Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of ...axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.
Display omitted
•Axonal growth trajectories are controlled with light by optogenetic Rac1 activation•Endogenous axon guidance programs are overridden to create novel circuitry in vivo•Defective axonal architecture is repaired, restoring long-range axonal connectivity•Functional synaptic connections are created following optogenetic axon guidance
Axons define the long-range connectivity of the nervous system. Using an optogenetic approach, Harris et al. direct axonal growth with light to engineer axonal architectures precisely and non-invasively. Axons can be guided across repulsive developmental barriers to create novel circuitry, and functional connectivity can be restored to defective circuits.
The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid ...and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.
Cellular barcoding to decipher clonal dynamics in disease Sankaran, Vijay G; Weissman, Jonathan S; Zon, Leonard I
Science (American Association for the Advancement of Science),
10/2022, Letnik:
378, Številka:
6616
Journal Article
Recenzirano
Odprti dostop
Cellular barcodes are distinct DNA sequences that enable one to track specific cells across time or space. Recent advances in our ability to detect natural or synthetic cellular barcodes, paired with ...single-cell readouts of cell state, have markedly increased our knowledge of clonal dynamics and genealogies of the cells that compose a variety of tissues and organs. These advances hold promise to redefine our view of human disease. Here, we provide an overview of cellular barcoding approaches, discuss applications to gain new insights into disease mechanisms, and provide an outlook on future applications. We discuss unanticipated insights gained through barcoding in studies of cancer and blood cell production and describe how barcoding can be applied to a growing array of medical fields, particularly with the increasing recognition of clonal contributions in human diseases.
The zebrafish is an excellent genetic and developmental model system used to study biology and disease. While the zebrafish model is associated with high fecundity, its reproductive potential has not ...been completely realized by scientists. One major issue is that embryo collection is inefficient. Here, we have developed an innovative breeding vessel designed to stimulate the natural reproductive behavior of the fish. This novel apparatus allows us to collect large numbers of developmentally synchronized embryos in brief and defined windows of time, and with minimal investments in labor and space. To demonstrate the efficacy of this approach, we placed three separate groups (n = 180) of fish in the vessel and allowed them to spawn for 10-minute intervals. During these trials, which were repeated three times, the fish produced 8600±917, 8400±794, and 6800±1997 embryos, respectively. This level of embryo production is nearly twice what we were able to achieve when using conventional crossing equipment with some of the same fish, and it required significantly less room and time to set up and break down. This system overcomes major space and labor restrictions inherent in spawning equipment currently used in the field, and will greatly accelerate efforts to improve the scale and throughput of experiments.
Since its introduction in early 1980s, the zebrafish (Danio rerio) has become an invaluable vertebrate animal model system to study many human disorders in almost all systems, from hepatic and brain ...pathology, to autoimmune and psychiatric disorders. Hematopoiesis between zebrafish and mammals is highly conserved, making the zebrafish an attractive model to study hematopoietic development and blood disorders. Unique attributes of the zebrafish include the ability to perform large-scale genetic and chemical screens in vivo, study development at the cellular level, and use transgenic fish to dissect mechanisms of disease or drug effects. This review summarizes major discoveries that helped define molecular control of hematopoiesis in vertebrates and specific contributions from studies in zebrafish.
Cell engineering has brought us tantalizingly close to the goal of deriving patient-specific hematopoietic stem cells (HSCs). While directed differentiation and transcription factor-mediated ...conversion strategies have generated progenitor cells with multilineage potential, to date, therapy-grade engineered HSCs remain elusive due to insufficient long-term self-renewal and inadequate differentiated progeny functionality. A cross-species approach involving zebrafish and mammalian systems offers complementary methodologies to improve understanding of native HSCs. Here, we discuss the role of conserved developmental timing processes in vertebrate hematopoiesis, highlighting how identification and manipulation of stage-specific factors that specify HSC developmental state must be harnessed to engineer HSCs for therapy.
Rowe et al. consider the current strategies aimed at engineering hematopoietic stem cells in vitro. They discuss the role of conserved developmental timing processes in vertebrate hematopoiesis and highlight how identification and manipulation of stage-specific factors that specify HSC developmental state may be harnessed to engineer HSCs for therapy.
Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt ...signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of β-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.
Zebrafish studies in the past two decades have made major contributions to our understanding of hematopoiesis and its associated disorders. The zebrafish has proven to be a powerful organism for ...studies in this area owing to its amenability to large-scale genetic and chemical screening. In addition, the externally fertilized and transparent embryos allow convenient genetic manipulation and in vivo imaging of normal and aberrant hematopoiesis. This review discusses available methods for studying hematopoiesis in zebrafish, summarizes key recent advances in this area, and highlights the current and potential contributions of zebrafish to the discovery and development of drugs to treat human blood disorders.
In vivo drug discovery in the zebrafish Peterson, Randall T; Zon, Leonard I
Nature reviews. Drug discovery,
200501, 2005-Jan, 2005-1-00, 20050101, Letnik:
4, Številka:
1
Journal Article
Recenzirano
Odprti dostop
The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which ...large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology.
PDF
