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"Collins, Michelle M"
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Electron paramagnetic resonance spectroscopy for analysis of free radicals in zebrafish
Electron paramagnetic resonance (EPR) is an excellent choice for detecting free radicals in biological samples. Biologically relevant radicals are extremely short-lived and cannot be detected directly, emphasizing the need for an appropriate compound to generate stable adducts that can be measured by EPR. Spin trapping with nitrone compounds like 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is a method commonly employed for detecting free radicals. However, due to the instability of nitrone radical adducts, using the cell-permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl pyrrolidine (CMH) appears to be a more effective approach within biological tissues. Here, we compare the use of DMPO and CMH to detect the most abundant reactive oxygen species radical, superoxide ( O 2 ⋅ - ), in zebrafish and present an optimized protocol for performing EPR with a CMH spin probe in both zebrafish hearts and larvae. Together, our data suggest that EPR using the CMH probe is a reliable method to detect O 2 ⋅ - in zebrafish pathologies linked to oxidative stress, such as cardiovascular diseases.
Journal Article
HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development
Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of
VEGFC
,
FLT4
, and
PROX1
during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that
hhex
is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian
HHEX
using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.
VEGFC, its receptor FLT4, and transcriptional effector PROX1 control formation of the lymphatic system but how is unclear. Here, the authors show that the transcription factor hematopoietically expressed homeobox (HHEX) regulates VEGFC, FLT4 and PROX1 in fish and mammals during angiogenic sprouting and lymphatic formation.
Journal Article
Modeling Human Cardiac Arrhythmias: Insights from Zebrafish
Cardiac arrhythmia, or irregular heart rhythm, is associated with morbidity and mortality and is described as one of the most important future public health challenges. Therefore, developing new models of cardiac arrhythmia is critical for understanding disease mechanisms, determining genetic underpinnings, and developing new therapeutic strategies. In the last few decades, the zebrafish has emerged as an attractive model to reproduce in vivo human cardiac pathologies, including arrhythmias. Here, we highlight the contribution of zebrafish to the field and discuss the available cardiac arrhythmia models. Further, we outline techniques to assess potential heart rhythm defects in larval and adult zebrafish. As genetic tools in zebrafish continue to bloom, this model will be crucial for functional genomics studies and to develop personalized anti-arrhythmic therapies.
Journal Article
Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish
Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.
Journal Article
Two major accretion epochs in M31 from two distinct populations of globular clusters
Large galaxies grow through the accumulation of dwarf galaxies
1
,
2
. In principle it is possible to trace this growth history via the properties of a galaxy’s stellar halo
3
–
5
. Previous investigations of the galaxy Messier 31 (M31, Andromeda) have shown that outside a galactocentric radius of 25 kiloparsecs the population of halo globular clusters is rotating in alignment with the stellar disk
6
,
7
, as are more centrally located clusters
8
,
9
. The M31 halo also contains coherent stellar substructures, along with a smoothly distributed stellar component
10
–
12
. Many of the globular clusters outside a radius of 25 kiloparsecs are associated with the most prominent substructures, but some are part of the smooth halo
13
. Here we report an analysis of the kinematics of these globular clusters. We find two distinct populations rotating perpendicular to each other. The rotation axis for the population associated with the smooth halo is aligned with the rotation axis for the plane of dwarf galaxies
14
that encircles M31. We interpret these separate cluster populations as arising from two major accretion epochs, probably separated by billions of years. Stellar substructures from the first epoch are gone, but those from the more recent second epoch still remain.
There are two distinct kinematic populations of globular clusters in Messier 31 (M31, the Andromeda galaxy) with rotation axes perpendicular to each other, suggesting that they arose from merger events separated by billions of years.
Journal Article
Correction: Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish
[This corrects the article DOI: 10.1371/journal.pgen.1007743.].
Journal Article
Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling
Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.
Journal Article
Pitx2c orchestrates embryonic axis extension via mesendodermal cell migration
Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.
Journal Article
Early sarcomere and metabolic defects in a zebrafish pitx2c cardiac arrhythmia model
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebra-fish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c
+/− fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c
−/− larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.
Journal Article