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1.
Cell ; 184(26): 6361-6377.e24, 2021 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-34875226

RESUMEN

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.


Asunto(s)
Área Hipotalámica Lateral/metabolismo , Hibridación Fluorescente in Situ , Animales , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Área Hipotalámica Lateral/citología , Imagenología Tridimensional , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuropéptidos/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN/metabolismo , RNA-Seq , Análisis de la Célula Individual , Transcripción Genética
2.
Cell ; 162(5): 1066-77, 2015 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-26317470

RESUMEN

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.


Asunto(s)
Envejecimiento/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Mutación , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Envejecimiento/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Núcleo Celular/química , Citoplasma/química , Humanos , Priones/química , Agregado de Proteínas , Estructura Terciaria de Proteína , Proteína FUS de Unión a ARN/metabolismo
3.
Nature ; 620(7974): 615-624, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37558872

RESUMEN

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.


Asunto(s)
Movimiento Celular , Morfogénesis , Células Fotorreceptoras , Retina , Animales , Humanos , Actomiosina/metabolismo , Competencia Celular , Diferenciación Celular , Movimiento Celular/fisiología , Proliferación Celular , Microtúbulos/metabolismo , Morfogénesis/fisiología , Organoides/citología , Organoides/embriología , Células Fotorreceptoras/citología , Células Fotorreceptoras/fisiología , Retina/citología , Retina/embriología , Pez Cebra/embriología
4.
Nature ; 588(7836): 106-111, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33116308

RESUMEN

The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin1,2. Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process3. Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs4, but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories5. Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.


Asunto(s)
Cíclidos/clasificación , Cíclidos/genética , Especiación Genética , Genoma/genética , Genómica , Simpatría/genética , Animales , Cíclidos/anatomía & histología , Femenino , Flujo Génico , Flujo Genético , Masculino , Preferencia en el Apareamiento Animal , Herencia Multifactorial/genética , Filogenia , Pigmentación/genética , Polimorfismo Genético
5.
Nature ; 583(7817): 578-584, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32699395

RESUMEN

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.


Asunto(s)
Adaptación Fisiológica/genética , Quirópteros/genética , Evolución Molecular , Genoma/genética , Genómica/normas , Adaptación Fisiológica/inmunología , Animales , Quirópteros/clasificación , Quirópteros/inmunología , Elementos Transponibles de ADN/genética , Inmunidad/genética , Anotación de Secuencia Molecular/normas , Filogenia , ARN no Traducido/genética , Estándares de Referencia , Reproducibilidad de los Resultados , Integración Viral/genética , Virus/genética
6.
PLoS Genet ; 19(7): e1010798, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37498820

RESUMEN

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.


Asunto(s)
Nematodos , Hielos Perennes , Humanos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Larva/genética , Larva/metabolismo , Filogenia
7.
Proc Natl Acad Sci U S A ; 120(7): e2201076120, 2023 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-36749728

RESUMEN

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.


Asunto(s)
Tortugas , Animales , Ecosistema , Dinámica Poblacional
8.
Mol Biol Evol ; 41(1)2024 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-38124445

RESUMEN

The escape of DNA from mitochondria into the nuclear genome (nuclear mitochondrial DNA, NUMT) is an ongoing process. Although pervasively observed in eukaryotic genomes, their evolutionary trajectories in a mammal-wide context are poorly understood. The main challenge lies in the orthology assignment of NUMTs across species due to their fast evolution and chromosomal rearrangements over the past 200 million years. To address this issue, we systematically investigated the characteristics of NUMT insertions in 45 mammalian genomes and established a novel, synteny-based method to accurately predict orthologous NUMTs and ascertain their evolution across mammals. With a series of comparative analyses across taxa, we revealed that NUMTs may originate from nonrandom regions in mtDNA, are likely found in transposon-rich and intergenic regions, and unlikely code for functional proteins. Using our synteny-based approach, we leveraged 630 pairwise comparisons of genome-wide microsynteny and predicted the NUMT orthology relationships across 36 mammals. With the phylogenetic patterns of NUMT presence-and-absence across taxa, we constructed the ancestral state of NUMTs given the mammal tree using a coalescent method. We found support on the ancestral node of Fereuungulata within Laurasiatheria, whose subordinal relationships are still controversial. This study broadens our knowledge on NUMT insertion and evolution in mammalian genomes and highlights the merit of NUMTs as alternative genetic markers in phylogenetic inference.


Asunto(s)
Genoma Mitocondrial , Genómica , Animales , Filogenia , Mitocondrias/genética , ADN Mitocondrial/genética , Mamíferos/genética , Análisis de Secuencia de ADN , Núcleo Celular/genética , Evolución Molecular
9.
Nat Methods ; 19(6): 696-704, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35361932

RESUMEN

Variant calling has been widely used for genotyping and for improving the consensus accuracy of long-read assemblies. Variant calls are commonly hard-filtered with user-defined cutoffs. However, it is impossible to define a single set of optimal cutoffs, as the calls heavily depend on the quality of the reads, the variant caller of choice and the quality of the unpolished assembly. Here, we introduce Merfin, a k-mer based variant-filtering algorithm for improved accuracy in genotyping and genome assembly polishing. Merfin evaluates each variant based on the expected k-mer multiplicity in the reads, independently of the quality of the read alignment and variant caller's internal score. Merfin increased the precision of genotyped calls in several benchmarks, improved consensus accuracy and reduced frameshift errors when applied to human and nonhuman assemblies built from Pacific Biosciences HiFi and continuous long reads or Oxford Nanopore reads, including the first complete human genome. Moreover, we introduce assembly quality and completeness metrics that account for the expected genomic copy numbers.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Nanoporos , Genoma , Genómica , Humanos , Análisis de Secuencia de ADN
10.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35042802

RESUMEN

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.


Asunto(s)
Secuencia de Bases/genética , Eucariontes/genética , Genómica/normas , Animales , Biodiversidad , Genómica/métodos , Humanos , Estándares de Referencia , Valores de Referencia , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas
11.
Cell ; 139(3): 623-33, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19879847

RESUMEN

The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.


Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Linaje de la Célula , Perfilación de la Expresión Génica , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans , Diferenciación Celular , Perfilación de la Expresión Génica/métodos
12.
Nature ; 559(7712): E2, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29795340

RESUMEN

In the originally published version of this Article, the sequenced axolotl strain (the homozygous white mutant) was denoted as 'D/D' rather than 'd/d' in Fig. 1a and the accompanying legend, the main text and the Methods section. The original Article has been corrected online.

13.
Nature ; 554(7690): 50-55, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29364872

RESUMEN

Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl (Ambystoma mexicanum) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3. However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3-/- and Pax7-/- mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies.


Asunto(s)
Ambystoma mexicanum/genética , Evolución Molecular , Genoma/genética , Genómica , Animales , ADN Intergénico/genética , Genes Esenciales/genética , Proteínas de Homeodominio/genética , Intrones/genética , Masculino , Ratones , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX7/genética , Picea/genética , Pinus/genética , Regeneración/genética , Retroelementos/genética , Secuencias Repetidas Terminales/genética
14.
BMC Bioinformatics ; 24(1): 288, 2023 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-37464285

RESUMEN

BACKGROUND:  PacBio high fidelity (HiFi) sequencing reads are both long (15-20 kb) and highly accurate (> Q20). Because of these properties, they have revolutionised genome assembly leading to more accurate and contiguous genomes. In eukaryotes the mitochondrial genome is sequenced alongside the nuclear genome often at very high coverage. A dedicated tool for mitochondrial genome assembly using HiFi reads is still missing. RESULTS:  MitoHiFi was developed within the Darwin Tree of Life Project to assemble mitochondrial genomes from the HiFi reads generated for target species. The input for MitoHiFi is either the raw reads or the assembled contigs, and the tool outputs a mitochondrial genome sequence fasta file along with annotation of protein and RNA genes. Variants arising from heteroplasmy are assembled independently, and nuclear insertions of mitochondrial sequences are identified and not used in organellar genome assembly. MitoHiFi has been used to assemble 374 mitochondrial genomes (368 Metazoa and 6 Fungi species) for the Darwin Tree of Life Project, the Vertebrate Genomes Project and the Aquatic Symbiosis Genome Project. Inspection of 60 mitochondrial genomes assembled with MitoHiFi for species that already have reference sequences in public databases showed the widespread presence of previously unreported repeats. CONCLUSIONS:  MitoHiFi is able to assemble mitochondrial genomes from a wide phylogenetic range of taxa from Pacbio HiFi data. MitoHiFi is written in python and is freely available on GitHub ( https://github.com/marcelauliano/MitoHiFi ). MitoHiFi is available with its dependencies as a Docker container on GitHub (ghcr.io/marcelauliano/mitohifi:master).


Asunto(s)
Genoma Mitocondrial , Filogenia , ARN , Eucariontes , Análisis de Secuencia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento
15.
Bioinformatics ; 37(5): 612-621, 2021 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-33031558

RESUMEN

MOTIVATION: Long tandem repeat expansions of more than 1000 nt have been suggested to be associated with diseases, but remain largely unexplored in individual human genomes because read lengths have been too short. However, new long-read sequencing technologies can produce single reads of 10 000 nt or more that can span such repeat expansions, although these long reads have high error rates, of 10-20%, which complicates the detection of repetitive elements. Moreover, most traditional algorithms for finding tandem repeats are designed to find short tandem repeats (<1000 nt) and cannot effectively handle the high error rate of long reads in a reasonable amount of time. RESULTS: Here, we report an efficient algorithm for solving this problem that takes advantage of the length of the repeat. Namely, a long tandem repeat has hundreds or thousands of approximate copies of the repeated unit, so despite the error rate, many short k-mers will be error-free in many copies of the unit. We exploited this characteristic to develop a method for first estimating regions that could contain a tandem repeat, by analyzing the k-mer frequency distributions of fixed-size windows across the target read, followed by an algorithm that assembles the k-mers of a putative region into the consensus repeat unit by greedily traversing a de Bruijn graph. Experimental results indicated that the proposed algorithm largely outperformed Tandem Repeats Finder, a widely used program for finding tandem repeats, in terms of sensitivity. AVAILABILITY AND IMPLEMENTATION: https://github.com/morisUtokyo/mTR.


Asunto(s)
Algoritmos , Secuenciación de Nucleótidos de Alto Rendimiento , Genoma Humano , Humanos , Repeticiones de Microsatélite , Análisis de Secuencia de ADN
16.
Nat Methods ; 15(12): 1090-1097, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30478326

RESUMEN

Fluorescence microscopy is a key driver of discoveries in the life sciences, with observable phenomena being limited by the optics of the microscope, the chemistry of the fluorophores, and the maximum photon exposure tolerated by the sample. These limits necessitate trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. In this work we show how content-aware image restoration based on deep learning extends the range of biological phenomena observable by microscopy. We demonstrate on eight concrete examples how microscopy images can be restored even if 60-fold fewer photons are used during acquisition, how near isotropic resolution can be achieved with up to tenfold under-sampling along the axial direction, and how tubular and granular structures smaller than the diffraction limit can be resolved at 20-times-higher frame rates compared to state-of-the-art methods. All developed image restoration methods are freely available as open source software in Python, FIJI, and KNIME.


Asunto(s)
Colorantes Fluorescentes/química , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Programas Informáticos , Animales , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestructura , Células HeLa , Humanos , Hígado/metabolismo , Hígado/ultraestructura , Fotones , Planarias/metabolismo , Planarias/ultraestructura , Retina/metabolismo , Retina/ultraestructura , Tribolium/metabolismo , Tribolium/ultraestructura , Pez Cebra/metabolismo
17.
Development ; 144(23): 4406-4421, 2017 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-29038308

RESUMEN

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.


Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Drosophila melanogaster/genética , Ecdisterona/farmacología , Ecdisterona/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes de Insecto , Discos Imaginales/citología , Discos Imaginales/efectos de los fármacos , Discos Imaginales/crecimiento & desarrollo , Insulina/farmacología , Insulina/fisiología , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Morfogénesis/fisiología , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Alas de Animales/efectos de los fármacos
18.
Opt Express ; 28(20): 29044-29053, 2020 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-33114810

RESUMEN

Estimation of optical aberrations from volumetric intensity images is a key step in sensorless adaptive optics for 3D microscopy. Recent approaches based on deep learning promise accurate results at fast processing speeds. However, collecting ground truth microscopy data for training the network is typically very difficult or even impossible thereby limiting this approach in practice. Here, we demonstrate that neural networks trained only on simulated data yield accurate predictions for real experimental images. We validate our approach on simulated and experimental datasets acquired with two different microscopy modalities and also compare the results to non-learned methods. Additionally, we study the predictability of individual aberrations with respect to their data requirements and find that the symmetry of the wavefront plays a crucial role. Finally, we make our implementation freely available as open source software in Python.

19.
Development ; 143(3): 540-6, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26700682

RESUMEN

Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.


Asunto(s)
Embrión no Mamífero/metabolismo , ARN/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Automatización , Membrana Celular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hibridación Fluorescente in Situ/métodos , ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Transcripción Genética
20.
PLoS Comput Biol ; 14(4): e1006079, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29652879

RESUMEN

Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables simulation of operational light-sheet microscopes by combining data from 105-106 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave-optical phenomena, and renders image-formation in light-sheet microscopy computationally tractable.


Asunto(s)
Microscopía Fluorescente/métodos , Microscopía Fluorescente/estadística & datos numéricos , Programas Informáticos , Biología Computacional , Simulación por Computador , Aumento de la Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/métodos , Imagenología Tridimensional/estadística & datos numéricos , Luz , Fenómenos Ópticos , Dispersión de Radiación
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