RESUMO
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.
Assuntos
Encéfalo , Neovascularização Fisiológica , Animais , Membrana Basal/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/citologia , Encéfalo/citologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Movimento Celular , Colágeno Tipo IV/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Meninges/citologia , Meninges/irrigação sanguínea , Meninges/metabolismo , Especificidade de Órgãos , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. RESULTS: We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. CONCLUSIONS: Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.
Assuntos
Vasos Linfáticos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Vasos Linfáticos/metabolismo , Linfangiogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto/metabolismoRESUMO
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.
Assuntos
Artrogripose/genética , Genes Letais , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Artrogripose/embriologia , Artrogripose/fisiopatologia , Consanguinidade , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Junção Neuromuscular/fisiopatologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Linhagem , Gravidez , Conformação Proteica , Receptores Nicotínicos/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.
Assuntos
Proliferação de Células , Proteínas Hedgehog/fisiologia , Retina/embriologia , Retina/crescimento & desenvolvimento , Via de Sinalização Wnt/fisiologia , Animais , Animais Geneticamente Modificados , Proliferação de Células/efeitos dos fármacos , Antagonismo de Drogas , Embrião não Mamífero , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Indóis/farmacologia , Modelos Biológicos , Organogênese/efeitos dos fármacos , Organogênese/genética , Organogênese/fisiologia , Oximas/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Retina/efeitos dos fármacos , Retina/metabolismo , Teratogênicos/farmacologia , Alcaloides de Veratrum/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Xenopus laevis/embriologiaRESUMO
Prdm12 is an epigenetic regulator expressed in developing and mature nociceptive neurons, playing a key role in their specification during neurogenesis and modulating pain sensation at adulthood. In vitro studies suggested that Prdm12 recruits the methyltransferase G9a through its zinc finger domains to regulate target gene expression, but how Prdm12 interacts with G9a and whether G9a plays a role in Prdm12's functional properties in sensory ganglia remain unknown. Here we report that Prdm12-G9a interaction is likely direct and that it involves the SET domain of G9a. We show that both proteins are largely co-expressed in dorsal root ganglia during early murine development, opening the possibility that G9a plays a role in DRG and may act as a mediator of Prdm12's function in the development of nociceptive sensory neurons. To test this hypothesis, we conditionally inactivated G9a in neural crest using a Wnt1-Cre transgenic mouse line. We found that the specific loss of G9a in the neural crest lineage does not lead to dorsal root ganglia hypoplasia due to the loss of somatic nociceptive neurons nor to the ectopic expression of the visceral determinant Phox2b as observed upon Prdm12 ablation. These findings suggest that Prdm12 function in the initiation of the nociceptive lineage does not critically involves its interaction with G9a.
Assuntos
Neurogênese , Nociceptores , Camundongos , Animais , Nociceptores/metabolismo , Neurogênese/fisiologia , Células Receptoras Sensoriais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Gânglios Espinais , Camundongos Transgênicos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
Prdm12 is a transcriptional regulator essential for the emergence of the somatic nociceptive lineage during sensory neurogenesis. The exact mechanisms by which Prdm12 promotes nociceptor development remain, however, poorly understood. Here, we report that the trigeminal and dorsal root ganglia hypoplasia induced by the loss of Prdm12 involves Bax-dependent apoptosis and that it is accompanied by the ectopic expression of the visceral sensory neuron determinants Phox2a and Phox2b, which is, however, not sufficient to impose a complete fate switch in surviving somatosensory neurons. Mechanistically, our data reveal that Prdm12 is required from somatosensory neural precursors to early post-mitotic differentiating nociceptive neurons to repress Phox2a/b and that its repressive function is context dependent. Together, these findings reveal that besides its essential role in nociceptor survival during development, Prdm12 also promotes nociceptor fate via an additional mechanism, by preventing precursors from engaging into an alternate Phox2 driven visceral neuronal type differentiation program.
RESUMO
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.
Assuntos
Barreira Hematoencefálica/fisiologia , Proteínas Ligadas por GPI/agonistas , Glioblastoma/terapia , Receptores Acoplados a Proteínas G/agonistas , Acidente Vascular Cerebral/terapia , Proteínas Wnt/genética , Via de Sinalização Wnt , Animais , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Receptores Frizzled/metabolismo , Glioblastoma/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , Sistema Nervoso/embriologia , Engenharia de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acidente Vascular Cerebral/metabolismo , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Xenopus laevis , Peixe-ZebraRESUMO
Functionally distinct classes of dorsal root ganglia (DRG) somatosensory neurons arise from neural crest cells (NCCs) in two successive phases of differentiation assumed to be respectively and independently controlled by the proneural genes Neurog2 and Neurog1. However, the precise role of Neurog2 during this process remains unclear, notably because no neuronal loss has been reported hitherto in Neurog2-/- mutants. Here, we show that at trunk levels, Neurog2 deficiency impairs the production of subsets of all DRG neuron subtypes. We establish that this phenotype is highly dynamic and reflects multiple defects in NCC-derived progenitors, including somatosensory-to-melanocyte fate switch, apoptosis, and delayed differentiation which alters neuronal identity, all occurring during a narrow time window when Neurog2 temporarily controls onset of Neurog1 expression and neurogenesis. Collectively, these findings uncover a critical period of cell fate plasticity and vulnerability among somatosensory progenitors and establish that Neurog2 function in the developing DRG is broader than initially envisaged.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Plasticidade Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/metabolismo , Animais , Diferenciação Celular/fisiologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Animais , Técnicas de Inativação de Genes/métodos , Loci Gênicos , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genética , Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Ligantes , Neovascularização Fisiológica , Ligação Proteica , Peixe-ZebraRESUMO
Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.
Assuntos
Antígenos CD/genética , Encéfalo/metabolismo , Caderinas/genética , Neovascularização Fisiológica/genética , Receptores de Lisoesfingolipídeo/genética , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , beta Catenina/genética , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Barreira Hematoencefálica/crescimento & desenvolvimento , Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/crescimento & desenvolvimento , Caderinas/metabolismo , Capilares/crescimento & desenvolvimento , Capilares/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Circulação Cerebrovascular/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/metabolismo , Proteína Vermelha FluorescenteRESUMO
Despite the critical role of endothelial Wnt/ß-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/ß-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/ß-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/ß-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.
Assuntos
Encéfalo/irrigação sanguínea , Proteínas Ligadas por GPI/metabolismo , Morfogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Primers do DNA/genética , Imunofluorescência , Gânglios Espinais/fisiologia , Hibridização In Situ , Luciferases , Análise em Microsséries , Microscopia Confocal , Mutagênese , Neurogênese/fisiologia , Reação em Cadeia da Polimerase , Análise de Célula Única , Imagem com Lapso de TempoRESUMO
The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.
Assuntos
Divisão Celular , Período de Replicação do DNA , Instabilidade Genômica , Retina/citologia , Células-Tronco/fisiologia , Transativadores/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Xenopus , Proteínas de Sinalização YAPRESUMO
Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.