LIN-44/Wnt Directs Dendrite Outgrowth through LIN-17/Frizzled in C. elegans Neurons

Nervous system function requires proper development of two functional and morphological domains of neurons, axons and dendrites. Although both these domains are equally important for signal transmission, our understanding of dendrite development remains relatively poor. Here, we show that in C. elegans the Wnt ligand, LIN-44, and its Frizzled receptor, LIN-17, regulate dendrite development of the PQR oxygen sensory neuron. In lin-44 and lin-17 mutants, PQR dendrites fail to form, display stunted growth, or are misrouted. Manipulation of temporal and spatial expression of LIN-44, combined with cell-ablation experiments, indicates that this molecule is patterned during embryogenesis and acts as an attractive cue to define the site from which the dendrite emerges. Genetic interaction between lin-44 and lin-17 suggests that the LIN-44 signal is transmitted through the LIN-17 receptor, which acts cell autonomously in PQR. Furthermore, we provide evidence that LIN-17 interacts with another Wnt molecule, EGL-20, and functions in parallel to MIG-1/Frizzled in this process. Taken together, our results reveal a crucial role for Wnt and Frizzled molecules in regulating dendrite development in vivo.


Introduction
Correct dendrite development is essential for the establishment of neuronal connectivity and, in sensory neurons, for the detection of external stimuli. However, the complexity and variety in morphology of dendrites has made the study of their development more challenging than that of axons. Previous findings have shown that some axon guidance molecules can also regulate dendrite development, often with opposing effects. For example, the guidance cue Slit can simultaneously repel axons and enhance dendrite branching and outgrowth in cortical neurons [1]. Similarly, Semaphorin 3A, a guidance molecule that acts through the Neuropilin-1 receptor, functions as both a chemorepellent for cortical axons and a chemoattractant for dendrites within the same neurons [2]. The differential response of axons and dendrites to Semaphorin 3A is mediated by asymmetric localization of a soluble guanylate cyclase to the dendrites [2]. In cultured hippocampal neurons, local elevation of cAMP and reduction of cGMP in undifferentiated neurites promotes axon formation and suppresses dendrite formation, whereas the reciprocal levels of these molecules have the opposite effects [3]. Interestingly, local upregulation of cAMP in a single neurite results in long-range inhibition of cAMP levels in all other neurites, suggesting a mechanism for the development of one axon and multiple dendrites and indicating that dendrite formation in this context is secondary to axon formation [3].
More recently, in vivo studies have uncovered molecules that regulate dendrite development independently of the axon. Sensory neurons in the head of C. elegans develop by anchoring their dendritic tips to the nose while the cell body migrates away, extending a dendrite (retrograde extension) [4]. In the C. elegans tail motor neuron, DA9, the extracellular guidance cue UNC-6/ Netrin controls the final extension of the dendrite in an axonindependent manner through its interaction with the receptor UNC-40/DCC [5]. In a different highly branched mechanosensory neuron, PVD, the cell-autonomous activity of the EFF-1 fusogen promotes branch retraction to retain a precise patterning of arbors during dendrite development [6]. In a Drosophila sensory neuron (vch'1), correct orientation of the dendrite is regulated by Netrin-A and its receptor Frazzled and is mediated by a migrating cap cell, which drags the tip of the dendrite into position [7]. In all these cases, however, the cell-intrinsic molecules involved in the initial stages of dendrite formation remain elusive.
In this study, we show that LIN-44/Wnt initiates and guides the development of the dendrite in the PQR oxygen sensory neuron, through a mechanism that occurs prior to and independently of the formation of the axon. In contrast to its role as a repellent in synapse formation and axon termination, in the context of PQR development LIN-44 acts as an attractant that is specific for the outgrowth of the dendrite. The effect of LIN-44 is mediated through the LIN-17 receptor, which functions in a cellautonomous manner. We also identify EGL-20/Wnt and MIG-1/Frizzled as crucial molecules in PQR dendrite development. Taken together, these findings show for the first time that Wnt signals and Frizzled receptors can promote dendrite-specific outgrowth in developing neurons in vivo.

Characterization of PQR Dendrite Development
PQR is an oxygen-sensory neuron with its cell body positioned in the posterior lumbar ganglion on the left side of the animal [26]. PQR extends a single axon anteriorly along the ventral nerve cord and a single dendrite posteriorly towards the tail ( Figures 1D, 2A). The tip of the dendrite, which is part of the left phasmid sensory organ, protrudes with its sensory cilia into the pseudocoelom. PQR is born post-embryonically, facilitating investigation of its development in newly hatched larvae. A gcy-36::GFP reporter was used as a selective marker for PQR, allowing visualization of its dendrite during development, starting at the L1 stage (see Materials and Methods).
PQR arises as a descendant of the QL neuroblast, and subsequently migrates towards the tail. We observed that upon reaching its final destination, at 5.5-6.5 h after hatching, PQR assumed a rounded or elliptical shape, without any neurites ( Figure 1A). At 6.5-7 h, dendrite formation began with lamellipodia-like extensions emerging on the dorsal-posterior region of the cell body, which had become elliptical or triangular in shape ( Figure 1B). At this stage, no other projections were present, indicating that dendrite outgrowth is initiated before outgrowth of the axon. At 7-7.5 h, the dorsal-posterior protrusion thinned and extended into a developing dendrite with a growth cone at its distal tip, and the cell body became rounder in shape ( Figure 1C). At the same time, the axon began to emerge from the ventral-anterior side of the cell, appearing as a small neurite that, unlike the dendrite, did not present a large growth cone at its tip. By 7.5 h, both the dendrite and axon were visible and continued to extend to their final positions until 18 h after hatching (L2/L3) ( Figure 1D). PQR subsequently maintained its morphology throughout adulthood (Figure 2A). Overall, our analysis demonstrates that the PQR dendrite forms by growth cone crawling and is initiated prior to axon outgrowth.

LIN-44/Wnt Regulates Dendrite Formation in PQR
We next used a candidate gene approach to discover the molecules regulating dendrite development in PQR. We found that animals mutant for LIN-44/Wnt presented severe defects, with PQR dendrites that were short, absent, or misrouted in the anterior direction ( Figure 2B-D, and quantified in 2E). The axon, however, appeared morphologically normal. These defects could arise from a dendrite-specific effect or a change in neuronal polarity whereby the identity of the neurites is compromised. To distinguish between these two possibilities we investigated whether there were any changes in the location of the presynaptic sites of PQR, which are normally on the axon. rab-3 encodes for a vesicleassociated Ras GTPase, which localizes to presynaptic densities [27,28]. Using a YFP::RAB-3 fusion protein expressed specifically in PQR (Pgcy-36::YFP::RAB-3), we found that the presynaptic sites in lin-44 mutants were largely located on the axon as in wild-type animals ( Figure 2F). This suggests that the identity of the neurites is unchanged and that the PQR defect of the lin-44 mutant is dendrite-specific.
Next, we tested if the PQR dendrite defect of lin-44 mutant animals could arise from an abnormal cell division in the precursor cell. However, we found that the asymmetric cell divisions of the PQR precursor occurred normally in the lin-44 mutant animals ( Figure S1), precluding this possibility.
Finally, we investigated whether the absent and short dendrite phenotypes we observed were generated either by excessive pruning or by direct outgrowth failure. Examination of early stages of PQR development in lin-44 mutants revealed that the dendrite often failed to form or fully extend (Table S1); we also observed animals with dendritic growth cones developing abnormally on the anterior side of the neuron, which would explain the anteriorly misrouted dendrites observed in adults (Table S1). Thus, our results indicate that LIN-44 acts at very early stages of PQR development by regulating proper formation of the growth cone and its extension.

LIN-44/Wnt Acts as an Attractant Cue for the Developing PQR Dendrite
The Wnt ligand LIN-44 is expressed in close proximity to the PQR neuron from four hypodermal cells (hyp-8, -9, -10, and -11) in the tip of the tail [29], a position posterior to the PQR dendrite ( Figure 3A). As the PQR dendrite grows towards the source of LIN-44, we hypothesized that this molecule might act instructively as an attractive cue for the developing dendrite. Alternatively, LIN-44 may act as a permissive cue, whereby its positional information is not essential for correct dendrite development. To distinguish between these two possibilities, we expressed LIN-44 ectopically from regions anterior to the PQR cell body in lin-44 mutant animals, using a version of LIN-44 genomic DNA that had

Author Summary
Neurons have distinct compartments, which include axons and dendrites. Both of these compartments are essential for communication between neurons, as signals are received by dendrites and transmitted by axons. Although dendrites are vital for neural connectivity, very little is known about how they are formed. Here, we have investigated how dendrites develop in vivo by examining an oxygen sensory neuron (PQR) in the nematode C. elegans. Using a genetic approach, we have discovered that Wnt proteins, a group of highly conserved secreted morphogens, interact with their canonical Frizzled receptors to control the development of the PQR dendrite. We show that Wnt molecules act as attractive signals to determine the initiation and direction of dendrite outgrowth. Interestingly, Wnt proteins act specifically on the dendrite without affecting the axon, suggesting that outgrowth of the dendrite can be regulated by distinct processes that are independent of axon formation. We predict that similar mechanisms may be in place in other species owing to the conserved roles of Wnt and Frizzled molecules in development.
been engineered to contain a secretion signal sequence to ensure proper secretion from cells that do not normally produce LIN-44 [16]. Transgenic lines were generated to express LIN-44 from the myo-2 promoter [30] in the pharynx (Pmyo-2::LIN-44), or from a short fragment of the cwn-1 promoter [21] in the intestine and head neurons (Pcwn-1::LIN-44) ( Figure 3A and Figures S2, S3). When compared to lin-44 mutant animals, transgenic animals expressing LIN-44 anterior to PQR displayed a decrease in the proportion of normal dendrites and an increase in the proportion of dendrites that were misrouted in the anterior direction, towards the ectopic source of LIN-44 ( Figure 3B and Figures S2, S3). On the contrary, expression of LIN-44 from its endogenous promoter (Plin-44::LIN-44) provided strong rescue of the PQR dendrite defect of lin-44 mutant animals ( Figure 3B).
We next examined the ectopic expression of LIN-44 from the myo-2 promoter in the wild-type background and found that it altered the normal development of the PQR dendrite ( Figure S4). Thus, the worsening of dendrite defects observed when LIN-44 is ectopically expressed from anterior regions suggests that LIN-44 has an instructive role in PQR dendrite development, whereby it acts as an attractive cue to direct the outgrowth of the dendrite.

A Pattern of LIN-44 Necessary for Dendrite Outgrowth Is Established During Embryogenesis
In wild-type C. elegans, the four tail hypodermal cells hyp-8, -9, -10, and -11 express LIN-44 throughout embryogenesis and larval stages [29]. In order to define the time period in which LIN-44 is required for normal PQR dendrite development we eliminated larval production of LIN-44 by laser ablation of the hyp-8, -9, -10, and -11 hypodermal cells. Remarkably, in adult animals that were laser-ablated as newly hatched L1 larvae, the PQR dendrite appeared to be largely unaffected ( Figure 4A) even though the ablations were performed several hours before PQR is born in the mid-L1 stage. This result indicates that LIN-44 expression from these hypodermal cells during embryogenesis is sufficient for the correct development of the PQR dendrite.
To further define the temporal requirement of LIN-44 we next utilized an inducible heat shock promoter to express LIN-44 (Phsp16-2::LIN-44) in a lin-44 mutant background at specific times during development. Heat shock-induced LIN-44 expression in newly hatched L1 animals partially rescued PQR dendrite defects ( Figure 4B and Figure S5). However, when animals were heat shocked later, at the time of dendrite outgrowth, no such rescue effect was observed ( Figure 4B), suggesting that LIN-44 expression is required prior to PQR dendrite outgrowth.
The hsp16-2 promoter drives expression broadly throughout the body of the animal, in cells that are both anterior and posterior to PQR [31]. Thus, the dendrite rescue we observed in heat shocked animals could indicate that LIN-44 plays a permissive role, or that the ligand is produced more efficiently from regions posterior to PQR. To further investigate this we expressed Phsp16-2::LIN-44 into a wild-type background and found that the ectopic expression of LIN-44 generated PQR defects similar to those of lin-44 mutants, confirming the instructive role of this molecule ( Figure  S6). Taken together, these results suggest that a molecular pattern of LIN-44 generated prior to PQR formation, during embryonic development and early L1, is both necessary and sufficient to instruct PQR dendrite outgrowth hours later, at which time the source of LIN-44 expression becomes dispensable.

LIN-17/Frizzled May Act as a Receptor for LIN-44
LIN-17 is a Frizzled molecule known to function as a receptor for LIN-44 in a variety of developmental processes [14,16,17,29,[32][33][34][35]. We found that lin-17 mutants had defects resembling those of lin-44, with PQR dendrites that were short, absent, and misrouted anteriorly ( Figure 5A). lin-17 mutants also presented a strong migration defect [18,19], with a high percentage (60% to 90%) of PQR neurons mispositioned in anterior regions of the body. Thus, our analysis was performed on those animals in which PQR was correctly positioned in order to eliminate any possible effect that the aberrant location may have had on PQR dendrite development. Importantly, lin-17 mutants, like lin-44 mutants, appeared to have largely normal localization of presynapses to the axon, as visualized using the YFP::RAB-3 fusion protein expressed specifically in PQR (Pgcy-36::YFP::RAB-3), eliminating the possibility of a switch in neurite identity ( Figure 2F).
In addition to testing known alleles of lin-17, we also performed a forward genetic screen and isolated a previously uncharacterized allele, vd002, consisting of a G to A transition in position 490 of the lin-17 gene that resulted in a cysteine residue being replaced by a tyrosine residue ( Figure 5A). The isolation of this mutant from an unbiased screen further supports the significance of lin-17 in this process.
To investigate whether there might be a genetic interaction between lin-17 and lin-44 with respect to PQR dendrite development, we next examined lin-17 lin-44 double mutants and found that the dendrite defects were qualitatively and quantitatively similar to those of lin-17 mutants ( Figure 6A). This indicates that these two molecules function in the same genetic pathway with respect to PQR dendrite development and strongly suggests that LIN-44 acts as a ligand for LIN-17 in this process.  Figure S7) [35]. Wnt signaling through the LIN-17 receptor could occur cell-autonomously within PQR or could result from interactions with the surrounding cells. We first tested whether LIN-17 acts cell-autonomously by expressing the wild-type lin-17 cDNA from the gcy-36 promoter, which is transcriptionally active in PQR during the final stages of its migration. This transgene failed to rescue the dendrite defects, despite being tested at a range of different concentrations (see Materials and Methods). We therefore questioned whether LIN-17 might be required in PQR at earlier stages, before the gcy-36 promoter is transcriptionally active. To test this possibility we used the egl-17 promoter that is highly and selectively expressed in the precursors of PQR during the L1 stage [36,37]  neurons (PHAL and PHBL) [26]. Recent results in different systems have demonstrated a role of the support cells in regulating dendrite development [4,7]. To determine if similar mechanisms were in place for PQR development, we next performed cellablation experiments whereby we selectively eliminated the socket cells or the socket cells together with the sheath cells. PQR morphology in ablated animals was largely normal, with only a small number of animals presenting short dendrites when left and right phasmid socket cells were ablated (3/15) or when left phasmid socket and left sheath cells were ablated (2/19). We never observed the penetrance and variety of defects of the lin-17 mutants. These results indicate that glial cells play a minor role in only the final stages of dendrite extension and suggest that LIN-17 does not have an effect on the PQR dendrite through these support cells (Table S2). In addition, ablations of the phasmid neurons PHA and PHB also had no effect on PQR dendrite development (Table S2), thereby providing further evidence that the function of LIN-17 in PQR dendrite development is unlikely to be mediated by the surrounding cells.
To further understand how LIN-17 acts on the PQR dendrite, we then asked at what stage in PQR development LIN-17 was visible on the cell membrane and how LIN-17 was distributed in PQR. Using a LIN-17::YFP functional fusion protein expressed under the control of the egl-17 promoter, we observed faint, relatively uniform localization of LIN-17 on the membrane of the QL.a cell as it was dividing into QL.aa and PQR (unpublished data). Following this division, the membrane-localized LIN-17::YFP in PQR decreased until it was barely visible at the time at which PQR had completed its posterior migration (unpublished data). This reduction in LIN-17::YFP appeared to be independent of down-regulation by the egl-17 promoter and is consistent with our previous results suggesting an early role for LIN-17 in regulating PQR dendrite outgrowth. We suggest that ubiquitous membrane-localization of LIN-17 may be required to detect the posterior source of Wnt ligand, which acts as the directional signal for the PQR dendrite.

Multiple Wnt and Frizzled Genes Regulate Dendrite Development
Multiple Wnt ligands and Frizzled receptors are known to function in basic developmental processes in C. elegans and have frequently been shown to have redundant or synergistic roles. Although lin-44 mutants present striking PQR dendrite defects, 32% of these animals still have the ability to sprout a normal PQR dendrite, suggesting the involvement of other molecules in this process. We therefore tested three other Wnt molecules-EGL-20, CWN-1, and CWN-2-for possible roles in PQR dendrite  formation. EGL-20 is expressed around the PQR cell body, in a group of epidermal and muscle cells near the anus [13,20], and CWN-1 and CWN-2 are expressed to a greater extent anteriorly in the intestine, body wall muscle, and neurons in the midbody and head regions, anterior to the PQR cell body [13,22,38]. No significant dendrite defects were observed in cwn-1 or cwn-2 single mutants. The cwn-1 cwn-2 double mutant presented a higher percentage of ectopic processes from the cell body, and dendrite branching, compared to the single mutants, but no absent-dendrite or dendrite-misrouting defects were observed (Table S3). This suggests that these molecules are less directly involved in development of the PQR dendrite, but are important to prevent the formation of ectopic processes. Although the loss of cwn-1 alone caused no significant dendrite defects on PQR, when combined with the lin-44 mutation it was able to enhance the dendrite misrouting defects of lin-44 mutants (Table S3). Thus, CWN-1 might have a minor and redundant role in PQR dendrite development.
As previously described, egl-20 mutants have a very strong Q cell migration defect [18][19][20] resulting in 97%-98% of animals having anteriorly positioned PQR neurons. Restricting our analysis to those animals with PQR correctly positioned, we found that only 7% of egl-20 animals developed a normal, fulllength dendrite, whereas the rest presented qualitatively similar defects to those of lin-44 and lin-17 animals, with absent, short, and anteriorly misrouted PQR dendrites ( Figure 6B). egl-20 mutants presented a higher proportion of anterior dendrites, as compared to lin-44 mutant animals ( Figure 6B), but the PQR dendrite phenotype of the egl-20 lin-44 double mutant did not display a significant worsening of defects when compared to the egl-20 single mutant. This suggests that egl-20 and lin-44 may interact to regulate PQR dendrite formation ( Figure 6B). Furthermore, the egl-20 lin-17 double mutant was no worse than either of the single mutants ( Figure 6C), suggesting that LIN-17 may act as a receptor for both EGL-20 and LIN-44.
Taken together, the above results indicate that egl-20 and lin-44 are the major regulators of PQR dendrite outgrowth, and appear to genetically interact, whereas cwn-1 plays only a minor role in the process. To determine the possible roles of other Frizzled receptors, we also studied PQR dendrite formation in cfz-2 and mig-1 mutants. cfz-2 mutants showed no significant defects, whereas mig-1 mutants presented 50% normal PQR dendrite ( Figure 6D, Table S3). Thus, LIN-17 appears to be the main Frizzled receptor regulating PQR dendrite formation. To analyze functional redundancy among the Frizzleds, we tested whether mig-1 could enhance the lin-17 defect. In the mig-1 lin-17 double mutant, there was almost a 2-fold increase in the absent-dendrite phenotype ( Figure 6D), indicating a possible parallel role of mig-1 in PQR dendrite formation.

Discussion
Dendrites, the specialized structures that allow neurons to receive sensory information from the environment and to relay signals to one another, must develop properly in order to build a functioning nervous system. Recent reports of dendrite morpho- genesis have advanced our understanding of dendrite sculpting and arborization [1,6,11,12,39], neuronal polarity [3,11,40], dendrite extension [4,5], and dendrite orientation [2,7]. To our knowledge, our study is the first to demonstrate that the initial outgrowth of a dendrite in vivo is controlled by Wnts and Frizzleds. Mutations in the Wnt ligands LIN-44 and EGL-20 and in the Frizzled receptors LIN-17 and MIG-1 cause a failure in dendrite development, resulting in dendrites that are absent, short, or misrouted. Our findings demonstrate that the Wnt ligand LIN-44 instructs the development of the dendrite through an attractive mechanism and is required prior to the initiation of dendrite outgrowth. This effect is likely to be mediated through the LIN-17 receptor, which acts cell-autonomously in PQR.
Our results suggest that posteriorly expressed LIN-44 acts as an attractive cue for the PQR dendrite. Ectopic expression of LIN-44 from the anterior side of PQR increases the tendency for dendrites to emerge and grow anteriorly, towards the source of LIN-44. This role of LIN-44 as an attractant in PQR dendrite development differs from its role as a repellent signal for synaptic clustering in the dorsal section of the DA9 motor neuron [16], highlighting the distinct effect of LIN-44 on these neighbouring neurons.
The partial rescue of PQR dendrite defects by ubiquitous expression of LIN-44 from the heat shock promoter could suggest a permissive role for LIN-44. However, a possible alternative interpretation is that local asymmetry of the ligand is generated, providing rescue when the concentration is higher on the posterior side of PQR. This conclusion is supported by the observations that a higher concentration of ligand (increased length of heat shock) is unable to increase the rescue, and that in the wild-type background heat shock-directed expression causes dendrite defects. To be fully functional, Wnts must undergo posttranslational modifications, sorting in the endoplasmic reticulum, and secretion from the cells where they are expressed [44]. It is possible that cells that do not normally express LIN-44 have lower efficiency in regulating the proper maturation and secretion of this Wnt molecule. Hence LIN-44 expression from the heat shock promoter may provide functional, secreted LIN-44 with variable efficiency depending on the tissue of expression.
Wnt patterning occurs during embryogenesis, at a time when many neurons are born. Our observation that PQR forms a normal dendrite following ablation of the tail hypodermal cells at the time of hatching suggests that embryonically expressed LIN-44 provides spatial information needed by the developing PQR several hours later. However, PQR remains receptive to heat shock misexpression of LIN-44 up until the dendrite begins developing. It is not known how stable Wnts are in C. elegans; however, in Drosophila the Wnt Wingless (Wg) and the morphogen Decapentaplegic (Dpp) are stable for about 3 h [45,46]. Wnts can also function at long distances. In C. elegans, for example, EGL-20 has been shown to direct cell migration across half the animal's body length [20,46]. Similarly in Drosophila, Wg can cover 10-20 cell diameters away from its source in the developing wing [47,48] spreading over a distance of about 50 mm in 30 min [46]. Our results showing an effect of LIN-44 when expressed in the pharynx from the promoter myo-2 in a region far from PQR also suggest a potential long range effect for this ligand.

LIN-17/Frizzled Regulates PQR Dendrite Development in a Cell-Autonomous Manner
Emerging evidence suggests that dendrites of sensory neurons are shaped in a variety of ways. In contrast to dendrite development by retrograde extension, or towing by associated cells [4,7], we and others [49] have observed that the dendrite of PQR forms by growth cone crawling, a mode of development more commonly seen in axons. In LIN-44 mutants, this growth cone often fails to form, preventing the outgrowth of a dendrite. Our results demonstrate that LIN-17, a receptor for LIN-44, cellautonomously regulates the initiation and outgrowth of the PQR dendrite. To our knowledge, a ligand-receptor pair that can specifically affect the development of a dendrite in this manner has not previously been described.
Interestingly, phasmid glia associated with the PQR dendrite do not have a major effect on its development. It has previously been shown that lin-44 and lin-17 mutants have defects in phasmid socket glia that arise due to disrupted polarity of the T cell precursor [29,35,50]. However, the aberrant structure of the phasmid in these mutants does not seem to be the main cause of dendrite defects, as ablation of these cells did not reproduce the mutant phenotypes. Notably, glia appear to have some involvement in the final extension of the dendrite, as some ablated animals had short dendrites. This is reminiscent of a previous study in which it was demonstrated that ablation of the sheath glia associated with the CEP sensory neuron in the head of C. elegans resulted in a failure of the sensory dendrite of this neuron to fully extend [51].
Different lines of evidence suggest that LIN-17, like LIN-44, may be required early in development to promote normal dendrite outgrowth. Cell-specific LIN-17 expression can rescue lin-17 dendrite defects if induced very early, before PQR is born, but has no such effect when induced later, once the cell has almost completed its migration. Furthermore, LIN-17::YFP expression from the rescuing egl-17 promoter appeared to become extremely faint or absent by the time the dendrite began to develop. This raises the interesting possibility that levels of LIN-17 receptor on the PQR cell surface are temporally regulated to elicit the appropriate response to Wnt ligands. We propose a model in which the LIN-17 receptor, present at low levels on the membrane of the PQR cell from the moment it is born, detects a posterior source of LIN-44 that signals the dendrite to emerge from the posterior side of the cell (Figure 7A,B). This initial specification of the site of dendrite outgrowth appears to be an important determinant of the subsequent direction of dendrite outgrowth. The tendency for lin-44 and lin-17 mutant dendrites to grow anteriorly from the PQR cell, rather than from random orientations (including dorsal or ventral), may imply the presence of an intrinsic anterior-posterior bias of the site and direction of PQR dendrite outgrowth controlled by Wnts and Frizzleds, or the existence of a dorso-ventral dendrite outgrowth controlled by other guidance molecules still unknown.

Multiple Wnt and Frizzled Molecules Coordinate Development of the PQR Dendrite
In C. elegans, Wnts are expressed in different regions along the anterior-posterior axis. These different Wnts have often been shown to have distinct effects on cells that are located in proximity to the respective source of Wnt expression. Our genetic studies suggest that, similar to LIN-44, the posteriorly expressed Wnt ligand EGL-20 also acts through the LIN-17 receptor to regulate PQR dendrite development ( Figure 7C whether EGL-20 plays an instructive role in this process remains unclear. Previous studies have also shown that both LIN-44/Wnt and EGL-20/Wnt can function through LIN-17/Frizzled; however, whether Frizzled receptors can simultaneously bind multiple Wnts, or whether Wnts can form homo-or hetero-dimers, remains unknown. The Wnt molecules CWN-1 and CWN-2 are both expressed more broadly in the body wall muscle, intestine, ventral cord neurons, and some head neurons [13,21,22,38]. Although these Wnts do not appear to directly regulate PQR dendrite development, our observation that a significant proportion of cwn-1 and cwn-2 mutants present ectopic processes on PQR suggests an indirect role in neurite pruning. This is consistent with recent findings that identify CWN-1 and CWN-2 as key regulators of developmental pruning of the head neuron AIM [21]. The MIG-1 receptor appears to act synergistically in a parallel pathway to LIN-17 ( Figure 7C). Notably, the increase in the percentage of the absent dendrite phenotype of the lin-17 mig-1 double mutant compared with the lin-17 mutant suggests a role for MIG-1 in regulating the ability of the neuron to send out a dendrite, regardless of its direction.
Wnt morphogens have diverse functions in developmental processes across species, yet how they act with such precision on a single cell within a closely wired nervous system remains enigmatic. As we and others have shown, spatio-temporal organization of Wnts and their Frizzled receptors must be tightly orchestrated. The challenge now will be to gain insight into how these molecules are patterned and how they can be interpreted differently by individual cells.

Strains and Genetics
Nematodes were cultured using standard methods [52].

Analysis of PQR Morphology
We analyzed PQR development in synchronized populations of anesthetized larvae (L1 stage) in a kyIs417(Pgcy-36::GFP) background. Animals were synchronized by collecting newly hatched animals, from a plate containing only eggs, every 10 min using M9 buffer. Synchronized animals were transferred to fresh plates and grown for 5-9 h at 22uC before imaging. Developmental stages were characterized in synchronized populations, with little variation among animals.
PQR morphology was scored at L4 or adult stages. Mutations in mig-1, lin-17, and egl-20 caused PQR migration defects, resulting in anterior (and in some cases posterior) mis-positioning of PQR. Given that this would cause PQR to be in a different position in relation to its normal surroundings, and importantly the source of LIN-44, we chose to score dendrite defects only in those animals where PQR had migrated to its normal position. The PQR dendrite was scored as short if it was less than three cell bodies in length.

Heat Shock Experiments
Wild-type and lin-44(n1792) mutant animals carrying the Phsp16-2::LIN-44 transgene were maintained at 18uC. As development at this temperature is slower than at 22uC as in Figure 1, dendrite outgrowth occurs at ,8 h rather than ,6.5 h. L1 animals were heat shock-induced at 33uC in a water bath for 30 min (or longer, where indicated) at different stages of development as indicated, following which they continued to grow at 18uC. Transgenic animals (lin-44; Phsp16-2::LIN-44) and non-transgenic controls (lin-44) were scored at the L4 stage or as young adults.

Microscopy and Cell Ablations
Animals were mounted on 4% agar pads and immobilized using tetramisole hydrochloride (0.01%-0.03%). Epifluorescence was used to visualize animals with a Zeiss Axioimager Z1 and a Zeiss Axioimager A1 microscope. A Photometrics camera, Cool snap HQ 2 , was used for imaging. Metamorph software was used to analyze the collected Z stacks. Developing stages of PQR were imaged using a Zeiss LSM510META confocal microscope and Zen 2008 software. An antifading agent, Dayco, was used in addition to tetramisole hydrochloride.
Laser ablations were performed in L1 animals carrying the kyIs417 transgene using a MicroPoint Laser System Basic Unit attached to a Zeiss Axio Imager A1 (Objective EC Plan-Neofluar 1006/1.30 Oil M27). Animals were ablated 0-1 h after hatching and were scored at the L4 stage. For ablations of phasmid glia and phasmid neurons, ablation success was determined at the L4 stage by soaking animals in DiI on slides for 2 h prior to scoring (DiI stains the phasmid neurons when these cells and the phasmid structure are unaltered [53][54][55]).

Statistical Analyses
Statistical analyses were performed using Primer of Biostatistics 3.01. Error of proportions was used to estimate variation within a single population. The Student's t test was used in all cases, except in those with multiple comparisons, for which the Bonferroni t test was used.