Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter

AbstractGenetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

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Red Light/Green Light, a Dual Fluorescent Protein Reporter System To Study Enhancer-Promoter Specificity in Drosophila

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.401033 ◽

2020 ◽

Vol 10 (3) ◽

pp. 985-997

Author(s):

Eric M. Camino ◽

Micheal L. Weinstein ◽

Mary P. List ◽

Jordan E. Vellky ◽

Mark Rebeiz ◽

...

Keyword(s):

Fluorescent Protein ◽

Red Light ◽

Control Element ◽

Gene Promoters ◽

Spatial And Temporal Patterns ◽

Reporter System ◽

Fluorescent Reporter ◽

Promoter Specificity ◽

Reporter Transgene

Enhancers activate gene transcription in spatial and temporal patterns by interactions with gene promoters. These elements typically reside distal to their target promoter, with which they must interact selectively. Additional elements may contribute to enhancer-promoter specificity, including remote control element sequences within enhancers, tethering elements near promoters, and insulator/boundary elements that disrupt off-target interactions. However, few of these elements have been mapped, and as a result, the mechanisms by which these elements interact remain poorly understood. One impediment is their method of study, namely reporter transgenes in which enhancers are placed adjacent to a heterologous promoter, which may circumvent mechanisms controlling enhancer-promoter specificity and long-range interactions. Here, we report an optimized dual reporter transgene system in Drosophila melanogaster that allows the simultaneous comparison of an enhancer’s ability to activate proximal and distal fluorescent reporter genes. Testing a panel of fluorescent transgenes in vivo, we found a two-protein combination that allows simultaneous measurement with minimal detection interference. We note differences among four tested enhancers in their ability to regulate a distally placed reporter transgene. These results suggest that enhancers differ in their requirements for promoter interaction and raise important practical considerations when studying enhancer function.

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In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽

10.1530/rep-13-0570 ◽

2014 ◽

Vol 147 (6) ◽

pp. 781-788 ◽

Cited By ~ 13

Author(s):

Franziska Schmerse ◽

Katja Woidacki ◽

Monika Riek-Burchardt ◽

Peter Reichardt ◽

Axel Roers ◽

...

Keyword(s):

Mast Cells ◽

Intravital Microscopy ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Specific Cell ◽

Enhanced Yellow Fluorescent Protein ◽

Two Photon Microscopy ◽

Two Photon ◽

Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

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In vivo imaging of liver injury and regeneration by functional two-photon microscopy

Zeitschrift für Gastroenterologie ◽

10.1055/s-0036-1597342 ◽

2016 ◽

Vol 54 (12) ◽

pp. 1343-1404

Author(s):

A Ghallab ◽

R Reif ◽

R Hassan ◽

AS Seddek ◽

JG Hengstler

Keyword(s):

Liver Injury ◽

In Vivo Imaging ◽

Two Photon Microscopy ◽

Two Photon

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Sensory Transmission is Bi-directionally Modulated by Astrocytic Ca2+ in Barrel Cortex of Behaving Mice

10.21203/rs.3.rs-199750/v1 ◽

2021 ◽

Author(s):

Simeng Gu ◽

Wei Wang ◽

Kuan Zhang ◽

Rou Feng ◽

Naling Li ◽

...

Keyword(s):

Sensory Input ◽

Barrel Cortex ◽

Synaptic Function ◽

Metabolic Clearance ◽

Sleep State ◽

Two Photon Microscopy ◽

Two Photon ◽

Sensory Transmission

Abstract Different effects of astrocyte during sleep and awake have been extensively studied, especially for metabolic clearance by the glymphatic system, which works during sleep and stops working during waking states. However, how astrocytes contribute to modulation of sensory transmission during sleep and awake animals remain largely unknown. Recent advances in genetically encoded Ca2+ indicators have provided a wealth of information on astrocytic Ca2+, especially in their fine perisynaptic processes, where astrocytic Ca2+ most likely affects the synaptic function. Here we use two-photon microscopy to image astrocytic Ca2+ signaling in freely moving mice trained to run on a wheel in combination with in vivo whole-cell recordings to evaluate the role of astrocytic Ca2+ signaling in different behavior states. We found that there are two kinds of astrocytic Ca2+ signaling: a small long-lasting Ca2+ increase during sleep state and a sharp widespread but short-long-lasting Ca2+ spike when the animal was awake (fluorescence increases were 23.2 ± 14.4% for whisker stimulation at sleep state, compared with 73.3 ± 11.7% for at awake state, paired t-test, p < 0.01). The small Ca2+ transients decreased extracellular K+, hyperpolarized the neurons, and suppressed sensory transmission; while the large Ca2+ wave enhanced sensory input, contributing to reliable sensory transmission in aroused states. Locus coeruleus activation works as a switch between these two kinds of astrocytic Ca2+ elevation. Thus, we show that cortical astrocytes play an important role in processing of sensory input. These two types of events appear to have different pharmacological sources and may play a different role in facilitating the efficacy of sensory transmission.

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Quantum Dots assisted and NIR-II Emissive In Vivo Two-photon microscopy

Photonics Research ◽

10.1364/prj.441471 ◽

2021 ◽

Author(s):

Huwei Ni ◽

Yalun Wang ◽

Tao Tang ◽

Wenbin Yu ◽

Dongyu Li ◽

...

Keyword(s):

Quantum Dots ◽

Two Photon Microscopy ◽

Two Photon

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Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2013.201 ◽

2013 ◽

Vol 34 (2) ◽

pp. 325-331 ◽

Cited By ~ 19

Author(s):

Kazuto Masamoto ◽

Hiroyuki Takuwa ◽

Chie Seki ◽

Junko Taniguchi ◽

Yoshiaki Itoh ◽

...

Keyword(s):

Adult Mouse ◽

Fluorescent Protein ◽

Vascular Endothelial Cells ◽

Three Dimensional ◽

Vascular Endothelial ◽

Two Photon Microscopy ◽

Two Photon ◽

Mouse Cortex ◽

Hypoxia Adaptation ◽

Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14 μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60 μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

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