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1.
Int J Mol Sci ; 25(5)2024 Feb 28.
Article in English | MEDLINE | ID: mdl-38474036

ABSTRACT

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.


Subject(s)
Rhabdomyosarcoma, Alveolar , Rhabdomyosarcoma , Humans , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Alveolar/metabolism , Rhabdomyosarcoma, Alveolar/pathology , Transforming Growth Factor beta , Transforming Growth Factor beta1 , Paired Box Transcription Factors/genetics , Epithelial-Mesenchymal Transition , Rhabdomyosarcoma/genetics , Oncogene Proteins, Fusion/genetics
2.
Front Cell Dev Biol ; 10: 941893, 2022.
Article in English | MEDLINE | ID: mdl-35846369

ABSTRACT

Adult neural stem and progenitor cells (aNSPCs) persist lifelong in teleost models in diverse stem cell niches of the brain and spinal cord. Fish maintain developmental stem cell populations throughout life, including both neuro-epithelial cells (NECs) and radial-glial cells (RGCs). Within stem cell domains of the brain, RGCs persist in a cycling or quiescent state, whereas NECs continuously divide. Heterogeneous populations of RGCs also sit adjacent the central canal of the spinal cord, showing infrequent proliferative activity under homeostasis. With the rise of the zebrafish (Danio rerio) model to study adult neurogenesis and neuroregeneration in the central nervous system (CNS), it has become evident that aNSPC proliferation is regulated by a wealth of stimuli that may be coupled with biological function. Growing evidence suggests that aNSPCs are sensitive to environmental cues, social interactions, nutrient availability, and neurotrauma for example, and that distinct stem and progenitor cell populations alter their cell cycle activity accordingly. Such stimuli appear to act as triggers to either turn on normally dormant aNSPCs or modulate constitutive rates of niche-specific cell cycle behaviour. Defining the various forms of stimuli that influence RGC and NEC proliferation, and identifying the molecular regulators responsible, will strengthen our understanding of the connection between aNSPC activity and their biological significance. In this review, we aim to bring together the current state of knowledge on aNSPCs from studies investigating the zebrafish CNS, while highlighting emerging cell cycle regulators and outstanding questions that will help to advance this fascinating field of stem cell biology.

3.
Sci Rep ; 11(1): 899, 2021 01 13.
Article in English | MEDLINE | ID: mdl-33441723

ABSTRACT

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.


Subject(s)
Microscopy, Electron/methods , Microscopy, Immunoelectron/methods , Stem Cells/classification , Animals , Cell Membrane , Humans , Microscopy, Electron, Transmission/methods , Microscopy, Fluorescence/methods , Radionuclide Imaging , Stem Cells/immunology , Stem Cells/metabolism , Workflow
4.
Sci Rep ; 9(1): 4420, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30872640

ABSTRACT

How diverse adult stem and progenitor populations regenerate tissue following damage to the brain is poorly understood. In highly regenerative vertebrates, such as zebrafish, radial-glia (RG) and neuro-epithelial-like (NE) stem/progenitor cells contribute to neuronal repair after injury. However, not all RG act as neural stem/progenitor cells during homeostasis in the zebrafish brain, questioning the role of quiescent RG (qRG) post-injury. To understand the function of qRG during regeneration, we performed a stab lesion in the adult midbrain tectum to target a population of homeostatic qRG, and investigated their proliferative behaviour, differentiation potential, and Wnt/ß-catenin signalling. EdU-labelling showed a small number of proliferating qRG after injury (pRG) but that progeny are restricted to RG. However, injury promoted proliferation of NE progenitors in the internal tectal marginal zone (TMZi) resulting in amplified regenerative neurogenesis. Increased Wnt/ß-catenin signalling was detected in TMZi after injury whereas homeostatic levels of Wnt/ß-catenin signalling persisted in qRG/pRG. Attenuation of Wnt signalling suggested that the proliferative response post-injury was Wnt/ß-catenin-independent. Our results demonstrate that qRG in the tectum have restricted capability in neuronal repair, highlighting that RG have diverse functions in the zebrafish brain. Furthermore, these findings suggest that endogenous stem cell compartments compensate lost tissue by amplifying homeostatic growth.


Subject(s)
Neural Stem Cells/cytology , Neurogenesis , Neuroglia/cytology , Neurons/cytology , Superior Colliculi/cytology , Tectum Mesencephali/cytology , Animals , Animals, Genetically Modified , Cell Proliferation , Neural Stem Cells/physiology , Neuroglia/physiology , Neurons/physiology , Superior Colliculi/physiology , Tectum Mesencephali/physiology , Wnt Signaling Pathway , Zebrafish , Zebrafish Proteins/metabolism
5.
Prog Neurobiol ; 170: 99-114, 2018 11.
Article in English | MEDLINE | ID: mdl-29902500

ABSTRACT

Neural stem and progenitor cells (NSPCs) are the primary source of new neurons in the brain and serve critical roles in tissue homeostasis and plasticity throughout life. Within the vertebrate brain, NSPCs are located within distinct neurogenic niches differing in their location, cellular composition, and proliferative behaviour. Heterogeneity in the NSPC population is hypothesized to reflect varying capacities for neurogenesis, plasticity and repair between different neurogenic zones. Since the discovery of adult neurogenesis, studies have predominantly focused on the behaviour and biological significance of adult NSPCs (aNSPCs) in rodents. However, compared to rodents, who show lifelong neurogenesis in only two restricted neurogenic niches, zebrafish exhibit constitutive neurogenesis across multiple stem cell niches that provide new neurons to every major brain division. Accordingly, zebrafish are a powerful model to probe the unique cellular and molecular profiles of NSPCs and investigate how these profiles govern tissue homeostasis and regenerative plasticity within distinct stem cell populations over time. Amongst the NSPC populations residing in the zebrafish central nervous system (CNS), proliferating radial-glia, quiescent radial-glia and neuro-epithelial-like cells comprise the majority. Here, we provide insight into the extent to which these distinct NSPC populations function and mature during development, respond to experience, and contribute to successful CNS regeneration in teleost fish. Together, our review brings to light the dynamic biological roles of these individual NSPC populations and showcases their diverse regenerative modes to achieve vertebrate brain repair later in life.


Subject(s)
Ependymoglial Cells/physiology , Epithelial Cells/physiology , Nerve Regeneration/physiology , Neural Stem Cells/physiology , Neuronal Plasticity/physiology , Neurons/physiology , Animals , Central Nervous System/growth & development , Central Nervous System/physiology , Zebrafish
6.
Nat Neurosci ; 20(6): 774-783, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28459441

ABSTRACT

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.


Subject(s)
Endothelial Cells/physiology , Meninges/blood supply , Neovascularization, Physiologic/physiology , Vascular Endothelial Growth Factors/physiology , Zebrafish Proteins/physiology , Zebrafish , Animals , Animals, Genetically Modified , Brain/blood supply , Brain/metabolism , Brain/physiology , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Female , Lipoproteins, LDL/metabolism , Male , Meninges/growth & development , Meninges/metabolism , Meninges/physiology , Signal Transduction/physiology , Vascular Endothelial Growth Factors/biosynthesis , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
7.
Zebrafish ; 14(6): 574-577, 2017 12.
Article in English | MEDLINE | ID: mdl-28296621

ABSTRACT

How distinct cell populations are distributed in three-dimensional space under homeostasis or following injury, neurodegeneration, or with senescence can teach us much about brain-wide patterns and signaling along the neuroaxis. Visualizing individual cell populations in the mature vertebrate central nervous system (CNS) has remained a challenge as a result of difficulty clearing adult brain tissue or limitations in imaging depth or resolution. We have developed a simple clearing and imaging pipeline optimally suited for the adult zebrafish brain to investigate changes in patterns of cell proliferation in wild-type and transgenic backgrounds that can easily be quantified and represented using FIJI and IMARIS software.


Subject(s)
Brain/anatomy & histology , Cell Proliferation , Image Processing, Computer-Assisted/methods , Tomography, Optical/veterinary , Zebrafish/anatomy & histology , Animals , Brain/growth & development , Software , Tomography, Optical/methods , Zebrafish/growth & development
8.
Front Neurosci ; 11: 750, 2017.
Article in English | MEDLINE | ID: mdl-29386991

ABSTRACT

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2'-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.

9.
Eur J Neurosci ; 40(11): 3591-607, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25231569

ABSTRACT

Teleost fishes retain populations of adult stem/progenitor cells within multiple primary sensory processing structures of the mature brain. Though it has commonly been thought that their ability to give rise to adult-born neurons is mainly associated with continuous growth throughout life, whether a relationship exists between the processing function of these structures and the addition of new neurons remains unexplored. We investigated the ultrastructural organisation and modality-specific neurogenic plasticity of niches located in chemosensory (olfactory bulb, vagal lobe) and visual processing (periventricular grey zone, torus longitudinalis) structures of the adult zebrafish (Danio rerio) brain. Transmission electron microscopy showed that the cytoarchitecture of sensory niches includes many of the same cellular morphologies described in forebrain niches. We demonstrate that cells with a radial-glial phenotype are present in chemosensory niches, while the niche of the caudal tectum contains putative neuroepithelial-like cells instead. This was supported by immunohistochemical evidence showing an absence of glial markers, including glial fibrillary acidic protein, glutamine synthetase, and S100ß in the tectum. By exposing animals to sensory assays we further illustrate that stem/progenitor cells and their neuronal progeny within sensory structures respond to modality-specific stimulation at distinct stages in the process of adult neurogenesis - chemosensory niches at the level of neuronal survival and visual niches in the size of the stem/progenitor population. Our data suggest that the adult brain has the capacity for sensory-specific modulation of adult neurogenesis and that this property may be associated with the type of stem cell present in the niche.


Subject(s)
Adult Stem Cells/physiology , Neurogenesis/physiology , Prosencephalon/physiology , Stem Cell Niche/physiology , Zebrafish/physiology , Adult Stem Cells/ultrastructure , Animals , Bromodeoxyuridine , Cell Count , Cell Survival/physiology , Female , Immunohistochemistry , Male , Microscopy, Electron, Transmission , Neural Stem Cells/physiology , Neural Stem Cells/ultrastructure , Neuroglia/physiology , Neuroglia/ultrastructure , Neurons/physiology , Neurons/ultrastructure , Olfactory Perception/physiology , Prosencephalon/ultrastructure , Taste Perception/physiology , Visual Perception/physiology
10.
Dev Neurobiol ; 74(11): 1053-77, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24753454

ABSTRACT

The social environment is known to modulate adult neurogenesis. Studies in mammals and birds have shown a strong correlation between social isolation and decreases in neurogenesis, whereas time spent in an enriched environment has been shown to restore these deficits and enhance neurogenesis. These data suggest that there exists a common adaptive response among neurogenic niches to each extreme of the social environment. We sought to further test this hypothesis in zebrafish, a social species with distinct neurogenic niches within primary sensory structures and telencephalic nuclei of the brain. By examining stages of adult neurogenesis, including the proliferating stem/progenitor population, their surviving cohort, and the resulting newly differentiated neuronal population, we show that niches residing in sensory structures are most sensitive to changes in the social context, and that social isolation or novelty are both capable of decreasing the number of proliferating cells while increasing the number of newborn neurons within a single niche. Contrary to observations in rodents, we demonstrate that social novelty, a form of enrichment, does not consistently rescue deficits in cell proliferation following social isolation, and that cortisol levels do not negatively regulate changes in adult neurogenesis, but are correlated with the social context. We propose that enhancement or suppression of adult neurogenesis in the zebrafish brain under different social contexts depends largely on the type of niche (sensory or telencephalic), experience from the preceding social environment, and occurs independently of changes in cortisol levels.


Subject(s)
Brain/cytology , Brain/physiology , Neurogenesis/physiology , Neurons/physiology , Social Environment , Stem Cell Niche/physiology , Animals , Brain/drug effects , Bromodeoxyuridine/metabolism , Cell Differentiation , Cell Proliferation , Exploratory Behavior , Female , Hydrocortisone/administration & dosage , Hydrocortisone/metabolism , Male , Neurogenesis/drug effects , Social Isolation , Stem Cell Niche/drug effects , Zebrafish
11.
J Comp Neurol ; 520(10): 2275-316, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22318736

ABSTRACT

A central goal of adult neurogenesis research is to characterize the cellular constituents of a neurogenic niche and to understand how these cells regulate the production of new neurons. Because the generation of adult-born neurons may be tightly coupled to their functional requirement, the organization and output of neurogenic niches may vary across different regions of the brain or between species. We have undertaken a comparative study of six (D, Vd, Vv, Dm, Dl, Ppa) periventricular zones (PVZs) harboring proliferative cells present in the adult forebrain of the zebrafish (Danio rerio), a species known to possess widespread neurogenesis throughout life. Using electron microscopy, we have documented for the first time the detailed cytoarchitecture of these zones, and propose a model of the cellular composition of pallial and subpallial PVZs, as well as a classification scheme for identifying morphologically distinct cell types. Immunolabeling of resin-embedded tissue confirmed the phenotype of three constitutively proliferating (bromodeoxyuridine [BrdU]+) cell populations, including a radial glial-like (type IIa) cell immunopositive for both S100ß and glutamine synthetase (GS). Our data revealed rostrocaudal differences in the density of distinct proliferative populations, and cumulative labeling studies suggested that the cell cycle kinetics of these populations are not uniform between PVZs. Although the peak numbers of differentiated neurons were generated after ~2 weeks among most PVZs, niche-specific decline in the number of newborn neurons in some regions occurred after 4 weeks. Our data suggest that the cytoarchitecture of neurogenic niches and the tempo of neuronal production are regionally distinct in the adult zebrafish forebrain.


Subject(s)
Adult Stem Cells/physiology , Cell Differentiation , Cerebral Ventricles/cytology , Neurogenesis/physiology , Prosencephalon/anatomy & histology , Adult Stem Cells/ultrastructure , Analysis of Variance , Animals , Bromodeoxyuridine/metabolism , Cell Count , Cell Cycle , Cell Movement/physiology , Cell Proliferation , ELAV Proteins/metabolism , Eye Proteins/metabolism , Glutamate-Ammonia Ligase/metabolism , Homeodomain Proteins/metabolism , Microscopy, Electron , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecule L1/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Sialic Acids/metabolism , Zebrafish/anatomy & histology
12.
J Exp Zool A Ecol Genet Physiol ; 315(5): 302-13, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21394929

ABSTRACT

The gas-filled swimbladder of teleost fishes provides hydrodynamic lift which counteracts the high density of other body tissues, and thereby allows the fish to achieve neutral buoyancy with minimal energy expenditure. In this study, we examined whether the absence of a constant direction gravitational vector affects the ontogeny of the swimbladder and buoyancy control in zebrafish (Danio rerio). We exposed fertilized eggs to simulated microgravity (SMG) in a closed rotating wall vessel with control eggs placed in a similar but nonrotating container. All eggs hatched in both groups. At 96 hr of postfertilization (hpf), all larvae were removed from the experimental and control vessels. At this point, 62% of the control larvae, but only 14% of SMG-exposed larvae, were observed to have inflated their swimbladder. In addition, the mean volume of the inflated swimbladders was significantly greater in the control larvae compared with larvae raised in SMG. After transfer to open stationary observation tanks, larvae with uninflated swimbladders in both groups swam to the surface to complete inflation, but this process was significantly delayed in larvae exposed to SMG. Initial differences in swimbladder inflation and volume between groups disappeared by 144 hpf. Furthermore, there were no apparent changes in patterns of development and maturation of swimbladder musculature, vasculature, or innervation resulting from SMG exposure at later stages of ontogeny. These data indicate that, despite a transient delay in swimbladder inflation in zebrafish larvae exposed to SMG, subsequent swimbladder development in these animals proceeded similarly to that in normal larvae.


Subject(s)
Air Sacs/growth & development , Swimming/physiology , Weightlessness , Zebrafish/growth & development , Animals , Body Weights and Measures , Larva/physiology , Statistics, Nonparametric , Zygote/physiology
13.
Zebrafish ; 7(1): 85-96, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20415646

ABSTRACT

Teleost fishes have body tissues that are denser than water, causing them to sink. Many teleosts therefore possess a gas-filled swimbladder that provides lift, allowing fish to attain neutral buoyancy. The importance of the swimbladder as a buoyancy aid during changing body sizes over ontogeny and its role in determining the swimming depth of fish remain unclear. In this study, we have used the zebrafish (Danio rerio) to investigate changes in the size and shape of the swimbladder during development and examine whether these changes affect the hydrostatic contribution of the swimbladder during swimming. Our results showed that swim-up behavior is critical for larvae to first inflate their swimbladder, decrease body density, and attain neutral buoyancy. Following inflation, we found a strong linear correlation between fish volume and swimbladder volume over ontogeny. This trend was supported by measures of the density of zebrafish, which was conserved within a narrow range between 1.00 +/- 0.001 and 0.996 +/- 0.001 g/cm(3) despite an increase in the swimming depth of zebrafish, which occurred upon transition to a double-chambered organ. Finally, we demonstrated that the contribution of the swimbladder keeps the fish within 1.7% of neutral buoyancy throughout larval development.


Subject(s)
Air Sacs/physiology , Swimming , Zebrafish/physiology , Animals , Behavior, Animal , Female , Larva/physiology , Male , Organ Size
14.
J Morphol ; 269(6): 666-73, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18302241

ABSTRACT

Many teleost fishes use a swimbladder, a gas-filled organ in the coelomic cavity, to reduce body density toward neutral buoyancy, thus minimizing the locomotory cost of maintaining a constant depth in the water column. However, for most swimbladder-bearing teleosts, the contribution of this organ to the attainment of neutral buoyancy has not been quantified. Here, we examined the quantitative contribution of the swimbladder to buoyancy and three-dimensional stability in a small cyprinid, the zebrafish (Danio rerio). In aquaria during daylight hours, adult animals were observed at mean depths from 10.1 +/- 6.0 to 14.2 +/- 5.6 cm below the surface. Fish mass and whole-body volume were linearly correlated (r(2) = 0.96) over a wide range of body size (0.16-0.73 g); mean whole-body density was 1.01 +/- 0.09 g cm(-3). Stereological estimations of swimbladder volume from linear dimensions of lateral X-ray images and direct measurements of gas volumes recovered by puncture from the same swimbladders showed that results from these two methods were highly correlated (r(2) = 0.85). The geometric regularity of the swimbladder thus permitted its volume to be accurately estimated from a single lateral image. Mean body density in the absence of the swimbladder was 1.05 +/- 0.04 g cm(-3). The swimbladder occupied 5.1 +/- 1.4% of total body volume, thus reducing whole-body density significantly. The location of the centers of mass and buoyancy along rostro-caudal and dorso-ventral axes overlapped near the ductus communicans, a constriction between the anterior and posterior swimbladder chambers. Our work demonstrates that the swimbladder of the adult zebrafish contributes significantly to buoyancy and attitude stability. Furthermore, we describe and verify a stereological method for estimating swimbladder volume that will aid future studies of the functions of this organ.


Subject(s)
Air Sacs/anatomy & histology , Swimming , Zebrafish/anatomy & histology , Animals , Body Weights and Measures , Female , Male , Pressure , Zebrafish/physiology
15.
Stem Cells ; 25(7): 1697-706, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17395777

ABSTRACT

Directing embryonic stem (ES) cells to differentiate into functional motoneurons has proven to be a strong technique for studying neuronal development as well as being a potential source of tissue for cell replacement therapies involving spinal cord disorders. Unfortunately, one of the mitogenic factors (i.e., sonic hedgehog agonist) used for directed differentiation is not readily available, and thus this technique has not been widely accessible. Here, we present a novel and simple method to derive motoneurons from ES cells using readily attainable reagents. ES cells were derived from a mouse in which enhanced green fluorescent protein (eGFP) was linked to a motoneuron specific promoter. The cells were plated onto a monolayer of 293 EcR-Shh cells that carry an integrated construct for the expression of sonic hedgehog (Shh) under ecdysone-inducible control. To initiate motoneuron differentiation, 293 EcR-Shh:ES cell cocultures were treated with ponasterone A (PA) and retinoic acid for 5 days. PA induces ecdysone, and thus drives Shh expression. To assess differentiation, putative ES cell-derived motoneurons were studied immunocytochemically and cultured on chick myotubes for functional analysis. We found that ES cells differentiated into eGFP+ cells that expressed transcription factors typical of motoneurons. Furthermore, ES cell-derived motoneurons were capable of forming functional connections with muscle fibers in vitro. Finally, when transplanted into the developing chick spinal cord, ES cell-derived motoneurons migrated to the ventral horn and projected axons to appropriate muscle targets. In summary, this simple treatment paradigm produces functional motoneurons that can be used for both developmental and preclinical studies. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Hedgehog Proteins/metabolism , Motor Neurons/cytology , Animals , Axons/drug effects , Axons/metabolism , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Separation , Cells, Cultured , Chickens , Coculture Techniques , Embryonic Stem Cells/drug effects , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Humans , LIM-Homeodomain Proteins , Mice , Motor Neurons/drug effects , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Receptors, Cholinergic/metabolism , Receptors, Steroid/metabolism , Stem Cell Transplantation , Transcription Factors , Tretinoin/pharmacology
16.
Prog Neurobiol ; 80(6): 281-307, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17218052

ABSTRACT

Adult neurogenesis has been identified in all vertebrate species examined thus far. However, an evolutionary trend towards a reduction in both the number of proliferation zones and the overall number of newborn cells has been revealed in more recent lineages of vertebrates, such as mammals. Adult neurogenesis, and in particular the characterization of adult neural stem cells in mammals has been the focus of intense research with the goal of developing new cell-based regenerative treatments for neurodegenerative diseases, spinal cord injury, and acute damage due to stroke. Conversely, most other vertebrate classes, which display widespread production of adult neurons, are not typically used as model systems in this context. A more profound understanding of the structural composition and the mechanisms that support proliferation zones in the mature brain have become critical for revealing how adult neural stem cells are maintained in these regions and how they regulate neurogenesis. In this review we argue that comprehensive analyses of adult neurogenesis in various vertebrate and invertebrate species will lead to a more complete understanding of the fundamental biology and evolution of adult neurogenesis and provide a better framework for testing hypotheses regarding the functional significance of this trait.


Subject(s)
Adult Stem Cells/cytology , Adult Stem Cells/physiology , Models, Neurological , Nerve Regeneration/physiology , Neurons/cytology , Neurons/physiology , Aging/pathology , Aging/physiology , Animals , Biological Evolution , Cell Differentiation , Cell Proliferation , Humans , Species Specificity
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