Netrin‐1/DCC‐mediated PLCγ1 activation is required for axon guidance and brain structure development

Coordinated expression of guidance molecules and their signal transduction are critical for correct brain wiring. Previous studies have shown that phospholipase C gamma1 (PLCγ1), a signal transducer of receptor tyrosine kinases, plays a specific role in the regulation of neuronal cell morphology and motility in vitro. However, several questions remain regarding the extracellular stimulus that triggers PLCγ1 signaling and the exact role PLCγ1 plays in nervous system development. Here, we demonstrate that PLCγ1 mediates axonal guidance through a netrin‐1/deleted in colorectal cancer (DCC) complex. Netrin‐1/DCC activates PLCγ1 through Src kinase to induce actin cytoskeleton rearrangement. Neuronal progenitor‐specific knockout of Plcg1 in mice causes axon guidance defects in the dorsal part of the mesencephalon during embryogenesis. Adult Plcg1‐deficient mice exhibit structural alterations in the corpus callosum, substantia innominata, and olfactory tubercle. These results suggest that PLCγ1 plays an important role in the correct development of white matter structure by mediating netrin‐1/DCC signaling.


Introduction
During development, several axon guidance molecules act as key regulators of neuronal wiring by inducing cytoskeleton rearrangement [1]. These guidance cues are perceived by specific receptors that are associated with diverse types of signal transducers that generate secondary messengers, thereby inducing axons to grow toward their proper destinations [2]. Netrin-1, a ligand for the deleted in colorectal cancer (DCC) receptor, functions as a guidance cue for migrating neuronal progenitors and axons in nervous system development by recruiting intracellular signaling complexes. To the best of our knowledge, DCC has not been proposed to function as a receptor tyrosine kinase (RTK), because DCC does not contain an intracellular catalytic domain, but contains three highly conserved protein-binding domains termed P1, P2, and P3 [3,4]. These domains mediate the assembly of various combinations of multiple signaling components such as the non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and Src family kinases [5][6][7][8], which are necessary for the integration of axon guidance cues. In particular, the dimerized P3 domain is important for recruiting focal adhesion kinase (FAK) and Src to the DCC complex [6,9]. These signaling components may contribute to cell motility by regulating the dynamics of the actin cytoskeleton [10,11]. Despite advances in the study of netrin-1/DCC signaling, little is known about how the intracellular DCC signaling complex is organized or how the cells translate the complicated instructions transmitted by this complex into actions. Recently, several in vitro studies have suggested the possibility that the netrin-1/DCC, a guidance cue, may be linked to PLCc1 signaling. Xie et al [12] have reported that netrin-1 can hydrolyze PIP2 in a DCC-dependent manner. This study showed that PLCc1 may be a potential messenger of netrin-1/DCC signaling; however, there is no direct evidence of a relationship between the DCC receptor and PLCc1 because receptor DCC does not contain an intracellular catalytic domain. Thus, it is unclear whether PLCc1 may be a downstream effector of netrin-1/DCC signaling, and if it is, how the netrin-1/DCC complex may regulate PLCc1 activity.
PLCc1 functions as a signal transducer that converts an extracellular stimulus into intracellular signals by generating secondary messengers, such as diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3) [13]. These messengers enable cells to respond to growth factors in a polarized manner, resulting in activities such as chemotactic migration [14]. Importantly, the transient calcium level gradient is essential for inducing a partial change in cytoskeletal structure. Previous studies have shown that the exposure of growth cones to a guidance cue gradient induces a corresponding gradient of elevated Ca 2+ concentrations [15,16]. In addition, chemically blocking Ca 2+ entry through transient receptor potential (TRP) channels can severely misroute axons; however, blocking Ca 2+ release from intracellular stores did not affect axon guidance because there were no asymmetric Ca 2+ gradients in the tip of the axon growth cone [1,17]. These studies suggest that DAG-mediated Ca 2+ influx is a key step in the induction of partial changes in the cytoskeleton structure. PLCc1 has been suggested to induce neurite outgrowth in response to RTKs, such as the fibroblast growth factor and hepatocyte growth factor receptors [18][19][20]. In addition, converging evidence implicates a disruption in PLCc1 signaling in many neurological disorders [21]. In this regard, RTK-mediated PLCc1 signaling is thought to play a pivotal role in the central nervous system development by regulating neuronal cell morphology and motility. Therefore, the regulation of PLCc1 activity by a specific ligand-receptor complex is an attractive model because it allows for the spatiotemporal regulation of PLCc1 activity. However, in vivo studies on the developmental role of PLCc1 have been limited because homozygous deletion of the Plcg1 gene causes an embryonically lethal vasculogenesis defect in mice.
In this study, to determine the function of PLCc1 in the developing brain, we used Nestin-Cre (Nes-Cre) transgenic mice to ablate Plcg1 in developing neuronal precursors. We found that netrin-1/ DCC signaling, a mediator of chemoattractant guidance cues, activates the lipase activity of PLCc1 through proto-oncogene tyrosineprotein kinase Src. Plcg1 f/f ;Nes-Cre embryos showed a severe axon guidance defect in the dorsal region of the mesencephalon, suggesting that PLCc1 may be involved in axon guidance in midbrain dopaminergic (mDA) neurons. This deficit persisted into adulthood, with structural alterations observed in the olfactory tubercle (OT) and substantia innominate (SI). Moreover, Plcg1-deficient mice exhibited diffused axon fibers in the corpus callosum (CC), suggesting that PLCc1 plays a role in axon extension and guidance by mediating netrin-1/DCC signaling and that disruption of PLCc1 signaling adversely affects nervous system development.
Our results indicate that PLCc1 is a crucial molecule mediating the directional movement of axons that are regulated by netrin-1/ DCC signaling during brain development.

Results and Discussion
PLCc1-deficient mouse embryos show a deficit in mesencephalic axon guidance Plcg1-deficient mice were generated by crossing a B6.Cg-Tg (Nes-Cre) 1 Kln/J mouse with a Plcg1-floxed mouse (Fig EV1). Unexpectedly, the brains of the Plcg1 f/f ;Nes-Cre mice (P140) in the C57BL/6J background were outwardly normal, with the Plcg1 f/f ;Nes-Cre and control brains showing few differences in gross morphology and cell division ( Fig EV2). However, we found that the deletion of Plcg1 caused diffused axon bundles in the superior colliculus during embryogenesis. The number of axon bundles decreased markedly, and they rarely reached the dorsal part of the mesencephalon (n = 5; t-test, **P < 0.005; Fig 1A). In control mice, longitudinally extending axon bundles were biased to the posterior part of the superior colliculus; however, the axon bundles of Plcg1 f/f ;Nes-Cre brains were evenly dispersed (n = 5; Fig 1B-D). Consequently, E12.5 Plcg1 f/f ;Nes-Cre brains exhibited tectal projection defects in the regions of the superior colliculus and the pretectal commissure. In mammals, the tectal projection structure from the ventral tegmental area (VTA) to the lateral habenula (LHb) is known to be involved in coordinating monoaminergic neurons in the central nervous system [22]. The axons of mDA neurons lacking DCC no longer innervate the LHb, terminating at its ventral border instead [23]. Similarly, we found a marked decrease in axonal density and axon dispersion in the embryonic superior colliculus as well as the pretectal commissure in Plcg1 f/f ;Nes-Cre brains. In addition, the ophthalmic nerve and dorsal ramus length were shortened by 58 and 44%, respectively, when compared with control embryos (Fig 1E-H). It is noteworthy that the observed mesencephalic pathway defect in Plcg1 f/f ;Nes-Cre embryos may stem from disrupted intracellular downstream signaling of the netrin-1/DCC pathway, because a netrin-1/DCC mutation also resulted in a malformed mesencephalic pathway [24]. Studies of heterozygous and homozygous DCC mutants have shown aberrant ventromedial dopaminergic neuronal migration, dorsal shifting of ventral striatal dopaminergic neuronal axon projections, the aberrant crossing of medial forebrain bundle fibers at the caudal diencephalic midline, and a reduction in prefrontal cortex dopaminergic neuronal innervation [25]. Thus, the disruption of PLCc1 signaling during mesencephalic neuron development can lead to a defect in white matter structure in the adult brain because mesencephalic axons establish wiring patterns that are maintained after development and throughout life [6]. During mouse embryogenesis, the mDA neurons mainly at A8-A10 share the enzyme, tyrosine hydroxylase (TH), which is involved in the synthesis of dopamine; however, they lack the enzymes needed to generate adrenaline or noradrenaline [7,26]. To determine whether the longitudinally extending axon bundles are dopaminergic, we performed double immunofluorescent staining using anti-TH and anti-DCC antibodies. DCC-positive axons were co-localized with THpositive axon bundles, confirming that the longitudinally extending axon bundles were dopaminergic ( Fig 1I).

Netrin-1/DCC regulates mesencephalic axon guidance via PLCc1
To establish the relationship between DCC and PLCc1, we tested whether netrin-1/DCC could affect the phosphorylation of PLCc1 on Y783 in primary mesencephalic neurons. We found that the level of PLCc1 phosphorylation (pY783) increased 30 min after treatment with recombinant netrin-1 protein (NTN1; Fig 2A). Furthermore, when DCC was knocked down with siRNA, we found that the PLCc1 pY783 level was decreased to the basal levels and this decrease was not attenuated when DCC-depleted mesencephalic neurons were treated with netrin-1 (50 ng/ml; Fig 2B). To test whether PLCc1 is involved in the netrin-1-mediated chemoattraction of axons, the ventral mesencephalon (VM, A9) was micro-dissected from Plcg1 f/f (Control) and Plcg1 f/f ;Nes-Cre mouse brains and co-cultured with 293T cells that had been transiently transfected with a netrin-1expressing vector (Fig 2D). We confirmed that netrin-1 was detected in both the cytoplasm and culture media soup of the 293T cells ( Fig 2C). Axons from the control mouse showed directionality toward the netrin-1-releasing 293T cells. By contrast, axons from the Plcg1 f/f ; Nes-Cre mouse did not show straight directionality to the netrin-1releasing 293T cells (Fig 2D), but displayed a more dispersed tendency (Fig 2E and F) (N = 4; n = 200). Most axons in the mutant VM were TH-positive and extended for shorter distances than those in the control (Fig 2G). In addition, netrin-1-induced neurite outgrowth did not occur properly in Plcg1-deficient mDA neurons ( Fig 2H). In culture, the mean length of the extending neurites from Plcg1 f/f ;Nes-Cre embryo was relatively short as compared to the control (Fig 2I-K). These results indicate that PLCc1-deficient neurons have a significant defect in mDA axonal attraction by netrin-1.

Netrin-1/DCC signaling induces PLCc1 Y783 phosphorylation by Src kinase
To understand the molecular mechanism underlying the netrin-1/ DCC catalytic activation of PLCc1, we performed a co-immunoprecipitation assay. These results showed that PLCc1 did not interact with the DCC receptor ( Fig 3A). To identify the site-specific kinases responsible for the phosphorylation of Y783 on PLCc1, we employed the LC-MS of anti-PLCc1 Immunoprecipitates obtained from primary mesencephalic neurons treated with netrin-1. This assay initially identified 960 proteins. Among them, we found a substantial number of proteins sharing a common profile with the negative controls (untreated and nonspecific-binding samples). We excluded these proteins from the initial profile and selected only the tyrosine kinases to identify candidates that could potentially interact with PLCc1. Two kinds of tyrosine kinase were identified: RET-protooncogene (NP_033076.2) and the neuronal proto-oncogene protein tyrosine kinase, Src (NP_033297.2). The Src kinase has been identified as a component of DCC [27]. To verify a direct interaction between PLCc1 and Src, we performed co-immunoprecipitation assays for these two proteins in primary cortical neurons. Rac1, a well-known interacting protein of PLCc1 [11,28], was used as a positive control. Immunoprecipitation with a PLCc1 antibody pulled down Src and Rac1 (Fig 3B). To test whether Src directly phosphorylated Y783, we performed an in vitro kinase assay with purified PLCc1 and mouse Src protein. The product was resolved by SDS-PAGE and immunoblotted with an anti-PLCc1 (pY783) antibody. The Src kinase directly phosphorylated the Y783 site of PLCc1 (Fig 3C and D). To further determine which tyrosine residue of PLCc1 was phosphorylated by Src, we tested the phosphorylation level of each residue in the presence of a Src-specific inhibitor (Src I). We found that only Y783, but not the other three tyrosine residues, was phosphorylated in response to netrin-1 treatment (Fig 3E). This result suggests Netrin-1/DCC regulates cellular motility by activating a variety of signaling molecules, including the small GTP-binding protein, Rac1, extracellular signal-regulated kinase (ERK), and Ca 2+ /calmodulindependent kinase IIa (CaMKIIa) [7]. Therefore, we tested whether ablation of Plcg1 affects the activity of these netrin-1-induced signaling pathways. Using a Rac1 activity assay, we found that netrin-1 could activate Rac1 in control cells; however, Rac1 activity was significantly impaired in the PLCc1-null neurons (Fig 3F and G). In addition, the netrin-1-induced phosphorylation of CaMKII and ERK was significantly reduced in PLCc1-null neurons, whereas the phosphorylation of FAK and Src remained unaltered (Fig 3H). These results suggest that the recruitment of Src and FAK to DCC is a prerequisite for the PLCc1 activation and it mediates the netrin-1/ DCC-induced regulation of cell motility.
PLCc1-deficient mouse brain exhibits a CC size reduction and misrouted axon bundles of the OT During neural development, mDA neurons extend their axons toward the anteromedial and ventral parts of the striatum, and then innervate the limbic system and neocortex, where these neurons constitute the mesocorticolimbic pathways. Based on our findings of an axonal guidance defect in Plcg1 f/f ;Nes-Cre embryos, we further characterized the structural changes in white matter tracts in the PLCc1-deficient adult brain. The most discernible phenotype was manifested in abnormally dispersed neural projections into the OT and SI (Fig 4B and D). These two structures are formed through embryonic development. The OT and SI are noted for the being innervated by mDA neurons from the VTA (Fig 4A). At E9.5, subplate neurons initially extend pioneer axons through the internal capsule that provide paths for follower axons that begin to appear [29]. At E11.5, VM neurons begin to extend their axons along the pioneer axon pathways to reach their telencephalic targets [30,31]. At E13.5, the axons pass longitudinally through the midbrain and diencephalon to form the medial forebrain bundle [30,31]. From E14.5 to E18.5, axon bundles reach the telencephalon and striatum, followed by innervation of the limbic system and neocortex [30][31][32][33].
As we observed, partial tract fibers appeared non-directional in the SI (white arrows), and PLCc1-depleted axons did not uniformly project toward the anterior part of the cortex in the mutant brain ( Fig 4B). In addition, the PLCc1-deficient tract fibers exhibited higher variance than that of the controls (n = 3 per genotype; oneway ANOVA; F-value 118.57; ***P < 0.001; Fig 4C). To determine whether Plcg1 inactivation in dopaminergic neurons actually leads to the mDA projection defect, we generated dopaminergic neuronspecific Plcg1 knockout mice by crossing the Plcg1-floxed strain with a Slc6a3 (DAT)-Cre transgenic line. In these mice, Plcg1 was specifically deleted in the TH-positive neurons (Fig 4F). Similar to the Plcg1 f/f ;Nes-Cre mouse brain, mDA neurons exhibited non-directional axon projections in the SI of Plcg1 f/f ;DAT-Cre mouse brains (yellow arrows; Fig 4D). To quantify the deviated axons, we divided the region of interest (ROI) into dorsal and ventral SI parts (> 0 to ≤ 1,350 and > 1,350 to ≤ 2,700) and measured fluorescence intensity in the ROI (red box). The axon fibers from Plcg1 f/f ;DAT-Cre mice were more widely distributed in the ventral part of the SI than in the control (n = 3 per genotype; one-way ANOVA; F-value 272.82, ***P < 0.001; Fig 4E). These observations suggest that the lack of response to netrin-1 signaling due to PLCc1 deficiency causes a structural change in the mDA system of the mouse brain.
In addition, we further characterized the structural changes in the CC in the PLCc1-deficient adult brain (Fig 4G and H). Previous studies have shown that mice lacking DCC exhibits severe neurodevelopmental defects in multiple central nervous system (CNS) commissures. In particular, netrin-1/DCC signaling exerts a significant influence on the development of the CC [1]. Based on our previous findings, we hypothesized that a deficiency in the netrin-1 response due to lack of PLCc1 may have an adverse effect on CC development. To test this hypothesis, we analyzed the structural changes in the CC in the PLCc1-deficient adult brain. We selected coronal sections from Plcg1 f/f and Plcg1 f/f ;Nes-Cre brains at the same rostrocaudal point to compare CC morphology between the genotypes. The CC in the Plcg1 f/f ;Nes-Cre mice had an oval shape owing to differences in CC size (Fig 4G). We acquired whole images of sagittal sections and delineated the CC to measure its area. The area of the CC in the Plcg1 f/f ;Nes-Cre mice was significantly smaller compared to that in the Plcg1 f/f mice (control: 1.18 AE 0.07; Plcg1  We compared control and Plcg1 f/f ;Nes-Cre mice by extracting quantitative parameters from tensor calculations for the CC area in each case (Fig 5A, C, and E). 6 of 11  Fig 5D). Finally, we measured the lengths of the tensor lines to evaluate changes in tensor line length between Plcg1 f/f ;Nes-Cre: 1,536.00 AE 203.51; Shapiro-Wilk and t-test, *P < 0.05; Fig 5F). We grouped the tensor line lengths into three categories: < 1.0, ≥ 1.0 to < 3.0, and ≥ 3.0 mm corresponding to short, medium, and long lengths, respectively. To test distribution normality, we applied the Shapiro-Wilk test to the three measurements [34]. Then, a t-test was performed to determine any differences between the Plcg1 f/f and Plcg1 f/f ;Nes-Cre mice. We observed that the number of tensors and volume in PLCc1-deficient mouse brains were decreased by 20 and 21%, respectively (n = 4) compared to the control values. In addition, the number of axon fibers in sections with FA values of ≥ 0.35 to ≤ 0.75 was decreased significantly, by 20% (n = 4). These data suggest that the CC axons of Plcg1 f/f ;Nes-Cre mice are more randomly diffused than those in control mice.
Recently, meaningful advances have been made toward revealing how neural networks are established during development; however, the precise signaling mechanisms that are involved in the formation of a typical wiring pattern are only partially understood. Our results have demonstrated that the regulation of neural development by netrin-1/DCC signaling may be mediated, at least in part, through the activation of PLCc1, which controls key steps in the axonal projection of mDA neurons as well as CC structural formation. In particular, mDA neurons have been implicated in various kinds of neurological disorders such as addiction, parkinsonism, schizophrenia, sleep abnormalities, and ADHD, with some evidence indicating that structural changes in neuronal networks may in part underlie the pathogenesis of these disorders [35][36][37][38][39]. Although axon guidance events are crucial for the correct development of mDA pathways, their precise role during the pathogenesis of mDA system-related disorders has not been established. Thus, further study assessing the causality between the structural defect in the PLCc1-null mDA system and behavioral phenotypes is required to better understand their potential contribution of axon guidance mechanisms to neurological disorders.

Animals
All animal experiments were performed using 8-week-old agematched, male C57/BL6 mice with neural progenitor-specific conditional knockout of Plcg1 (Plcg1 f/f ;Nes-Cre) or C57/BL6 mice expressing Cre recombinase under the control of the Slc6a3 (or DAT) promoter (Plcg1 f/f ;DAT-Cre); their floxed littermates were used as controls. All animal experiments were performed according to accepted international instructions for the use and implementation of such studies. All animal experiments were approved by the Institutional Animal Care and Use Committee at Ulsan National Institute of Science and Technology (UNIST-IACUC-14-005).

Conditioned medium
The conditioned medium used in Fig 3F-H

Mesencephalic neuron culture
Mesencephalic neurons were harvested and cultured as described previously [41]. Neuron preparations with a rate ≥ 95% on the trypan blue exclusion test were cultured further. The cells were seeded at a density of 4 × 10 5 cells per well on a poly-D-lysinecoated six-well plate and maintained in a humidified incubator at 37°C in an atmosphere of 5% CO 2 .

Post-processing and tensor line calculation
Region of interests for the CC were drawn on each RARE image for post-processing. Spline algorithms were applied to create smooth CC borderlines for each slice [42]. A total of 45 ROIs from all slices were interpolated to have an isotropic spatial resolution for the CC by cubic interpolation (ImageJ, version 1.48, National Institutes of Health). Diffusion Toolkit and TrackVis programs were used to calculate, visualize, and quantify the tensor lines. During the tensor calculation, corresponding ROIs from RARE images were used to delineate areas of the CC.

Rac1 activity assay
For netrin-1 treatment, 1.5 × 10 6 primary cortical neurons plated in 100-mm dishes. After 2 days of incubation, the cells were washed three times with serum-free neurobasal media. 8 h after serum starvation, the culture medium was changed to a conditioned medium with or without netrin-1. After 30 min, cells were lysed on ice in lysis buffer [50 mmol/l Tris (pH 7.2), 100 mmol/l NaCl, 5 mmol/l MgCl 2 , 1 mmol/l DTT, 10% glycerol, and 1% NP40] containing protease inhibitors, and Rac GTP pull-down assays were then performed according to the manufacturer's instructions (Abcam, Cambridge, UK).

Western blotting
Whole brain tissues and mesencephalic neurons were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer

Statistics
All statistical tests used in this study were performed two-sided. Statistical data are expressed as means AE s.e.m. At least three biological replicates were performed for all studies. Independent two-tailed t-test was used for analysis of differences between two groups. When comparing more than two groups, ANOVA was used followed by Holm-Sidak test or Bonferroni correction. For normal distribution, we applied the Shapiro-Wilk test. A P-value of less than 0.05 was considered statistically significant: *P < 0.05, **P < 0.005.
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