity of filopodial protrusion as described in Figure 4. (D–K) Representative images of growth cones as described in Figure 4. Arrows point to filopodia. The scale bar in (A) represents 5 μm.

FIGURE 10

Regulation of growth cone protrusion by UNC-6/Netrin and its receptors. (A) UNC-6/netrin inhibits VD growth cone protrusion through UNC-5 homodimers and UNC-5:UNC-40 heterodimers. (B) UNC-6/Netrin stimulates growth cone protrusion via UNC-40 homodimers. Work described here shows that UNC- 6/Netrin acts in parallel to the novel UNC-5B short isoform to stimulate growth cone protrusion in an UNC-40-independent manner.