RESUMO
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Assuntos
Divisão Celular , Polaridade Celular , Drosophila melanogaster , Células Epiteliais , Metáfase , Fuso Acromático , Estresse Mecânico , Animais , Metáfase/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fuso Acromático/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/citologia , Polaridade Celular/fisiologia , Padronização Corporal , Miosina Tipo II/metabolismo , Embrião não Mamífero/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Gastrulação/fisiologiaRESUMO
Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior. We previously found that the Y loops consist of regular clusters of nucleosomes, with an estimated median of 54 nucleosomes per cluster with wide variation.In this study, we report that the histone modifications H3K4me3, H3K27me3, and H3K36me3 are also clustered along the Y loops, with H3K4me3 more associated with diffuse chromatin compared to H3K27me3. These histone modifications form domains that can be stretches of Y loop chromatin micrometres long, or can be in short alternating domains. The different histone modifications are associated with different sizes of chromatin clusters and unique morphologies. Strikingly, a single chromatin cluster almost always only contains only one type of the histone modifications that were labelled, suggesting exclusivity, and therefore regulation at the level of individual chromatin clusters. The active mark H3K36me3 is more associated with actively elongating RNA polymerase II than H3K27me3, with polymerase often appearing on what are assumed to be looping regions on the periphery of chromatin clusters.These results provide a foundation for understanding the relationship between chromatin state, chromatin organisation, and transcription regulation - with potential implications for pause-release dynamics, splicing complex organisation and chromatin dynamics during polymerase progression along a gene.
Assuntos
Histonas , Nucleossomos , Animais , Histonas/metabolismo , Código das Histonas , Drosophila melanogaster/genética , Cromatina/genéticaRESUMO
Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants-c.1138G>A (p.G380R) and c.1948A>G (p.K650E)-which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor's age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.
Assuntos
Acondroplasia , Sêmen , Idoso , Humanos , Masculino , Acondroplasia/genética , Mutação , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Senescência CelularRESUMO
During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increase its occupancy at target sites in Notch-On conditions.
Assuntos
Proteínas de Ligação a DNA , Receptores Notch , Animais , Proteínas de Ligação a DNA/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Comportamento ApetitivoRESUMO
BigNeuron is an open community bench-testing platform with the goal of setting open standards for accurate and fast automatic neuron tracing. We gathered a diverse set of image volumes across several species that is representative of the data obtained in many neuroscience laboratories interested in neuron tracing. Here, we report generated gold standard manual annotations for a subset of the available imaging datasets and quantified tracing quality for 35 automatic tracing algorithms. The goal of generating such a hand-curated diverse dataset is to advance the development of tracing algorithms and enable generalizable benchmarking. Together with image quality features, we pooled the data in an interactive web application that enables users and developers to perform principal component analysis, t-distributed stochastic neighbor embedding, correlation and clustering, visualization of imaging and tracing data, and benchmarking of automatic tracing algorithms in user-defined data subsets. The image quality metrics explain most of the variance in the data, followed by neuromorphological features related to neuron size. We observed that diverse algorithms can provide complementary information to obtain accurate results and developed a method to iteratively combine methods and generate consensus reconstructions. The consensus trees obtained provide estimates of the neuron structure ground truth that typically outperform single algorithms in noisy datasets. However, specific algorithms may outperform the consensus tree strategy in specific imaging conditions. Finally, to aid users in predicting the most accurate automatic tracing results without manual annotations for comparison, we used support vector machine regression to predict reconstruction quality given an image volume and a set of automatic tracings.
Assuntos
Benchmarking , Microscopia , Microscopia/métodos , Imageamento Tridimensional/métodos , Neurônios/fisiologia , AlgoritmosRESUMO
While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription.
Assuntos
Drosophila , Microscopia , Masculino , Animais , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Nucleossomos/genética , Espermatócitos/metabolismo , Transcrição Gênica , Cromatina/genética , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviours and their subsequent compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline-dependent system in stress and related excessive behaviours, we hypothesised that neuroplastic changes in the LC may be associated with the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behaviour, and the ensuing development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behaviour was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268- neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behaviour, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice, while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+/zif268- LC neurons represents a signature of the tendency to develop compulsive coping behaviours.
Assuntos
Adaptação Psicológica , Comportamento Compulsivo , Locus Cerúleo , Animais , Masculino , Ratos , Regulação para Baixo , Locus Cerúleo/metabolismo , Neurônios/metabolismo , Ratos Sprague-Dawley , RNA Mensageiro/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We study the problem of deconvolution for light-sheet microscopy, where the data is corrupted by spatially varying blur and a combination of Poisson and Gaussian noise. The spatial variation of the point spread function of a light-sheet microscope is determined by the interaction between the excitation sheet and the detection objective PSF. We introduce a model of the image formation process that incorporates this interaction and we formulate a variational model that accounts for the combination of Poisson and Gaussian noise through a data fidelity term consisting of the infimal convolution of the single noise fidelities, first introduced in L. Calatroni et al. (SIAM J Imaging Sci 10(3):1196-1233, 2017). We establish convergence rates and a discrepancy principle for the infimal convolution fidelity and the inverse problem is solved by applying the primal-dual hybrid gradient (PDHG) algorithm in a novel way. Numerical experiments performed on simulated and real data show superior reconstruction results in comparison with other methods.
RESUMO
In vertebrate embryos the presomitic mesoderm becomes progressively segmented into somites at the anterior end while extending along the anterior-posterior axis. A commonly adopted model to explain how this tissue elongates is that of posterior growth, driven in part by the addition of new cells from uncommitted progenitor populations in the tailbud. However, in zebrafish, much of somitogenesis is associated with an absence of overall volume increase, and posterior progenitors do not contribute new cells until the final stages of somitogenesis. Here, we perform a comprehensive 3D morphometric analysis of the paraxial mesoderm and reveal that extension is linked to a volumetric decrease and an increase in cell density. We also find that individual cells decrease in volume over successive somite stages. Live cell tracking confirms that much of this tissue deformation occurs within the presomitic mesoderm progenitor zone and is associated with non-directional rearrangement. Taken together, we propose a compaction-extension mechanism of tissue elongation that highlights the need to better understand the role tissue intrinsic and extrinsic forces in regulating morphogenesis.
Assuntos
Mesoderma , Peixe-Zebra , Animais , Desenvolvimento Embrionário , Mesoderma/fisiologia , Morfogênese , SomitosRESUMO
Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection and is sensed primarily by RIG-I-like receptors (RLRs). Oligomerization of RLRs following binding to cytosolic dsRNA activates and nucleates self-assembly of the mitochondrial antiviral-signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context, MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling, and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interferon beta/metabolismo , Membranas Mitocondriais/metabolismo , Células 3T3/virologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Morte Celular/fisiologia , Citosol/fisiologia , Fibroblastos/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Knockout , Microscopia/métodos , Membranas Mitocondriais/virologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Domínios Proteicos , RNA de Cadeia Dupla/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Febre do Nilo Ocidental/metabolismoRESUMO
The reaggregation of dissociated cells to form organotypic structures provides an in vitro system for the analysis of the cellular interactions and molecular mechanisms involved in the formation of tissue architecture. The retina, an outgrowth of the forebrain, is a precisely layered neural tissue, yet the mechanisms underlying layer formation are largely unexplored. Here we describe the protocol to dissociate, re-aggregate, and culture zebrafish retinal cells from a transgenic, Spectrum of Fates, line where all main cell types are labelled with a combination of fluorescent proteins driven by fate-specific promoters. These cells re-aggregate and self-organize in just 48 h in minimal culture conditions. We also describe how the patterning in these aggregates can be analyzed using isocontour profiling to compare whether different conditions affect their self-organization.
Assuntos
Animais Geneticamente Modificados/metabolismo , Diferenciação Celular , Neurônios/citologia , Retina/citologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Agregação Celular , Proliferação de Células , Proteínas Luminescentes/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Peixe-Zebra/genéticaRESUMO
During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. Using a genetic clone-tracing method, we labelled early embryonic progenitors and found a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages using light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid-gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, the lineage of which varies in a species-specific manner due to vastly different rates of differentiation and growth.
Assuntos
Mesoderma/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/crescimento & desenvolvimento , Células-Tronco/citologia , Animais , Padronização Corporal , Divisão Celular , Linhagem da Célula , Rastreamento de Células , Gastrulação , Mesoderma/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Especificidade de Órgãos , Somitos/citologia , Somitos/metabolismo , Medula Espinal/citologia , Células-Tronco/metabolismo , Cauda , Peixe-ZebraRESUMO
During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that ß-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that ß-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate ß-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.
Assuntos
Actinas/metabolismo , Cones de Crescimento/metabolismo , Modelos Biológicos , Imagem Molecular , Neurogênese/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Xenopus laevisRESUMO
Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Although transient structures they are of fundamental importance as they generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about cellular dynamics underlying the morphological transitions during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi-photon imaging of transgenic chick embryos, where all cells express a membrane-bound GFP. We specifically focussed on the dynamic cellular changes in two principle regions within the somite, the medial and lateral domains, to investigate extensive morphological transformations. Furthermore, by using quantitative analysis and cell tracking, we capture for the first time a directed movement of dermomyotomal progenitor cells towards the rostro-medial domain of the dermomyotome, where skeletal muscle formation initiates.
Assuntos
Somitos/citologia , Animais , Diferenciação Celular/fisiologia , Embrião de Galinha , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Somitos/metabolismoRESUMO
Epithelial folding shapes embryos and tissues during development. Here, we investigate the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. We investigated the shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo. We demonstrate that formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. We find that epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity.
Assuntos
Actomiosina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteína Wnt1/metabolismo , Junções Aderentes/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Padronização Corporal , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epitélio/embriologia , Técnicas de Silenciamento de Genes , Genes de Insetos , Proteínas de Fluorescência Verde/genética , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Luminescentes/genética , Mutação , Miosina Tipo II/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/antagonistas & inibidores , Fosfatase de Miosina-de-Cadeia-Leve/genética , Transdução de Sinais , Proteína Wnt1/genéticaRESUMO
A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down.
Assuntos
Núcleo Celular/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Notch/metabolismo , Animais , Núcleo Celular/metabolismo , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Modelos Moleculares , Ligação Proteica , Receptores Notch/genética , Transdução de Sinais , Ativação TranscricionalRESUMO
To investigate the cell-cell interactions necessary for the formation of retinal layers, we cultured dissociated zebrafish retinal progenitors in agarose microwells. Within these wells, the cells re-aggregated within hours, forming tight retinal organoids. Using a Spectrum of Fates zebrafish line, in which all different types of retinal neurons show distinct fluorescent spectra, we found that by 48â h in culture, the retinal organoids acquire a distinct spatial organisation, i.e. they became coarsely but clearly laminated. Retinal pigment epithelium cells were in the centre, photoreceptors and bipolar cells were next most central and amacrine cells and retinal ganglion cells were on the outside. Image analysis allowed us to derive quantitative measures of lamination, which we then used to find that Müller glia, but not RPE cells, are essential for this process.