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1.
Dev Cell ; 59(3): 339-350.e4, 2024 Feb 05.
Article in English | MEDLINE | ID: mdl-38198889

ABSTRACT

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.


Subject(s)
Embryonic Development , Mitral Valve , Animals , Mice , Mitral Valve/abnormalities , Mitral Valve/metabolism , Retrospective Studies , Cell Differentiation
2.
ACS Photonics ; 10(12): 4104-4111, 2023 Dec 20.
Article in English | MEDLINE | ID: mdl-38145164

ABSTRACT

Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this light-efficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging.

3.
Glia ; 71(9): 2250-2265, 2023 09.
Article in English | MEDLINE | ID: mdl-37259810

ABSTRACT

Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRABATP1.0 , we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron-glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50-250 µm2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+ transients, and intracellular Ca2+ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca2+ -independent manners, thus potentially regulating hundreds of synapses all together.


Subject(s)
Astrocytes , Synapses , Mice , Animals , Astrocytes/metabolism , Synapses/metabolism , Neuroglia/metabolism , Neurons/metabolism , Adenosine Triphosphate/metabolism , Calcium/metabolism , Calcium Signaling/physiology
4.
PLoS Comput Biol ; 18(7): e1010211, 2022 07.
Article in English | MEDLINE | ID: mdl-35789212

ABSTRACT

Tridimensional microscopy and algorithms for automated segmentation and tracing are revolutionizing neuroscience through the generation of growing libraries of neuron reconstructions. Innovative computational methods are needed to analyze these neuronal traces. In particular, means to characterize the geometric properties of traced neurites along their trajectory have been lacking. Here, we propose a local tridimensional (3D) scale metric derived from differential geometry, measuring for each point of a curve the characteristic length where it is fully 3D as opposed to being embedded in a 2D plane or 1D line. The larger this metric is and the more complex the local 3D loops and turns of the curve are. Available through the GeNePy3D open-source Python quantitative geometry library (https://genepy3d.gitlab.io), this approach termed nAdder offers new means of describing and comparing axonal and dendritic arbors. We validate this metric on simulated and real traces. By reanalysing a published zebrafish larva whole brain dataset, we show its ability to characterize different population of commissural axons, distinguish afferent connections to a target region and differentiate portions of axons and dendrites according to their behavior, shedding new light on the stereotypical nature of neurites' local geometry.


Subject(s)
Neurons , Zebrafish , Algorithms , Animals , Axons/physiology , Neurites , Neurons/physiology
5.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Article in English | MEDLINE | ID: mdl-34740966

ABSTRACT

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.


Subject(s)
Cerebellum/growth & development , Animals , Calcium Signaling , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Purkinje Cells/physiology , Synapses/physiology
6.
J Vis Exp ; (159)2020 05 21.
Article in English | MEDLINE | ID: mdl-32510512

ABSTRACT

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.


Subject(s)
Astrocytes/cytology , Cerebral Cortex/cytology , Electroporation/methods , Animals , Color , Female , Mice , Neurogenesis
7.
Neuron ; 107(4): 617-630.e6, 2020 08 19.
Article in English | MEDLINE | ID: mdl-32559415

ABSTRACT

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.


Subject(s)
Gene Expression , Gene Transfer Techniques , Neural Stem Cells , Transgenes , Animals , Cell Lineage , Genetic Vectors , Humans , Mice
8.
Glia ; 68(9): 1729-1742, 2020 09.
Article in English | MEDLINE | ID: mdl-32073702

ABSTRACT

Astrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID.


Subject(s)
Astrocytes , Intellectual Disability , Cytoskeletal Proteins/genetics , Humans , Intellectual Disability/genetics , Neuroglia , Neurons
9.
Nat Commun ; 10(1): 4884, 2019 10 25.
Article in English | MEDLINE | ID: mdl-31653848

ABSTRACT

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.


Subject(s)
Astrocytes/cytology , Cell Differentiation , Cerebral Cortex/cytology , Animals , Astrocytes/metabolism , Cell Lineage , Cell Plasticity , Cell Proliferation , Clone Cells/cytology , Mice
10.
Cell Rep ; 29(2): 437-452.e4, 2019 10 08.
Article in English | MEDLINE | ID: mdl-31597102

ABSTRACT

The somatotopic motor-neuron projections onto their cognate target muscles are essential for coordinated movement, but how that occurs for facial motor circuits, which have critical roles in respiratory and interactive behaviors, is poorly understood. We report extensive molecular heterogeneity in developing facial motor neurons in the mouse and identify markers of subnuclei and the motor pools innervating specific facial muscles. Facial subnuclei differentiate during migration to the ventral hindbrain, where neurons with progressively later birth dates-and evolutionarily more recent functions-settle in more-lateral positions. One subpopulation marker, ETV1, determines both positional and target muscle identity for neurons of the dorsolateral (DL) subnucleus. In Etv1 mutants, many markers of DL differentiation are lost, and individual motor pools project indifferently to their own and neighboring muscle targets. The resulting aberrant activation patterns are reminiscent of the facial synkinesis observed in humans after facial nerve injury.


Subject(s)
DNA-Binding Proteins/metabolism , Facial Muscles/embryology , Facial Muscles/innervation , Motor Neurons/physiology , Transcription Factors/metabolism , Animals , Cell Movement , Female , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Male , Mice, Mutant Strains , Mutation/genetics , Repressor Proteins/metabolism , Transcription, Genetic
11.
Nat Commun ; 10(1): 2160, 2019 May 09.
Article in English | MEDLINE | ID: mdl-31073140

ABSTRACT

Affiliation 4 incorrectly read 'University of the Basque Country (Ikerbasque), University of the Basque Country and Donostia International Physics Center, San Sebastian 20018, Spain.'Also, the affiliations of Ignacio Arganda-Carreras with 'IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain' and 'Donostia International Physics Center (DIPC), San Sebastian, 20018, Spain' were inadvertently omitted.Additionally, the third sentence of the first paragraph of the Results section entitled 'Multicontrast organ-scale imaging with ChroMS microscopy' incorrectly read 'For example, one can choose lambda1 = 850 and lambda2 = 110 nm for optimal two-photon excitation of blue and red chromophores.'. The correct version reads 'lambda2 = 1100 nm' instead of 'lambda2 = 110 nm'. These errors have now been corrected in the PDF and HTML versions of the Article.

12.
Nat Commun ; 10(1): 1662, 2019 04 10.
Article in English | MEDLINE | ID: mdl-30971684

ABSTRACT

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.


Subject(s)
Cerebral Cortex/diagnostic imaging , Imaging, Three-Dimensional/methods , Luminescent Proteins/chemistry , Microscopy, Fluorescence, Multiphoton/methods , Neuroimaging/methods , Animals , Astrocytes/metabolism , Cerebral Cortex/cytology , Color , Dependovirus , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , HEK293 Cells , Humans , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Nestin/genetics , Neuroanatomical Tract-Tracing Techniques/methods , Parvovirinae/genetics , Pyramidal Cells/metabolism , Transfection
13.
Neuron ; 102(1): 159-172.e7, 2019 04 03.
Article in English | MEDLINE | ID: mdl-30824354

ABSTRACT

Adult neural stem cells and multiciliated ependymal cells are glial cells essential for neurological functions. Together, they make up the adult neurogenic niche. Using both high-throughput clonal analysis and single-cell resolution of progenitor division patterns and fate, we show that these two components of the neurogenic niche are lineally related: adult neural stem cells are sister cells to ependymal cells, whereas most ependymal cells arise from the terminal symmetric divisions of the lineage. Unexpectedly, we found that the antagonist regulators of DNA replication, GemC1 and Geminin, can tune the proportion of neural stem cells and ependymal cells. Our findings reveal the controlled dynamic of the neurogenic niche ontogeny and identify the Geminin family members as key regulators of the initial pool of adult neural stem cells.


Subject(s)
Astrocytes/cytology , Ependyma/cytology , Ependymoglial Cells/cytology , Neural Stem Cells/cytology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Astrocytes/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Lineage , DNA Replication , Electroporation , Embryo, Mammalian , Ependymoglial Cells/metabolism , Geminin/metabolism , Mice , Neural Stem Cells/metabolism
14.
Light Sci Appl ; 7: 12, 2018.
Article in English | MEDLINE | ID: mdl-30839589

ABSTRACT

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.

15.
EMBO J ; 35(24): 2658-2670, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27797819

ABSTRACT

Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.


Subject(s)
Cell Proliferation , Epidermal Cells , Hair Follicle/cytology , Keratinocytes/physiology , Stem Cells/physiology , Animals , Cell Differentiation , Cytological Techniques , Mice , Models, Theoretical , Skin/cytology , Spatio-Temporal Analysis
16.
Glia ; 63(4): 699-717, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25530205

ABSTRACT

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single-out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre-lox recombination, previously used for visualizing axons and neurons. We used tamoxifen-inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single-cell resolution.


Subject(s)
Luminescent Proteins/metabolism , Nerve Fibers, Myelinated/metabolism , Oligodendroglia/cytology , Optic Nerve/cytology , Staining and Labeling/methods , Animals , Fluorescent Antibody Technique/methods , Luminescent Proteins/genetics , Mice , Mice, Transgenic , Oligodendroglia/metabolism , Recombination, Genetic , Stochastic Processes , Tamoxifen/administration & dosage , Transgenes
17.
Stem Cells ; 32(12): 3046-54, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25113584

ABSTRACT

Lineage tracing is an essential tool to study stem cell fate. Although traditional lineage tracing techniques have considerably advanced our understanding of stem cell behavior, they pose significant limitations for identification and longitudinal tracking of the progeny of individual stem cells, to compare their behaviors. This is of importance given the well-established heterogeneity among stem cells both in terms of potentialities and proliferative capacities. The recent development of multicolor genetic reporters addressable to specific cell populations largely overcomes these issues. These new "rainbow" technologies provide increased resolution in clonal identification and offer the possibility to study the relative distribution, contacts, tiled arrangement, and competitive interactions among cells or groups of cells of the same type.


Subject(s)
Cell Differentiation/physiology , Cell Lineage/physiology , Genes, Reporter/physiology , Homeostasis/physiology , Stem Cells/cytology , Animals , Cell Lineage/genetics , Cells, Cultured , Genes, Reporter/genetics , Humans , Stem Cells/metabolism
18.
Neuron ; 81(3): 505-20, 2014 Feb 05.
Article in English | MEDLINE | ID: mdl-24507188

ABSTRACT

We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.


Subject(s)
Brain/cytology , Cell Lineage/physiology , Neuroglia/physiology , Neurons/physiology , Spinal Cord/cytology , Stem Cells/physiology , Age Factors , Animals , Animals, Newborn , Brain/embryology , Brain/growth & development , Cell Differentiation/physiology , Cell Movement/physiology , Chick Embryo , Colorimetry , Electroporation , Embryo, Mammalian , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Transgenic , Neurogenesis/physiology , Spinal Cord/embryology , Stem Cells/cytology , Time Factors , Transposases/physiology
19.
Curr Biol ; 23(1): 21-31, 2013 Jan 07.
Article in English | MEDLINE | ID: mdl-23177476

ABSTRACT

BACKGROUND: The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS: When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS: The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.


Subject(s)
Blastomeres/cytology , Embryonic Development , Animals , Blastomeres/physiology , Cell Lineage , Female , Luminescent Proteins/analysis , Male , Mice , Mice, Transgenic , Recombination, Genetic , Red Fluorescent Protein
20.
Nat Methods ; 9(8): 815-8, 2012 Jul 08.
Article in English | MEDLINE | ID: mdl-22772730

ABSTRACT

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.


Subject(s)
Color , Microscopy, Fluorescence, Multiphoton/methods , Photons , Animals , Cerebral Cortex/cytology , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Fluorescence , Lasers , Mice , Time Factors
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