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• W2891934139 abstract "Scientific Report17 September 2018free access Source DataTransparent process Netrin-1/DCC-mediated PLCγ1 activation is required for axon guidance and brain structure development Du-Seock Kang orcid.org/0000-0002-3688-1087 School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea College of Life Science & Bioengineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon, Korea Search for more papers by this author Yong Ryoul Yang School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Cheol Lee School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author BumWoo Park School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Kwang IL Park School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Jeong Kon Seo UNIST Central Research Facility, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Young Kyo Seo School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author HyungJoon Cho School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Lucio Cocco Cellular Signaling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy Correction added on 6 May 2019, after first online publication: the author name has been corrected. Search for more papers by this author Pann-Ghill Suh Corresponding Author [email protected] orcid.org/0000-0002-9811-756X School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Du-Seock Kang orcid.org/0000-0002-3688-1087 School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea College of Life Science & Bioengineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon, Korea Search for more papers by this author Yong Ryoul Yang School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Cheol Lee School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author BumWoo Park School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Kwang IL Park School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Jeong Kon Seo UNIST Central Research Facility, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Young Kyo Seo School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author HyungJoon Cho School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Lucio Cocco Cellular Signaling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy Correction added on 6 May 2019, after first online publication: the author name has been corrected. Search for more papers by this author Pann-Ghill Suh Corresponding Author [email protected] orcid.org/0000-0002-9811-756X School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea Search for more papers by this author Author Information Du-Seock Kang1,2, Yong Ryoul Yang1, Cheol Lee1, BumWoo Park1, Kwang IL Park1, Jeong Kon Seo3, Young Kyo Seo1, HyungJoon Cho1, Lucio Cocco4 and Pann-Ghill Suh *,1 1School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Korea 2College of Life Science & Bioengineering, Korea Advanced Institute of Science & Technology (KAIST), Daejeon, Korea 3UNIST Central Research Facility, Ulsan National Institute of Science and Technology, Ulsan, Korea 4Cellular Signaling Laboratory, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy *Corresponding author. Tel: +82 52 217 2621; E-mail: [email protected] EMBO Rep (2018)19:e46250https://doi.org/10.15252/embr.201846250 Correction(s) for this article Netrin-1/DCC-mediated PLCγ1 activation is required for axon guidance and brain structure development06 May 2019 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis Netrin-1 activates PLCγ1 via Src kinase, which is crucial for axon guidance and corpus callosum and mDA system development. PLCγ1 promotes axon extension and guidance by mediating netrin-1/DCC signaling. Netrin-1 activates the lipase activity of PLCγ1 through phosphorylation of the Y783-residue by Src kinase. Disruption of PLCγ1 signaling adversely affects corpus callosum structural and mDA system development. 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 5-8, 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 PLCγ1 signaling. Xie et al 12 have reported that netrin-1 can hydrolyze PIP2 in a DCC-dependent manner. This study showed that PLCγ1 may be a potential messenger of netrin-1/DCC signaling; however, there is no direct evidence of a relationship between the DCC receptor and PLCγ1 because receptor DCC does not contain an intracellular catalytic domain. Thus, it is unclear whether PLCγ1 may be a downstream effector of netrin-1/DCC signaling, and if it is, how the netrin-1/DCC complex may regulate PLCγ1 activity. PLCγ1 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 Ca2+ concentrations 15, 16. In addition, chemically blocking Ca2+ entry through transient receptor potential (TRP) channels can severely misroute axons; however, blocking Ca2+ release from intracellular stores did not affect axon guidance because there were no asymmetric Ca2+ gradients in the tip of the axon growth cone 1, 17. These studies suggest that DAG-mediated Ca2+ influx is a key step in the induction of partial changes in the cytoskeleton structure. PLCγ1 has been suggested to induce neurite outgrowth in response to RTKs, such as the fibroblast growth factor and hepatocyte growth factor receptors 18-20. In addition, converging evidence implicates a disruption in PLCγ1 signaling in many neurological disorders 21. In this regard, RTK-mediated PLCγ1 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 PLCγ1 activity by a specific ligand–receptor complex is an attractive model because it allows for the spatiotemporal regulation of PLCγ1 activity. However, in vivo studies on the developmental role of PLCγ1 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 PLCγ1 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 PLCγ1 through proto-oncogene tyrosine-protein kinase Src. Plcg1f/f;Nes-Cre embryos showed a severe axon guidance defect in the dorsal region of the mesencephalon, suggesting that PLCγ1 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 PLCγ1 plays a role in axon extension and guidance by mediating netrin-1/DCC signaling and that disruption of PLCγ1 signaling adversely affects nervous system development. Our results indicate that PLCγ1 is a crucial molecule mediating the directional movement of axons that are regulated by netrin-1/DCC signaling during brain development. Results and Discussion PLCγ1-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 Plcg1f/f;Nes-Cre mice (P140) in the C57BL/6J background were outwardly normal, with the Plcg1f/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 Plcg1f/f;Nes-Cre brains were evenly dispersed (n = 5; Fig 1B–D). Consequently, E12.5 Plcg1f/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 Plcg1f/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 Plcg1f/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 PLCγ1 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 TH-positive axon bundles, confirming that the longitudinally extending axon bundles were dopaminergic (Fig 1I). Click here to expand this figure. Figure EV1. Brain volume, weight, and cell number did not differ between control and Plcg1f/f;Nes-Cre mice A, B. No differences were found in the weight or volume of the adult mouse brains at 12 weeks after birth (n = 4; scale bar = 5 mm). C, D. Levels of phospho-histone H3 (pH3), a marker of the mitotic index, and the total numbers of cells in regions of interest were nearly identical in the two groups of mice (n = 4). Data information: Data are presented as the mean ± s.e.m. NS, not significant; t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Pattern of phospholipase C gamma1 (PLCγ1) expression in the mouse brain In situ hybridization of PLCγ1 in a whole-mount wild-type mouse embryo at 11.5 days (E11.5; scale bar = 5 mm). PLCγ1 was expressed in the subventricular zone (a) and spinal cord (b) (n = 6). At 4 days after birth (P4), PLCγ1 was expressed mainly in the cortex (c), hippocampus (d), subventricular zone (e), cerebellum (f), olfactory bulb (g), and medulla (h) (n = 3; scale bar = 1 mm). A coronal section of a wild-type mouse brain at 12 weeks after birth (scale bar: 1 mm). PLCγ1 was expressed mainly in the cortex (I), hippocampus (j), and medial habenula (k) (n = 3). (a'–k' are enlarged figures of a–k). At P4, PLCγ1 from total mouse brain lysate (Plcg1f/f in lanes 1–3; Plcg1f/f;Nes-Cre in lanes 4–6) was immunoblotted using an antibody directed against PLCγ1, as described previously (n = 3). In situ hybridization of PLCγ1 in a Plcg1f/f;Nes-Cre mouse brain at P4 (n = 3; scale bar = 1 mm). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Phospholipase C gamma1 (PLCγ1)-deficient mouse embryos show a deficit in mesencephalic axon guidance A. Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus are shown magnified in each inset (scale bar = 200 μm). B. Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm). Green and red arrow heads mark the reference point of ROI, respectively. C, D. The axons passing through the ROI were quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005). Data are presented as the mean ± s.e.m. E. Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and ophthalmic nerves (white and yellow boxes). Scale bar: 200 μm. F, G. Enlarged views of the ophthalmic nerve (yellow arrowheads) and dorsal ramus (white arrowheads; scale bar: 50 μm). H. Nerve length measurements; Op, ophthalmic nerve; DR, dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m. I. Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm. Download figure Download PowerPoint Netrin-1/DCC regulates mesencephalic axon guidance via PLCγ1 To establish the relationship between DCC and PLCγ1, we tested whether netrin-1/DCC could affect the phosphorylation of PLCγ1 on Y783 in primary mesencephalic neurons. We found that the level of PLCγ1 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 PLCγ1 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 PLCγ1 is involved in the netrin-1-mediated chemoattraction of axons, the ventral mesencephalon (VM, A9) was micro-dissected from Plcg1f/f (Control) and Plcg1f/f;Nes-Cre mouse brains and co-cultured with 293T cells that had been transiently transfected with a netrin-1-expressing 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 Plcg1f/f;Nes-Cre mouse did not show straight directionality to the netrin-1-releasing 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 Plcg1f/f;Nes-Cre embryo was relatively short as compared to the control (Fig 2I–K). These results indicate that PLCγ1-deficient neurons have a significant defect in mDA axonal attraction by netrin-1. Figure 2. Phospholipase C gamma1 (PLCγ1) mediates netrin-1/DCC signaling In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities (n = 3). Data are presented as the mean ± s.e.m. Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3). Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lysate, #3 and #4: NTN-1-transfected supernatant. Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The yellow lines indicate the boundaries of the 293T cell aggregates. The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4). Schematic diagrams of axon extensions in (D). The graphs were drawn according to the vector values between the borders of the cell aggregates and the mesencephalic tissues. Boxplot representations of the mean angle of the axon extension in (E). Bottom and top whiskers, minima and maxima; bottoms and tops of the rectangles; horizontal lines: medians. Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200, t-test, **P < 0.005). Each experimental set was performed in quadruplicate. Data are presented as the mean ± s.e.m. Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue (scale bar = 200 μm). Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500 μm). Schematic of the method for the measurement of neurite lengths. Mean length of neurites. Bottom and top whiskers, minima and maxima; bottoms and tops of the rectangles; central lines: medians (F-value: 1.82, t-test, **P < 0.005). Source data are available online for this figure. Source Data for Figure 2 [embr201846250-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint Netrin-1/DCC signaling induces PLCγ1 Y783 phosphorylation by Src kinase To understand the molecular mechanism underlying the netrin-1/DCC catalytic activation of PLCγ1, we performed a co-immunoprecipitation assay. These results showed that PLCγ1 did not interact with the DCC receptor (Fig 3A). To identify the site-specific kinases responsible for the phosphorylation of Y783 on PLCγ1, we employed the LC-MS of anti-PLCγ1 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 PLCγ1. Two kinds of tyrosine kinase were identified: RET-proto-oncogene (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 PLCγ1 and Src, we performed co-immunoprecipitation assays for these two proteins in primary cortical neurons. Rac1, a well-known interacting protein of PLCγ1 11, 28, was used as a positive control. Immunoprecipitation with a PLCγ1 antibody pulled down Src and Rac1 (Fig 3B). To test whether Src directly phosphorylated Y783, we performed an in vitro kinase assay with purified PLCγ1 and mouse Src protein. The product was resolved by SDS–PAGE and immunoblotted with an anti-PLCγ1 (pY783) antibody. The Src kinase directly phosphorylated the Y783 site of PLCγ1 (Fig 3C and D). To further determine which tyrosine residue of PLCγ1 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 specific regulation of Src at Y783. Taken together, these results show that netrin-1 induces phosphorylation of PLCγ1 at Y783 by Src kinase. Figure 3. Netrin-1/DCC induces specific phosphorylation of phospholipase C gamma1 (PLCγ1) on Y783 by neuronal proto-oncogene tyrosine kinase Src Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3). Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3). In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. Quantification of the band intensities in (C) (N = 3). Data are presented as the mean ± s.e.m. Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3). Rac1 activity assay (N = 3). Quantification of Rac1 activity levels in (F). Data are presented as the mean ± s.e.m. Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing netrin-1. Source data are available online for this figure. Source Data for Figure 3 [embr201846250-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint 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 Ca2+/calmodulin-dependent kinase IIα (CaMKIIα) 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 PLCγ1-null neurons (Fig 3F and G). In addition, the netrin-1-induced phosphorylation of CaMKII and ERK was significantly reduced in PLCγ1-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 PLCγ1 activation and it mediates the netrin-1/DCC-induced regulation of cell motility. PLCγ1-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 Plcg1f/f;Nes-Cre embryos, we further characterized the structural changes in white matter tracts in the PLCγ1-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 30-33. As we observed, partial tract fibers appeared non-directional in the SI (white arrows), and PLCγ1-depleted axons did not uniformly project toward the anterior part of the cortex in the mutant brain (Fig 4B). In addition, the PLCγ1-deficient tract fibers exhibited higher variance than that of the controls (n = 3 per genotype; one-way 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 neuron-specific 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 Plcg1f/f;Nes-Cre mouse brain, mDA neurons exhibited non-directional axon projections in the SI of Plcg1f/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 Plcg1f/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 PLCγ1 deficiency causes a structural change in the mDA system of the mouse brain. Figure 4. Plcg1 deletion reduces the volume of the corpus callosum and causes abnormal axon projection into the olfactory tubercle Schematic of the main phenotype (CC, corpus callosum; OT, olfactory tubercle; S" @default.
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• W2891934139 title "Netrin‐1/<scp>DCC</scp>‐mediated<scp>PLC</scp>γ1 activation is required for axon guidance and brain structure development" @default.
• W2891934139 cites W1861860658 @default.
• W2891934139 cites W1920903668 @default.
• W2891934139 cites W1964802316 @default.
• W2891934139 cites W1973525545 @default.
• W2891934139 cites W1982830599 @default.
• W2891934139 cites W1984917376 @default.
• W2891934139 cites W1987670066 @default.
• W2891934139 cites W1990378871 @default.
• W2891934139 cites W1996407849 @default.
• W2891934139 cites W2003890389 @default.
• W2891934139 cites W2007491346 @default.
• W2891934139 cites W2009125613 @default.
• W2891934139 cites W2017296316 @default.
• W2891934139 cites W2021718929 @default.
• W2891934139 cites W2023416141 @default.
• W2891934139 cites W2024732305 @default.
• W2891934139 cites W2047701792 @default.
• W2891934139 cites W2052480977 @default.
• W2891934139 cites W2061304971 @default.
• W2891934139 cites W2064591722 @default.
• W2891934139 cites W2067557095 @default.
• W2891934139 cites W2069582700 @default.
• W2891934139 cites W2070153588 @default.
• W2891934139 cites W2071656982 @default.
• W2891934139 cites W2073305444 @default.
• W2891934139 cites W2073784532 @default.
• W2891934139 cites W2086458272 @default.
• W2891934139 cites W2087965446 @default.
• W2891934139 cites W2093371001 @default.
• W2891934139 cites W2094712734 @default.
• W2891934139 cites W2096445305 @default.
• W2891934139 cites W2104992905 @default.
• W2891934139 cites W2116903886 @default.
• W2891934139 cites W2125737877 @default.
• W2891934139 cites W2129722759 @default.
• W2891934139 cites W2135805788 @default.
• W2891934139 cites W2146297126 @default.
• W2891934139 cites W2151237279 @default.
• W2891934139 cites W2152243296 @default.
• W2891934139 cites W2161621335 @default.
• W2891934139 cites W2168745915 @default.
• W2891934139 cites W2730554772 @default.
• W2891934139 doi "https://doi.org/10.15252/embr.201846250" @default.
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