RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport

The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.


Introduction
There is great diversity in the structure and complexity of dendritic arbors across neuron types, and establishing the correct dendritic morphology is critical for the proper connectivity and function of neural circuits. A developing dendritic arbor must target a specific receptive field, adopt the appropriate neuron-specific architecture, and avoid overlapping in connectivity with itself and neighboring dendrites. A number of extrinsic cues and intrinsic mechanisms help orchestrate the formation of these complex neuronal morphologies, including transcriptional programs, extracellular guidance cues, and contact-dependent repulsive molecules that mediate self-avoidance [1][2][3][4][5].
Dendritic arbor development requires tremendous cellular growth and likely has specialized membrane trafficking demands. Little is known about how the transport of branching factors and membrane components is coordinated across a large, polarized neuron. Dendrites are more sensitive than axons in their reliance on the membrane supply from secretory pathways [2,6], and they have distinct transport needs. For example, a set of dendritic arbor reduction (dar) genes have been identified which are required for dendritic arbor outgrowth but not axon growth. These dar genes are important for ER-to-Golgi transport [6]. In addition, the Rab GTPases, a conserved family of small GTPase proteins that regulate membrane identity and vesicle trafficking [7][8][9], are likely important for the polarization and outgrowth of neurites, though their precise role in both axonal and dendritic development remains unclear [10].
One of these small GTPases, Rab10, has been shown to mediate membrane trafficking in several polarized cell types, including neurons [11][12][13][14][15][16][17][18]. The importance of Rab10 for endosomal sorting and endocytic recycling has been demonstrated in Drosophila epithelial cells [15] as well as in C. elegans neurons [16][17] and intestinal epithelial cells [11][12][13][14]. In hippocampal neurons, Rab10 is required for directional membrane trafficking in the growing axon [19][20], and it has been shown to directly bind the anterograde motor kinesin1 via the adaptor protein JIP1 [21]. In addition to sorting and trafficking cargo to the appropriate destinations, a growing neurite must appropriately dock and release membrane and protein cargoes. The docking of Rab10-postive vesicles is required for axon outgrowth in hippocampal neurons [22]. Rab10 has also been linked to the exocyst, a secretory complex responsible for polarized exocytosis [23][24][25].
The exocyst is an octameric complex that was initially identified in yeast for its role in membrane addition during bud outgrowth, and it has since been shown to have conserved functions in polarized growth across several cell types [25][26][27][28]. In Drosophila, the sec5 component of the exocyst complex is required to establish polarity in the oocyte [26], and the sec15 component is also required for the polarized delivery of photoreceptors [28]. Both Rab10 and the exocyst complex are required for branch outgrowth in the Drosophila trachea [25]. Taken together, these data suggest that both the exocyst complex and RAB-10 support outgrowth in polarized cells; however, the roles of RAB-10 and the exocyst have never been examined in a growing dendrite.
In Caenorhabditis elegans, the PVD sensory neuron forms elaborate dendritic arbors that are organized into orthogonal tiers of characteristic menorah-shaped units [29][30]. The PVD neuron has proved to be useful in the study of the molecular mechanisms of dendrite morphogenesis. Importantly, the dendritic branches of PVD maintain self-avoidance [31], a key feature across neural networks. Extracellular cues are essential for PVD dendritic morphogenesis: the branching receptor DMA-1, expressed in PVD, instructs spatially-restricted growth of dendritic branches via interaction with the SAX-7/MNR-1 adhesion complex [32][33][34]. Previous work has also identified various intracellular processes important for PVD dendritic morphology, including transcription factors that instruct cell fate [29,35], microtubule-associated proteins [36], and membrane fusion proteins that prevent over-branching [37]. However, the role of polarized membrane trafficking in the growing PVD dendrite remains uncharacterized.
In this work, we identified a novel role for the small GTPase RAB-10 in patterning the dendritic arbor of PVD. We demonstrate that both RAB-10 and the exocyst complex are required for proper outgrowth and patterning of the PVD dendritic arbor. RAB-10 and EXOC-8 are also required for the appropriate localization of branching factors such as DMA-1. Additionally, we found that the molecular motors kinesin and dynein are important for anterior-posterior patterning of the PVD dendritic arbor. The PVD dendritic outgrowth phenotype was also observed in a recently published report [38], and our work further identifies a role for RAB-10 in dendritic patterning beyond its role in promoting outgrowth. We propose that RAB-10 regulates dendrite morphogenesis by balancing anterograde and retrograde transport via molecular motors.

wy787 disrupts dendritic arbor morphology
We visualized the morphology of the PVD neurons by expressing membrane-bound-GFP under the control of a cell-specific promoter (ser2prom3:myrGFP) (Fig 1A and 1B) [29]. In wild-type animals, one anterior and one posterior primary dendritic branch extend from the cell body during the L2 larval stage. Dense menorahs consisting of a series of orthogonal 2°, 3°, and 4°branches emerge from the anterior and posterior 1°dendrites as the animal progresses through subsequent larval stages (L2-L4) (Fig 1A, 1B and 1E). To understand the molecular mechanisms of dendritic branching, we performed a forward genetic screen to identify mutations that affect the dendritic morphology of PVD. From this screen, we isolated individuals with the fully penetrant and recessive mutant allele wy787, which show a severe reduction in the number of dendritic branches (Fig 1C and 1D). wy787 mutant animals also have several non-neuronal phenotypes, including intestinal vacuoles and reduced brood size. In wy787 mutants, the number of secondary branches is reduced from 42 ± 6 in wild-type to 28 ± 9 in wy787, and the number of quaternary branches is reduced from 114 ± 15 to 17 ± 10 (S1 Table). In addition to the reduction in branch number, we noticed a shift in branch distribution. In wy787 mutants, considerably more branches are found in the distal anterior region of the 1°d endrite, where wild-type animals typically grow few branches (Fig 1B and 1D). To quantify the distribution of menorah, we divided the entire PVD dendritic arbor into four regions: the region posterior to the cell body (designated as "-1") and three equal-length regions in the anterior dendrite (designated as "+1, +2 and +3"). We defined a "branch complexity" index to characterize the completeness of menorahs in each region. This branch complexity metric weights three characteristics equally: (1) the number of secondary branches, (2) the percentage of secondary branches that form a tertiary branch, (3) and the average number of quaternary branches per tertiary branch ( Fig 1G). In wy787 mutants, branch complexity is severely reduced in the +2, +1, and -1 regions but increased in the +3 region. (Fig 1C, 1D and 1H). The severe decrease in branch complexity in the +1 and -1 regions is the result of a near-total loss of 3°and 4°branches in the +1 and -1 regions (S1 Table). The increase in branch complexity in the anterior +3 region led us to hypothesize that the branching defects cannot be entirely explained by an overall lack of branching activity, but are instead the result of abnormal distribution of branching activity along the anterior-posterior axis.
The lack of branches in the posterior regions (+1 and -1) could be due to a defect in 2°b ranch outgrowth or stabilization. We distinguished between these possibilities by examining dendrite morphology during earlier developmental time points. In the L2 and L3 larval stages, wild-type and wy787 showed no significant difference in the number of secondary filopodia (Fig 1F), which suggests that wy787 could be defective in branch-stabilization. However, in wy787 the posterior 2°branches are often shorter than they are in wild-type and often do not grow in the correct orthogonal orientation. These defective branches cannot reach the tertiary branch-point (Fig 1C), a region enriched for the branch-promoting complex, MNR-1/SAX-7, that supports tertiary branch outgrowth [33]. By the L4 stage, wy787 had significantly fewer 2°b ranches, and those 2°branches that reached the tertiary line in the posterior regions (+1 and -1) failed to form 3°branches (Fig 1D and 1F). This developmental failure is consistent with a defect in both stabilization and outgrowth of 2°branches as well as both initiation and maintenance defects in 3°and 4°branches. This defective branching in the posterior regions is reminiscent of the phenotype of the branching receptor mutant dma-1 [32][33].

RAB-10 is required cell-autonomously for dendritic arbor morphology
We mapped and cloned the wy787 allele and identified the causal lesion as a mutation in the small GTPase RAB-10. wy787 contains a point mutation that results in a substitution of a conserved residue (A66V) in the GTP-binding domain of RAB-10 (S1 Fig) and can be rescued by rab-10 genomic DNA (S1 Fig) and rab-10 cDNA (Fig 2B). To confirm that RAB-10 is required for dendritic arbor growth, we also obtained the putative null allele ok1494, which has a 663 base pair deletion in the rab-10 coding region and should cause a complete loss of function of this gene (Fig 2E). This deletion allele recapitulated the PVD dendritic phenotype of wy787 with an even more severe reduction in posterior branch complexity and a more anterior shift in the +3 region compared with the wy787 allele ( Fig 2C).
To determine whether RAB-10 functions cell-autonomously within PVD to pattern the dendritic arbor, we expressed RAB-10 under the control of two PVD-specific promoters (ser2-prom3 and pdes2short) [30] in the wy787 and ok1494 mutant backgrounds. Expression of RAB-10 in PVD rescued the posterior reduction in branch complexity (Fig 2B, 2D, 2F and 2G), while muscle-specific expression of RAB-10 failed to rescue the dendritic phenotype of rab-10 mutants (S1 Fig). Taken together, these data suggest that RAB-10 functions cell-autonomously to regulate the dendritic branch distribution of PVD neurons.
The GTP-bound state of RAB-10 is required for dendritic arbor development RAB-10 is a member of the Rab family of GTP-binding proteins. These conserved proteins help to control membrane identity and regulate intracellular vesicle trafficking [7][8][9]. A typical Rab GTPase adopts two conformations: an active GTP-bound form and an inactive GDPbound form. Guanine nucleotide exchange factors (GEFs) help activate Rabs by catalyzing the exchange of GDP for GTP, and GTPase activating proteins (GAPs) accelerate the GTPase activity of Rabs [7]. The missense mutation in wy787, A66V, is two amino acids away from the GTP binding site of RAB-10 and likely interferes with the innate activity of RAB-10.
GTP-binding sites and GTPase sites are highly conserved across Rab GTPases in many species (S1 Fig), and it has been well established that these sites can be mutated to generate dominant-negative and constitutively active forms of Rab GTPases [11][12][13][14][17][18]20,23]. To assess whether the GTPase activity of RAB-10 is required for normal PVD development, we generated the constitutively active and inactive forms of RAB-10. The constitutively active mutant, Q68L, alters the GTPase site to prevent GTP-hydrolysis, thus locking RAB-10 in its active conformation. The inactive form, T23N, prevents GTP-binding and should result in an inactive RAB-10. Using a PVD-specific promoter, we expressed either the active or inactive form of RAB-10 in both wild-type and the rab-10 (ok1494) mutant background. In the wild-type background, the putative GDP-locked (T23N) of RAB-10 likely acts as a dominant negative. We observed a moderate reduction in posterior branch complexity in 30% of animals, and 1 in 50 animals have a branching phenotype indistinguishable from that of rab-10 (S1 Fig). Interestingly, overexpression of rab-10(GDP) also causes an anterior shift in branch complexity. This provides further support for the cell-autonomous action of RAB-10.
The putative GTP-locked form (Q68L) of RAB-10 rescued the dendritic morphology defect of rab-10 ( Fig 3A and 3C), though the rescue was not as robust as that of the wild-type form of RAB-10. The dominant-negative RAB-10(GDP) not only failed to rescue the dendritic defect but also further reduced branch complexity in the anterior region (Fig 3B and 3D). This is likely a dominant negative effect caused by sequestration of GEFs that interact with RAB-10 and one or more additional small G proteins. Together, these results support the notion that RAB-10 functions cell-autonomously as a GTPase to regulate PVD dendrite development. The DennD4 protein has been shown to act as the GEF for the mammalian RAB10 protein [39]. We asked whether the worm DennD4 homolog, denn-4, is also required for PVD morphogenesis. Since the existing denn-4 alleles result in early lethality, preventing us from studying PVD morphology, we used somatic CRISPR-Cas9 to generate a conditional denn-4 knockout ( Fig 4A). We used a heat shock promoter (Phsp16.2) to drive the expression of CAS9 and an sgRNA designed to target denn-4, a method that has been proven effective in generating somatic mosaics [40]. Phsp16.2 is active in many tissues, including neurons [41]. Indeed, knocking down denn-4 with this strategy caused a reduction of branch complexity in the -1 and +1 regions in 21% of animals ( Fig 4C-4E). While this phenotype is milder than the branch reduction seen in rab-10, it is qualitatively similar, suggesting that denn-4 acts as a GEF for RAB-10 and to promote dendrite morphogenesis. In order to establish the relationship between denn-4 and rab-10, we knocked down denn-4 using somatic CRISPR-Cas9 while simultaneously overexpressing RAB-10(GTP) in PVD. Overexpression of RAB-10(GTP) reduced the penetrance of denn-4 dendritic phenotypes to 6% (Fig 4D). Since Phsp16.2 likely knocks down denn-4 in many tissues, we cannot determine whether denn-4 functions cell- autonomously within PVD. However, suppression of the denn-4 phenotype by PVD-specific expression of RAB-10(GTP) supports the notion that denn-4 plays a role in dendritic development within PVD, and that denn-4 acts as a GEF of rab-10, as suggested by studies in non-  RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport neuronal cells [39]. Together, these data argue strongly that the GTPase activity of RAB-10 is required to regulate dendrite morphogenesis in PVD.
Mutations in the exocyst complex genes exoc-8 and sec-5 cause dendritic morphology defects similar to rab-10 RAB-10 has been shown to function with the exocyst complex in vesicle trafficking and docking [23][24][25]. Therefore, we tested whether rab-10 acts via the exocyst to regulate dendrite branch distribution in PVD. We examined PVD morphology in strains carrying mutations in each of several conserved exocyst genes, including exoc-8 and sec-5. The exoc-8(ok2523) allele is a 1474 base pair deletion and a putative null [24]. The sec-5(pk2358) allele introduces an early stop codon at position 389, and has been described as a non-null, strong loss-of-function allele [42]. We found that mutations in either exoc-8(ok2523) or sec-5(pk2358) resulted in a posterior loss of dendritic branch complexity, nearly phenocopying rab-10 mutants (Fig 5A  and S2 Fig). The reduction in secondary branches in the +2 region was less dramatic in the exoc-8(ok2523) mutant than in the rab-10 mutants (S1 Table), but significant reduction in branch complexity was observed in the +2, +1, and -1 regions (Fig 5A and 5F). sec-5(pk2358) displayed a less severe but qualitatively similar phenotype (S2 Fig). No significant 2°branch loss was observed in sec-5 (S1 Table), but 4°branches were reduced in the +2, +1, and -1 regions (S1 Table). To test whether the exocyst complex functions cell-autonomously to pattern the PVD dendrite, we expressed exoc-8 using a PVD specific promoter in the exoc-8 (ok2523) background. Expressing exoc-8 in PVD rescued the dendritic morphology defect ( Fig  5B and 5F), suggesting that exoc-8 also functions cell-autonomously.
The striking anterior shift of dendritic complexity observed in rab-10 was not observed in exoc-8(ok2523) or sec-5(pk2358) mutants ( Fig 5A and S2 Fig). While both rab-10 and the exocyst are required for branching in the normal +2,+1, and -1 regions, this indicates that rab-10's role in anterior-posterior patterning may not require the exocyst components. We investigated the relationship between rab-10 and the exocyst with two genetic interaction experiments. Since rab-10;exoc-8 animals do not survive [24], we overexpressed the dominant-negative RAB-10(GDP) in exoc-8 to assess the double mutant phenotype. Overexpression of RAB-10 (GDP) in exoc-8 further reduced branch complexity to rab-10 levels and also caused an anterior shift in branch complexity, phenocopying rab-10 ( Fig 5E). The exoc-8;RAB-10(GDP) phenotype was indistinguishable from that of rab-10 and not stronger than the rab-10 phenotype (Fig 5G), suggesting that rab-10 and the exocyst function in a common pathway. To further explore this genetic relationship, we overexpressed RAB-10(GTP) in the exoc-8 background. RAB-10(GTP) was able to rescue the +2, +1, and -1 reduction in branch complexity caused by the exoc-8 mutation (Fig 5C and 5F), suggesting that rab-10 acts downstream of exoc-8. Together these data demonstrate that both rab-10 and the exocyst act cell-autonomously to pattern the PVD dendrite. rab-10 likely has exoc-8 independent functions in anterior-posterior patterning, but these genetic interaction data indicate that rab-10 and the exocyst function in a common pathway to promote proximal dendritic growth.

DMA-1 localization is altered in rab-10 mutants
The lack of stabilized dendritic branches, despite secondary filopodia outgrowth, in the rab-10 mutants is similar to phenotypes found in the dma-1 mutants [32][33]. We have previously demonstrated that DMA-1 functions cell autonomously in PVD as the branching receptor that responds to extrinsic SAX-7/MNR-1 guidance cues to promote 3°and 4°branch formation as well as stabilization of 2°branches [33]. We observed that in dma-1 mutants, all 4°and most 3°b ranches are lost in all regions of PVD (S3 Fig and S1 Table). Since DMA-1 is a transmembrane protein and functions as a receptor on the plasma membrane, we hypothesized that rab-10 and exoc-8 might regulate the membrane localization of DMA-1.
To understand how RAB-10 and EXOC-8 regulate DMA-1 localization, we expressed a DMA-1::GFP fusion protein under the control of a PVD specific promoter in wild-type, exoc-8 and rab-10 animals. In wild-type animals, DMA-1::GFP displays a predominantly diffuse staining pattern throughout the entire PVD dendrite, which likely represents DMA-1 localization on the plasma membrane. DMA-1::GFP can also be observed as punctate structures that likely represent secretory and endocytic vesicles (Fig 6A). The majority of DMA-1 puncta co-localize with the late endosomal marker RAB-7, suggesting that they represent RAB-7-positive vesicles targeted for lysosomal degradation (S4 Fig). This co-localization is not disrupted in rab-10, but RAB-7 forms larger accumulations than the normal wild-type puncta in the primary dendrite of rab-10 (S4 Fig). A small portion of the DMA-1 puncta overlap with RAB-10 puncta (S4 Fig). In rab-10, the diffuse component of DMA-1::GFP appears absent from the posterior dendrite (+2, +1 and -1 regions) and the DMA-1::GFP puncta form larger accumulations than in wildtype, similar to the change seen in RAB-7 localization (Fig 6B and S3 Fig and S4 Fig). In the anterior (+3) region of PVD, where the full dendritic arbor forms normally, DMA-1::GFP localization appeared largely normal in the rab-10 mutant (Fig 6B). Since both exoc-8 and rab-10 are required for posterior dendritic growth, we also examined DMA-1::GFP localization in exoc-8. Similar defects in DMA-1::GFP localization were observed, with a decrease in diffusive staining in the +1 region and large accumulations of intracellular material in the primary and defective secondary branches. (Fig 6C).
To further examine this localization defect, we used fluorescence recovery after bleaching (FRAP) assays to examine the lateral mobility of DMA-1::GFP. We reasoned that the DMA-1 on the plasma membrane should be more diffusive compared with the DMA-1 trapped in intracellular membrane organelles. We focused on the primary dendrite in the +1 region because this region showed pronounced differences between wild-type and rab-10 in both DMA-1::GFP localization and dendritic arbor formation. Indeed, wild-type animals showed a more complete and faster recovery of fluorescence than in the rab-10 mutants (Fig 6D-6F), further supporting the notion that DMA-1 fails to be inserted into the plasma membrane in the posterior dendrite of the rab-10 mutants.
In dma-1 mutants, the PVD dendrite is almost completely devoid of 2°, 3°, and 4°branches [32][33][34] (S3 Fig), making the branching defect more severe than that of rab-10. This implies that DMA-1 function is not completely dependent on rab-10. Conversely, if rab-10 controls PVD dendrite morphogenesis only by regulating DMA-1, we would predict that the dma-1; rab-10 double mutant should phenocopy the dma-1 mutant. In the dma-1;rab-10 double mutant, 3°and 4°branches are lost in all regions of PVD, including the distal regions (S3 Fig  and S1 Table), and in this regard it is indistinguishable from the dma-1 single mutant. However, the double mutant has fewer 2°branches than either rab-10 or dma-1 alone (S3 Fig and  S1 Table). This further reduction in 2°branches suggests that rab-10 may regulate a larger cohort of proteins, which includes DMA-1, that are important for branching activity. To assess rab-10's general role in distributing membrane proteins, we also examined the exogenous membrane protein mcd8.3 in both wild-type and rab-10 animals and observed a similar phenomenon. More accumulations of large puncta, which likely represent intracellular protein, occurred in rab-10 than in wild-type, and the diffuse proportion was reduced. This defect was most notable in the incomplete secondary branches (S3 Fig). The normal subcellular localization of DMA-1 and excessive branches in the anterior dendrite of the rab-10 mutant argue strongly that RAB-10 is not essential for the function of DMA-1. Instead, RAB-10 is required for correctly distributing "branching activity" along the anterior-posterior axis of the primary dendrite. This branching activity includes both DMA-1 and other factors required for dendrite branching. Even in the wild-type animals, the branches are not evenly distributed across the entire 1°dendrite: the anterior +3 region contains far fewer branches compared with the +2 or +1 region, suggesting a regulated distribution of branching activity (Fig 1B and 1H). It is therefore plausible that RAB-10 is critical to establish this distribution.

Microtubule organization is uniform along the A-P axis of PVD
Since microtubule-based transport is essential for organelle and protein localization in large cells like PVD, we characterized the microtubule organization along the primary dendrite. Using the established EBP-2::GFP marker [43] to visualize the growing tips of microtubules in different regions of PVD, we found that microtubules in the anterior primary dendrite (+1, +2 and +3 regions) are predominantly oriented minus-end out with respect to the soma, with the minus end pointing to the distal anterior dendrite (Fig 7A and 7C). This is consistent with the literature that suggests most invertebrate dendrites adopt minus-end out microtubule organization [44][45][46]. Surprisingly, microtubules in the posterior dendrites (-1 region) are mostly plus-end out (Fig 7A and 7C), mimicking the organization of polarity in axons (Fig 7A and  7C). This microtubule organization dictates that a retrograde minus-end-directed motor, such as dynein, should move processively from the posterior-most dendrite to the anterior-most dendrite. Conversely, an anterograde plus-end-directed motor, such as kinesin-1, would move from the anterior-most dendrite to the posterior-most region.
We examined the PVD phenotype in dhc-1 and unc-116/kinesin-1 mutants to directly test if major molecular motor systems are involved in PVD dendrite branching, possibly by transporting vesicles carrying DMA-1 and other dendrite outgrowth factors into the developing dendrite. Similar to previous a report, we found that the dhc-1 mutant lacked dendritic branches specifically in the anterior region [36]. This is consistent with what we would expect based on microtubule organization in PVD, because dynein transports cargoes towards the anterior dendrite. Reduced dynein activity in the dhc-1 mutant might result in the specific reduction of branching activity in the distal anterior dendrite. Strikingly, consistent with our predictions, the posterior dendritic branches showed no reduction in complexity in the dhc-1 mutant (Fig 8B).
Next, we examined the unc-116/kinesin-1 mutant for PVD dendrite morphology defects. Similar to previous reports, we found that a partial-loss-of-function unc-116 allele (e2310) caused a severe lack of anterior branches [36]. This phenotype is especially pronounced in the distal anterior region (Fig 8C). Superficially, this result is not consistent with our model; UNC-116/kinesin-1 should traffic cargoes toward the posterior part of the cell. However, our previous experiments in the neuron DA9 showed that UNC-116 is required for establishing microtubule polarity in the dendrite in addition to its known function in trafficking organelle cargoes. In the unc-116 mutant, the DA9 dendrite adopts an axon-like "plus-end distal" organization, while the axonal microtubules are not affected [43]. To test if the unc-116 mutant also alters microtubule polarity in the PVD dendrite, we visualized EBP-2::GFP comets in anterior and posterior primary dendrites. Similar to the result seen in the DA9 dendrite, the microtubules in the anterior PVD dendrites are reversed in orientation compared to the wild-type controls: the microtubules of the anterior dendrite switch from minus-end distal in wild-type to predominantly plus-end distal in the unc-116 mutant (Fig 7B, 7C and 7D). Interestingly, posterior dendrites in the mutant maintain the plus-end distal orientation, similar to the wild-type control (Fig 7B, 7C and 7D). As a result of the polarity change in the partial-loss-of-function allele, kinesin/UNC-116 should traffic cargoes to the distal anterior and distal posterior dendrite, while dynein should traffic cargoes toward the cell body ( Fig 7D). Therefore, in the unc-116 mutants, both the reversal in microtubule polarity and the reduced activity of the kinesin/ UNC-116 motor might contribute to the dendritic phenotype. It is conceivable that the reduced kinesin activity, normal dynein activity, and plus-end distal microtubule orientation leads to a specific loss of branching cargo from the anterior distal dendrite.

RAB-10 regulates the dendritic trafficking of branching factors along the A-P axis
Together, these data demonstrate that microtubules in both anterior and posterior primary dendrite in wild-type animals are oriented such that the vast majority of minus-ends of microtubules point to the anterior end of the animal (Fig 7A). The dramatic anterior shift of dendritic arbor observed in rab-10 suggested that RAB-10 could be responsible for patterning the dendritic arbor by directing protein trafficking along the A-P axis. It has previously been demonstrated that vertebrate Rab10 interacts with kinesin-1 to regulate axonal vesicle transport in the growing axon of hippocampal neurons [21]. Based on the anterior bias in branch distribution observed in the rab-10 mutants, we hypothesized that RAB-10 promotes the transport of branching activity towards the plus ends of microtubules. To further test this hypothesis, we explored the genetic interaction between rab-10 and unc-116. The reversed polarity of microtubules in the anterior dendrite of unc-116 mutants provided a framework to test the potential cooperation between rab-10 and unc-116. If the anterior shift of PVD dendritic branches seen in rab-10 occurred as a result of a failure of plus-end directed trafficking, then we would expect the branching defect to be reversed by switching microtubule polarity: our hypothesis predicts that the double mutants should lack distal anterior dendrites but have branches in the anterior +1 and posterior -1 region. Indeed, we found that the double mutant showed reduced branch complexity in the +3 region compared with that of rab-10 single mutants (Fig 8E). In striking contrast to the rab-10 single mutants, which have their highest branch complexity in the anterior "+3" region, the double mutant has the highest branch complexity in the posterior "+1" and "-1" regions, where the microtubule minus-ends are directed (Fig 8E and 8G).
To further explore the relationship between rab-10 and the molecular motors, we examined the effect of knocking down dynein in the rab-10 background. In our model, the minus-enddirected dynein motor is predicted to be responsible for distributing branch activity to the distal anterior dendrite, and our hypothesis predicts that the loss of dynein would suppress the anterior-shifted branches of rab-10. We utilized the previously described somatic   (Figs 8B, 9A and 9D). The conditional dhc-1 knockout in the rab-10 background resulted in a complete loss of branches from the distal anterior region (Fig 9C and 9E) in 57% of animals, consistent with our hypothesis that dynein is required for distributing distal branching activity. These genetic interaction experiments between RAB-10/UNC-116 and RAB-10/DHC-1 provide compelling support for RAB-10's role in regulating motor-assisted transport of factors required for branching activity, and this suggests that RAB-10 is responsible for determining not just outgrowth, but also the distribution of branches.
We propose that RAB-10 plays dual functions in regulating dendritic branching. On one hand, it regulates dendritic trafficking of branching factors towards the plus-ends of microtubules along the A-P axis, likely through association with UNC-116/kinesin-1. The factors regulated by RAB-10 include the branching receptor DMA-1 and other unidentified membrane components. In addition, once the vesicles are delivered to the proper domain of the plasma membrane, RAB-10 is required to dock and fuse membrane vesicles through its interaction with the exocyst complex.

Discussion
Many intracellular processes must work together during the construction of an elaborately branched, polarized neuron. The polarity of neurites must be appropriately established to allow directional transport, and factors required for dendrite growth and branching must be correctly sorted into transport vesicles. Molecular motors then transport these cargoes to appropriate subcellular loci, where vesicle docking and membrane exocytosis ensue. Disrupting any of these processes could have profound effects on dendritic morphogenesis.
We identified novel roles for the small GTPase RAB-10 and the exocyst complex in dendrite morphogenesis of the C. elegans PVD neuron using genetic approaches. Additionally, we found that the molecular motors kinesin and dynein are important for anterior-posterior patterning of the PVD dendritic arbor, and demonstrated genetic interactions between both RAB-10/kinesin-1 and RAB-10/dynein in patterning the anterior-posterior distribution of branches. We propose a two-part role for RAB-10 in dendrite morphogenesis. First, RAB-10 acts to pattern the dendritic arbor by promoting the trafficking of branching factors toward the plus-ends of microtubules. Second, RAB-10 promotes dendritic outgrowth by mediating docking and exocytosis of protein cargoes.

Polarized trafficking underlies branch patterning in the PVD dendrite
Dramatic dendritic morphogenesis defects were observed in the posterior regions of PVD in the rab-10 mutant, which were also independently described in a recent report [38]. However, our detailed analysis of PVD morphology in rab-10 mutants revealed an additional anteriorposterior patterning defect that has not been described previously. While branches are lost from the posterior dendrite, more exuberant branching is observed in the anterior distal dendrite compared with wild-type dendrites. This anterior shift of the dendritic arbor indicates that RAB-10 is not merely responsible for branch outgrowth but also for regulating the distribution of branching activity along the dendrite, a process we hypothesized to be mediated by molecular motors. Indeed, we found dendrite morphology defects in both dynein and kinesin-1 mutants. Based on the MT polarity in each genotype and their corresponding dendritic morphology phenotypes, we propose that RAB-10 potentiates UNC-116/kinesin-1 mediated transport, which moves cargoes from the anterior to posterior dendrite. This model is further supported by the observed phenotype in the unc-116; rab-10 double mutant, which showed a suppression of the posterior branching defects seen in rab-10.
Our work indicates that PVD achieves its stereotyped dendritic branching pattern by balancing anterograde and retrograde transport of branching factors, which requires the correct establishment of microtubule polarity, the appropriate balance of motor activity, and the correct association of transport cargoes with these motors. We propose a model in which RAB-10 is required for this balance of motor activity. First, microtubule polarity in the anterior and posterior primary dendrites dictates that kinesins, such as UNC-116, traffic membrane organelles from the anterior to posterior dendrite. Dynein does the opposite. In wild-type animals, these two motors have a particular equilibrium of activity, which results in the "normal" distribution of branching factors. Second, the kinesin activity is slightly stronger than the dynein activity in PVD, which sets up a subtle posterior-anterior gradient with fewer branches in the distal anterior dendrite. Third, RAB-10 promotes kinesin-1 mediated trafficking. In the rab-10 mutants, this model predicts that kinesin-1-mediated transport of branching factors is reduced, and dynein activity dominates, resulting in a shift of the distribution of branching factors towards the anterior dendrite. This shift is dependent on microtubule polarity, as demonstrated by the unc-116; rab-10 double mutant, where microtubule polarity is reversed. In this mutant, dynein functions as a retrograde motor, which rescues dendritic branches in the posterior region.
We demonstrate a genetic interaction between RAB-10 and kinesin-1, and a physical interaction has been previously shown in hippocampal axons, where Rab10 mediates anterograde trafficking by associating with kinesin-1 and its adaptor protein JIP1 [21]. In PVD, RAB-10 may similarly regulate the balance of motor activity by directly activating UNC-116. Another possibility is that RAB-10's role in balancing motor activity arises from a preferential association of RAB-10-positive plasmalemmal precursor vesicles with kinesin over dynein or that RAB-10 is involved in the appropriate sorting of kinesin and dynein cargos. Additionally, it has previously been shown that reduction of either dynein or kinesin-1 activity in Drosophila sensory neurons results in the loss of distal dendrites, suggesting that across many species, balancing motor activity is necessary to distribute branching components across developing dendrites [47]. Future research will focus on further characterization of the relationship between RAB-10 and the molecular motors to establish the nature of the genetic interactions between RAB-10/UNC-116 and RAB-10/dynein in the growing PVD dendrite.

Docking and secretion of branching factors supports dendritic outgrowth
In addition to RAB-10's role in patterning the PVD dendrite, we have also demonstrated that RAB-10 is required for branch outgrowth and the subcellular localization of transmembrane proteins such as DMA-1. This suggests a second role for RAB-10, in which RAB-10 and the exocyst coordinate docking and secretion of membrane and protein cargoes.
RAB-10 has previously been shown to work in conjunction with the exocyst complex in both endocytic trafficking and tracheal outgrowth [23][24][25]. In the PVD dendrite, we found that the loss of two exocyst complex proteins, EXOC-8 and SEC-5, results in reduced dendritic complexity in the posterior regions of PVD, nearly phenocopying rab-10. We also found that the constitutively active RAB-10(GTP) was able to rescue the dendritic phenotype of exoc-8, suggesting that rab-10 and exoc-8 act in a common pathway to support dendritic branch formation. Additionally, we demonstrated that a transmembrane protein responsible for dendrite growth and branching, DMA-1, was disrupted in rab-10 mutants. These results suggest that RAB-10 and the exocyst complex play a role in the secretion of proteins that control dendritic outgrowth and branching. The requirement of RAB-10 and the exocyst for dendritic outgrowth and membrane protein localization were also described in a recent report [38], which adds independent support to our discovery that RAB-10 and the exocyst are required for dendritic branch morphogenesis. We therefore propose that RAB-10 and the exocyst act together to control dendritic outgrowth by supporting docking and exocytosis of protein and membrane cargoes at appropriate subcellular dendritic domains.
Taken together, our data provide support for new roles for the small GTPase RAB-10 in dendritic outgrowth and patterning. RAB-10 coordinates the distribution of branching factors along the A-P axis of the growing PVD dendrite, and in conjunction with the exocyst complex, RAB-10 allows for the docking and secretion of branching factors such as DMA-1. Our work provides novel insight into the regulation of branch distribution by RAB-10 and demonstrates that the regulation of dendritic trafficking by Rab GTPases is of critical importance in the establishment of complex dendritic arbors.

Isolation and mapping of the wy787 mutant allele
The wy787 allele was isolated from an F2 semiclonal screen of 3,000 haploid genomes in the PVD::myrGFP strain. Worms were mutagenized with 50mM ethyl methanesulfonate. The rab-10 locus was identified with SNP mapping, fosmid rescue, and sequencing.

Fluorescence microscopy
Images were captured in live animals using a Plan-Apochromat 63x/1.4 objective on a Zeiss LSM710 confocal microscope or using a Plan-Apochromat 63x/1.4 objective on a Zeiss Axio Observer Z1 microscope equipped with a Yokagawa spinning disk head. For still images, worms were immobilized using 10 mM Levamisole (Sigma-Aldrich) in M9 on 2% agarose pads. For FRAP imaging of DMA-1::GFP and time-lapse imaging of EBP-2::GFP, worms were soaked in 0.1% tricane/0.01% levamisole in M9 for 20 minutes and were then mounted on 5% agarose pads with 0.05μg of polysterene microspheres (Polysceinces). Video for EBP-2::GFP comet analysis was acquired at 8 frames per second.

Quantification of dendritic arbor
Images were straightened with the ImageJ software (US National Institutes of Health) using the primary dendrite as a reference. In L4 animals, the PVD dendrite was divided into four segments (three equal-length anterior segments, and one posterior segment) and the number of secondary and quaternary branches per segment was counted. Quaternary branches were assigned to a segment based upon the location of the secondary branch forming the base of the menorah from which the quaternary branch originated. Any protrusion from the primary dendrite was classified as a secondary branch, and any correctly-oriented protrusion from the tertiary dendrite was classified as a quaternary branch. A "branch complexity" index, defined in Fig 1, was computed for each segment. This term weights each branch order and is normalized to an "ideal" segment. In L2 and L3 animals, only the total number of secondary branches was tabulated.

Somatic CRISPR-Cas9 experiments
CRISPR-Cas9 constructs were designed according to previously established methods [40]. Phsp16.2, a heat-shock promoter expressed in many tissues, was used to drive Cas9 expression. Worms were synchronized via bleaching and L1 plates were heat shocked at 33°C for two hours. The efficiency of the CRISPR-Cas9 editing was determined by observing >30 animals of each genotype. For the denn-4 experiment, which had~20% efficiency, animals with the CRISPR-Cas9 array and an observed PVD phenotype were selected for imaging and quantification, in comparison to wild-type controls without the array from the same plate. For the dhc-1 experiment, all animals with the CRISPR-Cas9 array were included for imaging and quantification, without pre-screening for neuronal phenotype, because the dhc-1 sgRNA had a much higher efficiency (~60%). Animals from multiple replicates of each CRISPR experiment were pooled for the quantification.

FRAP analysis
FRAP experiments were performed using the Zeiss Axio Observer Z1 microscope equipped with a Yokagawa spinning disk head. A small region of primary dendrite just anterior to the cell body was bleached for 1000ms. Recovery images were taken every second for five minutes.

Statistical analysis
Branch complexity numbers per segment were compared across all genotypes using the 2-way ANOVA with post-hoc Tukey's multiple comparisons test. Similarly, branch complexity numbers were compared across all genotypes within each somatic CRISPR experiment using the 2-way ANOVA with post-hoc Tukey's multiple comparisons test. FRAP data were compared using 2-way ANOVA. Developmental differences in secondary branches between wild-type and wy787 were assessed using multiple t-tests.  Table. PVD branch numbers by genotype. The average number of branches per subcellular compartment and the total number of branches are reported for each genotype. Wild-type is listed first, with other genotypes listed in order of increasing total quaternary branches. (XLSX)