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• W3196210988 abstract "•Ang1 and Ang2 are expressed in cerebellar neural and endothelial cells, respectively•The angiopoietin receptor Tie2 is expressed in blood vessels and Purkinje cells•Tie2 signaling regulates PC dendritic morphogenesis in a cell-autonomous manner•PCs network functionality is altered in PC specific Tie2-deficient mice Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function. Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function. Research of the last decade has shown that factors involved in the development of the nervous system are also required to regulate blood vessel growth and guidance, and vice versa. Furthermore, the intercellular communication between vascular and neural cells (neural progenitors, neurons, and glia) is crucial for the proper development and function of the central and peripheral nervous systems (Paredes et al., 2018Paredes I. Himmels P. Ruiz de Almodóvar C. Neurovascular Communication during CNS Development.Dev. Cell. 2018; 45: 10-32Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar; Segarra et al., 2019Segarra M. Aburto M.R. Hefendehl J. Acker-Palmer A. Neurovascular Interactions in the Nervous System.Annu. Rev. Cell Dev. Biol. 2019; 35: 615-635Crossref PubMed Scopus (21) Google Scholar). However, the molecular determinants that both systems use to communicate are poorly understood. While most studies in this context have focused on characterizing particular aspects of this neurovascular crosstalk in the developing cortex, spinal cord, and retina (Paredes et al., 2018Paredes I. Himmels P. Ruiz de Almodóvar C. Neurovascular Communication during CNS Development.Dev. Cell. 2018; 45: 10-32Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), the cerebellum, and how neuro-vascular mechanisms might regulate dendritic morphogenesis, has been largely unexplored. Cerebellar function has been linked to sensory-motor processing (D’Angelo et al., 2011D’Angelo E. Mazzarello P. Prestori F. Mapelli J. Solinas S. Lombardo P. Cesana E. Gandolfi D. Congi L. The cerebellar network: from structure to function and dynamics.Brain Res. Brain Res. Rev. 2011; 66: 5-15Crossref PubMed Scopus (68) Google Scholar), as well as to the coordination of emotions (e.g., reward seeking and anxiety) and diverse aspects of cognition (e.g., spatial learning and navigation) (Adamaszek et al., 2017Adamaszek M. D’Agata F. Ferrucci R. Habas C. Keulen S. Kirkby K.C. Leggio M. Mariën P. Molinari M. Moulton E. et al.Consensus Paper: Cerebellum and Emotion.Cerebellum. 2017; 16: 552-576Crossref PubMed Scopus (208) Google Scholar; Koziol et al., 2014Koziol L.F. Budding D. Andreasen N. D’Arrigo S. Bulgheroni S. Imamizu H. Ito M. Manto M. Marvel C. Parker K. et al.Consensus paper: the cerebellum’s role in movement and cognition.Cerebellum. 2014; 13: 151-177Crossref PubMed Scopus (550) Google Scholar; Wang and Zoghbi, 2001Wang V.Y. Zoghbi H.Y. Genetic regulation of cerebellar development.Nat. Rev. Neurosci. 2001; 2: 484-491Crossref PubMed Scopus (373) Google Scholar). In mice, the formation of the cerebellum starts during embryonic stages and continues postnatally (Butts et al., 2014Butts T. Green M.J. Wingate R.J. Development of the cerebellum: simple steps to make a ‘little brain’.Development. 2014; 141: 4031-4041Crossref PubMed Scopus (131) Google Scholar). The mature cerebellum contains several types of excitatory and inhibitory neurons, organized in a three-layered structure: from outside to inside—the molecular layer (ML), the Purkinje cell layer (PCL), and the internal granular layer (IGL) (Butts et al., 2014Butts T. Green M.J. Wingate R.J. Development of the cerebellum: simple steps to make a ‘little brain’.Development. 2014; 141: 4031-4041Crossref PubMed Scopus (131) Google Scholar). PCs represent the main GABAergic cell type, integrating motor, and sensory information derived from an array of parallel fibers and a single climbing fiber input. The PC soma is positioned in the PCL (a single cell layer), and its extensively branched dendritic tree expands into the ML. PC dendrites project in a planar fashion, oriented in parallel to the anterior-posterior axis of the brain (Leto et al., 2016Leto K. Arancillo M. Becker E.B. Buffo A. Chiang C. Ding B. Dobyns W.B. Dusart I. Haldipur P. Hatten M.E. et al.Consensus Paper: Cerebellar Development.Cerebellum. 2016; 15: 789-828Crossref PubMed Scopus (182) Google Scholar). PC dendritic morphogenesis starts postnatally and proceed during a long period of time up to at least 3 months of age (McKay and Turner, 2005McKay B.E. Turner R.W. Physiological and morphological development of the rat cerebellar Purkinje cell.J. Physiol. 2005; 567: 829-850Crossref PubMed Scopus (182) Google Scholar). It is regulated by cellular mechanisms involving dendritic growth, branching, dendritic self-avoidance, retraction processes, and maturation, which assures precise integration of inputs and propagation of signals (Takeo et al., 2015Takeo Y.H. Kakegawa W. Miura E. Yuzaki M. RORα Regulates Multiple Aspects of Dendrite Development in Cerebellar Purkinje Cells In Vivo.J. Neurosci. 2015; 35: 12518-12534Crossref PubMed Scopus (31) Google Scholar). Several signaling molecules have been described to regulate PC morphogenesis, among them Slit/Robo signaling (Gibson et al., 2014Gibson D.A. Tymanskyj S. Yuan R.C. Leung H.C. Lefebvre J.L. Sanes J.R. Chédotal A. Ma L. Dendrite self-avoidance requires cell-autonomous slit/robo signaling in cerebellar purkinje cells.Neuron. 2014; 81: 1040-1056Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), Protocadherins (Ing-Esteves et al., 2018Ing-Esteves S. Kostadinov D. Marocha J. Sing A.D. Joseph K.S. Laboulaye M.A. Sanes J.R. Lefebvre J.L. Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.J. Neurosci. 2018; 38: 2713-2729Crossref PubMed Scopus (40) Google Scholar; Lefebvre et al., 2012Lefebvre J.L. Kostadinov D. Chen W.V. Maniatis T. Sanes J.R. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system.Nature. 2012; 488: 517-521Crossref PubMed Scopus (281) Google Scholar; Toyoda et al., 2014Toyoda S. Kawaguchi M. Kobayashi T. Tarusawa E. Toyama T. Okano M. Oda M. Nakauchi H. Yoshimura Y. Sanbo M. et al.Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity.Neuron. 2014; 82: 94-108Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), tropomyosin receptor kinase C (TrkC) (Huang et al., 2014Huang G.J. Edwards A. Tsai C.Y. Lee Y.S. Peng L. Era T. Hirabayashi Y. Tsai C.Y. Nishikawa S. Iwakura Y. et al.Ectopic cerebellar cell migration causes maldevelopment of Purkinje cells and abnormal motor behaviour in Cxcr4 null mice.PLoS ONE. 2014; 9: e86471Crossref PubMed Scopus (21) Google Scholar; Joo et al., 2014Joo W. Hippenmeyer S. Luo L. Neurodevelopment. Dendrite morphogenesis depends on relative levels of NT-3/TrkC signaling.Science. 2014; 346: 626-629Crossref PubMed Scopus (65) Google Scholar), and thyroid hormone receptor α1 (TRα1) (Heuer and Mason, 2003Heuer H. Mason C.A. Thyroid hormone induces cerebellar Purkinje cell dendritic development via the thyroid hormone receptor alpha1.J. Neurosci. 2003; 23: 10604-10612Crossref PubMed Google Scholar), among others. Remarkably, at the same time as PCs are extending and positioning their dendritic trees, blood vessels are growing and branching in close vicinity. This also occurs within an environment of glia cells (Buffo and Rossi, 2013Buffo A. Rossi F. Origin, lineage and function of cerebellar glia.Prog. Neurobiol. 2013; 109: 42-63Crossref PubMed Scopus (99) Google Scholar). If and how growing blood vessels, glia cells, and neurons communicate during cerebellum formation to assure proper PC dendritic morphogenesis remains unknown. The angiopoietin-Tie signaling pathway is crucial for blood vessel formation, assembly, maturation, and homeostasis (Augustin et al., 2009Augustin H.G. Koh G.Y. Thurston G. Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system.Nat. Rev. Mol. Cell Biol. 2009; 10: 165-177Crossref PubMed Scopus (996) Google Scholar). In ECs, Tie2 is the main protein tyrosine kinase receptor that is activated by angiopoietin ligands. Its activation is tightly regulated by the ratio of Ang1/Ang2 expression. While Ang1-induced signaling mediates EC survival and maturation, Ang2 can either counteract Ang1 or induce Tie2 activation in a context-dependent manner (Augustin et al., 2009Augustin H.G. Koh G.Y. Thurston G. Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system.Nat. Rev. Mol. Cell Biol. 2009; 10: 165-177Crossref PubMed Scopus (996) Google Scholar). Tie2 activation is further regulated by the presence or absence of its co-receptor Tie1 (Hansen et al., 2010Hansen T.M. Singh H. Tahir T.A. Brindle N.P. Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface.Cell. Signal. 2010; 22: 527-532Crossref PubMed Scopus (85) Google Scholar; Savant et al., 2015Savant S. La Porta S. Budnik A. Busch K. Hu J. Tisch N. Korn C. Valls A.F. Benest A.V. Terhardt D. et al.The Orphan Receptor Tie1 Controls Angiogenesis and Vascular Remodeling by Differentially Regulating Tie2 in Tip and Stalk Cells.Cell Rep. 2015; 12: 1761-1773Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Concerning CNS development, several studies (mainly in vitro) suggest a potential role of the angiopoietins in regulating neuronal progenitor proliferation, differentiation, migration, survival, and branching (Bai et al., 2009Bai Y. Cui M. Meng Z. Shen L. He Q. Zhang X. Chen F. Xiao J. Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway.Biochem. Biophys. Res. Commun. 2009; 378: 296-301Crossref PubMed Scopus (28) Google Scholar; Kosacka et al., 2005Kosacka J. Figiel M. Engele J. Hilbig H. Majewski M. Spanel-Borowski K. Angiopoietin-1 promotes neurite outgrowth from dorsal root ganglion cells positive for Tie-2 receptor.Cell Tissue Res. 2005; 320: 11-19Crossref PubMed Scopus (44) Google Scholar; Marteau et al., 2011Marteau L. Pacary E. Valable S. Bernaudin M. Guillemot F. Petit E. Angiopoietin-2 regulates cortical neurogenesis in the developing telencephalon.Cereb. Cortex. 2011; 21: 1695-1702Crossref PubMed Scopus (20) Google Scholar; Rosa et al., 2010Rosa A.I. Gonçalves J. Cortes L. Bernardino L. Malva J.O. Agasse F. The angiogenic factor angiopoietin-1 is a proneurogenic peptide on subventricular zone stem/progenitor cells.J. Neurosci. 2010; 30: 4573-4584Crossref PubMed Scopus (50) Google Scholar; Ward et al., 2005Ward N.L. Putoczki T. Mearow K. Ivanco T.L. Dumont D.J. Vascular-specific growth factor angiopoietin 1 is involved in the organization of neuronal processes.J. Comp. Neurol. 2005; 482: 244-256Crossref PubMed Scopus (37) Google Scholar). However, the in vivo characterization of their effects on neurons is still missing. In addition, in vivo proof of whether the receptor Tie2 is expressed in certain neuronal types, and if the observed effects occur via direct signaling to Tie2 in neurons is lacking. In this study, we describe a cell-autonomous role for Tie2 in PCs as a modifier of dendritic morphogenesis. Using in vivo mouse genetics for Ang1, Ang2, and Tie2, we identify the Ang-Tie2 pathway as a neural-vascular-PC intercellular communication axis that regulates PC dendritic morphogenesis, with Tie2 acting in a PC cell-autonomous fashion. Unbiased analysis of the PC translatome indicates that the absence of Tie2 in PCs leads to deregulated expression of proteins involved in cytoskeleton dynamics and neurite branching. At the functional level, we show that Tie2-deficient PCs present altered network electrophysiological properties. We analyzed the temporal expression of angiopoietin ligands and their Tie2 receptor in the entire developing cerebellum. In the first postnatal week, Ang1 mRNA expression levels increased and peaked at P7. Subsequently, mRNA levels gradually diminished (Figure S1A). Consistently, in situ hybridization (ISH) showed that Ang1 mRNA was detected in the white matter (WM) of the cerebellum as well as in the IGL (Figure 1A). Ang1 mRNA expression declined in later stages (Figures S1A and S1B). To determine the cellular source of Ang1 we analyzed an available single cell RNA sequencing (scRNA-seq) dataset of the developing murine cerebellum (Carter et al., 2018Carter R.A. Bihannic L. Rosencrance C. Hadley J.L. Tong Y. Phoenix T.N. Natarajan S. Easton J. Northcott P.A. Gawad C. A Single-Cell Transcriptional Atlas of the Developing Murine Cerebellum.Curr. Biol. 2018; 28: 2910-2920.e2Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In this dataset, Ang1 expression was found in the majority of GABAergic interneurons, in pial vessels and in a subpopulation of astrocytes (Figure S1C). To verify these results, we performed ISH combined with immunofluorescent (IF) staining on postnatal cerebellar tissue at different developmental stages (from P3 to P21). Consistent with the analysis of Ang1 expression using the scRNA-seq dataset, Ang1 mRNA colocalized with the GABAergic interneuron lineage marker Pax2 (Fleming et al., 2013Fleming J.T. He W. Hao C. Ketova T. Pan F.C. Wright C.C.V. Litingtung Y. Chiang C. The Purkinje neuron acts as a central regulator of spatially and functionally distinct cerebellar precursors.Dev. Cell. 2013; 27: 278-292Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Maricich and Herrup, 1999Maricich S.M. Herrup K. Pax-2 expression defines a subset of GABAergic interneurons and their precursors in the developing murine cerebellum.J. Neurobiol. 1999; 41: 281-294Crossref PubMed Scopus (192) Google Scholar; Figure 1B). Ang1 expression also colocalized with GFAP-expressing astrocytes located in the WM and the IGL (Figure 1C), which is consistent with previously published data (Baldwin et al., 2001Baldwin M.E. Catimel B. Nice E.C. Roufail S. Hall N.E. Stenvers K.L. Karkkainen M.J. Alitalo K. Stacker S.A. Achen M.G. The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.J. Biol. Chem. 2001; 276: 19166-19171Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The temporal course of Ang2 mRNA expression in the whole cerebellum was less dynamic (Figure S1D). Expression of Ang2 mRNA was found in ECs at all developmental stages analyzed, as previously described (Fiedler et al., 2006Fiedler U. Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. Witzenrath M. Rosseau S. Suttorp N. et al.Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.Nat. Med. 2006; 12: 235-239Crossref PubMed Scopus (701) Google Scholar; Figure 1D). Next, we analyzed the expression of the angiopoietin receptor Tie2 using a previously described Tie2-GFP reporter mouse line (De Palma et al., 2005De Palma M. Venneri M.A. Galli R. Sergi Sergi L. Politi L.S. Sampaolesi M. Naldini L. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors.Cancer Cell. 2005; 8: 211-226Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar). GFP expression was not just restricted to blood vessels of the postnatal cerebellum but was also observed in cerebellar PCs at P7 and P9 (Figure 1E; Figure S1E) and adult (Figure S1E). Of note, GFP signal was also detected in pyramidal neurons of the developing hippocampus (Figure S1F). Expression of Ang1 mRNA was also observed in the developing hippocampus (Figure S1G). To confirm the expression of Tie2 in PCs, we made use of the RiboTag mouse line, in which a modified ribosomal protein Rpl22 is tagged with an HA epitope and expressed in a Cre-dependent manner (Sanz et al., 2009Sanz E. Yang L. Su T. Morris D.R. McKnight G.S. Amieux P.S. Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.Proc. Natl. Acad. Sci. USA. 2009; 106: 13939-13944Crossref PubMed Scopus (477) Google Scholar). We crossed a PC-specific Cre driver line (Pcp2Cre; Barski et al., 2000Barski J.J. Dethleffsen K. Meyer M. Cre recombinase expression in cerebellar Purkinje cells.Genesis. 2000; 28: 93-98Crossref PubMed Scopus (135) Google Scholar) with RiboTag mice to assure specific expression in PCs (from here on Pcp2Cre:Rpl22HA/HA). The temporal expression of Cre recombinase in the driver line and its functionality was assessed by ISH of Cre mRNA as well as by using the ROSA-mTmG reporter line (from here on Pcp2Cre:mTmG) at different postnatal stages (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2101) Google Scholar). These approaches showed that Cre expression and activity started around P7 in a small number of PCs and that the majority of PCs expressed functional Cre recombinase by P21 (Figures S1H and S1I). Considering the temporal expression of Cre recombinase in PCs, we immunoprecipitated the HA-Rpl22 subunit together with its associated mRNA from cerebellar lysates of Pcp2Cre:Rpl22HA/HA animals at different time points starting from P10 and analyzed expression of Tie2 in the associated mRNA. Tie2 mRNA was detectable after ribosomal pull-down in all tested stages (Figure S1J). Finally, to verify that Tie2 mRNA was expressed in PCs, we crossed Pcp2Cre:Rpl22HA/HA mice with Tie2flox/flox animals (Savant et al., 2015Savant S. La Porta S. Budnik A. Busch K. Hu J. Tisch N. Korn C. Valls A.F. Benest A.V. Terhardt D. et al.The Orphan Receptor Tie1 Controls Angiogenesis and Vascular Remodeling by Differentially Regulating Tie2 in Tip and Stalk Cells.Cell Rep. 2015; 12: 1761-1773Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) to generate triple-transgenic Pcp2Cre:Tie2flox/flox:Rpl22HA/HA mice, in which a reduction of Tie2 mRNA should be detected after HA-Rpl22 pull-down, when compared to Pcp2Cre:Rpl22HA/HA animals. Indeed, Tie2 mRNA expression was reduced in the triple-transgenic mice as early as P10 (Figure S1M), confirming the expression of Tie2 in PCs. Taken together, the time points when Tie2 and its ligands Ang1 and Ang2 were expressed in the developing cerebellum coincided with the critical period of PC dendritic morphogenesis. This suggests that the angiopoietin-Tie2 signaling axis could be an intercellular communication mechanism via which neural cells, ECs, and PCs crosstalk to regulate PC development. As Tie2 is expressed in PCs during this developmental period, this also suggests that Tie2 might exert a cell-autonomous function in PCs. The expression of Tie2 in PCs led us to investigate whether Tie2 regulated the dendritic development of these neurons in vivo, in a cell-autonomous manner. For this, we analyzed PC dendritic morphogenesis in Pcp2Cre+/−:Tie2flox/flox mice (in which Tie2 was specifically deleted in PCs; termed from hereon Pcp2Cre:Tie2flox/flox) and control littermates (Pcp2Cre−/−:Tie2flox/flox; termed from hereon Tie2flox/flox). Pcp2Cre:Tie2flox/flox animals showed no obvious differences in cerebellar anatomy, density of PC soma, or bodyweight when compared to control mice (Figures 2A–2D). PC dendritic branching was analyzed by stereotactic injection of low concentrations of AAV serotype 8 encoding YFP (AAV8-YFP, which has a tropism for PCs; Gibson et al., 2014Gibson D.A. Tymanskyj S. Yuan R.C. Leung H.C. Lefebvre J.L. Sanes J.R. Chédotal A. Ma L. Dendrite self-avoidance requires cell-autonomous slit/robo signaling in cerebellar purkinje cells.Neuron. 2014; 81: 1040-1056Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) into the cerebellum of P7, P42, or 6-month-old mice, allowing us to reconstruct PC dendritic arbors at the single-cell level upon imaging (Figures S2A–S2C). Mice were sacrificed 2 weeks post-injection, and the number of PC dendritic branches as well as the total dendritic length were analyzed (see STAR Methods and Figure S2 for further details of the analysis). This revealed that Pcp2Cre:Tie2flox/flox mice lost dendritic complexity over time. While initially the number of branches was slightly increased at P21 (Figures 2E–2G), PC dendritic complexity at later stages was characterized by a reduced number of both dendritic branches and total dendritic length (Figures 2H–2M). Notably, we also observed changes in the ordered array of the dendritic field (Figures 2E, 2H, and 2K). Interestingly, similar defects in dendritic morphogenesis were observed in heterozygous Pcp2Cre:Tie2+/flox animals (Figures S3A–S3C and S3G–S3I). Pcp2Cre+/− mice (from here on Pcp2Cre; Pcp2Cre−/− animals from here on referred to as WT) were analyzed as an additional control. These mice did not show any differences in cerebellar anatomy, the density of PC soma and bodyweight, or in PC dendritic morphology (Figures S4A–S4G), indicating that it is the absence of Tie2 in PCs that leads to the observed dendritic branching phenotype and not the expression of Cre recombinase. In summary, our analysis revealed that Tie2 in PCs contributes to the regulation of dendritic morphogenesis in a cell-autonomous manner. The fact that Pcp2Cre:Tie2flox/flox mice showed a slight increase in PC branching at P21, which is not maintained at later developmental stages, suggests that a compensatory mechanism might be active in early development, but this is not sufficient to compensate for the absence of Tie2. Next, we asked whether changes in the expression of the Tie2 ligands Ang1 and Ang2 would also impair PC dendritic morphogenesis. To investigate the role of Ang1 in the developing cerebellum, we crossed Ang1flox/flox mice (provided by Susan E. Quaggin [Jeansson et al., 2011Jeansson M. Gawlik A. Anderson G. Li C. Kerjaschki D. Henkelman M. Quaggin S.E. Angiopoietin-1 is essential in mouse vasculature during development and in response to injury.J. Clin. Invest. 2011; 121: 2278-2289Crossref PubMed Scopus (305) Google Scholar]) with the Nestin-Cre driver line (Tronche et al., 1999Tronche F. Kellendonk C. Kretz O. Gass P. Anlag K. Orban P.C. Bock R. Klein R. Schütz G. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.Nat. Genet. 1999; 23: 99-103Crossref PubMed Scopus (1390) Google Scholar). Nestin-Cre recombines in all cells derived from Nestin-expressing neural progenitors (Wojcinski et al., 2017Wojcinski A. Lawton A.K. Bayin N.S. Lao Z. Stephen D.N. Joyner A.L. Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin+ progenitors.Nat. Neurosci. 2017; 20: 1361-1370Crossref PubMed Scopus (46) Google Scholar), including interneurons and astrocytes, where we detected Ang1 expression during cerebella development. Ang1 deletion was confirmed by ISH (Figure S5A) and qPCR (Figures S5B and S5D). Genetic inactivation of Ang1 expression did not affect Ang2 mRNA levels (Figures S5C and S5E). The overall anatomy of the cerebellum and the density of PC soma were unchanged in NesCre:Ang1+/flox and NesCre:Ang1flox/flox animals (Figures 3A–3C), despite the fact that both genotypes showed a reduction in bodyweight at P21 compared to their Cre-negative littermates (Figure 3D). Using the same AAV8 approach, we then studied PC dendritic development in NesCre:Ang1+/flox and NesCre:Ang1flox/flox animals. PCs showed lower dendritic complexity already at P21, with a reduced number of dendritic branches in both genotypes, and reduced total dendritic length in NesCre:Ang1flox/flox animals, when compared to control littermates (Figures S5F–S5H). This phenotype was even more severe at P56 (Figures 3E–3G). Similarly, dendritic length of in vivo CA1 hippocampal neurons of NesCre:Ang1flox/flox pups (measured from Golgi staining) was also reduced compared to control mice (Figures S5I and S5J). These data suggested that Ang1-Tie2 signaling in Tie2-expressing neurons might be required to promote the development of dendritic branches. To test this hypothesis in PCs, we used an in vitro approach using cerebellar slice cultures. Cerebellar slices from P6 mice were cultured with or without recombinant Ang1 for 3 days in vitro (DIV). Analysis of PC dendrites showed that Ang1 stimulation resulted in a significant increase in both the number of branches and branch length (Figures 3H–3J). Taken together, these data show that Ang1 promoted dendritic growth and branching in CNS neurons. In vivo, neural loss of Ang1 led to a reduction in PC dendritic complexity, which recapitulated the phenotype observed in PC-specific Tie2 knockout mice. Based on the expression pattern of Ang1, our data suggest that direct binding of neural-derived Ang1 to Tie2 in PCs might induce a signaling cascade to regulate dendritic development. We next studied whether Ang2 would also contribute to PC dendritic development using homo- and heterozygous Ang2 full knockout mice (from hereon Ang2+/− and Ang−/−) (Gale et al., 2002Gale N.W. Thurston G. Hackett S.F. Renard R. Wang Q. McClain J. Martin C. Witte C. Witte M.H. Jackson D. et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1.Dev. Cell. 2002; 3: 411-423Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Reduction of cerebellar Ang2 mRNA was confirmed in whole-tissue lysates of P21 mice (Figure S5K). Levels of Ang1 mRNA were unchanged in Ang2+/− and Ang2−/− animals (Figure S5L). The overall anatomy of the cerebellum, including the thickness of the ML as well as the density of PC somas, was unchanged (Figures 3K–3M). However, Ang2−/− mice had significantly reduced bodyweight at P21 compared to their WT littermates (Figure 3N). At P21, a significant reduction in the number of dendritic branches as well as the total dendritic length was observed in Ang2−/− animals but not in Ang2+/− mice (Figures S5M–S5O). At P56, both Ang2+/− and Ang2−/− animals showed a strong reduction of dendritic arborization (Figures 3O–3Q). Thus, Ang2 deficiency also resulted in reduced dendritic complexity of PCs. Altogether, the data suggested that the angiopoietin-Tie2 signaling axis contributed to the development and long-term maintenance of PC dendrites. In PCs, dendritic self-avoidance (repulsion between branches of the same neuron) and acquisition of dendritic planarity are crucial steps to achieve proper morphology and functionality (Ing-Esteves et al., 2018Ing-Esteves S. Kostadinov D. Marocha J. Sing A.D. Joseph K.S. Laboulaye M.A. Sanes J.R. Lefebvre J.L. Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.J. Neurosci. 2018; 38: 2713-2729Crossref PubMed Scopus (40) Google Scholar; Lefebvre et al., 2012Lefebvre J.L. Kostadinov D. Chen W.V. Maniatis T. Sanes J.R. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system.Nature. 2012; 488: 517-521Crossref PubMed Scopus (281) Google Scholar; Toyoda et al., 2014Toyoda S. Kawaguchi M. Kobayashi T. Tarusawa E. Toyama T. Okano M. Oda M. Nakauchi H. Yoshimura Y. Sanbo M. et al.Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity.Neuron. 2014; 82: 94-108Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This process can occur independently of dendritic branching (Gibson et al., 2014Gibson D.A. Tymanskyj S. Yuan R.C. Leung H.C. Lefebvre J.L. Sanes J.R. Chédotal A. Ma L. Dendrite s" @default.
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