RESUMO
Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevent the widespread adoption of these methods. Here, we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hr in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, samples processed with EZ Clear can be subjected to downstream applications, such as tissue embedding and cryosectioning followed by standard histology or immunofluorescent staining without loss of fluorescence signal from endogenous or synthetic reporters. Furthermore, we demonstrate that wholemount adult mouse brains processed with EZ Clear can be successfully immunolabeled for fluorescent imaging while still retaining signal from endogenous fluorescent reporters. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adapt and implement in diverse imaging modalities in biomedical research.
Assuntos
Corantes , Imageamento Tridimensional , Animais , Encéfalo/diagnóstico por imagem , Imageamento Tridimensional/métodos , Camundongos , Solventes , Coloração e RotulagemRESUMO
Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Remodelação Vascular , Saco Vitelino , Animais , Artérias , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Remodelação Vascular/genética , Saco Vitelino/metabolismoRESUMO
Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. Yet efficient delivery of CRISPR to the lung remains a challenge. The NIH Somatic Cell Genome Editing (SCGE) Consortium is developing safe and effective methods for genome editing in disease tissues. Methods developed by consortium members are independently validated by the SCGE small animal testing center to establish rigor and reproducibility. We have developed and validated a dual adeno-associated virus (AAV) CRISPR platform that supports effective editing of a lox-stop-lox-Tomato reporter in mouse lung airway. After intratracheal injection of the AAV serotype 5 (AAV5)-packaged S. pyogenes Cas9 (SpCas9) and single guide RNAs (sgRNAs), we observed â¼19%-26% Tomato-positive cells in both large and small airways, including club and ciliated epithelial cell types. This highly effective AAV delivery platform will facilitate the study of therapeutic genome editing in the lung and other tissue types.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Edição de Genes/métodos , Pulmão , Camundongos , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos TestesRESUMO
SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
Assuntos
Instabilidade Cromossômica , Dano ao DNA , Variação Genética , Anormalidades Musculoesqueléticas/patologia , NF-kappa B/genética , Osteocondrodisplasias/patologia , Adolescente , Adulto , Alelos , Animais , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Estudos de Associação Genética , Humanos , Camundongos , Camundongos Knockout , Anormalidades Musculoesqueléticas/genética , Osteocondrodisplasias/genética , Sequenciamento do Exoma , Adulto Jovem , Peixe-ZebraRESUMO
Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn et al., 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath et al., 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Wynn et al., 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk-sac-derived Embryonic Macrophages (EMs) in vitro to generate a new platform for mechanistic studies of EM differentiation. To accomplish this goal, we isolated pure (>98%) EGFP+ populations by flow cytometry from embryonic day 9.5 (E9.5) Csf1r-EGFP+/tg mice, then evaluated the angiogenic potential of EMs relative to Bone Marrow-Derived Macrophages (BMDMs). We found that EMs expressed more pro-angiogenic and less pro-inflammatory macrophage markers than BMDMs. EMs also promoted more endothelial cell (EC) cord formation in vitro, as compared to BMDMs in a manner that required direct cell-to-cell contact. Importantly, EMs preferentially matured into microglia when co-cultured with mouse Neural Stem/Progenitor Cells (NSPCs). In conclusion, we have established a protocol to isolate and propagate EMs in vitro, have further defined specialized properties of yolk-sac-derived macrophages, and have identified EM-EC and EM-NSPC interactions as key inducers of EC tube formation and microglial cell maturation, respectively.
Assuntos
Células Precursoras Eritroides/fisiologia , Macrófagos/fisiologia , Células Progenitoras Mieloides/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Técnicas de Cocultura/métodos , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/citologia , Camundongos/embriologia , Fenótipo , Saco Vitelino/citologiaRESUMO
Apela (also known as Elabela, Ende, and Toddler) is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.
Assuntos
Proteínas de Transporte/metabolismo , Perda do Embrião/genética , Perda do Embrião/patologia , Mesoderma/embriologia , Mesoderma/metabolismo , Penetrância , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Apelina/metabolismo , Receptores de Apelina/metabolismo , Antígeno CD11b/metabolismo , Proteínas de Transporte/química , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário , Células Endoteliais/metabolismo , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Mutação/genética , Células Mieloides/metabolismo , Miocárdio/patologia , Hormônios Peptídicos , Peptídeos/química , Fenótipo , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima/genética , Remodelação VascularRESUMO
Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.
Assuntos
Modelos Animais de Doenças , Técnicas de Inativação de Genes , Animais , Feminino , Doenças Genéticas Inatas , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
Assuntos
Anormalidades Múltiplas/genética , Endopeptidases/genética , Deficiência Intelectual/genética , Adolescente , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Deleção de Genes , Humanos , Masculino , Camundongos , Linhagem , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Convulsões/genéticaRESUMO
FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.
RESUMO
The transcription factor Sox2 is both necessary and sufficient for the generation of sensory regions of the inner ear. It regulates expression of the Notch ligand Jag1 in prosensory progenitors, which signal to neighboring cells to up-regulate Sox2 and sustain prosensory identity. However, the expression pattern of Sox2 in the early inner ear is very broad, suggesting that Sox2-expressing progenitors form a wide variety of cell types in addition to generating the sensory regions of the ear. We used Sox2-CreER mice to follow the fates of Sox2-expressing cells at different stages in ear development. We find that Sox2-expressing cells in the early otocyst give rise to large numbers of non-sensory structures throughout the inner ear, and that Sox2 only becomes a truly prosensory marker at embryonic day (E)11.5. Our fate map reveals the organ of Corti derives from a central domain on the medial side of the otocyst and shows that a significant amount of the organ of Corti derives from a Sox2-negative population in this region.
Assuntos
Orelha Interna/citologia , Células-Tronco Neurais/citologia , Órgão Espiral/embriologia , Fatores de Transcrição SOXB1/análise , Animais , Antígenos de Diferenciação/análise , Linhagem da Célula , Orelha Interna/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genes Reporter , Imageamento Tridimensional , Proteína Jagged-1/biossíntese , Proteína Jagged-1/genética , Proteínas Luminescentes/análise , Camundongos , Camundongos Transgênicos , Órgão Espiral/citologia , Receptores Notch/fisiologia , Transdução de Sinais/fisiologiaRESUMO
A fundamental principle governing organ size and function is the fine balance between cell proliferation and cell differentiation. Here, we identify RONIN (THAP11) as a key transcriptional regulator of retinal progenitor cell (RPC) proliferation. RPC-specific loss of Ronin results in a phenotype strikingly similar to that resulting from the G1- to S-phase arrest and photoreceptor degeneration observed in the Cyclin D1 null mutants. However, we determined that, rather than regulating canonical cell-cycle genes, RONIN regulates a cohort of mitochondrial genes including components of the electron transport chain (ETC), which have been recently implicated as direct regulators of the cell cycle. Coincidentally, with premature cell-cycle exit, Ronin mutants exhibited deficient ETC activity, reduced ATP levels, and increased oxidative stress that we ascribe to specific loss of subunits within complexes I, III, and IV. These data implicate RONIN as a positive regulator of mitochondrial gene expression that coordinates mitochondrial activity and cell-cycle progression.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas Repressoras/genética , Células-Tronco/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Diferenciação Celular , Proliferação de Células , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Embrião de Mamíferos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , Células Fotorreceptoras de Vertebrados/citologia , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Transcrição GênicaRESUMO
The role of the Hippo signaling pathway in cranial neural crest (CNC) development is poorly understood. We used the Wnt1(Cre) and Wnt1(Cre2SOR) drivers to conditionally ablate both Yap and Taz in the CNC of mice. When using either Cre driver, Yap and Taz deficiency in the CNC resulted in enlarged, hemorrhaging branchial arch blood vessels and hydrocephalus. However, Wnt1(Cre2SOR) mutants had an open cranial neural tube phenotype that was not evident in Wnt1(Cre) mutants. In O9-1 CNC cells, the loss of Yap impaired smooth muscle cell differentiation. RNA-sequencing data indicated that Yap and Taz regulate genes encoding Fox transcription factors, specifically Foxc1. Proliferation was reduced in the branchial arch mesenchyme of Yap and Taz CNC conditional knockout (CKO) embryos. Moreover, Yap and Taz CKO embryos had cerebellar aplasia similar to Dandy-Walker spectrum malformations observed in human patients and mouse embryos with mutations in Foxc1. In embryos and O9-1 cells deficient for Yap and Taz, Foxc1 expression was significantly reduced. Analysis of Foxc1 regulatory regions revealed a conserved recognition element for the Yap and Taz DNA binding co-factor Tead. ChIP-PCR experiments supported the conclusion that Foxc1 is directly regulated by the Yap-Tead complex. Our findings uncover important roles for Yap and Taz in CNC diversification and development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Face/embriologia , Crista Neural/embriologia , Fosfoproteínas/metabolismo , Crânio/embriologia , Animais , Apoptose/genética , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Perda do Embrião/patologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hemorragia/patologia , Hidrocefalia/embriologia , Hidrocefalia/patologia , Mandíbula/patologia , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Defeitos do Tubo Neural/patologia , Fenótipo , Análise de Sequência de RNA , Transdução de Sinais , Transativadores , Proteínas de Sinalização YAPRESUMO
UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.
Assuntos
Adenocarcinoma/patologia , Colágeno/metabolismo , Fibroblastos/patologia , Neoplasias Pulmonares/patologia , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Células Tumorais Cultivadas/patologia , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Neoplasias Experimentais , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Both chemical and mechanical stimuli can dramatically influence cell behavior. By optimizing the signals cells experience, it may be possible to control the behavior of therapeutic cell populations. In this work, biomimetic geometries of adhesive ligands, which recapitulate the morphology of mature cells, are used to direct human mesenchymal stem cell (HMSC) differentiation toward a desired lineage. Specifically, adipocytes cultured in 2D are imaged and used to develop biomimetic virtual masks used in laser scanning lithography to form patterned fibronectin surfaces. The impact of adipocyte-derived pattern geometry on HMSC differentiation is compared to the behavior of HMSCs cultured on square and circle geometries, as well as adipocyte-derived patterns modified to include high stress regions. HMSCs on adipocyte mimetic geometries demonstrate greater adipogenesis than HMSCs on the other patterns. Greater than 45% of all HMSCs cultured on adipocyte mimetic patterns underwent adipogenesis as compared to approximately 19% of cells on modified adipocyte patterns with higher stress regions. These results are attributed to variations in cytoskeletal tension experienced by cells on the different protein micropatterns. The effects of geometry on adipogenesis are mitigated by the incorporation of a cytoskeletal protein inhibitor; exposure to this inhibitor leads to increased adipogenesis on all patterns examined.
Assuntos
Células-Tronco Mesenquimais , Adipócitos , Biomimética , Diferenciação Celular , Células Cultivadas , HumanosRESUMO
Successful engineering of complex organs requires improved methods to promote rapid and stable vascularization of artificial tissue scaffolds. Toward this goal, tissue engineering strategies utilize the release of pro-angiogenic growth factors, alone or in combination, from biomaterials to induce angiogenesis. In this study we have used intravital microscopy to define key, dynamic cellular changes induced by the release of pro-angiogenic factors from polyethylene glycol diacrylate hydrogels transplanted in vivo. Our data show robust macrophage recruitment when the potent and synergistic angiogenic factors, PDGFBB and FGF2 were used as compared with VEGF alone and intravital imaging suggested roles for macrophages in endothelial tip cell migration and anastomosis, as well as pericyte-like behavior. Further data from in vivo experiments show that delivery of CSF1 with VEGF can dramatically improve the poor angiogenic response seen with VEGF alone. These studies show that incorporating macrophage-recruiting factors into the design of pro-angiogenic biomaterial scaffolds is a key strategy likely to be necessary for stable vascularization and survival of implanted artificial tissues.
Assuntos
Indutores da Angiogênese/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Macrófagos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-sis/farmacologia , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Becaplermina , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis , Macrófagos/fisiologia , Camundongos , Alicerces TeciduaisRESUMO
Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/ß-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with membrane protrusions or filopodia extending from the ECs. Flk1-myr::mCherry+ EC membrane particles were observed on and around ECs as well as within macrophages. Electron microscopy studies confirmed that they were in phagosomes within macrophages, indicating that the macrophages engulfed the membrane particles. Interestingly, EC plasma membrane uptake by PM macrophages did not correlate with apoptosis and was found shortly after vessel formation at mid-gestation stages in the embryo; long before vessel regression begins during postnatal development. Additionally, genetic ablation of macrophages showed that EC membrane particles were still shed in the absence of macrophages suggesting that macrophages do not induce the formation or release of EC microparticles. These studies have uncovered a novel event during programmed capillary regression in which resident macrophages scavenge endothelial cell microparticles released from the PM vessels. This finding suggests that there may be an initial disruption in vessel homeostasis embryonically as the PM forms that may underlie its ultimate regression postnatally.
Assuntos
Micropartículas Derivadas de Células/imunologia , Olho/irrigação sanguínea , Macrófagos/imunologia , Fagocitose/imunologia , Pupila/fisiologia , Animais , Capilares , Membrana Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Células Endoteliais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
The ability to visually observe angiogenesis and lymphangiogenesis simultaneously and repeatedly in living animals would greatly enhance our understanding of the inter-dependence of these processes. To generate a mouse model that allows such visualization via in vivo fluorescence imaging, we crossed Prox1-GFP mice with Flk1::myr-mCherry mice to generate Prox1-GFP/Flk1::myr-mCherry mice, in which lymphatic vessels emit green fluorescence and blood vessels emit red fluorescence. Corneal neovascularization was induced in these mice using three injury models: implantation of a vascular endothelial growth factor (VEGF) pellet, implantation of a basic fibroblast growth factor (bFGF) pellet, and alkali burn injury. Vessel growth was observed in vivo by stereomicroscopy on days 0, 3, 7 and 10 after pellet implantation or alkali injury as well as in flat-mounted corneas via confocal microscopy after the final in vivo imaging time point. We observed blood and lymphatic vessel growth in all three models, with the most significant growth occurring from days 0-7. Upon VEGF stimulation, the growth kinetics of blood and lymphatic vessels were similar. Blood vessels exhibited similar growth patterns in VEGF- and bFGF-stimulated corneas. Alkali burn injury induced robust angiogenesis and lymphangiogenesis. The intrinsic fluorescence of blood and lymphatic endothelial cells in Prox1-GFP/Flk1::myr-mCherry mice permitted simultaneous in vivo imaging of angiogenesis and lymphangiogenesis. This allowed us to differentiate the processes as well as observe their inter-dependence, and will be valuable in development of therapies targeting angiogenesis and/or lymphangiogenesis.
Assuntos
Lesões da Córnea/patologia , Neovascularização da Córnea , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/fisiologia , Proteínas Luminescentes/metabolismo , Linfangiogênese , Proteínas Supressoras de Tumor/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Álcalis/toxicidade , Animais , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Lesões da Córnea/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Vermelha FluorescenteRESUMO
Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature.
Assuntos
Adenocarcinoma/irrigação sanguínea , Vasos Sanguíneos/patologia , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína Supressora de Tumor p53/deficiência , Via de Sinalização Wnt , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Hipóxia Celular , Rastreamento de Células/métodos , Feminino , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Xenoenxertos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lentivirus/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Transdução Genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genética , Via de Sinalização Wnt/genéticaRESUMO
The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-of-function experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wild-type littermates, implying that malignant progression was dependent specifically upon tumor cell-derived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen.
Assuntos
Adenocarcinoma/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proteínas da Matriz Extracelular/genética , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The vasculature of an organism has the daunting task of connecting all the organ systems to nourish tissue and sustain life. This complex network of vessels and associated cells must maintain blood flow, but constantly adapt to acute and chronic changes within tissues. While the vasculature has been studied for over a century, we are just beginning to understand the processes that regulate its formation and how genetic hierarchies are influenced by mechanical and metabolic cues to refine vessel structure and optimize efficiency. As we gain insights into the developmental mechanisms, it is clear that the processes that regulate blood vessel development can also enable the adult to adapt to changes in tissues that can be elicited by exercise, aging, injury, or pathology. Thus, research in vessel development has provided tremendous insights into therapies for vascular diseases and disorders, cancer interventions, wound repair and tissue engineering, and in turn, these models have clearly impacted our understanding of development. Here we provide an overview of the development of the vascular system, highlighting several areas of active investigation and key questions that remain to be answered.