RESUMEN
Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.
Asunto(s)
Encéfalo , Neovascularización Fisiológica , Animales , Membrana Basal/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/citología , Encéfalo/citología , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Movimiento Celular , Colágeno Tipo IV/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliales/metabolismo , Células Endoteliales/citología , Meninges/citología , Meninges/irrigación sanguínea , Meninges/metabolismo , Especificidad de Órganos , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a "hit-and-run" adeno-associated virus-assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.
Asunto(s)
Barrera Hematoencefálica/fisiología , Proteínas Ligadas a GPI/agonistas , Glioblastoma/terapia , Receptores Acoplados a Proteínas G/agonistas , Accidente Cerebrovascular/terapia , Proteínas Wnt/genética , Vía de Señalización Wnt , Animales , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Receptores Frizzled/metabolismo , Glioblastoma/metabolismo , Ligandos , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Sistema Nervioso/embriología , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Accidente Cerebrovascular/metabolismo , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Xenopus laevis , Pez CebraRESUMEN
Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.
Asunto(s)
Artrogriposis/genética , Genes Letales , Mutación , Proteínas de Complejo Poro Nuclear/genética , Alelos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Artrogriposis/embriología , Artrogriposis/fisiopatología , Consanguinidad , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Proteínas Musculares/metabolismo , Unión Neuromuscular/fisiopatología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/deficiencia , Linaje , Embarazo , Conformación Proteica , Receptores Nicotínicos/metabolismo , Homología de Secuencia de Aminoácido , Pez Cebra/anomalías , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Wnt signaling is key to many developmental, physiological, and disease processes in which cells seem able to discriminate between multiple Wnt ligands. This selective Wnt recognition or "decoding" capacity has remained enigmatic because Wnt/Frizzled interactions are largely incompatible with monospecific recognition. Gpr124 and Reck enable brain endothelial cells to selectively respond to Wnt7. We show that Reck binds with low micromolar affinity to the intrinsically disordered linker region of Wnt7. Availability of Reck-bound Wnt7 for Frizzled signaling relies on the interaction between Gpr124 and Dishevelled. Through polymerization, Dishevelled recruits Gpr124 and the associated Reck-bound Wnt7 into dynamic Wnt/Frizzled/Lrp5/6 signalosomes, resulting in increased local concentrations of Wnt7 available for Frizzled signaling. This work provides mechanistic insights into the Wnt decoding capacities of vertebrate cells and unravels structural determinants of the functional diversification of Wnt family members.