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Published in final edited form as: Dev Biol. 2016 Oct 13;420(1):60–66. doi: 10.1016/j.ydbio.2016.10.010

Transition between synaptic branch formation and synaptogenesis is regulated by the lin-4 microRNA

Yan Xu 1, Christopher C Quinn 1,*
PMCID: PMC5841448  NIHMSID: NIHMS824568  PMID: 27746167

Abstract

Axonal branch formation and synaptogenesis are sequential events that are required for the establishment of neuronal connectivity. However, little is known about how the transition between these two events is regulated. Here, we report that the lin-4 microRNA can regulate the transition between branch formation and synaptogenesis in the PLM axon of C. elegans. The PLM axon grows a collateral branch during the early L1 stage and undergoes synaptogenesis during the late L1 stage. Loss of the lin-4 microRNA disrupts synaptogenesis during the late L1 stage, suggesting that lin-4 promotes synaptogenesis. Conversely, the target of lin-4, the LIN-14 transcription factor, promotes PLM branch formation and inhibits synaptogenesis during the early L1 stage. Moreover, we present genetic evidence suggesting that synaptic vesicle transport is required for PLM branch formation and that the role of LIN-14 is to promote transport of synaptic vesicles to the region of future branch growth. These observations provide a novel mechanism whereby lin-4 promotes the transition from branch formation to synaptogenesis by repressing the branch-promoting and synaptogenesis-inhibiting activities of LIN-14.

Keywords: Axon Branching, Synaptogenesis, microRNA, lin-4, LIN-14

Introduction

Neuronal connectivity is achieved through sequential developmental steps including axon specification, axon outgrowth, synaptic branch formation, synaptogenesis and synaptic remodeling. Temporal control is required to ensure the orderly transition between each of these sequential steps. Moreover, defects in the temporal control of neuronal development are linked to neurodevelopmental disorders (Amir et al., 1999, Chan et al., 1996, Clement et al., 2012, Sarnat and Flores-Sarnat, 2013, Wan et al., 1999). For example, mutations in genes that cause intellectual disability in humans can cause premature synapse formation in mice. Thus, disruptions in the timing of neurodevelopmental steps may cause defects in neuronal connectivity that underlie neurodevelopmental disorders. However, the mechanisms that regulate temporal control of neuronal development are largely unknown. In particular, little is known about how the transition between sequential steps is regulated.

Although mechanisms regulating the transition between steps of axonal development are unknown, mechanisms have been identified that can regulate the transition between stage-specific cell division programs (Ambros and Horvitz, 1984, Lee et al., 1993, Moss, 2007, Rougvie, 2001, Wightman et al., 1993). In C. elegans, development proceeds through a series of four larval stages (L1,L2,L3, and L4). Heterochronic genes regulate the transition between each of these stages. For example, the heterochronic gene lin-14 encodes a transcription factor that promotes L1 specific cell fates and inhibits L2 specific cell fates. At the end of the L1 stage, the lin-4 microRNA represses LIN-14, thereby allowing the transition from L1 specific cell fates to L2 specific cell fates. In a similar fashion, the hbl-1 heterochronic gene encodes a transcription factor that promotes L2 specific cell fates and inhibits L3 specific cell fates (Abbott et al., 2005). The mir-48, mir-84 and mir-241 microRNAs repress hbl-1 to promote the transition between the L2 and L3 cell fates. Thus, transitions between stage-specific cell division programs are mediated by microRNAs that repress transcription factors that promote one developmental step, while inhibiting the subsequent developmental step.

The roles of lin-4 and LIN-14 in regulating transitions between stage specific cell division programs prompted us to ask if they might also regulate transitions between the sequential steps of neuronal development. Here, we report that lin-4 and LIN-14 can regulate the transition from branch formation to synaptogenesis in the PLM axon of C. elegans. The PLM normally grows a branch during the early L1 stage and synaptogenesis occurs in the late L1 stage. Our results support a model where LIN-14 regulates the localization of synaptic vesicles to promote branch formation and inhibit synaptogenesis in the early L1 stage. In the late L1 stage, lin-4 promotes the transition from branch formation to synaptogenesis by repressing the branch-promoting and synaptogenesis-inhibiting activities of LIN-14.

Results

Temporal regulation of synaptogenesis in the PLM axon branch

We are using the PLM neuron of C. elegans to study the process of synaptic branch formation and synaptogenesis. The two PLM neurons belong to a class of 6 touch receptor neurons, which mediate the response to light touch (Ernstrom and Chalfie, 2002). The cell body of each PLM resides in the tail ganglia and extends its axon anteriorly during the embryonic period (Figure 1A). After hatching, the PLM axon extends a single collateral branch that forms a cluster of synaptic connections in the ventral nerve cord (Marcette et al., 2014, Schaefer et al., 2000). To characterize the temporal relationship between branch formation and synaptogenesis, we examined branch growth and localization of synaptic vesicles in populations of synchronized worms. In this experiment we used a mec-7::rfp transgene to visualize the PLM axon and branch, as well as a mec-7::gfp::rab-3 transgene as a marker for synaptic vesicles. We found that the PLM branch forms during the early L1 stage, and is present in nearly all individuals by six hours after hatching (Figure 1B). At this time point, RAB-3 is not localized within the synaptic region, but rather is clustered at the branch point (Figure 1C–E). Recruitment of RAB-3 into the synaptic region occurs in the late L1 stage and reaches full penetrance at 12 hours after hatching (Figure 1F–I). These results suggest that synaptogenesis is not completed immediately after branch formation, but rather is temporally restricted to the late L1 stage.

Figure 1.

Figure 1

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Developmental timing of PLM synaptic branch formation and synaptogenesis. (A) Diagram of the PLM neuron. The PLM extends an axon laterally that terminates in the midbody. A single collateral branch extends ventrally and forms a cluster of synapses in the ventral nerve cord (depicted in green). (B) The PLM axon grows a synaptic branch in nearly all individuals by 6 hours after hatching. (C–E) At 6 hours after hatching, GFP::RAB-3 is localized to the branch point (see arrowhead in E), but is excluded from the synaptic region. (F–H) At 12 hours after hatching, GFP::RAB-3 is localized to the synaptic region (see arrowhead in H). Labeling of the PLM axon by a mec-7::rfp transgene is shown in C and F. Localization of GFP::RAB-3 is shown in D and G. Merged images are shown in E and H. (I) Localization of GFP::RAB-3 in the synaptic region was observed in nearly all individuals by 12 hours after hatching. Scale bars are 5μm. For all observations n>20. Error bars represent standard error of the proportion.

The lin-4 microRNA can temporally regulate PLM synaptogenesis

To identify a mechanism that can temporally regulate PLM synaptogenesis, we searched for candidate mutations that alter the timing of RAB-3 recruitment into the PLM synaptic region. We considered the lin-4 microRNA as a candidate because its expression, as measured in either whole worm lysate or in touch receptor neurons, is low during the early L1 stage and high during the late L1 stage (Feinbaum and Ambros, 1999, Zou et al., 2012), suggesting that it could be responsible for the temporal regulation of PLM synaptogenesis. Null mutants in lin-4 have egg-laying defects, precluding accurate synchronization. Therefore, we used conditional CRISPR against lin-4 restricted to the touch receptor neurons, which we call lin-4(cKO). In this experiment, we used the mec-7 promoter to restrict expression of CAS-9 to touch receptor neurons in worms that were also expressing two sgRNAs targeted upstream and downstream of the lin-4 sequence. In wild type, we found that RAB-3 was recruited to the PLM synaptic region by the late L1 stage at 12 hours after hatching in nearly all individuals (Figures 1I, 2A–C, 2G). By contrast, in lin-4(cKO) mutants at 12 hours after hatching, we found that 32% of PLM axon branches failed to recruit RAB-3 to the synaptic region. Examination of lin-4(cKO) mutants at various time points indicated that RAB-3 was recruited to the PLM synaptic region of nearly all individuals by the early L2 stage at 18 hours after hatching (Figure 2D–G), representing a delay of about 6 hours relative to wild type. These observations suggest that lin-4 functions cell autonomously in the PLM neuron to promote the proper timing of synaptogenesis during the late L1 stage.

Figure 2.

Figure 2

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Loss of lin-4 function delays recruitment of RAB-3 to the synaptic region. (A–C) Example of a wild type PLM branch at 12 hours after hatching, showing localization of GFP::RAB-3 to the synaptic region (see arrowhead in C). (D–F) Example of lin-4(cKO) mutant branch at 12 hours after hatching, showing a failure to recruit GFP::RAB-3 into the synaptic region (see arrowhead in F). Labeling of the PLM axon by a mec-7::rfp transgene is shown in A and D. Localization of GFP::RAB-3 is shown in B and E. Merged images are shown in C and F. (G) In wild type PLM axons, GFP::RAB-3 is recruited to the synaptic region in nearly all individuals by 12 hours after hatching. In lin-4(cKO) a failure to recruit GFP::RAB-3 to the synaptic region was observed at 12 hours after hatching. At later time points, the penetrance of defects in GFP::RAB-3 recruitment to the synaptic region was decreased, reaching wild type levels by 18 hours after hatching. (H) In indivduals that did recruit GFP::RAB-3 to the synaptic region, the size of the GFP::RAB-3 cluster was reduced at all time points in lin-4(cKO) mutatnts relative to wild type. Scale bars are 5μm. For all observations n>33. Error bars represent standard error of the proportion.

The lin-4 microRNA promotes growth of the PLM synaptic varicosity

The PLM synapses are closely spaced within the PLM synaptic region, such that they appear as a single cluster, which is called the synaptic varicosity. To further assess the effect of lin-4 loss of function on synaptogenesis, we examined the size of the synaptic varicosity at time points throughout the L1 and early L2 stage. We found that the size of the PLM synaptic varicosity was reduced at each time point in lin-4(cKO) mutants relative to wild type (Figure 2H). To determine if these early defects in the size of the synaptic varicosity persist in later stages, we also examined L4 stage worms. We found that in the L4 stage, the size of the synaptic varicosity was also reduced in lin-4(cKO) mutants relative to wild type (Figure 3A–C). Since analysis of L4 stage animals does not require synchronization, we were also able to test the effect of the lin-4(e912) null allele on the size of the synaptic varicosity. We found that this null allele also reduces the size of the PLM synaptic varicosity relative to wild type. Together, these observations suggest that lin-4 functions cell autonomously to promote growth of the PLM synaptic varicosity.

Figure 3.

Figure 3

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Loss of lin-4 function disrupts PLM synapse formation (A) Example of wild type PLM synaptic clusters labeled with GFP::RAB-3 at the L4 stage. (B) Example of lin-4 null mutant PLM synaptic clusters labeled with GFP::RAB-3 at the L4 stage. (C) Loss of function in lin-4 or gain of function in lin-14 causes a decrease in the size of the PLM synaptic cluster. Alleles used were the lin-4(e912) null and lin-14(n355) gain of function. The lin-4(cKO) is a conditional CRISPR construct that deletes lin-4 within the touch receptor neurons. This construct expresses sgRNAs upstream and downstream of lin-4 and uses the mec-7 promoter to restrict CAS-9 expression to the touch receptor neurons. Scale bars are 5μm. For all observations n>23. Error bars represent standard error of the mean. Asterisk indicates statistically significant difference relative to wild type, t-test (p<0.0005).

LIN-14 inhibits synaptogenesis

In the late L1 stage, the lin-4 microRNA binds to the mRNA encoding the LIN-14 transcription factor and represses its expression (Arasu et al., 1991, Lee et al., 1993, Wightman et al., 1993). Thus, LIN-14 expression is high in the early L1 stage, but is reduced in the late L1 stage. To examine the role of the LIN-14 transcription factor in synaptogenesis, we examined the PLM synaptic varicosity in lin-14(n355) gain of function mutants. This gain of function allele is missing its lin-4 binding sites, thus rendering it insensitive to regulation by lin-4. If lin-4 represses LIN-14 to regulate synaptogenesis, we would expect that the lin-14(n355) gain of function mutation would phenocopy the small synaptic varicosity phenotype of the lin-4 null mutant. Indeed, we found that the size of the PLM synaptic clusters were reduced in lin-14(n355) mutants (Figure 3C). These observations support a model where LIN-14 is an inhibitor of synaptogenesis and lin-4 promotes synaptogenesis by repressing LIN-14.

LIN-14 promotes branch formation

To further explore the role of LIN-14 in the development of the PLM synaptic branch, we examined the PLM branch in lin-14 loss of function mutants. We found that the PLM branch was often absent in the lin-14(n355n679) and lin-14(n179) loss of function mutants (Figure 4A–C). To determine if lin-14 functions cell autonomously to regulate the PLM branch, we also used conditional CRISPR to disrupt lin-14. In this experiment, we used the mec-7 promoter to express CAS-9 in the touch receptor neurons in worms also expressing two sgRNAs targeting lin-14. We found that these lin-14(cKO) mutants also caused a missing PLM branch phenotype, suggesting that lin-14 functions cell-autonomously to regulate the PLM branch.

Figure 4.

Figure 4

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LIN-14 promotes branch formation. (A) Example of wild type PLM axon with branch at 6 hours after hatching. (B) Example of lin-14(n355n679) mutant PLM axon with a missing branch at 6 hours after hatching. (C) Loss of lin-14 function causes a missing PLM branch in the L4 stage. The lin-14(cKO) allele is a conditional CRISPR construct that disrupts lin-14 in the touch receptor neurons by using the mec-7 promoter to express CAS-9 in touch receptor neurons along with two sgRNAs against lin-14. (D) Branch growth is delayed in lin-14(n355n679) loss of function mutants. In wild type the PLM grows a branch in nearly all individuals by 6 hours after hatching (see Figure 1B). In lin-14(n355n679) mutants, branches were not observed until 24 hours after hatching. The lin-14(n179) mutants were grown at 22°C, all other mutants were grown at 20°C. For data in C, n>200 for each genotype. Scale bars are 5μm. For data in D, n>40 for each time point. Error bars represent the standard error of the proportion. Asterisk indicates statistically significant difference relative to wild type, z-test for proportions (p<0.0001).

The absence of PLM branches in the lin-14 loss of function mutants could be explained by either a defect in branch formation or a defect in branch stability. To address this question, we used the strong loss of function allele, lin-14(n355n679) to examine the timing of branch defects. In wild type, the PLM branch is present in nearly all individuals by 6 hours after hatching (Figure 1B). By contrast, in the lin-14(n355n679) mutants, we did not observe any PLM branches during the L1 stage at 6, 10 and 16 hours after hatching (Figure 4D). At 24 hours after hatching, we found that a small portion of PLM axons did have a branch. The portion of PLM axons that had a branch was unchanged at 48 hours after hatching. Together, these results suggest that loss of lin-14 function causes a defect in branch formation.

LIN-14 regulates localization of synaptic vesicles prior to branch formation

To investigate the role of LIN-14 in PLM branch formation, we compared the localization of RAB-3 in lin-14 loss of function mutants relative to wild type. For this experiment we used worms that were 4 hours past hatching, which is the time point just prior to branch formation. In wild type, we observed that RAB-3 was localized to puncta in the mid section of the axon, in the neighborhood of where branch formation would be expected to occur (Figure 5A). By contrast, in lin-14(n355n679) and lin-14(n179) loss of function mutants, RAB-3 was most often localized to clusters within the posterior portion of the PLM axon (Figure 5B–C). These observations suggest that LIN-14 can promote localization of synaptic vesicles to the region of branch formation during the period prior to PLM branch formation.

Figure 5.

Figure 5

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LIN-14 promotes localization of the RAB-3 synaptic vesicle marker prior to branch formation. (A) Example of wild type PLM axon at 4 hours past hatching. GFP::RAB-3 is labeled in green and the axon is labeled in red. In wild type, GFP::RAB-3 is localized to clusters in the middle portion of the axon (arrowheads). (B) Example of lin-14(n355n679) loss of function mutant PLM axon at 4 hours past hatching. GFP::RAB-3 is mislocalized to clusters in the posterior portion of the axon (arrowheads). (C) Percentage of PLM axons that have GFP::RAB-3 clusters localized to the posterior third of the PLM axon. The lin-14(n179) mutants were grown at 22°C, all other mutants were grown at 20°C. Error bars represent the standard error of the proportion. Scale bars are 5μm. Posterior is to the left. For all observations n>30.

Synaptic vesicle trafficking is required for PLM branch formation

Since synaptic vesicles are mislocalized in lin-14 mutants, we wondered if synaptic vesicle trafficking is required for the formation of the PLM branch. To address this question, we tested loss of function mutations in unc-104 and syd-2. UNC-104 is a motor protein that is required for transportation of synaptic vesicles (Hall and Hedgecock, 1991). SYD-2 is a liprin-α protein that functions in synapse assembly, but also promotes synaptic vesicle trafficking by binding to UNC-104 and regulating its motility (Miller et al., 2005, Wagner et al., 2009). In the PLM axon, loss of function mutations in either unc-104 or syd-2 cause the accumulation of synaptic vesicles in the posterior portion of the axon (Zheng et al., 2014). Therefore, if synaptic vesicle trafficking is required for branch formation, we expect that loss of function mutations in unc-104 and syd-2 should cause a missing branch phenotype. Indeed, we found that the PLM branch was absent in a portion of both unc-104 and syd-2 loss of function mutants (Figure 6). We also tested mutations in nab-1 and elks-1, two genes that are involved in synapse assembly, but not synaptic vesicle transport (Chia et al., 2013, Chia et al., 2012, Dai et al., 2006). We found that branch formation was normal in both elks-1 and nab-1 mutants. To determine if synaptic vesicle transport is required in a cell-autonomous manner we used a mec-7::unc-104 transgene to express UNC-104 in the touch receptor neurons. Consistent with the idea that UNC-104 functions within the PLM neuron to promote branch formation, we found that the mec-7::unc-104 transgene rescues the branch formation defects in unc-104 mutants (Figure 6).

Figure 6.

Figure 6

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Synaptic vesicle trafficking is required for PLM branch formation. The PLM axon branch was absent in a portion of syd-2(ok217) and unc-104(e1265) null mutants. By contrast, mutations in elks-1 and nab-1, genes that promote active zone development, did not exhibit any PLM branch defects. PLM branch defects were rescued by mec-7::unc-104 transgenes, suggesting that vesicle transport functions in a cell-autonomous manner to promote PLM branch formation. All observations were made at the L4 stage. Error bars represent the standard error of the proportion. For each observation n>150. Asterisk indicates statistically significant difference relative to wild type, z-test for proportions (p<0.0001).

Discussion

Many of the signaling components that regulate individual steps of axonal development have been identified (Colon-Ramos et al., 2007, Norris and Lundquist, 2011, Quinn and Wadsworth, 2008, Stavoe and Colon-Ramos, 2012, Hallam et al., 2002, Holbrook et al., 2012, Owald et al., 2010, Owald et al., 2012, Patel et al., 2006, Wentzel et al., 2013, Xu and Quinn, 2015, Xu et al., 2015, Xu and Quinn, 2012). However, little is known about how the transitions between these steps are regulated. Here, we describe a mechanism that can regulate the transition between branch growth and synaptogenesis in the PLM touch receptor neuron. We present evidence that the LIN-14 transcription factor induces branch formation by promoting synaptic vesicle transport during the time prior to branch formation. Moreover, our evidence suggests that LIN-14 can also inhibit synaptogenesis during the early L1 stage and that LIN-14 must be repressed by lin-4 for normal synapse formation to occur in the late L1 stage. Our observations support a model where lin-4 triggers the transition from branch growth to synaptogenesis by repressing the branch growth-promoting and synaptogenesis-inhibiting activities of LIN-14 (Figure 7).

Figure 7.

Figure 7

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Model for the role of lin-4 in promoting the transition from branch formation to synaptogenesis. (A) During the early L1 stage, LIN-14 promotes branch formation and inhibits synaptogenesis. (B) During the late L1 stage, the lin-4 microRNA represses LIN-14, thereby derepressing synaptogenesis and inhibiting branch formation.

Defects in temporal control of synapse development underlie neurodevelopmental disorders

A key finding of this study is the identification of a mechanism that can temporally regulate synaptogenesis. Knowledge of this mechanism is important because failures in the temporal regulation of synaptogenesis are associated with neurodevelopmental disorders. For example, mutation of SYNGAP1 results in premature synapse development and cognitive defects in mouse (Clement et al., 2012) and are associated with intellectual disability, autism and epilepsy in humans (Hamdan et al., 2011, Hamdan et al., 2009, Krepischi et al., 2010). Likewise, mutations in MeCP2 can cause Rett syndrome, intellectual disability, seizures and microcephaly in humans (Amir et al., 1999, Wan et al., 2000). Studies of mice deficient in MeCP2 have revealed premature synaptogenesis, suggesting that temporal misregulation of synaptogenesis is likely a key feature of Rett syndrome. Moreover, precocious and late synaptogenesis have been observed in holoprosencephaly in humans, a disorder that causes seizures and mental retardation (Sarnat and Flores-Sarnat, 2013). In addition to timing defects, we also observed that loss of lin-4 function causes a persistent decrease in the size of the synaptic varicosity. This phenotype may also be related to neurodevelopmental disorders. For example, a recent study using patient-derived iPS cells has indicated that mutations in DISC1 that are associated with schizophrenia can cause a reduction in the density of synapses as well as defects in presynaptic function (Wen et al., 2014).

Roles for developmental timing genes in cell division and neuronal development

The roles of lin-4 and LIN-14 in regulating the transition between branch growth and synaptogenesis are reminiscent of their roles in regulating the transition between stage-specific cell division programs. During axon development, LIN-14 promotes branch growth, but inhibits the subsequent step of synaptogenesis. Repression of LIN-14, by the lin-4 microRNA, triggers the end of branch growth and the beginning of synaptogenesis. During cell division, LIN-14 promotes L1 specific cell division programs, but inhibits L2 specific cell division programs. Repression of LIN-14, by the lin-4 microRNA, triggers the progression from L1 stage-specific cell division programs to L2 stage-specific cell division programs (Ambros and Horvitz, 1987, Lee et al., 1993, Moss, 2007, Rougvie, 2001, Wightman et al., 1993). In both axon development and cell division, the transition between developmental steps is promoted by a microRNA that represses a transcription factor that promotes one step, while inhibiting the subsequent step.

We speculate that other genes implicated in regulating transitions between stage specific cell division programs might also regulate different transitions between stages of neuronal development. For example, the mir-48, mir-84 and mir-241 microRNAs function together to promote the transition between the L2 and L3 stage specific cell division programs (Abbott et al., 2005). These microRNAs are thought to repress the HBL-1 transcription factor, which promotes L2 cell fates and inhibits L3 fates. We speculate that these microRNAs and HBL-1 could also be involved in regulating neuronal developmental steps that occur after synaptogenesis. Supporting this idea, HBL-1 can promote synaptic remodeling in DD motor neurons (Thompson-Peer et al., 2012), a developmental step that occurs after synaptogenesis. Thus, we hypothesize that HBL-1 may regulate the transition between synaptogenesis and synaptic remodeling.

Other roles for lin-4 and LIN-14 in neuronal development

Roles for lin-4 and LIN-14 have been identified for several other aspects of neuronal development (Hallam and Jin, 1998, Olsson-Carter and Slack, 2010, Zou et al., 2012). For example, in the HSN neuron, lin-4 can promote axon extension by repressing expression of LIN-14 and LIN-28 (Olsson-Carter and Slack, 2010). Previous work has also indicated that lin-4 and LIN-14 can inhibit netrin-mediated axon guidance by regulating the localization of UNC-40 (Zou et al., 2012). However, the transcriptional targets of LIN-14 that regulate these processes have not yet been identified. We have also been unable to identify the transcriptional targets of LIN-14 that are able to regulate branch formation and synaptogenesis. Therefore, the identification of LIN-14 targets that can regulate neuronal development will be an important area for future investigations.

The function of lin-4 and LIN-14 in the regulation of neuronal development seems to be quite different in different neurons. For example, in the PLM neuron, LIN-14 promotes synaptogenesis. By contrast, in the DD motor neurons, LIN-14 is not required for synaptogenesis, but rather is required for synaptic remodeling {Hallam, 1998 #728}. Furthermore, in the HSN, LIN-14 regulates the timing of axon extension (Olsson-Carter and Slack, 2010). However, in the AVM and PLM it does not regulate axon extension but rather the strength of axon guidance signaling in AVM (Zou et al., 2012) as well as branch formation and synaptogenesis in the PLM. One possibility is that LIN-14 regulates a common set of downstream genes that cause different results in different contexts. Alternatively, LIN-14 might function combinatorially with other transcription factors to regulate different genes in different neurons. In support of the later possibility, recent work has indicated that LIN-14 collaborates with the UNC-30 transcription factor to achieve spatiotemporal control of OIG-1 expression in DD motor neurons (Howell et al., 2015).

Role of LIN-14 in promoting synaptic vesicle trafficking prior to branch formation

We have found that the RAB-3 synaptic vesicle marker is mislocalized to the posterior portion of the PLM axon in lin-14 mutants, suggesting that LIN-14 may promote synaptic vesicle trafficking prior to branch formation. Moreover, we have also found that mutations in genes that are required for synaptic vesicle trafficking cause PLM branch formation defects. Interestingly, prior to branch formation in wild type worms, RAB-3 is clustered to puncta in the region where future branch formation is expected to occur. Moreover, RAB-3 is clustered to the branch point in newly formed PLM branches. Taken together, these observations suggest that LIN-14 may promote branch formation by regulating the trafficking of synaptic vesicles to the region of future branch formation. Consistent with our findings, a recent study of branch formation in the C. elegans HSN neuron has also found that synaptic vesicles cluster in the region of branch formation (Chia et al., 2013). However, in this system mutations in unc-104 and syd-2 produce only subtle defects in branch formation, suggesting that vesicle trafficking may not be required for branch formation in the HSN neuron. The difference in the requirement between the HSN and PLM for vesicle trafficking is likely to reflect additional redundant mechanisms for branch formation in the HSN axon. In the PLM, these redundant mechanisms may be absent or weaker, thereby allowing for defects to be observed in unc-104 and syd-2 mutants.

Materials and Methods

Genetics

Genetic manipulations were carried out using standard procedures. For all experiments the N2 Bristol genetic background was used. All strains were maintained at 20°C, except where noted. All strains were maintained on NGM plates seeded with OP50 bacteria. Alleles used were: lin-4(e912), lin-14(n355), lin-14(n179), lin-14(n355n679). Each of these mutant alleles were obtained from the Caenorhabditis Genetics Center (CGC).

Transgenically encoded fluorescent markers

The jsIs973 and jsIs821 transgenes were obtained from Michael Nonet. The jsIs973 transgene encodes mec-7::rfp and was used to observe the PLM axon and branch. The jsIs821 transgene encodes mec-7::gfp::rab-3 and was used to observe the localization of GFP::RAB-3.

Conditional CRISPR mutants

The lin-4(cKO) mutants were created by microinjection of plasmids encoding mec-7::cas-9 at 5 ng/ul, u6::lin-4_sgRNA#1 at 25 ng/ul, u6::lin-4_sgRNA#2 at 25 ng/ul and odr-1::rfp at 50 ng/ul. The sequence for lin-4_sgRNA#1 was GTCTCTGGGAGTGATGGGG. The sequence for lin-4_sgRNA#2 was AAATGAACCTTTTATTCGG. Stable transgenic arrays were obtained using standard procedures. To confirm the expected deletion size of around 880 bp, we lysed worms carrying the CRISPR transgene and carried out two rounds of PCR. We observed a single band of around 1600 bp in wild type worms and a 700 bp band in worms carrying the CRISPR transgene, indicating the presence of the expected deletion. The lin-14(cKO) mutants were generated in the same way, except that the sgRNAs were as follows: ACGGAACTGCTAAGGCTGG and GTGGGGCAACACGGAGTGG.

Analysis of Phenotypes

For experiments involving synchronized populations, adults were allowed to lay eggs onto a plate for a period of 1 hour. After egg laying, the plates were incubated for 12 hours at 20°C to allow the eggs to hatch. After hatching, larvae were observed at the indicated times after hatching. The size of PLM synaptic clusters were analyzed by imaging the GFP::RAB-3 signal at a preset exposure time and measuring the length of the synaptic cluster using the Zeiss AxioVision software. Recruitment of GFP::RAB-3 into the synaptic region was determined by imaging the axonal RFP signal and the GFP::RAB-3 signal using preset exposure times and recruitment was scored if the GFP::RAB-3 signal covered at least 25% of the length of the synaptic region. For the PLM GFP::RAB-3 posterior clustering phenotype, the axonal RFP signal and the GFP::RAB-3 signal were imaged using preset exposure times. The posterior clustering phenotype was scored if any of the GFP-RAB-3 clusters were in the posterior 1/3 of the PLM axon.

Highlights.

  • The lin-4 microRNA promotes synaptogenesis.

  • The LIN-14 transcription factor inhibits synaptogenesis and promotes branch formation.

  • LIN-14 promotes synaptic vesicle transport prior to branch formation.

  • Genes implicated in synaptic vesicle transport are required for branch formation.

Acknowledgments

We thank Michael Nonet, Guangshuo Ou and the Caenorhabditis Genetics Center (funded by NIH P40 OD010440) for strains and reagents. We also thank Allison Abbott for advice and discussion relating to microRNAs.

Funding

This work was funded by NIH grants 1R03NS091983 and 1R03NS081361 to CCQ. Additional funding came from a Research Growth Initiative grant #101X263 from the University of Wisconsin-Milwaukee (UWM) to CCQ, startup funding from UWM to CCQ, and a Shaw Scientist Award from the Greater Milwaukee Foundation to CCQ.

All strains and reagents are available upon request.

Footnotes

Competing Interests

The authors declare no competing interests.

Author Contributions

Both authors contributed to the experiments, analysis and writing of the manuscript.

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