RESUMO
Transcription factor Foxd3 has been described in model systems as a key member of the pluripotency network in mice as well as being involved in the formation of many critical vertebrate cell types in vivo. Yet virtually nothing is known about roles of FOXD3 in human development and conflicting reports exist regarding its expression in human embryonic stem cells (hESCs). We find that FOXD3 is expressed at both the RNA and protein levels in undifferentiated hESCs and report a Foxd3 expression domain in paraxial mesoderm derivatives of wild-type mouse embryos. Furthermore, increasing FOXD3 activity in hESCs is sufficient for rapid and specific generation of mesenchymal cell types of the paraxial mesoderm, even under pluripotency maintenance conditions. Gene expression diagnostic of chondroblasts, skeletal myoblasts, osteoblasts, and adipoblast is observed within 48 hours of FOXD3 induction, as are morphological and genetic hallmarks of epithelial-to-mesenchymal transition. FOXD3-overexpressing cells can be maintained for several passages, while downregulation of the transgene leads to further differentiation. Loss-of-function also leads to differentiation, toward endoderm and mesoderm. Taken together, these data indicate that a balance of FOXD3 activity is required to maintain pluripotency.
Assuntos
Células-Tronco Embrionárias/citologia , Endoderma/citologia , Fatores de Transcrição Forkhead/metabolismo , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismoRESUMO
Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1 / ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2 . Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.
RESUMO
The specification of left-right asymmetry is an evolutionarily conserved developmental process in vertebrates. The interplay between two TGFß ligands, Derrière/GDF1 and Xnr1/Nodal, together with inhibitors such as Lefty and Coco/Cerl2, have been shown to provide the signals that lead to the establishment of laterality. However, molecular events leading to and following these signals remain mostly unknown. We find that APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, is induced by TGFß signaling, and that its activity is necessary to specify the left-right axis in Xenopus and zebrafish embryos. Surprisingly, we find that APOBEC2 selectively inhibits Derrière, but not Xnr1, signaling. The inhibitory effect is conserved, as APOBEC2 blocks TGFß signaling, and promotes muscle differentiation, in a mammalian myoblastic cell line. This demonstrates for the first time that a putative RNA/DNA editing enzyme regulates TGFß signaling and plays a major role in development.
Assuntos
Padronização Corporal , Citidina Desaminase/metabolismo , Desenvolvimento Embrionário , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Edição de RNA , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Citidina Desaminase/genética , DNA/genética , DNA/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
N95 respirator face masks serve as effective physical barriers against airborne virus transmission, especially in a hospital setting. However, conventional filtration materials, such as nonwoven polypropylene fibers, have no inherent virucidal activity, and thus, the risk of surface contamination increases with wear time. The ability of face masks to protect against infection can be likely improved by incorporating components that deactivate viruses on contact. We present a facile method for covalently attaching antiviral quaternary ammonium polymers to the fiber surfaces of nonwoven polypropylene fabrics that are commonly used as filtration materials in N95 respirators via ultraviolet (UV)-initiated grafting of biocidal agents. Here, C12-quaternized benzophenone is simultaneously polymerized and grafted onto melt-blown or spunbond polypropylene fabric using 254 nm UV light. This grafting method generated ultrathin polymer coatings which imparted a permanent cationic charge without grossly changing fiber morphology or air resistance across the filter. For melt-blown polypropylene, which comprises the active filtration layer of N95 respirator masks, filtration efficiency was negatively impacted from 72.5 to 51.3% for uncoated and coated single-ply samples, respectively. Similarly, directly applying the antiviral polymer to full N95 masks decreased the filtration efficiency from 90.4 to 79.8%. This effect was due to the exposure of melt-blown polypropylene to organic solvents used in the coating process. However, N95-level filtration efficiency could be achieved by wearing coated spunbond polypropylene over an N95 mask or by fabricating N95 masks with coated spunbond as the exterior layer. Coated materials demonstrated broad-spectrum antimicrobial activity against several lipid-enveloped viruses, as well as Staphylococcus aureus and Escherichia coli bacteria. For example, a 4.3-log reduction in infectious MHV-A59 virus and a 3.3-log reduction in infectious SuHV-1 virus after contact with coated filters were observed, although the level of viral deactivation varied significantly depending on the virus strain and protocol for assaying infectivity.
Assuntos
Compostos de Amônio , Vírus , Antivirais/farmacologia , Máscaras , Respiradores N95 , Polímeros/farmacologia , PolipropilenosRESUMO
Particulate respirators such as N95s are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID-19 pandemic. To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3 M 8211-N95 particulate respirators inoculated with SARS-CoV-2. A germicidal UVC device to deliver tailored UVC dose was developed and test coupons (2.5 cm2) of the 3 M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 s) by fixing the distance between the lamp and the test coupon to 15.2 cm while providing an exposure of at least 5.43 mWcm-2. Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator test coupons after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. An average of 4.38 × 103 PFU ml-1 (SD 772.68) was recovered from untreated test coupons while 4.44 × 102 PFU ml-1 (SD 203.67), 4.00 × 102 PFU ml-1 (SD 115.47), 1.56 × 102 PFU ml-1 (SD 76.98) and 4.44 × 101 PFU ml-1 (SD 76.98) was recovered in exposures 2, 6, 18 and 54 s per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mW cm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFU ml-1) on N95 respirator test coupons when irradiated for 120 s per side or longer suggesting 3.5 log reduction in 240 s of irradiation, 1.3 J cm-2. A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 test coupons inoculated with SARS-CoV-2 for 120 s per side resulted in 3.5 log reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 during the COVID-19 pandemic.
Assuntos
COVID-19/prevenção & controle , Descontaminação/instrumentação , Respiradores N95/virologia , SARS-CoV-2/efeitos da radiação , Raios Ultravioleta , Animais , COVID-19/virologia , Chlorocebus aethiops , Descontaminação/economia , Desenho de Equipamento , Reutilização de Equipamento , Células HEK293 , Humanos , SARS-CoV-2/isolamento & purificação , Fatores de Tempo , Células VeroRESUMO
Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.
Assuntos
Canais Iônicos/genética , Cálculos Renais/veterinária , Osteogênese/genética , Proteínas Quinases/genética , Peixe-Zebra , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , Sequência de Bases , Osso e Ossos/anatomia & histologia , Mapeamento Cromossômico , DNA Complementar/genética , Técnicas Histológicas , Hibridização In Situ , Canais Iônicos/metabolismo , Rim/metabolismo , Cálculos Renais/genética , Larva/metabolismo , Dados de Sequência Molecular , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Análise de Sequência de DNA , Canais de Cátion TRPM , Proteínas de Peixe-Zebra/genéticaRESUMO
The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.
Assuntos
Melanóforos/citologia , Crista Neural/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Alelos , Animais , Apoptose , Caspase 3 , Caspases/análise , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Mapeamento Cromossômico , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Melanóforos/química , Melanóforos/fisiologia , Mutação/genética , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Cells in the pluripotent state have the ability to self-renew indefinitely and to differentiate to all the cells of the embryo. These cells provide an in vitro window into development, including human development, as well as holding extraordinary promise for cell-based therapies in regenerative medicine. The recent demonstration that somatic cells can be reprogrammed to the pluripotent state has raised the possibility of patient and disease-specific induced pluripotent cells. In this article, we review the molecular underpinning of pluripotency. We focus on the transcriptional and signaling networks that underlie the state of pluripotency and control differentiation. In general, the action of each of the molecular components and pathways is dose and context dependent highlighting the need for a systems approach to understanding pluripotency.
Assuntos
Células-Tronco Embrionárias/citologia , Animais , Diferenciação Celular , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Medicina Regenerativa , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The neural crest generates multiple cell types during embryogenesis but the mechanisms regulating neural crest cell diversification are incompletely understood. Previous studies using mutant zebrafish indicated that foxd3 and tfap2a function early and differentially in the development of neural crest sublineages. Here, we show that the simultaneous loss of foxd3 and tfap2a function in zebrafish foxd3(zdf10);tfap2a(low) double mutant embryos globally prevents the specification of developmentally distinct neural crest sublineages. By contrast, neural crest induction occurs independently of foxd3 and tfap2a function. We show that the failure of neural crest cell diversification in double mutants is accompanied by the absence of neural crest sox10 and sox9a/b gene expression, and that forced expression of sox10 and sox9a/b differentially rescues neural crest sublineage specification and derivative differentiation. These results demonstrate the functional necessity for foxd3 and tfap2a for neural crest sublineage specification and that this requirement is mediated by the synergistic regulation of the expression of SoxE family genes. Our results identify a genetic regulatory pathway functionally discrete from the process of neural crest induction that is required for the initiation of neural crest cell diversification during embryonic development.
Assuntos
Linhagem da Célula/fisiologia , Crista Neural/fisiologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/fisiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Crista Neural/embriologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.
Assuntos
Diferenciação Celular , Cromatóforos/metabolismo , Melanóforos/metabolismo , Animais , Padronização Corporal , Tamanho Celular , Mapeamento Cromossômico , Modelos Biológicos , Modelos Genéticos , Mutação , Crista Neural/metabolismo , Fenótipo , Fatores de Tempo , Peixe-ZebraRESUMO
The vertebrate neural crest is a pluripotent cell population that generates a large variety of cell types, including peripheral neurons, cartilage and pigment cells. Mechanisms that control the patterning of the neural crest toward specific cell fates remain only partially understood. Zebrafish homozygous for the sympathetic mutation 1 (sym1) have defects in a subset of neural crest derivatives, such as peripheral neurons, glia and cartilage, but retain normal numbers of melanocytes. The sym1 mutation is a nucleotide deletion that disrupts the forkhead DNA-binding domain of the foxd3 gene, which encodes a conserved winged-helix transcription factor. We show that sym1 mutants have normal numbers of premigratory neural crest cells, but these cells express reduced levels of snai1b and sox10, implicating foxd3 as an essential regulator of these transcription factors in the premigratory neural crest. The onset of neural crest migration is also delayed in sym1 mutants, and there is a reduction in the number of migratory trunk neural crest cells, particularly along the medial migration pathway. TUNEL analysis revealed aberrant apoptosis localized to the hindbrain neural crest at the 15-somite stage, indicating a critical role for foxd3 in the survival of a subpopulation of neural crest cells. These results show that foxd3 selectively specifies premigratory neural crest cells for a neuronal, glial or cartilage fate, by inducing the expression of lineage-associated transcription factors in these cells and regulating their subsequent migration.