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. Author manuscript; available in PMC: 2013 Dec 3.
Published in final edited form as: Nat Neurosci. 2011 Dec 4;15(1):57–63. doi: 10.1038/nn.2978

The transmembrane leucine-rich repeat protein DMA-1 promotes dendrite branching and growth in C. elegans

Oliver W Liu 1, Kang Shen 1
PMCID: PMC3848871  NIHMSID: NIHMS331891  PMID: 22138642

Abstract

Dendrites often adopt complex branched structures. The development and organization of these arbors fundamentally determine the potential input and connectivity of a given neuron. The cell-surface receptors that control dendritic branching remain poorly understood. Here, we show that in Caenorhabditis elegans, a previously uncharacterized transmembrane protein containing extracellular leucine-rich repeat (LRR) domains, which we name DMA-1 (Dendrite-Morphogenesis-Abnormal), promotes dendrite branching and growth. Sustained expression of dma-1 is found only in the elaborately branched sensory neurons PVD and FLP. Genetic analysis showed that loss of dma-1 causes much reduced dendritic arbors while overexpression of dma-1 results in excessive branching. Forced expression of dma-1 in neurons with simple dendrites was sufficient to promote ectopic branching. Animals lacking dma-1 are defective in sensing harsh touch. DMA-1 is the first transmembrane LRR protein to be implicated in dendritic branching and expands the breadth of roles played by LRR receptors in nervous system development.

Dendritic arbors adopt diverse branched morphologies of varying complexity that are characteristic for a given neuron type 1,2. The organization of the dendritic arbors is fundamental to the connectivity and function of a neuron, determining both the potential receptive field and the sampling and processing of signals within that field 3. For example, in the mammalian retina, there are about 12 types of retinal ganglion cells (RGCs) and 30 types of amacrine cells 1. Each RGC and amacrine cell type exhibits a characteristic combination of dendritic field size, branch complexity, and dendritic targeting area. An essential step in the development of these complex dendrite morphologies is the regulation of dendrite branching.

While fundamental to the shape and connectivity of the nervous system, the mechanisms that regulate dendrite branching are not well understood, though a variety of pathways have been implicated in the process. In mammals, reduced activity of the transcriptional factors NeuroD and Crest, which are widely expressed in the nervous system, results in decreased dendritic branching and growth in certain cell types 4,5. Secreted ligands, like Semaphorin 3A, can promote dendritic branching and spine maturation in mouse cortical neurons 6.

The majority of our understanding of the molecular mechanisms involved in dendritic branching comes from studies of the dendritic arborization neurons found in Drosophila melanogaster. The study of the four classes of dendritic arborization neurons, which vary by complexity of branching, has helped illuminate important dendrite organizational principles such as the ability to identify and avoid sister dendrites from the same neuron (self-avoidance), and the ability of dendrites from neurons of the same cell-type to minimally overlap (tiling) 7,8. Genetic screens utilizing dendritic arborization neurons have identified intracellular pathways that regulate levels of branching, including mechanisms involving transcriptional regulation 9,10, actin and microtubule-dependent trafficking 11,12, secretion/endocytosis 13,14, signal transduction 15,16, and RNA localization 17. These pathways function, in large part, to mobilize the cellular machinery needed for dendrite growth 8. Conspicuously absent from our molecular understanding of dendrite branching has been the identity of cell surface proteins promoting this process. Organized dendrite branching must ultimately be defined and instructed by extracellular interactions, yet the cell-surface receptors involved in activating dendritic branching pathways remain, for the most part, unknown.

In the model nematode Caenorhabditis elegans, where most neurons are morphologically simple, two sets of sensory neurons, PVD in the body and FLP in the head, adopt complex, highly branched and highly organized dendrite structures with features that resemble the “self-avoidance” and “tiling” principles seen in dendritic arborization neurons 18,19. The genetic and experimental tractability of C. elegans make the PVD and FLP neurons attractive, novel systems in which to study mechanisms that regulate dendrite branching.

The immense complexity of nervous system development is made possible by a multitude of receptor-ligand interactions on the surfaces of neurons 20,21. Transmembrane LRR (leucine-rich repeat) proteins often are expressed exclusively in the nervous system, and extracellular LRR domains can serve as protein-protein interaction motifs, suggesting that they participate in important receptor-ligand interactions 22,23. Indeed, specific roles for transmembrane LRR proteins have recently been uncovered in synaptogenesis and dendrite targeting 24-27. Still, the functions of most neuronally expressed transmembrane LRR proteins remain unknown. Here, in order to identify potential functions of transmembrane LRR proteins in the C. elegans nervous sytem, we initiated an effort to determine the expression and localization pattern of every type-1 transmembrane LRR protein in the C. elegans genome. Through this analysis, we identified a gene we call dma-1 (T10G9.3; dendrite morphogenesis abnormal-1), which demonstrated sustained expression in only two sets of neurons, PVD and FLP, suggesting a model where this cell-surface receptor acts as an intrinsic factor to promote dendrite branching and growth. We provide additional evidence supporting this model, which expands the breadth of known functions played by LRR proteins in nervous system development, and demonstrates the importance of cell-surface interactions in controlling dendrite branching.

Results

Neuronally expressed transmembrane LRR proteins

In C. elegans, 17 genes are predicted to encode type-1 transmembrane proteins with extracellular LRR domains 22. In order to determine the expression and localization patterns of these LRR proteins, we systematically generated tagged transgenes utilizing a precise fosmid recombineering technique which preserves upstream, downstream, and intronic genome sequences 28 (Fig. S1). This approach allowed us to accurately determine the in vivo expression of these proteins since we maintained all likely regulatory sequences in the tagged transgene. Using this strategy, we identified six transmembrane LRR proteins that are expressed predominantly in the nervous system (Table S1).

When examining these expression patterns, we noted that the previously uncharacterized gene T01G9.3, which we have named dma-1, is expressed in a unique subset of neurons. dma-1 is predicted to encode a 603 aa transmembrane protein with an extracellular region composed of 15 LRR motifs and a short intracellular region (73 aa) with no obvious domains (Fig. 1a). Sustained expression of DMA-1 is found most prominently in two pairs of sensory neurons, PVD and FLP (Fig. 1b–c). Notably, these neurons are by far the most highly branched neurons in C. elegans, extending elaborate dendrite arbors that envelop the body and head respectively 18,19,29. The remaining 298 neurons in C. elegans contain few or no branches. Expression of DMA-1 can be detected in PVD neurons immediately after the cells are born during the L2 larval stage, but before extensive branching of these neurons has initiated (Fig. S2a). DMA-1 can also be detected transiently in other head neurons (including the SIA, SIB, SMB and/or SMD classes of neurons based on the presence of processes along the sub-lateral cords), ventral cord neurons, and the vulva during larval and early adult stages (Fig. S2b–d) This provocative expression pattern of DMA-1 in primarily PVD and FLP prompted us to investigate potential roles for dma-1 in dendrite branching and morphogenesis.

Figure 1.

Figure 1

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DMA-1 is expressed in highly branched neurons

a, Schematic of DMA-1 protein structure including signal peptide (SP), leucine rich repeat N-terminal domain (LRR-NT), leucine-rich repeats (LRR), leucine rich repeat C-terminal domain (LRR-CT), and transmembrane (TM). b-c, Confocal images of adult worms expressing DMA-1 ∷GFP under the control of its endogenous promoter. Strong expression is seen in the highly branched PVD and FLP neurons. Background autofluorescence is due to gut granules. Scale bars equal 20μm.

dma-1 mutants are defective in dendrite morphogenesis

We examined PVD morphology using a cell specific marker (ser2prom3∷myrGFP) that clearly labels the entire neuron (Fig. 2a). PVD neurons (PVDR and PVDL) are generated post-embryonically in the lateral posterior body during the L2 larval stage. Only recently has the extent of dendritic branching in PVD been appreciated 18,19,29,30. The neurons send out three primary (1°) processes: anteriorly- and posteriorly-directed dendritic processes, which run laterally along the worm body, and a ventrally-directed axon, which enters the ventral nerve cord as a commissure and then migrates anteriorly along the nerve cord. During the subsequent larval stages (late L2 – L4), a series of orthogonal secondary (2°), tertiary (3°), and quaternary (4°) level branches emerges resulting in an array of branched structural units resembling candelabras which decorate the initial 1° dendritic processes (Figs. 2b and S3a). 2° processes can branch prior to reaching the 3° level, creating secondary prime (2°′) branches. Multiple organizing principles seen in other models of dendritic branching, such as Drosophila melanogaster dendritic arborization neurons, are evident in the development of PVD, including regions of restricted branching, self-avoidance between sister dendrites, and rough tiling between PVD and FLP branches 19. In PVD, the frequency of 2°′ branching between the 1° and 3° levels is low, and adjacent 3° processes do not overlap even as they grow towards each other (Fig. 2b). To determine the role of dma-1 in PVD dendrite development, we generated a null allele, wy686, by deleting a region of the genome from ∼3kb upstream of the dma-1 start site to the 3′ end of the gene using the recently developed MosDel technique 31. When we crossed the PVD marker into the dma-1(wy686) mutant background strain and examined L4 animals, we observed a fully penetrant dendrite branching phenotype (Fig. 2c). While the anterior 1° dendritic process showed normal outgrowth in the majority of animals (the remaining animals displayed some reduction in dendrite length, Fig. S3b), candelabra structures never developed along the 1° branch. 2° branches grew minimally and often branched and/or appeared to be fused with adjacent 2° branches before reaching the 3° level. 3° and 4° branches were largely absent. The posterior 1° dendritic process exhibited similar loss of candelabra formation and highly penetrant growth defects (Fig. S3c). Interestingly, the ventral axon appeared morphologically normal, suggesting that dma-1 is specifically required for development of dendrites. dma-1 knockdown by feeding RNAi caused similar dendrite branching phenotypes in ∼50% of animals (Fig. S4).

Figure 2. dma-1 positively regulates PVD branching.

Figure 2

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a, PVD dendrites envelop the entire worm body. PVD can be labeled in its entirety with a cell specific marker (PVD∷GFP). b, Schematic of a stereotypical PVD neuron. PVD extends primary (1°) dendritic processes anteriorly and posteriorly from the cell body and a ventrally directed axonal process. Orthogonal branching (2°, 3°, 4°) generates a well-ordered array of candelabra-shaped arbors that demonstrates regulated branching and self-avoidance. c, Deletion of dma-1 (the dma-1(wy686) allele) results in a fully-penetrant dendrite morphology defect in PVD. d, By the L3 larval stage, PVD has established 2° and 3° branching. e, dma-1(wy686) mutant L3 worms display easily quantifiable branching and growth defects. f, The density of 2° branching was determined by counting the number of 2° branches per 100 um of the anterior 1° process for a given worm. The distribution of densities was then compared for a wildtype and mutant population of worms. dma-1(wy686) mutant worms demonstrated a broad range of defects in 2° branch density with an average decrease of 44%. g, The average length of 2° branches decreased 47% compared to wild-type. All error bars shown represent s.e.m. *** p < 0.0001, t-test. Scale bars equal 20μm.

The qualitative defect in dendrite morphologies we observed in dma-1 (wy686) mutants could be due to decreases in the amount of 2° branching and/or aberrant outgrowth of these 2° branches. In order to quantitatively measure these effects, we chose to study mid-L3 larval stage worms. By mid-L3, in wildtype PVD neurons, stable 2° branches have developed and are in the process of 3° and 4° branch elaboration (Fig. S2a). As a result, this time point offers high resolution for observing defects in 2° branch formation and outgrowth, allowing us to easily measure both the frequency and length of 2° branches (Fig. 2d–e). We found that in dma-1(wy686), the frequency of 2° branches decreased 44% compared to wild type. Furthermore, the average length of 2° branches decreased by 47% (Fig. 2f–g). Thus, the abnormal dendrite morphology in dma-1(wy686) is due to a combination of decreased levels of 2° branching as well as decreased outgrowth of these 2° processes, which results in complete loss of higher order branches.

DMA-1 cell autonomously regulates dendrite morphogenesis

The expression analysis described above demonstrated that dma-1 is strongly expressed in PVD and not in neighboring neurons or tissue, suggesting that DMA-1 functions cell autonomously in PVD to promote branching. Consistent with this model, we could rescue the PVD morphology defects in dma-1(wy686) by expressing dma-1 cDNA from a PVD-specific promoter (Fig. 3a–b). We generated two lines in which ∼80% of animals demonstrated at least partial rescue (Fig. 3c). In fully rescued animals (56% and 61%), dendrite outgrowth, dendrite branching, and formation of candelabra structures were indistinguishable from wild-type, demonstrating that dma-1 acts cell autonomously in PVD neurons to promote dendrite branching and growth.

Figure 3.

Figure 3

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DMA-1 functions cell autonomously

a-b, The dendrite morphology defect in dma-1(wy686) mutant animals can be cell-autonomously rescued by PVD-specific expression of dma-1. c, Quantification of cell-autonomous rescue of branching defects by cell-specific expression of dma-1. Two dma-1(wy686) lines carrying arrays expressing PVD∷dma-1 were generated. Worms carrying the array were categorized based on PVD morphology as mutant, partial PVD rescue, or full PVD rescue. Partial PVD rescue displayed some candelabra formation. Full PVD rescue was indistinguishable from wildtype PVD. d, Subcellular localization of GFP-tagged DMA-1 in PVD. DMA-1∷GFP is found in all dendrite processes, but not in the axon. Scale bars equal 20μm.

To determine the subcellular localization of dma-1 within PVD, we expressed a GFP-tagged dma-1 transgene using a PVD-specific promoter (Fig. 3d). We found all dendritic processes to be labeled with this tagged protein and single slice imaging of the cell body confirmed that DMA-1∷GFP can be found on the cell surface (Fig. S5). We also observed numerous bright puncta in the cell body and dendritic processes. Notably, the PVD axon was not well labeled by DMA-1∷GFP, indicating that DMA-1 is a dendrite-specific protein, consistent with DMA-1 playing a direct role in regulating dendrite morphogenesis. Similar to PVD, the FLP neurons are highly branched (Fig. 4a) and express dma-1 (Fig. 1c). We found that FLP branching is severely compromised in the dma-1(wy686) mutants (Fig. 4b) and that this phenotype can be rescued by FLP-specific expression of dma-1 (Figs. 4c and S6). Thus dma-1 functions cell autonomously in both sets of highly branched neurons in worms to promote arborization.

Figure 4. dma-1 also regulates FLP branching.

Figure 4

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a, FLP neurons in the head elaborate highly branched dendrites similar to PVD. b, Deletion of dma-1 results in a fully-penetrant dendrite morphology defect in FLP as well. c, This defect can be cell-autonomously rescued by FLP-specific expression of dma-1. Scale bars equal 20μm.

DMA-1 overexpression causes excessive dendrite branching

To determine whether the activity of DMA-1 is rate-limiting for dendritic branching, we examined the effects of dma-1 overexpression in wild-type animals. We found that dma-1 overexpression resulted in excessive branching of PVD (Fig. 5a–b). Instead of the well-arrayed candelabra structures found in wild-type, animals carrying the dma-1 overexpression array developed tangled networks of dendritic branches. While the general organization of 1°, 2°, 3°, and 4° level branches could be distinguished, uncontrolled dendrite branching and growth were evident. The frequency of 2° branching along the 1° processes increased by 58%. The amount of 2°′ branching in the regions between the 1° and 3° processes increased ∼7 fold (Fig. 5c–d). The majority of these 2° and 2°′ processes did not grow orthogonally to the 1° processes indicating that these branches were not properly guided to the 3° branch level. Finally, the characteristic self-avoidance of sister dendrites seen in wildtype worms was lost in worms overexpressing dma-1. 2° and 2°′ processes often intersected and/or fused, while almost all 3° processes overlapped with adjacent 3° processes (Fig. 5b). To quantify this loss of self-avoidance, we calculated an Avoidance Index (AI) by comparing the number of visible gaps between 3° branches to the number of candelabra in a given worm (Fig. 5e). In wild-type worms, this ratio is ∼1 (1.00 +/− 0.02 s.e.m.) since there is a gap between each candelabra. As 3° branches overlap, the AI decreases. In worms overexpressing dma-1, we observed a dramatic reduction in the AI (0.20 +/− 0.04 s.e.m.) demonstrating a severe defect in 3° self-avoidance.

Figure 5. dma-1 overexpression causes excessive branching.

Figure 5

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a, By the L4 larval stage, PVD displays complete candelabra structures. b, Overexpression of dma-1 results in excessive PVD branching. c-d, Quantification demonstrates significant increases in both 2° and 2°’ branching. b,e, Overexpression of dma-1 also results in a dramatic loss of sister dendrite self-avoidance between 3° level branches as measured by Avoidance Index. All error bars shown represent s.e.m. ** p < 0.001, *** p < 0.0001, t-test. Background autofluorescence is due to gut granules. Scale bars equal 20μm.

Expression of a truncated version of DMA-1 that lacks the transmembrane and cytoplasmic domains (dma-1Δtm) from a PVD-specific promoter did not rescue PVD morphology defects in dma-1(wy686) animals or result in overbranching in wild-type worms, indicating that the transmembrane domain of DMA-1 is required for its function in PVD branching (data not shown).

Ectopic expression of DMA-1 induces branching

Consistent with our conclusions from the loss-of-function experiments, the overexpression phenotypes demonstrate a role for dma-1 in activation of pathways involved in dendrite branching and growth. To further test this hypothesis, we asked if expression of dma-1 was sufficient to induce branching in neurons that normally do not branch extensively. We chose two sets of sensory neurons, PDE and PLM, which 1) display simple architectures with a single branch point, thus demonstrating that they have the potential to branch, 2) do not express detectable levels of dma-1, and 3) send out processes that are closely positioned to PVD processes so that the extracellular environment is likely to be conducive to DMA-1 receptor activation (Fig. 6a, b, e). In both cases, we found that cell-specific expression of dma-1 results in ectopic branching of these neurons in 25-30% of animals (Fig. 6d, g). Interestingly, in PDE, orthogonal ectopic branches appeared at a stereotyped position in the commissure of PDE, corresponding to the region where 3° level branches emerge in PVD (Fig. 6c), suggesting that an activating ligand is localized to discrete locations in the worm. In PLM, ectopic branches are seen along the sublateral process, which runs parallel to 3° processes of PVD (Fig. 6f). Thus, in morphologically simple neurons neighboring PVD, ectopic expression of dma-1 is sufficient to activate branching pathways in certain extracellular environments. Expression of dma-1Δtm in PDE or PLM did not result in ectopic branching (multiple transgenic lines tested), indicating that ectopic branching observed in PDE and PLM requires functional DMA-1 and is not due simply to overexpression of a non-specific LRR protein (Fig. 6d, g). Ectopic expression of full length dma-1 in OLL head neurons did not result in increased dendritic complexity in these neurons, supporting the hypothesis that activation of DMA-1 is context-dependent within the animal (data not shown).

Figure 6. Expression of dma-1 in morphologically simple neurons can induce ectopic branching.

Figure 6

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a, b, e PDE and PLM are morphologically simple sensory neurons with processes that run adjacent to PVD dendrites and can be labeled with cell-specific markers. c, Expression of dma-1 in PDE causes orthogonal ectopic branching (arrows) at a stereotyped position in the PDE commissure, reminiscent in location and direction of PVD 3° branching. d, Ectopic branching at this stereotyped position is seen in 25% of animals carrying the Ex[PDE∷dma-1] array, but not in animals carrying Ex[PDE∷dma-1Δtm] arrays. f, Expression of dma-1 in PLM causes ectopic branching along the PLM process which runs laterally along the worm. g, Ectopic branching along PLM is seen in 29% of animals that carry the Ex[PLM∷dma-1] array, but no increase in seen in animals carrying Ex[PLM∷dma-1Δtm] arrays. *** p < 0.001, chi-squared test. Scale bars equal 20μm.

Loss of dma-1 causes defects in harsh touch response

PVD has been shown to play a role in responding to multiple stimuli including strong mechanical stimulation (harsh touch) 32,33. The similarity in form and function between PVD and multi-dendritic nociceptors from other organisms suggests that the highly branched dendritic morphology of PVD plays an important role in its ability to sense stimuli. As dma-1 mutant animals demonstrate greatly reduced PVD branching, we tested if loss of dma-1 also results in a defect in PVD sensory function.

Animals respond to prodding in the midsection of the body (vulva harsh touch) by initiating forwards or backwards locomotion. This behavior is dependent on PVD and the touch receptor neurons (ALM, AVM, PLM, PVM). PVD function is typically assessed in a mec-4 mutant background, which lacks functioning touch receptor neurons. As controls, we found that mec-4(u253) and dma-1(wy686) single mutant animals demonstrate wild-type responses to harsh touch (∼90% of worms initiate backing), while only 20% of mec-3(e1338) mutant animals, which lack function in both PVD and the touch receptor neurons, respond to harsh touch (Fig. 7). Additional neurons involved in sensing harsh touch likely account for the remaining response measured in mec-3(e1338) animals 33. We found that dma-1(wy686) mec-4(u253) double mutant animals display an intermediate defect in response to harsh touch (∼64% of worms initiate backing). Expression of dma-1 from a PVD-specific promoter in dma-1(wy686) mec-4(u253) double mutant animals partially rescues the sensory defect in these animals indicating that DMA-1 functions cell autonomously in PVD to affect the response to harsh touch and is consistent with the hypothesis that the dendrite morphology of PVD is critical in determining the sensory input to the neuron and thus its function (Fig. 7). The lack of complete rescue of the harsh touch response defect in these animals is consistent with partial rescue of the PVD morphology defect in animals expressing PVD-specific DMA-1.

Figure 7. dma-1 mutants display defects in response to harsh touch.

Figure 7

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Animals with the indicated genotypes were assayed for response to harsh touch. The mec-4(u253) mutation disables the touch receptor neurons (TRNs) allowing for the function of PVD in the harsh touch response to be isolated and assayed alone. As controls, mec-4(u253) and dma-1(wy686) single mutants responded similarly to WT. mec-4(u253) dma-1(wy686) double mutant animals demonstrated a significant defect in harsh touch response. As a control, the mec-3(e1338) mutation disables both the TRNs and PVD. Cell-specific expression of dma-1 in PVD partially rescued the sensory defect detected in mec-4(u253) dma-1(wy686) double mutant animals. *** p < 0.001, chi-squared test

Discussion

Using a systematic fosmid-tagging approach to simultaneously characterize expression and sub-cellular localization patterns, we endeavored to identify the genomic complement of cell-surface LRR receptor proteins important in the development and function of the nervous system. Our goals for this approach were to not only simply determine which transmembrane LRR proteins are expressed in the nervous system, but to also use the precise identity of the neurons in these expression patterns to deduce aspects of the protein function. Here, with DMA-1, we present an instance where, indeed, the unique expression pattern of the protein provided remarkable insight into its role in regulating dendrite branching.

A pattern of sustained expression in primarily PVD and FLP has not been previously reported for any other gene in C. elegans (http://genome.sfu.ca/gexplore/) 34. Genes involved in mechanosensation, such as mec-3 and mec-10, are expressed in PVD and FLP, but also in the six touch receptor neurons (ALML, ALMR, PLML, PLMR, AVM, PVM), suggesting that DMA-1 is not involved in mechanosensation in general. Instead, this pattern intimates a role for DMA-1 in the property unique to PVD and FLP in the C. elegans nervous system: highly branched dendrites. Our genetic characterization of dma-1 supports a model where DMA-1 functions as an intrinsic factor on the cell-surface of dendrites which can activate dendrite branching and growth pathways. Loss-of-function mutants suffer severe reductions in PVD and FLP dendrite branching and growth. These defects are rescued in the mutant by PVD or FLP cell-specific expression of dma-1, demonstrating DMA-1 acts cell autonomously to regulate this phenotype. Strikingly, overexpression of dma-1 was sufficient to generate a highly overbranched PVD morphology, indicating that the level of DMA-1 is rate-limiting for generating dendritic branches. This property is similar to what has been observed in D. melanogaster dendritic arborization neurons where expression levels of the transcription factor CUT determine the degree of dendritic branching. Similarly, with DMA-1, we have identified a factor intrinsic to PVD whose expression level determines the level of PVD dendritic branching. Instead of a transcription factor, however, we have identified a cell surface receptor, suggesting that in PVD, one rate-limiting control of dendrite branching is the processing of external cues by surface receptors.

Dendritic morphology is regulated both by growth promoting and growth inhibiting factors. In D. melanogaster, self avoidance is elegantly mediated by the strict homotypic binding properties of different splice variants of the cell adhesion molecule DSCAM 35. Recent time lapse imaging of PVD 3° branches in C. elegans suggest that self-avoidance in PVD is also initiated by branch-branch contact 19. Upon dma-1 overexpression, we observed a loss of self avoidance between the PVD 3° branches in addition to overbranching, indicating that in these animals, the mechanisms to regulate self-avoidance are diminished and/or are no longer sufficient. As DMA-1 promotes dendritic growth, as well as branching, it is possible the growth promoting signals provided by overexpressed DMA-1 overpowers contact dependent mechanisms that control 3° length.

The specific expression pattern of DMA-1 prompted us to ask whether DMA-1 is sufficient to generate dendritic branches. We observed ectopic branches in PDE and PLM when we ectopically expressed DMA-1 in these morphologically simple neurons, providing additional evidence that DMA-1 is sufficient to at least partially activate dendrite branching pathways. Of note, the degree of ectopic branching on PDE and PLM is clearly lower than the amount of branching usually seen in PVD and FLP, indicating that we have not fully recapitulated the molecular pathways involved in DMA-1 mediated branching. It is possible that, although PDE and PLM processes are often in close proximity to PVD processes, they still do not receive all the appropriate external cues/binding partners that activate DMA-1 in PVD. It is also possible that PDE and PLM do not have sufficient levels of intracellular factors, such as high levels and distal localization of golgi and endosomes, necessary for building a highly branched structure. Intriguingly, ectopic branches in PDE were found at a highly stereotyped location on the PDE commissure, suggesting that the activating partner of DMA-1 is spatially regulated and that DMA-1 is involved in local cell-cell interactions that activate branching. This would make DMA-1 the first contact dependent receptor known to promote dendritic branching. The stereotyped nature of dendritic structures in general has long been appreciated, suggesting a requirement for close interaction between a neuron and its surrounding tissue for proper spatial regulation of dendrite branching 1,2. Yet, the surface proteins involved in cell-cell interactions that activate dendritic branching are not well established. Diffusible factors, such as Slit or semaphorins, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) have been shown to influence aspects of dendrite morphology, but these are not dependent on cell-cell contact 6,36,37. The immunoglobulin superfamily protein Turtle has been suggested to restrict dendrite branching in low complexity dendritic arborization neurons in Drosophila 38. The identification of proteins that interact with DMA-1 will be an important step in determining the extracellular mechanism of DMA-1 activation.

Besides PVD and FLP, expression of DMA-1 can also be transiently detected in other head neurons and ventral cord motor neurons. These neurons do not display extensive branching, suggesting that they perhaps utilize DMA-1 primarily to regulate growth of processes. Interestingly, besides FLP, the head neurons that display the most consistent expression of DMA-1 elaborate processes that grow along the dorsal and ventral sub-lateral cords, which are the same boundaries along which the tertiary branches of PVD grow. These data suggest that DMA-1 may play a role in regulating growth and branching along the sub-lateral boundaries and support the model that the activating ligand of DMA-1 may be localized in these regions.

Based on sequence alone, the identity of a DMA-1 binding partner is difficult to predict. The leucine-rich repeat structural motif forms a curved solenoid structure that provides a surface suited for many types of protein-protein interactions 39. For example, LRR domains have been shown to interact with the Ig domains in Robo 40, the LNS domains of neurexin 24, and the N-terminal domain of human E-cadherin 41. While the variety of potential interactors may make identification of LRR binding partners more difficult, it is precisely this combination of breadth and specificity that makes LRR transmembrane proteins particularly intriguing candidates to be mediators of the complexity of nervous system development and function. DMA-1 is the first transmembrane LRR protein shown to function in nervous system development in C. elegans and is the first transmembrane LRR protein implicated in dendritic branching. A close homolog to DMA-1 exists in flies (reduced ocelli) which is expressed throughout the nervous system 42, suggesting that interactions involving this class of extracellular LRR transmembrane proteins will be conserved in nervous system development of other species.

Methods

Strains and genetics

Worms were rasied on OP50 Escherichia coli-seeded nematode growth medium (NGM) plates at 20°C. N2 Bristol was used as the wild-type reference strain. Strain IE22994 containing the ttTi22997 transposon insertion upstream of dma-1 was obtained from L. Segalat. CX6365 rrf-1(pk1426) was obtained from C. Kenyon. TV8378 pha-1(e2123ts) was obtained from A. Fire. TU253 mec-4(u253) and CB1338 mec-3(e1338) were obtained from the Caenorhabditiis Genetics Center. Strain TV9696 dma-1(wy686∷unc-119+)I, was generated using the MosDel 31 approach starting with strain IE22994.

Constructs and transgenes

Fosmids containing C. elegans genomic sequence (Geneservice) were manipulated using a previously described fosmid recombineering approach 28. All other plasmid constructs were generated using yeast homologous recombination in a pRS413 backbone 44. The ser2prom3 (PVD), Pmec-3 (FLP, PLM), Pdat-1 (PDE) promoters were used for cell-specific expression. The following transgenic extrachromosomal arrays were generated via injection using standard techniques 45: wyEx3282(Pdma-1∷dma-1∷GFP∷SL2∷mcherry (fosmid)), wyEx3355(ser2prom3∷myrGFP), wyEx4004(ser2prom3∷dma-1#1), wyEx4005(ser2prom3∷dma-1#2), wyEx4280(Pmec-3∷myrGFP), wyEx4290(Pmec-3∷ dma-1), wyEx4288(Pdma-1∷dma-1(fosmid)), wyEx3974(Pdat-1∷GFP), wyEx4281(Pdat-1∷dma-1), wyEx4289(Pmec-3∷dma-1), wyEx4286(ser2prom3∷dma-1∷GFP). Podr-1∷RFP (20ng/μl), Pmyo-2∷mcherry (2ng/μl), Punc-122∷RFP (20ng/μl), and Ppha-1∷pha-1 (50ng/μl) were used as co-injection markers. Additional transgenic lines containing tagged extracellular LRR proteins are listed in Supplementary Table 1.

Fluorescence microscopy and confocal imaging

Images of fluorescent proteins were captured in live C. elegans using a Plan-Apochromat 63×/1.4 objective on a Zeiss LSM710 confocal microscope. Worms were immobilized using 10mM levamisole (Sigma). Z-stacks were collected and the maximum projection was used for additional analysis.

Quantification of branching

To measure the frequency of 2° and 2°′ branches, we always used the 1° dendritic process anterior of the PVD cell body for analysis. A 2° branch was defined as any distinguishable protrusion that extended away from the 1° process. The frequency of 2° branching was then calculated as the number of 2° branches per 100μm of the 1° dendritic process. 2°′ branches were defined as any branch that resulted from a branching event off a 2° process before reaching the 3° level. Again, frequency was measured per 100μm of the 1° dendritic process. To calculate the 3° branch Avoidance Index (AI), we counted the number of mature 2° branches (2° branches that extend 3° branches) and the number of distinct gaps between 3° branches. The AI is the ratio of gaps to mature 2° branches. All lengths were measured using ImageJ software.

RNA interference

Feeding RNAi was performed as previously described using the enhanced RNAi strain rrf3(pk14246)II and constructs from the Ahringer Lab library 46.

Harsh touch response

The assay to measure the response to harsh touch was performed as previously described 32. A positive response was scored as movements of at least one body length in response to stimulus, though the speed of these movements can be variable in different mutant backgrounds.

Statistical analysis

Student's t-test and chi-square analysis were used to compare populations as indicated in the figure legends.

Supplementary Material

1

Supplementary Figure 1: Dual-tag strategy for determining expression pattern and subcellular localization of transmembrane receptors containing extracellular LRR domains using fosmid recombineering.

Supplementary Table 1: Spreadsheet listing all C. elegans genes in the genome predicted to encode Type I transmembrane proteins containing extracellular LRR domains, their domain structure, and expression patterns as determined by our fosmid recombineering approach.

Supplementary Figure 2: While DMA-1∷GFP is dominantly expressed in PVD and FLP, especially by the adult stage, DMA-1∷GFP expression in additional head and ventral cord neurons was detected during larval and early adult stages. a, DMA-1 expression was detected in the PVD cell body shortly after PVD are born (L2 larval stage) before any dendrite outgrowth or branching has occurred. Weaker DMA-1∷GFP expression can be seen in ventral cord motorneurons (asterisks) and in the sub-lateral cords (arrowheads). b, Examples of early larval stage animals (before PVD birth) that displayed transient expression of DMA-1∷GFP in ventral cord motorneurons (asterisks) and the sub-lateral cords (arrowheads). Expression was variable from animal to animal, seen in both commissural and non-commissural motorneurons, and was mostly undetectable by the early adult stage. c, In the head, expression of DMA-1∷GFP was detected during development in additional head neurons besides FLP. Of these neurons, the strongest expression was detected in neurons that run around the nerve ring and extend processes along the sub-lateral cords (likely some combination of the SIA, SIB, SMB and/or SMD classes of neurons). A number of additional head neurons displayed inconsistent DMA-1∷GFP expression from animal to animal. Again, expression in these additional neurons was transient and mostly undetectable by the early adult stage. d, Strong expression DMA-1∷GFP was detected in vulva cells during the L3-L4 larval stages.

Supplementary Figure 3: a, Development of PVD morphology during larval development. PVD is born in L2 and the axon and 1° dendritic processes extend anteriorly and posteriorly. By late L2/early L3, 2° dendritic processes begin to grow out orthogonally from the 1° process. By L3, 3° dendritic processes grow anteriorly and posteriorly from the ends of the 2° processes. By L4, the 4° processes have emerged orthogonal to the 3° processes, establishing the candelabra structure. b,_dma-1(wy686) mutants display variable defects in the length of the 1° dendritic processes of PVD. The direction of outgrowth was usually not affected. Dendrites were categorized as normal length, slight growth defect (>50% of normal WT length), or severe growth defect (<50% normal WT length). Severe growth defects in the anterior 1° process was observed in ∼10% of animals. c, Defects in the length of the posterior 1° process were more pronounced, with ∼70% of animals displaying a severe growth defect.

Supplementary Figure 4: a, Feeding RNAi against dma-1 results in similar dendrite branching phenotypes observed in the null mutant. b, Worms fed dma-1 dsRNA were categorized based on PVD morphology as having no defect, moderate PVD branching defect, or severe PVD branching defect.

Supplementary Figure 5: A thin slice image of the PVD cell body expressing DMA-1∷GFP. Bright GFP localization can be seen in intracellular membrane structures that are likely to be golgi/ER. The plasma membrane is also clearly visible, indicating that DMA-1∷GFP is a cell-surface protein. The dotted line outlines the main cell body of PVD.

Supplementary Figure 6: A dma-1(wy686) line carrying an array expressing FLP∷dma-1 rescued defects in FLP branching. Worms were categorized by FLP morphology as mutant, partial FLP rescue, or full FLP rescue. A FLP∷GFP marker was used to label FLP. Rescue of this phenotype was not as robust as that seen for PVD. This could be due, in part, to differences in the timing and expression levels of promoters used to drive dma-1 cell-specific expression.

Acknowledgments

We thank C. Gao and Y. Fu for technical assistance, C. Chen, K. Mizumoto, P. Chia and A. Hellman for critical reading of the manuscript, and the Shen laboratory for helpful discussion. We also would like to thank the Hobert and the Jorgensen laboratories for sharing plasmids and expertise regarding fosmid recombineering and MosDel. This work is supported by grants from the Howard Hughes Medical Institute and the National Institutes of Health to K.S., and a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund to O.W.L. O.W.L. conducted the experiments. K.S. supervised the project. O.W.L. and K.S. wrote the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplementary Figure 1: Dual-tag strategy for determining expression pattern and subcellular localization of transmembrane receptors containing extracellular LRR domains using fosmid recombineering.

Supplementary Table 1: Spreadsheet listing all C. elegans genes in the genome predicted to encode Type I transmembrane proteins containing extracellular LRR domains, their domain structure, and expression patterns as determined by our fosmid recombineering approach.

Supplementary Figure 2: While DMA-1∷GFP is dominantly expressed in PVD and FLP, especially by the adult stage, DMA-1∷GFP expression in additional head and ventral cord neurons was detected during larval and early adult stages. a, DMA-1 expression was detected in the PVD cell body shortly after PVD are born (L2 larval stage) before any dendrite outgrowth or branching has occurred. Weaker DMA-1∷GFP expression can be seen in ventral cord motorneurons (asterisks) and in the sub-lateral cords (arrowheads). b, Examples of early larval stage animals (before PVD birth) that displayed transient expression of DMA-1∷GFP in ventral cord motorneurons (asterisks) and the sub-lateral cords (arrowheads). Expression was variable from animal to animal, seen in both commissural and non-commissural motorneurons, and was mostly undetectable by the early adult stage. c, In the head, expression of DMA-1∷GFP was detected during development in additional head neurons besides FLP. Of these neurons, the strongest expression was detected in neurons that run around the nerve ring and extend processes along the sub-lateral cords (likely some combination of the SIA, SIB, SMB and/or SMD classes of neurons). A number of additional head neurons displayed inconsistent DMA-1∷GFP expression from animal to animal. Again, expression in these additional neurons was transient and mostly undetectable by the early adult stage. d, Strong expression DMA-1∷GFP was detected in vulva cells during the L3-L4 larval stages.

Supplementary Figure 3: a, Development of PVD morphology during larval development. PVD is born in L2 and the axon and 1° dendritic processes extend anteriorly and posteriorly. By late L2/early L3, 2° dendritic processes begin to grow out orthogonally from the 1° process. By L3, 3° dendritic processes grow anteriorly and posteriorly from the ends of the 2° processes. By L4, the 4° processes have emerged orthogonal to the 3° processes, establishing the candelabra structure. b,_dma-1(wy686) mutants display variable defects in the length of the 1° dendritic processes of PVD. The direction of outgrowth was usually not affected. Dendrites were categorized as normal length, slight growth defect (>50% of normal WT length), or severe growth defect (<50% normal WT length). Severe growth defects in the anterior 1° process was observed in ∼10% of animals. c, Defects in the length of the posterior 1° process were more pronounced, with ∼70% of animals displaying a severe growth defect.

Supplementary Figure 4: a, Feeding RNAi against dma-1 results in similar dendrite branching phenotypes observed in the null mutant. b, Worms fed dma-1 dsRNA were categorized based on PVD morphology as having no defect, moderate PVD branching defect, or severe PVD branching defect.

Supplementary Figure 5: A thin slice image of the PVD cell body expressing DMA-1∷GFP. Bright GFP localization can be seen in intracellular membrane structures that are likely to be golgi/ER. The plasma membrane is also clearly visible, indicating that DMA-1∷GFP is a cell-surface protein. The dotted line outlines the main cell body of PVD.

Supplementary Figure 6: A dma-1(wy686) line carrying an array expressing FLP∷dma-1 rescued defects in FLP branching. Worms were categorized by FLP morphology as mutant, partial FLP rescue, or full FLP rescue. A FLP∷GFP marker was used to label FLP. Rescue of this phenotype was not as robust as that seen for PVD. This could be due, in part, to differences in the timing and expression levels of promoters used to drive dma-1 cell-specific expression.

NIHMS331891-supplement-1.pdf (1.2MB, pdf)

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