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1.
Cell ; 160(1-2): 241-52, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25594182

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK:


Assuntos
Endotélio/fisiologia , Células-Tronco Hematopoéticas/citologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Divisão Celular , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/fisiologia , Endotélio/citologia , Células-Tronco Hematopoéticas/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nicho de Células-Tronco , Células Estromais/citologia , Células Estromais/metabolismo , Peixe-Zebra/fisiologia
2.
PLoS Biol ; 22(4): e3002590, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38683849

RESUMO

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.


Assuntos
Encéfalo , Pericitos , Fatores de Transcrição , Proteínas de Peixe-Zebra , Animais , Encéfalo/metabolismo , Encéfalo/embriologia , Diferenciação Celular , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Mesoderma/metabolismo , Mesoderma/citologia , Crista Neural/metabolismo , Crista Neural/citologia , Pericitos/metabolismo , Pericitos/citologia , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
3.
Development ; 150(18)2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37708300

RESUMO

Arteriovenous malformations (AVMs) develop where abnormal endothelial signalling allows direct connections between arteries and veins. Mutations in RASA1, a Ras GTPase activating protein, lead to AVMs in humans and, as we show, in zebrafish rasa1 mutants. rasa1 mutants develop cavernous AVMs that subsume part of the dorsal aorta and multiple veins in the caudal venous plexus (CVP) - a venous vascular bed. The AVMs progressively enlarge and fill with slow-flowing blood. We show that the AVM results in both higher minimum and maximum flow velocities, resulting in increased pulsatility in the aorta and decreased pulsatility in the vein. These hemodynamic changes correlate with reduced expression of the flow-responsive transcription factor klf2a. Remodelling of the CVP is impaired with an excess of intraluminal pillars, which is a sign of incomplete intussusceptive angiogenesis. Mechanistically, we show that the AVM arises from ectopic activation of MEK/ERK in the vein of rasa1 mutants, and that cell size is also increased in the vein. Blocking MEK/ERK signalling prevents AVM initiation in mutants. Alterations in venous MEK/ERK therefore drive the initiation of rasa1 AVMs.


Assuntos
Malformações Arteriovenosas , Peixe-Zebra , Humanos , Animais , Malformações Arteriovenosas/genética , Veias , Proteínas Ativadoras de GTPase , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteína p120 Ativadora de GTPase/genética
4.
Proc Natl Acad Sci U S A ; 119(35): e2121333119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994645

RESUMO

SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1, but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2, functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2, thereby modulating stroke risk.


Assuntos
Fatores de Transcrição Forkhead , Pericitos , Acidente Vascular Cerebral , Animais , DNA Intergênico/genética , DNA Intergênico/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Variação Estrutural do Genoma/genética , Humanos , Pericitos/metabolismo , Polimorfismo de Nucleotídeo Único , Risco , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Ativação Transcricional/genética
5.
Clin Genet ; 105(4): 386-396, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38151336

RESUMO

Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.


Assuntos
Hidropisia Fetal , Motivo Estéril alfa , Feminino , Humanos , Hidropisia Fetal/diagnóstico por imagem , Hidropisia Fetal/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Receptor EphB4/genética , Receptor EphB4/metabolismo
6.
Microvasc Res ; 151: 104610, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37739214

RESUMO

Images contain a wealth of information that is often under analyzed in biological studies. Developmental models of vascular disease are a powerful way to quantify developmentally regulated vessel phenotypes to identify the roots of the disease process. We present vessel Metrics, a software tool specifically designed to analyze developmental vascular microscopy images that will expedite the analysis of vascular images and provide consistency between research groups. We developed a segmentation algorithm that robustly quantifies different image types, developmental stages, organisms, and disease models at a similar accuracy level to a human observer. We validate the algorithm on confocal, lightsheet, and two photon microscopy data in a zebrafish model expressing fluorescent protein in the endothelial nuclei. The tool accurately segments data taken by multiple scientists on varying microscopes. We validate vascular parameters such as vessel density, network length, and diameter, across developmental stages, genetic mutations, and drug treatments, and show a favorable comparison to other freely available software tools. Additionally, we validate the tool in a mouse model. Vessel Metrics reduces the time to analyze experimental results, improves repeatability within and between institutions, and expands the percentage of a given vascular network analyzable in experiments.


Assuntos
Software , Peixe-Zebra , Camundongos , Animais , Humanos , Algoritmos , Núcleo Celular , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos
7.
Oncology ; : 1-8, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39265556

RESUMO

INTRODUCTION: Early phase clinical trials (EPCTs) enable access to novel therapies for patients who have exhausted standard of care treatment and contribute a crucial role in drug development and research. Culturally and linguistically diverse (CALD) or socially disadvantaged patients have notably lower rates of participation in these trials. We aimed to characterise the social and cultural demographics of patients enrolled on an EPCT in South Western Sydney. METHODS: We conducted a 10-year retrospective review of patients enrolled on a EPCT at Liverpool Hospital. CALD patients were defined as those born overseas or whose preferred language was other than English. The patient residential address was used to calculate distance travelled, and the Index of Relative Socioeconomic Disadvantage (IRSD) and Index of Relative Socioeconomic Advantage and Disadvantage (IRSAD) scores were calculated and used as a surrogate for socioeconomic status (SES). RESULTS: Our study included 233 patients across 39 EPCTs. Ninety-one patients (39%) were identified as CALD. The median IRSD and IRSAD scores were 941 and 944, respectively, with 62.7-67.4% of patients residing in an area with greater disadvantage compared to the median of Australia. The median distance travelled was 17 kilometres with only 12% of participants travelling more than 50 km. CALD patients were more likely to reside in an area of low SES (OR 3.4, 95% CI: 1.8-6.5, p < 0.01) and travelled shorter median distances (10 vs. 23 km) when compared to non-CALD patients. CONCLUSION: Our study cohort contained a lower proportion of CALD patients and a higher SES than what we might have expected from our local population. Furthermore, there was a trend toward greater SES disadvantage (lower IRSD/IRSAD scores) for the CALD population. This study provides novel Australian data to support the underrepresentation of culturally diverse or disadvantaged patients on EPCTs. Future efforts should be made to reduce barriers to participation and improve equity in clinical trial participation.

8.
PLoS Genet ; 17(8): e1009769, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424892

RESUMO

Vessel growth integrates diverse extrinsic signals with intrinsic signaling cascades to coordinate cell migration and sprouting morphogenesis. The pro-angiogenic effects of Vascular Endothelial Growth Factor (VEGF) are carefully controlled during sprouting to generate an efficiently patterned vascular network. We identify crosstalk between VEGF signaling and that of the secreted ligand Semaphorin 3fb (Sema3fb), one of two zebrafish paralogs of mammalian Sema3F. The sema3fb gene is expressed by endothelial cells in actively sprouting vessels. Loss of sema3fb results in abnormally wide and stunted intersegmental vessel artery sprouts. Although the sprouts initiate at the correct developmental time, they have a reduced migration speed. These sprouts have persistent filopodia and abnormally spaced nuclei suggesting dysregulated control of actin assembly. sema3fb mutants show simultaneously higher expression of pro-angiogenic (VEGF receptor 2 (vegfr2) and delta-like 4 (dll4)) and anti-angiogenic (soluble VEGF receptor 1 (svegfr1)/ soluble Fms Related Receptor Tyrosine Kinase 1 (sflt1)) pathway components. We show increased phospho-ERK staining in migrating angioblasts, consistent with enhanced Vegf activity. Reducing Vegfr2 kinase activity in sema3fb mutants rescues angiogenic sprouting. Our data suggest that Sema3fb plays a critical role in promoting endothelial sprouting through modulating the VEGF signaling pathway, acting as an autocrine cue that modulates intrinsic growth factor signaling.


Assuntos
Neovascularização Fisiológica/genética , Semaforinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Movimento Celular , Células Endoteliais/metabolismo , Endotélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese , Neovascularização Fisiológica/fisiologia , Receptores Notch/metabolismo , Semaforinas/genética , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/farmacologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
9.
Development ; 147(10)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32423977

RESUMO

The thin endothelial wall of a newly formed vessel is under enormous stress at the onset of blood flow, rapidly acquiring support from mural cells (pericytes and vascular smooth muscle cells; vSMCs) during development. Mural cells then develop vasoactivity (contraction and relaxation) but we have little information as to when this first develops or the extent to which pericytes and vSMCs contribute. For the first time, we determine the dynamic developmental acquisition of vasoactivity in vivo in the cerebral vasculature of zebrafish. We show that pericyte-covered vessels constrict in response to α1-adrenergic receptor agonists and dilate in response to nitric oxide donors at 4 days postfertilization (dpf) but have heterogeneous responses later, at 6 dpf. In contrast, vSMC-covered vessels constrict at 6 dpf, and dilate at both stages. Using genetic ablation, we demonstrate that vascular constriction and dilation is an active response. Our data suggest that both pericyte- and vSMC-covered vessels regulate their diameter in early development, and that their relative contributions change over developmental time.


Assuntos
Músculo Liso Vascular/embriologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Animais Geneticamente Modificados , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Encéfalo/embriologia , Células Endoteliais/fisiologia , Endotélio Vascular/embriologia , Inativação Gênica , Metronidazol/farmacologia , Contração Muscular/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Vasodilatação/efeitos dos fármacos
10.
PLoS Genet ; 15(5): e1008163, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31091229

RESUMO

As small regulatory transcripts, microRNAs (miRs) act as genetic 'fine tuners' of posttranscriptional events, and as genetic switches to promote phenotypic switching. The miR miR26a targets the BMP signalling effector, smad1. We show that loss of miR26a leads to hemorrhage (a loss of vascular stability) in vivo, suggesting altered vascular differentiation. Reduction in miR26a levels increases smad1 mRNA and phospho-Smad1 (pSmad1) levels. We show that increasing BMP signalling by overexpression of smad1 also leads to hemorrhage. Normalization of Smad1 levels through double knockdown of miR26a and smad1 rescues hemorrhage, suggesting a direct relationship between miR26a, smad1 and vascular stability. Using an in vivo BMP genetic reporter and pSmad1 staining, we show that the effect of miR26a on smooth muscle differentiation is non-autonomous; BMP signalling is active in embryonic endothelial cells, but not in smooth muscle cells. Nonetheless, increased BMP signalling due to loss of miR26a results in an increase in acta2-expressing smooth muscle cell numbers and promotes a differentiated smooth muscle morphology. Similarly, forced expression of smad1 in endothelial cells leads to an increase in smooth muscle cell number and coverage. Furthermore, smooth muscle phenotypes caused by inhibition of the BMP pathway are rescued by loss of miR26a. Taken together, our data suggest that miR26a modulates BMP signalling in endothelial cells and indirectly promotes a differentiated smooth muscle phenotype. Our data highlights how crosstalk from BMP-responsive endothelium to smooth muscle is important for smooth muscle differentiation.


Assuntos
Células Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Endotélio , Regulação da Expressão Gênica/genética , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/genética , Proteína Smad1/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
11.
Int J Mol Sci ; 23(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36076925

RESUMO

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disease caused by heterozygous missense mutations within the gene encoding for the nuclear envelope protein transmembrane protein 43 (TMEM43). The disease is characterized by myocyte loss and fibro-fatty replacement, leading to life-threatening ventricular arrhythmias and sudden cardiac death. However, the role of TMEM43 in the pathogenesis of ACM remains poorly understood. In this study, we generated cardiomyocyte-restricted transgenic zebrafish lines that overexpress eGFP-linked full-length human wild-type (WT) TMEM43 and two genetic variants (c.1073C>T, p.S358L; c.332C>T, p.P111L) using the Tol2-system. Overexpression of WT and p.P111L-mutant TMEM43 was associated with transcriptional activation of the mTOR pathway and ribosome biogenesis, and resulted in enlarged hearts with cardiomyocyte hypertrophy. Intriguingly, mutant p.S358L TMEM43 was found to be unstable and partially redistributed into the cytoplasm in embryonic and adult hearts. Moreover, both TMEM43 variants displayed cardiac morphological defects at juvenile stages and ultrastructural changes within the myocardium, accompanied by dysregulated gene expression profiles in adulthood. Finally, CRISPR/Cas9 mutants demonstrated an age-dependent cardiac phenotype characterized by heart enlargement in adulthood. In conclusion, our findings suggest ultrastructural remodeling and transcriptomic alterations underlying the development of structural and functional cardiac defects in TMEM43-associated cardiomyopathy.


Assuntos
Displasia Arritmogênica Ventricular Direita , Proteínas de Membrana , Miocárdio , Adulto , Animais , Displasia Arritmogênica Ventricular Direita/genética , Heterozigoto , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Miocárdio/patologia , Peixe-Zebra/genética
12.
Australas Psychiatry ; 29(6): 617-624, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34192474

RESUMO

OBJECTIVES: People with mental illness may be vulnerable to psychological distress and reduced well-being during the COVID-19 pandemic. The aim of this study was to assess psychosocial and lifestyle predictors of distress and well-being in people with mental illness during the pandemic. METHOD: People with mental illness who participated in an exercise programme prior to the pandemic were invited to complete surveys about mental health and lifestyle corresponding to before and during the pandemic. RESULTS: Social support reduced, alcohol intake increased, and sleep quality and diet worsened during the pandemic, contributing to distress. Psychological distress was associated with the two or more mental illnesses, and negatively associated with having a physical disease. Better diet appeared to protect against increases in distress; loneliness hindered improvements in well-being. CONCLUSIONS: Healthy lifestyle programmes designed to improve social connection may improve health for people with mental illnesses during and after the COVID-19 pandemic.


Assuntos
COVID-19 , Transtornos Mentais , Humanos , Estilo de Vida , Transtornos Mentais/epidemiologia , Pandemias , SARS-CoV-2 , Qualidade do Sono
13.
Australas Psychiatry ; 29(2): 175-179, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33380159

RESUMO

OBJECTIVES: People with mental illness may be vulnerable to decline in mental health and reduced physical activity because of the COVID-19 pandemic and associated restrictions. The aim of this study was to inform the design of physical activity interventions for implementation under these conditions to improve/maintain well-being and physical activity in this population. METHODS: People with mental illness who had participated in a physical activity program prior to the pandemic were invited to complete a survey about the impact of COVID-19 on mental health and physical activity and their preferences for engaging in a physical activity program under pandemic-related restrictions. RESULTS: More than half the 59 respondents reported worse mental health and lower physical activity during the pandemic. The preferred format for a physical activity program was one-on-one exercise instruction in-person in a park. Program components endorsed as helpful included incentivization, provision of exercise equipment and fitness devices, and daily exercise programs. About a third of the participants reported limitations in using technology for a physical activity program. CONCLUSIONS: In-person exercise support is preferred by people with mental illnesses during pandemic-related restrictions. Enablement strategies such as providing equipment and self-monitoring devices should be utilized; assistance may be needed to incorporate the use of technology in exercise programs.


Assuntos
COVID-19/psicologia , Terapia por Exercício/métodos , Terapia por Exercício/psicologia , Transtornos Mentais/psicologia , Transtornos Mentais/terapia , Preferência do Paciente/psicologia , Adulto , COVID-19/epidemiologia , COVID-19/prevenção & controle , Estudos Transversais , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Preferência do Paciente/estatística & dados numéricos , Distanciamento Físico , Inquéritos e Questionários
14.
Dev Biol ; 453(1): 34-47, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199900

RESUMO

Vascular smooth muscle of the head derives from neural crest, but developmental mechanisms and early transcriptional drivers of the vSMC lineage are not well characterized. We find that in early development, the transcription factor foxc1b is expressed in mesenchymal cells that associate with the vascular endothelium. Using timelapse imaging, we observe that foxc1b expressing mesenchymal cells differentiate into acta2 expressing vascular mural cells. We show that in zebrafish, while foxc1b is co-expressed in acta2 positive smooth muscle cells that associate with large diameter vessels, it is not co-expressed in capillaries where pdgfrß positive pericytes are located. In addition to being an early marker of the lineage, foxc1 is essential for vSMC differentiation; we find that foxc1 loss of function mutants have defective vSMC differentiation and that early genetic ablation of foxc1b or acta2 expressing populations blocks vSMC differentiation. Furthermore, foxc1 is expressed upstream of acta2 and is required for acta2 expression in vSMCs. Using RNA-Seq we determine an enriched intersectional gene expression profile using dual expression of foxc1b and acta2 to identify novel vSMC markers. Taken together, our data suggests that foxc1 is a marker of vSMCs and plays a critical functional role in promoting their differentiation.


Assuntos
Diferenciação Celular , Embrião não Mamífero/citologia , Fatores de Transcrição Forkhead/metabolismo , Cabeça/irrigação sanguínea , Cabeça/embriologia , Músculo Liso Vascular/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Endotélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericitos/metabolismo , Transcriptoma/genética , Regulação para Cima , Peixe-Zebra/genética
15.
Adv Exp Med Biol ; 1109: 33-51, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30523588

RESUMO

The zebrafish is an outstanding model for studying vascular biology in vivo. Pericytes and vascular smooth muscle cells can be imaged as they associate with vessels and provide stability and integrity to the vasculature. In zebrafish, pericytes associate with the cerebral and trunk vasculature on the second day of development, as assayed by pdgfrß and notch3 markers. In the head, cerebral pericytes are neural crest derived, except for the pericytes of the hindbrain vasculature, which are mesoderm derived. Similar to the hindbrain, pericytes on the trunk vasculature are also mesoderm derived. Regardless of their location, pericyte development depends on a complex interaction between blood flow and signalling pathways, such as Notch, SONIC HEDGEHOG and BMP signalling, all of which positively regulate pericyte numbers.Pericyte numbers rapidly increase as development proceeds in order to stabilize both the blood-brain barrier and the vasculature and hence, prevent haemorrhage. Consequently, compromised pericyte development results in compromised vascular integrity, which then evolves into detrimental pathologies. Some of these pathologies have been modelled in zebrafish by inducing mutations in the notch3, foxc1 and foxf2 genes. These zebrafish models provide insights into the mechanisms of disease as associated with pericyte biology. Going forward, these models may be key contributors in elucidating the role of vascular mural cells in regulating vessel diameter and hence, blood flow.


Assuntos
Vasos Sanguíneos/citologia , Pericitos/citologia , Peixe-Zebra , Animais , Barreira Hematoencefálica , Miócitos de Músculo Liso
16.
Dev Biol ; 409(1): 114-128, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26477558

RESUMO

Despite considerable interest in angiogenesis, organ-specific angiogenesis remains less well characterized. The vessels that absorb nutrients from the yolk and later provide blood supply to the developing digestive system are primarily venous in origin. In zebrafish, these are the vessels of the Sub-intestinal venous plexus (SIVP) and they represent a new candidate model to gain an insight into the mechanisms of venous angiogenesis. Unlike other vessel beds in zebrafish, the SIVP is not stereotypically patterned and lacks obvious sources of patterning information. However, by examining the area of vessel coverage, number of compartments, proliferation and migration speed we have identified common developmental steps in SIVP formation. We applied our analysis of SIVP development to obd mutants that have a mutation in the guidance receptor PlexinD1. obd mutants show dysregulation of nearly all parameters of SIVP formation. We show that the SIVP responds to a unique combination of pathways that control both arterial and venous growth in other systems. Blocking Shh, Notch and Pdgf signaling has no effect on SIVP growth. However Vegf promotes sprouting of the predominantly venous plexus and Bmp promotes outgrowth of the structure. We propose that the SIVP is a unique model to understand novel mechanisms utilized in organ-specific angiogenesis.


Assuntos
Padronização Corporal , Intestinos/irrigação sanguínea , Veias/anatomia & histologia , Veias/embriologia , Peixe-Zebra/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular , Proliferação de Células , Embrião não Mamífero/anatomia & histologia , Camundongos , Mutação/genética , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ducto Vitelino/anatomia & histologia , Ducto Vitelino/embriologia , Proteínas de Peixe-Zebra/metabolismo
17.
Dev Biol ; 414(2): 181-92, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27126199

RESUMO

Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/fisiologia , Neovascularização Fisiológica/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Artérias/embriologia , Movimento Celular , Técnicas de Inativação de Genes , Proteínas com Homeodomínio LIM/deficiência , Proteínas com Homeodomínio LIM/genética , Mesoderma , Morfolinos/genética , Morfolinos/toxicidade , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Mensageiro/genética , Receptores Notch/fisiologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Veias/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
18.
Development ; 141(12): 2473-82, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24917502

RESUMO

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. We know little about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. Here, we show that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. We propose a novel, tissue-autonomous mechanism of organ cohesion, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.


Assuntos
Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/fisiologia , Semaforinas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Axônios/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Morfogênese , Proteínas do Tecido Nervoso/genética , Prosencéfalo/embriologia , Receptores de Superfície Celular/genética , Semaforinas/genética , Transdução de Sinais , Células-Tronco/citologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
19.
Nanomedicine ; 13(3): 999-1010, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27993727

RESUMO

Nanoparticle (NP) interactions with biological tissues are affected by the size, shape and surface chemistry of the NPs. Here we use in vivo (zebrafish) and in vitro (HUVEC) models to investigate association of quantum dots (QDs) with endothelial cells and the effect of fluid flow. After injection into the developing zebrafish, circulating QDs associate with endothelium and penetrate surrounding tissue parenchyma over time. Amino-functionalized QDs cluster, interact with cells, and clear more rapidly than carboxy-functionalized QDs in vivo, highlighting charge influences. QDs show stronger accumulation in slow-flowing, small caliber venous vessels than in fast-flowing high caliber arterial vessels. Parallel-plate flow experiments with HUVEC support these findings, showing reduced QD-EC association with increasing flow. In vivo, flow arrest after nanoparticle injection still results in venous accumulation at 18 h. Overall our results suggest that both QD charge and blood flow modulate particle-endothelial cell interactions.


Assuntos
Vasos Sanguíneos/fisiologia , Células Endoteliais/metabolismo , Pontos Quânticos/metabolismo , Resinas Acrílicas/administração & dosagem , Resinas Acrílicas/metabolismo , Resinas Acrílicas/toxicidade , Aminação , Animais , Velocidade do Fluxo Sanguíneo , Vasos Sanguíneos/efeitos dos fármacos , Ácidos Carboxílicos/administração & dosagem , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Pontos Quânticos/administração & dosagem , Pontos Quânticos/toxicidade , Peixe-Zebra
20.
J Neurosci ; 35(39): 13430-47, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26424889

RESUMO

Imprinted genes are dosage sensitive, and their dysregulated expression is linked to disorders of growth and proliferation, including fetal and postnatal growth restriction. Common sequelae of growth disorders include neurodevelopmental defects, some of which are indirectly related to placental insufficiency. However, several growth-associated imprinted genes are also expressed in the embryonic CNS, in which their aberrant expression may more directly affect neurodevelopment. To test whether growth-associated genes influence neural lineage progression, we focused on the maternally imprinted gene Zac1. In humans, either loss or gain of ZAC1 expression is associated with reduced growth rates and intellectual disability. To test whether increased Zac1 expression directly perturbs neurodevelopment, we misexpressed Zac1 in murine neocortical progenitors. The effects were striking: Zac1 delayed the transition of apical radial glial cells to basal intermediate neuronal progenitors and postponed their subsequent differentiation into neurons. Zac1 misexpression also blocked neuronal migration, with Zac1-overexpressing neurons pausing more frequently and forming fewer neurite branches during the period when locomoting neurons undergo dynamic morphological transitions. Similar, albeit less striking, neuronal migration and morphological defects were observed on Zac1 knockdown, indicating that Zac1 levels must be regulated precisely. Finally, Zac1 controlled neuronal migration by regulating Pac1 transcription, a receptor for the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP). Pac1 and Zac1 loss- and gain-of-function presented as phenocopies, and overexpression of Pac1 rescued the Zac1 knockdown neuronal migration phenotype. Thus, dysregulated Zac1 expression has striking consequences on neocortical development, suggesting that misexpression of this transcription factor in the brain in certain growth disorders may contribute to neurocognitive deficits. Significance statement: Altered expression of imprinted genes is linked to cognitive dysfunction and neuropsychological disorders, such as Angelman and Prader-Willi syndromes, and autism spectrum disorder. Mouse models have also revealed the importance of imprinting for brain development, with chimeras generated with parthenogenetic (two maternal chromosomes) or androgenetic (two paternal chromosomes) cells displaying altered brain sizes and cellular defects. Despite these striking phenotypes, only a handful of imprinted genes are known or suspected to regulate brain development (e.g., Dlk1, Peg3, Ube3a, necdin, and Grb10). Herein we show that the maternally imprinted gene Zac1 is a critical regulator of neocortical development. Our studies are relevant because loss of 6q24 maternal imprinting in humans results in elevated ZAC1 expression, which has been associated with neurocognitive defects.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Genes Supressores de Tumor/fisiologia , Neocórtex/citologia , Neurônios/fisiologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/embriologia , Neuritos/fisiologia , Neuritos/ultraestrutura , Neurônios/ultraestrutura , Gravidez , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Fatores de Transcrição/genética
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