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1.
Nat Commun ; 11(1): 5499, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127892

RESUMO

The epiblast, which provides the foundation of the future body, is actively reshaped during early embryogenesis, but the reshaping mechanisms are poorly understood. Here, using a 3D in vitro model of early epiblast development, we identify the canonical Wnt/ß-catenin pathway and its central downstream factor Esrrb as the key signalling cascade regulating the tissue-scale organization of the murine pluripotent lineage. Although in vivo the Wnt/ß-catenin/Esrrb circuit is dispensable for embryonic development before implantation, autocrine Wnt activity controls the morphogenesis and long-term maintenance of the epiblast when development is put on hold during diapause. During this phase, the progressive changes in the epiblast architecture and Wnt signalling response show that diapause is not a stasis but instead is a dynamic process with underlying mechanisms that can appear redundant during transient embryogenesis.


Assuntos
Diapausa/fisiologia , Células-Tronco Embrionárias/metabolismo , Receptores Estrogênicos/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Camadas Germinativas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Estrogênicos/genética , beta Catenina/genética
2.
J Plant Res ; 133(6): 751-763, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33033876

RESUMO

The YABBY family is a class of plant-specific transcription factors comprising a typical N-terminal C2C2-type zinc finger domain and a C-terminal helix-loop-helix YABBY domain. YABBY transcription factors play important roles in multiple biological processes, including polarity establishment in plant leaves, the formation and development of reproductive organs, the response to plant hormone signals, resistance to stress, crop breeding and agricultural production. The aim of this review is to summarize our current understanding of the roles, functions and value of the YABBY family in plants, with particular emphasis on new insights into the molecular and physiological mechanisms involved in the YABBY-mediated modulation of polarity establishment, morphogenesis and development, and phytohormone and stress responses in plants. In addition, we propose that this transcription factor family presents great value and potential for research, application and development in crop breeding and agricultural production in the future.


Assuntos
Morfogênese , Reguladores de Crescimento de Planta/fisiologia , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas , Fatores de Transcrição , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Nature ; 585(7826): 574-578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939089

RESUMO

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.


Assuntos
Homeostase , Intestinos/embriologia , Morfogênese , Organoides/embriologia , Tecidos Suporte , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Cryptosporidium parvum/patogenicidade , Células-Tronco Embrionárias Humanas/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Intestinos/citologia , Intestinos/parasitologia , Intestinos/patologia , Camundongos , Modelos Biológicos , Organoides/citologia , Organoides/parasitologia , Organoides/patologia , Regeneração , Medicina Regenerativa , Células-Tronco , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual
4.
Proc Natl Acad Sci U S A ; 117(37): 23073-23084, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873638

RESUMO

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Culina/metabolismo , Hipocampo/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Morfogênese/fisiologia , Animais , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Dendritos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Neurogênese/fisiologia , Células Piramidais/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
5.
Urologiia ; (4): 144-150, 2020 Sep.
Artigo em Russo | MEDLINE | ID: mdl-32897029

RESUMO

Despite significant advances in andrology, the problem of penile cavernous fibrosis remains not fully understood. Many studies on the epidemiology of erectile dysfunction have been published, but consensus on the place and role of this pathology in the structure of sexual disorders has not yet been reached. The data obtained at different time intervals and in different geographical areas are strikingly different. Also, the role of organic disorders in the penis, including fibroplastic changes, in certain etiological factors has not been determined. In addition, the relationship between etiological factors and morphological changes in penile tissues is disputed due to the small amount of data obtained from the pathohistological study of human penis biopsies. This review is devoted to the systematization of epidemiological data and etiological factors of cavernous fibrosis, the definition of the relationship between them, the analysis of clinical and experimental studies, which study the relationship between the degree of severity of damaging agents and the formation of typical fibrogenic reactions.


Assuntos
Disfunção Erétil , Induração Peniana , Fibrose , Humanos , Masculino , Morfogênese , Ereção Peniana , Pênis
6.
PLoS One ; 15(9): e0238955, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32997668

RESUMO

Cell shape change is one of the driving forces of animal morphogenesis, and the model organism Caenorhabditis elegans has played a significant role in analyzing the underlying mechanisms involved. The analysis of cell shape change requires quantification of cellular shape descriptors, a method known as cellular morphometry. However, standard C. elegans live imaging methods limit the capability of cellular morphometry in 3D, as spherical aberrations generated by samples and the surrounding medium misalign optical paths. Here, we report a 3D live imaging method for C. elegans embryos that minimized spherical aberrations caused by refractive index (RI) mismatch. We determined the composition of a refractive index matching medium (RIMM) for C. elegans live imaging. The 3D live imaging with the RIMM resulted in a higher signal intensity in the deeper cell layers. We also found that the obtained images improved the 3D cell segmentation quality. Furthermore, our 3D cellular morphometry and 2D cell shape simulation indicated that the germ cell precursor P4 had exceptionally high cortical tension. Our results demonstrate that the RIMM is a cost-effective solution to improve the 3D cellular morphometry of C. elegans. The application of this method should facilitate understanding of C. elegans morphogenesis from the perspective of cell shape changes.


Assuntos
Caenorhabditis elegans/anatomia & histologia , Imageamento Tridimensional/métodos , Refratometria/métodos , Animais , Pesos e Medidas Corporais/métodos , Caenorhabditis elegans/embriologia , Forma Celular/fisiologia , Células Germinativas , Morfogênese
7.
Proc Natl Acad Sci U S A ; 117(34): 20943-20949, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32817465

RESUMO

The reiterative process of lateral root (LR) formation is widespread and underlies root system formation. However, early LR primordium (LRP) morphogenesis is not fully understood. In this study, we conducted both a clonal analysis and time-lapse experiments to decipher the pattern and sequence of pericycle founder cell (FC) participation in LR formation. Most commonly, LRP initiation starts with the specification of just one FC longitudinally. Clonal and anatomical analyses suggested that a single FC gradually recruits neighboring pericycle cells to become FCs. This conclusion was validated by long-term time-lapse live-imaging experiments. Once the first FC starts to divide, its immediate neighbors, both lengthwise and laterally, are recruited within the hour, after which they recruit their neighboring cells within a few hours. Therefore, LRP initiation is a gradual, multistep process. FC recruitment is auxin-dependent and is abolished by treatment with a polar auxin transport inhibitor. Furthermore, FC recruitment establishes a morphogenetic field where laterally peripheral cells have a lower auxin response, which is associated with a lower proliferation potential, compared to centrally located FCs. The lateral boundaries of the morphogenetic field are determined by phloem-adjacent pericycle cells, which are the last cells to be recruited as FCs. The proliferation potential of these cells is limited, but their recruitment is essential for root system formation, resulting in the formation of a new vascular connection between the nascent and parent root, which is crucial for establishing a continuous and efficient vascular system.


Assuntos
Arabidopsis/genética , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Meristema/metabolismo , Morfogênese/genética , Organogênese Vegetal/fisiologia , Floema/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
PLoS Comput Biol ; 16(8): e1008049, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822341

RESUMO

Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a "template-based search" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.


Assuntos
Biologia Computacional/métodos , Processamento de Imagem Assistida por Computador/métodos , Morfogênese/fisiologia , Algoritmos , Animais , Divisão Celular/fisiologia , Mineração de Dados/métodos , Drosophila/citologia , Drosophila/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Feminino , Masculino , Microscopia Confocal , Fatores de Tempo
9.
PLoS Genet ; 16(8): e1008820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750048

RESUMO

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Morfogênese , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutação com Perda de Função , Ligação Proteica , Transporte Proteico , Proteínas de Ligação a Tacrolimo/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento
10.
Nat Commun ; 11(1): 3910, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764693

RESUMO

SARS-CoV-2, a ß-coronavirus, has rapidly spread across the world, highlighting its high transmissibility, but the underlying morphogenesis and pathogenesis remain poorly understood. Here, we characterize the replication dynamics, cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison, HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion, apoptosis, destruction of epithelium integrity, cilium shrinking and beaded changes are observed in the plaque regions. Taken together, our results provide important insights into SARS-CoV-2 cell tropism, replication and morphogenesis.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Células Epiteliais/virologia , Morfogênese/fisiologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Betacoronavirus/patogenicidade , Linhagem Celular , Células Cultivadas , Efeito Citopatogênico Viral , Células Epiteliais/patologia , Humanos , Pandemias , Sistema Respiratório/patologia , Tropismo , Replicação Viral
11.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 49(1): 90-99, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32621417

RESUMO

Neurons are the structural and functional unit of the nervous system. Precisely regulated dendrite morphogenesis is the basis of neural circuit assembly. Numerous studies have been conducted to explore the regulatory mechanisms of dendritic morphogenesis. According to their action regions, we divide them into two categories: the intrinsic and extrinsic regulators of neuronal dendritic morphogenesis. Intrinsic factors are cell type-specific transcription factors, actin polymerization or depolymerization regulators and regulators of the secretion or endocytic pathways. These intrinsic factors are produced by neuron itself and play an important role in regulating the development of dendrites. The extrinsic regulators are either secreted proteins or transmembrane domain containing cell adhesion molecules. They often form receptor-ligand pairs to mediate attractive or repulsive dendritic guidance. In this review, we summarize recent findings on the intrinsic and external molecular mechanisms of dendrite morphogenesis from multiple model organisms, including Caenorhabditis elegans, Drosophila and mice. These studies will provide a better understanding on how defective dendrite development and maintenance are associated with neurological diseases.


Assuntos
Dendritos , Neurônios , Animais , Caenorhabditis elegans/citologia , Camundongos , Morfogênese , Doenças do Sistema Nervoso/fisiopatologia , Neurônios/citologia , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 11(1): 3516, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665580

RESUMO

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Feminino , Humanos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/genética
13.
BMC Evol Biol ; 20(1): 92, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727367

RESUMO

BACKGROUND: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classification of Gastrostyla-like species, a taxonomically difficult group of hypotrichs with a common ventral cirral pattern but various dorsal and ontogenetic patterns, is poorly understood. Hence, systematic relationships within this group and with other taxa in the subclass Hypotrichia remain unresolved. RESULTS: 18S rRNA gene sequence of a new Gastrostyla-like taxon was obtained. Phylogenetic analyses based on the 18S rRNA gene sequences indicate that this ciliate represents a new genus that is closely related to Heterourosomoida and Kleinstyla within the oxytrichid clade of the Hypotrichia. However, the position of this cluster remains unresolved. All three genera deviate from the typical oxytrichids by their incomplete (or lack of) dorsal kinety fragmentation during morphogenesis. Morphology and morphogenesis of this newly discovered form, Heterogastrostyla salina nov. gen., nov. spec., are described. Heterogastrostyla nov. gen., is characterised as follows: more than 18 fronto-ventral-transverse cirri, cirral anlagen V and VI develop pretransverse cirri, and dorsal ciliature in Urosomoida-like pattern. CONCLUSIONS: Similar to the CEUU-hypothesis about convergent evolution of urostylids and uroleptids, we speculate that the shared ventral cirral patterns of Gastrostyla-like taxa might have resulted from convergent evolution.


Assuntos
Cilióforos/classificação , Classificação , Salinidade , Solo , Animais , Sequência de Bases , Núcleo Celular/genética , DNA Ribossômico/genética , Hypotrichida/classificação , Hypotrichida/genética , Funções Verossimilhança , Morfogênese/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Especificidade da Espécie
14.
J Vis Exp ; (161)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32716381

RESUMO

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.


Assuntos
Bioensaio/métodos , Candida albicans/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Hifas/crescimento & desenvolvimento , Morfogênese , Animais , Candida albicans/efeitos dos fármacos , Ceco/microbiologia , Cefoperazona/farmacologia , Feminino , Proteínas Fúngicas/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Hifas/efeitos dos fármacos , Masculino , Metaboloma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Morfogênese/efeitos dos fármacos
15.
Nat Commun ; 11(1): 3377, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632100

RESUMO

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.


Assuntos
Glândulas Mamárias Humanas/metabolismo , Morfogênese/genética , Mutação , Comunicação Parácrina/genética , Biomimética/métodos , Linhagem Celular , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Glândulas Mamárias Humanas/irrigação sanguínea , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Fenótipo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
16.
Nucleic Acids Res ; 48(15): 8374-8392, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619237

RESUMO

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.


Assuntos
Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Humanos , Morfogênese/genética , Fator de Transcrição Sp1/genética , TATA Box/genética , Peixe-Zebra/genética
17.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510485

RESUMO

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.


Assuntos
Arabidopsis/citologia , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microtúbulos/metabolismo , Imagem Molecular , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Sobrevivência Celular , Parede Celular/metabolismo , Celulose/metabolismo , Meristema/citologia , Meristema/crescimento & desenvolvimento , Morfogênese
18.
Trends Plant Sci ; 25(8): 719-722, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513584

RESUMO

How the plant cell wall expands and forms shapes is a long-standing mystery. Traditional thought is that turgor pressure drives these processes. However, a recent study by Haas and colleagues shows for the first time that the expansion of pectin homogalacturonan nanofilaments drives morphogenesis without turgor pressure in plant epidermal cells.


Assuntos
Pectinas , Células Vegetais , Parede Celular , Células Epidérmicas , Morfogênese
19.
PLoS Genet ; 16(6): e1008849, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516352

RESUMO

Cohesin, a multisubunit protein complex, is required for holding sister chromatids together during mitosis and meiosis. The recruitment of cohesin by the sister chromatid cohesion 2/4 (SCC2/4) complex has been extensively studied in Saccharomyces cerevisiae mitosis, but its role in mitosis and meiosis remains poorly understood in multicellular organisms, because complete loss-of-function of either gene causes embryonic lethality. Here, we identified a weak allele of Atscc2 (Atscc2-5) that has only minor defects in vegetative development but exhibits a significant reduction in fertility. Cytological analyses of Atscc2-5 reveal multiple meiotic phenotypes including defects in chromosomal axis formation, meiosis-specific cohesin loading, homolog pairing and synapsis, and AtSPO11-1-dependent double strand break repair. Surprisingly, even though AtSCC2 interacts with AtSCC4 in vitro and in vivo, meiosis-specific knockdown of AtSCC4 expression does not cause any meiotic defect, suggesting that the SCC2-SCC4 complex has divergent roles in mitosis and meiosis. SCC2 homologs from land plants have a unique plant homeodomain (PHD) motif not found in other species. We show that the AtSCC2 PHD domain can bind to the N terminus of histones and is required for meiosis but not mitosis. Taken together, our results provide evidence that unlike SCC2 in other organisms, SCC2 requires a functional PHD domain during meiosis in land plants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose/genética , Dedos de Zinco PHD/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Técnicas de Silenciamento de Genes , Genoma de Planta/genética , Mutação com Perda de Função , Mitose/genética , Morfogênese/genética , Mutagênese , Plantas Geneticamente Modificadas , Polinização/genética , Sequenciamento Completo do Genoma
20.
PLoS One ; 15(6): e0234375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555682

RESUMO

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.


Assuntos
Rim/anormalidades , Rim/efeitos dos fármacos , Quercetina/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Vimentina/metabolismo , beta Catenina/química , beta Catenina/genética
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