Mural norrin/β-catenin signaling regulates Lama2 expression to promote neurovascular unit assembly

Neurovascular unit (NVU) assembly and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are critical for these processes, yet which signaling pathway(s) regulate their expression remains unknown. Here we show that mural cells have active Norrin/β-catenin signaling during central nervous system development. Bulk RNA sequencing and validation using P10 and P14 wild-type versus Apcdd1-/- retinas reveal that Lama2 (Laminin-α2 chain) mRNA and protein levels are increased in mutant vasculature undergoing higher Norrin/β-catenin signaling. Mural cells are the main source of Lama2, and β-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes including Aquaporin-4 (a water channel in astrocyte endfeet) and Integrin-α6 (a laminin receptor) are upregulated in Apcdd1-/- retinas following higher Lama2 vBM deposition. Thus, the Norrin/β-catenin pathway regulates Lama2 expression in mural cells to promote NVU assembly and neurovascular barrier maturation. SUMMARY Biswas et al., demonstrate that Norrin/β-catenin signaling is active in CNS mural cells and regulates Lama2 deposition in the vascular basement membrane, promoting neurovascular unit assembly and blood-CNS barrier maturation.

[1]  M. Corada,et al.  Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/β-catenin signaling , 2020, Development.

[2]  D. Agalliu,et al.  Neuronal and glial regulation of CNS angiogenesis and barriergenesis , 2020, Development.

[3]  S. Sloan,et al.  Astrocyte‐to‐astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation , 2019, Glia.

[4]  J. Nathans,et al.  Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood–brain barrier and blood–retina barrier development and maintenance , 2018, Proceedings of the National Academy of Sciences.

[5]  Ying Sun,et al.  Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types , 2018, Scientific Data.

[6]  Yao Yao,et al.  Laminins and their receptors in the CNS , 2018, Biological reviews of the Cambridge Philosophical Society.

[7]  Shweta Varshney,et al.  Laminin‐dystroglycan signaling regulates retinal arteriogenesis , 2018, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  Koji Ando,et al.  A molecular atlas of cell types and zonation in the brain vasculature , 2018, Nature.

[9]  K. Plate,et al.  Functional morphology of the blood-brain barrier in health and disease , 2018, Acta Neuropathologica.

[10]  T. Cutforth,et al.  The Wnt Inhibitor Apcdd1 Coordinates Vascular Remodeling and Barrier Maturation of Retinal Blood Vessels , 2017, Neuron.

[11]  D. Antonetti,et al.  The inner blood-retinal barrier: Cellular basis and development , 2017, Vision Research.

[12]  C. Iadecola.  The Neurovascular Unit Coming of Age: A Journey through Neurovascular Coupling in Health and Disease , 2017, Neuron.

[13]  Jie J. Zheng,et al.  Modulating the wnt signaling pathway with small molecules , 2017, Protein science : a publication of the Protein Society.

[14]  I. Agalliu,et al.  Endothelial Wnt/β-catenin signaling reduces immune cell infiltration in multiple sclerosis , 2017, Proceedings of the National Academy of Sciences.

[15]  J. Gautam,et al.  The role of pericytic laminin in blood brain barrier integrity maintenance , 2016, Scientific Reports.

[16]  Evan Z. Macosko,et al.  Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets , 2015, Cell.

[17]  Xin Zhang,et al.  Development of astrocytes in the vertebrate eye , 2014, Developmental dynamics : an official publication of the American Association of Anatomists.

[18]  J. Nathans,et al.  Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling. , 2014, Developmental cell.

[19]  J. Nathans,et al.  Canonical WNT signaling components in vascular development and barrier formation. , 2014, The Journal of clinical investigation.

[20]  E. Huang,et al.  Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer , 2014, Nature Neuroscience.

[21]  S. Strickland,et al.  Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity , 2014, Nature Communications.

[22]  R. Nusse,et al.  The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction. , 2014, Cold Spring Harbor perspectives in biology.

[23]  S. Strickland,et al.  Ablation of astrocytic laminin impairs vascular smooth muscle cell function and leads to hemorrhagic stroke , 2013, The Journal of cell biology.

[24]  E. Huang,et al.  Foxc1 is required by pericytes during fetal brain angiogenesis , 2013, Biology Open.

[25]  William J. Brunken,et al.  Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina , 2013, Development.

[26]  J. Nathans,et al.  Norrin/Frizzled4 Signaling in Retinal Vascular Development and Blood Brain Barrier Plasticity , 2012, Cell.

[27]  S. Feil,et al.  Norrin stimulates cell proliferation in the superficial retinal vascular plexus and is pivotal for the recruitment of mural cells. , 2012, Human molecular genetics.

[28]  Lois E. H. Smith,et al.  Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice , 2012, PloS one.

[29]  K. M. Baeten,et al.  Extracellular matrix and matrix receptors in blood–brain barrier formation and stroke , 2011, Developmental neurobiology.

[30]  A. Reichenbach,et al.  Genetic Deletion of Laminin Isoforms β2 and γ3 Induces a Reduction in Kir4.1 and Aquaporin-4 Expression and Function in the Retina , 2011, PloS one.

[31]  A. Hadjantonakis,et al.  A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse , 2010, BMC Developmental Biology.

[32]  Bengt R. Johansson,et al.  Pericytes regulate the blood–brain barrier , 2010, Nature.

[33]  Z. Yablonka-Reuveni,et al.  LRP5 Is Required for Vascular Development in Deeper Layers of the Retina , 2010, PloS one.

[34]  A. Brivanlou,et al.  APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex , 2010, Nature.

[35]  C. Delporte,et al.  Aquaporin expression in blood-retinal barrier cells during experimental autoimmune uveitis , 2010, Molecular vision.

[36]  J. Nathans,et al.  Norrin, Frizzled-4, and Lrp5 Signaling in Endothelial Cells Controls a Genetic Program for Retinal Vascularization , 2009, Cell.

[37]  D. Rice,et al.  TSPAN12 Regulates Retinal Vascular Development by Promoting Norrin- but Not Wnt-Induced FZD4/β-Catenin Signaling , 2009, Cell.

[38]  Calvin J Kuo,et al.  Wnt/β-catenin signaling is required for CNS, but not non-CNS, angiogenesis , 2009, Proceedings of the National Academy of Sciences.

[39]  A. McMahon,et al.  Canonical Wnt Signaling Regulates Organ-Specific Assembly and Differentiation of CNS Vasculature , 2008, Science.

[40]  K. Plate,et al.  Wnt/β-catenin signaling controls development of the blood–brain barrier , 2008, The Journal of cell biology.

[41]  K. Sekiguchi,et al.  Ligand-binding specificities of laminin-binding integrins: A comprehensive survey of laminin–integrin interactions using recombinant α3β1, α6β1, α7β1 and α6β4 integrins , 2006 .

[42]  A. Reichenbach,et al.  Atypical gliosis in Müller cells of the slowly degenerating rds mutant mouse retina. , 2006, Experimental eye research.

[43]  C. Grimm,et al.  Role of the Norrie disease pseudoglioma gene in sprouting angiogenesis during development of the retinal vasculature. , 2005, Investigative ophthalmology & visual science.

[44]  P. Heutink,et al.  Mutations in Col4a1 Cause Perinatal Cerebral Hemorrhage and Porencephaly , 2005, Science.

[45]  B. Nico,et al.  The role of aquaporin-4 in the blood–brain barrier development and integrity: Studies in animal and cell culture models , 2004, Neuroscience.

[46]  J. Nathans,et al.  Vascular Development in the Retina and Inner Ear Control by Norrin and Frizzled-4, a High-Affinity Ligand-Receptor Pair , 2004, Cell.

[47]  K. Tryggvason,et al.  Laminin isoforms in tumor invasion, angiogenesis and metastasis. , 2002, Seminars in cancer biology.

[48]  B. Engelhardt,et al.  Endothelial Cell Laminin Isoforms, Laminins 8 and 10, Play Decisive Roles in T Cell Recruitment across the Blood–Brain Barrier in Experimental Autoimmune Encephalomyelitis , 2001, The Journal of cell biology.

[49]  E. Engvall,et al.  Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models. , 1998, The Journal of clinical investigation.

[50]  D. Schuppan,et al.  Characterization of integrin receptors in normal and neoplastic human brain. , 1993, The American journal of pathology.

[51]  J. B. C. de Andrade,et al.  Hemorrhagic Stroke , 2021, Neurocritical Care for Neurosurgeons.

[52]  M. Karsdal,et al.  Laminins , 2019, Biochemistry of Collagens, Laminins and Elastin.

[53]  K. Sekiguchi,et al.  Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins. , 2006, Matrix biology : journal of the International Society for Matrix Biology.