RESUMO
In this Spotlight, we hear first-hand accounts from five scientists and educators who use microscopy and imaging to engage, entertain, educate and inspire new audiences with science and the field of developmental biology in particular. The 'voices' that follow each convey each authors' own personal take on why microscopy is such a powerful tool for capturing the minds, and the hearts, of scientists, students and the public alike. They discuss how microscopy and imaging can reveal new worlds, and improve our communication and understanding of developmental biology, as well as break down barriers and promote diversity for future generations of scientific researchers.
Assuntos
Microscopia , Animais , Humanos , Retratos como AssuntoRESUMO
In the mature vertebrate nervous system, central and peripheral nervous system (CNS and PNS, respectively) GLIA myelinate distinct motor axon domains at the motor exit point transition zone (MEP TZ). How these cells preferentially associate with and myelinate discrete, non-overlapping CNS versus PNS axonal segments, is unknown. Using in vivo imaging and genetic cell ablation in zebrafish, we demonstrate that radial glia restrict migration of PNS glia into the spinal cord during development. Prior to development of radial glial endfeet, peripheral cells freely migrate back and forth across the MEP TZ. However, upon maturation, peripherally located cells never enter the CNS. When we ablate radial glia, peripheral glia ectopically migrate into the spinal cord during developmental stages when they would normally be restricted. These findings demonstrate that radial glia contribute to both CNS and PNS development and control the unidirectional movement of glial cell types across the MEP TZ early in development. GLIA 2016. GLIA 2016;64:1138-1153.
Assuntos
Movimento Celular/fisiologia , Sistema Nervoso/citologia , Neuroglia/fisiologia , Medula Espinal/citologia , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/embriologia , Neuroglia/ultraestrutura , Medula Espinal/embriologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Neurogênese/fisiologia , Neurônios/fisiologia , Medula Espinal/citologia , Fatores Etários , Animais , Animais Geneticamente Modificados , Apoptose/genética , Diferenciação Celular , Proliferação de Células/genética , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Locomoção/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Medula Espinal/embriologia , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína Vermelha FluorescenteRESUMO
Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.
Assuntos
Células Ependimogliais/metabolismo , Cinesinas/metabolismo , Tubo Neural/embriologia , Neurogênese/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Apoptose/genética , Caspase 3/biossíntese , Contagem de Células , Proliferação de Células , Cisteína/análogos & derivados , Cisteína/farmacologia , Células Ependimogliais/citologia , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
UNLABELLED: The Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity. RESULTS: WAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil. CONCLUSIONS: Whether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.
Assuntos
Poluição por Petróleo/efeitos adversos , Petróleo/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/crescimento & desenvolvimento , Desastres , Embrião não Mamífero/anormalidades , Embrião não Mamífero/embriologia , Monitoramento Ambiental , Golfo do México , Cabeça/anormalidades , Cabeça/embriologia , Cabeça/crescimento & desenvolvimento , Modelos Animais , Atividade Motora , Petróleo/análise , Poluentes Químicos da Água/análise , Peixe-Zebra/anormalidadesRESUMO
Peripheral neuropathy is a common side effect of a number of pharmaceutical compounds, including several chemotherapy drugs. Among these are vincristine sulfate, a mitotic inhibitor used to treat a variety of leukemias, lymphomas, and other cancers, and bortezomib, a 26S proteasome inhibitor used primarily to treat relapsed multiple myeloma and mantle cell lymphoma. To gain insight into the mechanisms by which these compounds act, we tested their effects in zebrafish. Vincristine or bortezomib given during late embryonic development caused significant defects at both behavioral and cellular levels. Intriguingly, the effects of the two drugs appear to be distinct. Vincristine causes uncoordinated swimming behavior, which is coupled with a reduction in the density of sensory innervation and overall size of motor axon arbors. Bortezomib, in contrast, increases the duration and amplitude of muscle contractions associated with escape swimming, which is coupled with a preferential reduction in fine processes and branches of sensory and motor axons. These results demonstrate that zebrafish is a convenient in vivo assay system for screening potential pharmaceutical compounds for neurotoxic side effects, and they provide an important step toward understanding how vincristine and bortezomib cause peripheral neuropathy.
Assuntos
Antineoplásicos/efeitos adversos , Axônios/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Ácidos Borônicos/efeitos adversos , Pirazinas/efeitos adversos , Vincristina/efeitos adversos , Animais , Axônios/patologia , Bortezomib , Imuno-Histoquímica , Larva/efeitos dos fármacos , Peixe-ZebraRESUMO
The formation of the central nervous system depends on the coordinated development of neural and glial cell types that arise from a common precursor. Using an existing group of zebrafish mutants generated by viral insertion, we performed a "shelf-screen" to identify genes necessary for astroglial development and axon scaffold formation. We screened 274 of 315 viral insertion lines using antibodies that label axons (anti-Acetylated Tubulin) and astroglia (anti-Gfap) and identified 25 mutants with defects in gliogenesis, glial patterning, neurogenesis, and axon guidance. We also identified a novel class of mutants affecting radial glial cell numbers. Defects in astroglial patterning were always associated with axon defects, supporting an important role for axon-glial interactions during axon scaffold development. The genes disrupted in these viral lines have all been identified, providing a powerful new resource for the study of axon guidance, glio- and neurogenesis, and neuron-glial interactions during development of the vertebrate CNS.
Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Axônios/metabolismo , Desenvolvimento Embrionário/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
During embryonic development of bilaterally symmetrical organisms, neurons send axons across the midline at specific points to connect the two halves of the nervous system with a commissure. Little is known about the cells at the midline that facilitate this tightly regulated process. We exploit the conserved process of vertebrate embryonic development in the zebrafish model system to elucidate the identity of cells at the midline that may facilitate postoptic (POC) and anterior commissure (AC) development. We have discovered that three different gfap+ astroglial cell morphologies persist in contact with pathfinding axons throughout commissure formation. Similarly, olig2+ progenitor cells occupy delineated portions of the postoptic and anterior commissures where they act as multipotent, neural progenitors. Moreover, we conclude that both gfap+ and olig2+ progenitor cells give rise to neuronal populations in both the telencephalon and diencephalon; however, these varied cell populations showed significant developmental timing differences between the telencephalon and diencephalon. Lastly, we also showed that fli1a+ mesenchymal cells migrate along the presumptive commissure regions before and during midline axon crossing. Furthermore, following commissure maturation, specific blood vessels formed at the midline of the POC and immediately ventral and parallel to the AC. This comprehensive account of the cellular populations that correlate with the timing and position of commissural axon pathfinding has supported the conceptual modeling and identification of the early forebrain architecture that may be necessary for proper commissure development.
Assuntos
Prosencéfalo , Peixe-Zebra , Animais , Axônios/metabolismo , Neurônios/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.
Assuntos
Transtorno Autístico/tratamento farmacológico , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Animais , Animais Geneticamente Modificados , Transtorno Autístico/genética , Modelos Animais de Doenças , Estrogênios/uso terapêutico , Genisteína/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Fenótipo , Fitoestrógenos/farmacologia , Psicotrópicos/farmacologia , Psicotrópicos/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/genética , Transtornos da Transição Sono-Vigília/tratamento farmacológico , Transtornos da Transição Sono-Vigília/genética , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Peixe-ZebraRESUMO
Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.
Assuntos
Transplante de Células/métodos , Transferência Embrionária/métodos , Gástrula/citologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/transplante , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Gástrula/metabolismo , Microinjeções/métodos , Microscopia Confocal , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Quimeras de Transplante/embriologiaRESUMO
Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial ;bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a ;channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Prosencéfalo/embriologia , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Morfogênese , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Transdução de Sinais , Transativadores/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Functional morphology has benefited greatly from the input of techniques and thinking from other disciplines. This has been especially productive in situations where each discipline has made significant contributions to a particular research topic. A combination of methodologies from functional morphology and developmental biology has allowed us to characterize feeding mechanics of first-feeding larval zebrafish (Danio rerio). Contrary to kinematic patterns commonly seen in adult teleosts, larval zebrafish showed no lateral abduction during the expansive phase of a suction-feeding event. Instead, dorsoventral expansion of the buccal chamber, more typical of patterns seen in primitive fishes, characterized the expansive phase. Moreover, a pronounced preparatory phase during which the buccal chamber is constricted by the protractor hyoideus was consistently seen in first-feeding larval kinematics. Key kinematic variables associated with first feeding correlated significantly with the hydrodynamic regime as measured by the Reynolds number. Using the tools of both functional morphology and developmental biology we have not only determined which cranial muscles are important for successful feeding but also uncovered important physiological differences in muscle structure. Muscles necessary for the rapid dorsoventral expansion of the head are composed primarily of fast-twitch fibers while those involved in more tonic contractions such as hyoid protraction have more slow-twitch muscle fibers. While most evolutionary developmental studies have examined mechanisms responsible for large evolutionary changes in morphology, we propose that the type of data uncovered in functional studies can lead to the generation of hypotheses concerning the developmental mechanisms responsible for smaller intra- and/or interspecific changes.
RESUMO
The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.