Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) ...are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish,
, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications.
Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to ...induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation.
Background & Aims The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate ...host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.
Human brain organoids are a valuable research tool for studying brain development, physiology, and pathology. Yet, a host of potential ethical concerns are inherent in their creation. There is a ...growing group of bioethicists who acknowledge the moral imperative to develop brain organoid technologies and call for caution in this research. Although a relatively new technology, brain organoids and their uses are already being discussed in media literature. Media literature informs the public and policymakers but has the potential for utopian or dystopian distortions. Thus, it is important to understand how this technology is portrayed to the public.
To investigate how brain organoids are displayed to the public, we conducted a systematic review of media literature indexed in the Nexis Uni database from 2013-2019. News and media source articles passing exclusion criteria (n = 93) were scored to evaluate tone and relevant themes. Themes were validated with a pilot sample before being applied to the dataset. Thematic analysis assessed article tone, reported potential for the technology, and the scientific, social, and ethical contexts surrounding brain organoids research.
Brain organoid publications became more frequent from 2013 to 2019. We observed increases in positively and negatively toned articles, suggesting growing polarization. While many sources discuss realistic applications of brain organoids, others suggest treatment and cures beyond the scope of the current technology. This could work to overhype the technology and disillusion patients and families by offering false hope. In the ethical narrative we observe a preoccupation with issues such as development of artificial consciousness and "humanization" of organoid-animal chimeras. Issues of regulation, ownership, and accuracy of the organoid models are rarely discussed.
Given the power that media have to inform or misinform the public, it is important this literature provides an accurate and balanced reflection of the therapeutic potential and associated ethical issues regarding brain organoid research. Our study suggests increasing polarization, coupled with misplaced and unfounded ethical concern. Given the inhibitory effects of public fear or disillusion on research funding, it is important media literature provides an accurate reflection of brain organoids.
Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor Wang, Yu; Chandra, Rashmi; Samsa, Leigh Ann ...
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
04/2011, Letnik:
300, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder ...contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) (Ca(2+)(i)). The increase in Ca(2+)(i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.
The Neuregulin-1 (Nrg1) signaling pathway has been widely implicated in many aspects of heart development including cardiac trabeculation. Cardiac trabeculation is an important morphogenetic process ...where clusters of ventricular cardiomyocytes extrude and expand into the lumen of the ventricular chambers. In mouse, Nrg1 isoforms containing an immunoglobulin-like (IgG) domain are essential for cardiac trabeculation through interaction with heterodimers of the epidermal growth factor-like (EGF-like) receptors ErbB2/ErbB4. Recent reports have underscored the importance of Nrg1 signaling in cardiac homeostasis and disease, however, placental development has precluded refined evaluation of the role of this pathway in mammals. ErbB2 has been shown to have a developmentally conserved role in cardiac trabeculation in zebrafish, a vertebrate model organism with completely external development, but the requirement for Nrg1 has not been examined. We found that among the multiple Nrg1 isoforms, the IgG domain-containing, type I Nrg1 (nrg1-I) is the only isoform detectable in the heart. Then, using CRISPR/Cas9 gene editing, we targeted the IgG domain of Nrg1 to produce novel alleles, nrg1nc28 and nrg1nc29, encoding nrg1-I and nrg1-II truncations. Our results indicated that zebrafish deficient for nrg1-I developed trabeculae in an ErbB2-dependent manner. Further, these mutants survive to reproductive adulthood with no overt cardiovascular defects. We also found that additional EGF-like ligands were expressed in the zebrafish heart during development of trabeculae. Together, these results suggest that Nrg1 is not the primary effector of trabeculation and/or that other EGF-like ligand(s) activates the ErbB2/ErbB4 pathway, either through functioning as the primary ligand or acting in a redundant manner. Overall, our work provides an example of cross-species differences in EGF family member requirements for an evolutionary conserved process.
Collaborative group learning and peer teaching are robust active learning techniques. Students and instructors interact with technology extensively in their lives and in the classroom. Technology ...facilitates collaborative group learning by enabling synchronous interaction with digital documents and immediate access to information. Though it is widely accepted that group learning is an improvement to traditional lectures, challenges in the design, execution, and evaluation of group learning can be a barrier to implementing this pedagogy in the higher education classroom. Divide and Conquer is a simple, easy-to-use, and modern technique that faculty and instructors can use to rapidly transform traditional lecture content into collaborative small group learning and peer-teaching experiences. Students are divided into groups that complete instructor-prescribed activities on a shared Google Slide deck, and then teach the class what they learned. This technique can be used to explore a range of topics including science and non-science content and is particularly amenable to self-contained, related mini-research topics (i.e. the lowest level of organization on the outline of a lecture). This innovative technique was inspired primarily by the Jigsaw technique. However, it is distinct in that it deliberately builds technology skills and includes a class-level presentation. It is recommended for any higher education classroom across disciplines.
Vortex Dynamics in Trabeculated Embryonic Ventricles Battista, Nicholas A; Douglas, Dylan R; Lane, Andrea N ...
Journal of cardiovascular development and disease,
01/2019, Letnik:
6, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Proper heart morphogenesis requires a delicate balance between hemodynamic forces, myocardial activity, morphogen gradients, and epigenetic signaling, all of which are coupled with genetic regulatory ...networks. Recently both in vivo and in silico studies have tried to better understand hemodynamics at varying stages of veretebrate cardiogenesis. In particular, the intracardial hemodynamics during the onset of trabeculation is notably complex-the inertial and viscous fluid forces are approximately equal at this stage and small perturbations in morphology, scale, and steadiness of the flow can lead to significant changes in bulk flow structures, shear stress distributions, and chemical morphogen gradients. The immersed boundary method was used to numerically simulate fluid flow through simplified two-dimensional and stationary trabeculated ventricles of 72, 80, and 120 h post fertilization
zebrafish embryos and
-inhibited embryos at seven days post fertilization. A 2D idealized trabeculated ventricular model was also used to map the bifurcations in flow structure that occur as a result of the unsteadiness of flow, trabeculae height, and fluid scale ( R e ). Vortex formation occurred in intertrabecular regions for biologically relevant parameter spaces, wherein flow velocities increased. This indicates that trabecular morphology may alter intracardial flow patterns and hence ventricular shear stresses and morphogen gradients. A potential implication of this work is that the onset of vortical (disturbed) flows can upregulate Notch1 expression in endothelial cells in vivo and hence impacts chamber morphogenesis, valvulogenesis, and the formation of the trabeculae themselves. Our results also highlight the sensitivity of cardiac flow patterns to changes in morphology and blood rheology, motivating efforts to obtain spatially and temporally resolved chamber geometries and kinematics as well as the careful measurement of the embryonic blood rheology. The results also suggest that there may be significant changes in shear signalling due to morphological and mechanical variation across individuals and species.
Cholecystokinin (CCK) is secreted by neuroendocrine cells comprising 0.1%-0.5% of the mucosal cells in the upper small intestine. Using CCK promoter-driven green fluorescent protein (GFP) expression ...in transgenic mice, we have applied immunofluorescence techniques to analyze the morphology of CCK cells. GFP and CCK colocalize in neuroendocrine cells with little aberrant GFP expression. CCK-containing cells are either flask- or spindle-shaped, and in some cells, we have found dendritic processes similar to pseudopods demonstrated for gut somatostatin-containing D cells. Most pseudopods are short, the longest process visualized extending across three cells. Pseudopods usually extend to adjacent cells but some weave between neighboring cells. Dual processes have also been observed. Three-dimensional reconstructions suggest that processes are not unidirectional and thus are unlikely to be involved in migration of CCK cells from the crypt up the villus. Abundant CCK immunostaining is present in the pseudopods, suggesting that they release CCK onto the target cell. In order to identify the type of cells being targeted, we have co-stained sections with antibodies to chromogranin A, trefoil factor-3, and sucrase-isomaltase. CCK cell processes almost exclusively extend to sucrase-isomaltase-positive enterocytes. Thus, CCK cells have cellular processes possibly involved in paracrine secretion.
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