Name: stem cell-like xenopus embryonic explants to study early neural developmental features in vitro and in vivo
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Description: Watch this Scientific Journal Video about Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo at JoVE.com

The author thanks Hugo Juraver-Geslin, Marion Wassef and Anne H&#233;l&#232;ne Monsoro-Burq for their help and advice, and the Animal Facility of the Institut Curie. The author thanks Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the "Association pour la Recherche sur le Cancer" (ARC 4972 and ARC 5115; FRC DOC20120605233 and LABEX Memolife) and the Fondation Pierre Gilles de Gennes (FPGG0039).

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The authors have nothing to disclose.

התפתחות עצבית היא בניצוחו של גומלין מורכב בין תוכניות סלולריות התפתחותיים ואותות מהרקמות הסובבות (נבדקו ב3,31,32). כאן אנו מתארים קבוצה של פרוטוקולים שניתן להשתמש בX. laevis עובר לחקור חיצוני וגורמים פנימיים מעורבים בקביעת גורל עצבית והמורפוגנזה עצבית במבח...

מערכת העצבים של בעלי החוליות עולה מהצלחת העצבית כשכבת אחידה של תאי neuroepithelial. הבנה כיצד תוכניות התפתחותיות מושרות, מקודדות, והוקמו במהלך אזורית של הצלחת העצבית היא, כיום, מטרה עיקרית בביולוגיה התפתחותית. בהשוואה למערכות אחרות, עובר Xenopus ניסוי נוח הוא מודל של בחירה לניתוח שלבים מוקדמים של התפתחות עצבית 1,2. זה קל להשיג מספר גדול של עוברים, ופיתוח חיצוני נותן גישה לצעדים הראשונים של neurulation 3. כלים רבים זמינים כדי לתפעל באופן ניסיוני Xenopus laevis (laevis X.) התפתחות עוברית. מיקרו הזרקה של mRNAs או morpholinos (MO), כוללים שיטות פעולה מושרה, יחד עם כלים ביוכימיים ותרופתיים, מאפשר מבוקר רווח של פונקציה (GOF) ואובדן התפקוד (LOF) ושינוי ספציפי של מסלולי איתות 4,5. בלההאאקטודרם גג stocoel, הממוקם סביב קוטב החיה של blastula, או עובר gastrula בשלב מוקדם מאוד, ומכונה &quot;בעלי החיים קאפ &#39;(AC), הוא מקור לתאי pluripotent שניתן לתכנת על ידי מניפולציה של ביטוי גנים לפני explants הכנה. בכתב יד זה הם פרוטוקולים מפורטים לשימוש X. explants laevis AC לבדוק במבחנה ובמנגנונים מולקולריים vivo ותהליכים תאיים התפתחות עצבית בסיסית. טכניקה מוצגת, המאפשרת תצפית יפה של דפוסי ביטוי גנים בצינור עצבי ראשן צפרדעים, צעד ראשוני בזיהוי רמזי נחישות גורל. בעוד התצפית של רקמות רכוב שטוחות נפוצה במחקר של עובר חומוס 6, זה לא תואר כראוי בצפרדעים. מניפולציה של ביטוי גנים על ידי הזרקת mRNA או MO סינטטיים ללסטומרים של 2 או 4 עובר שלב תא מאפשרת תכנות של ACexplants 4. לדוגמא עיכוב של מסלול חלבון העצם המוךפו&quot;גנטי (BMP) על ידי ביטוי של אנטי-BMP גורם הבחור!, נותן זהות עצבית לתאי AC 3. הפרוטוקול מפורט לביצוע חשיפה מבוקר זמן ומקומי של explants AC לרמזים חיצוניים באמצעות מגע ישיר עם חרוז שרף אניוני. לבסוף טכניקה מתוארת לבדיקת תכונות ההתפתחותי של אבות עצביים in vivo על ידי השתלה של explants המעורב הוכן מהתאים נפרדים מתוכנת ניתקו וקשורים-מחדש. עובר הצפרדע הוא מודל רב עוצמה כדי לחקור את ההתפתחות עצבית חוליות מוקדמות. שילוב מניפולציה של ביטוי גנים לexplant תרבויות במבחנה מספק מידע חשוב בחקר האזורי neuroepithelium, התפשטות, והמורפוגנזה 7-12. תכנות של explants AC מותר פיתוח של לב פונקציונלי vivo לשעבר 13,14. השימוששל explant השתלת 15 הובילו לזיהוי של בורר תעתיק המינימלי התרמה תכנית בידול הרכס העצבי 16. Limitans Zona intrathalamica (ZLI) הוא מרכז איתות שמפריש סוניק קיפוד (ששש) כדי לשלוט על הצמיחה ואזורית של המוח הקדמי הזנב. כאשר נחשפו ברציפות שלשש, תאי neuroepithelial coexpressing גני שלושה שעתוק גורם - כמו-barH homeobox-2 (barhl2), orthodenticle-2 (otx2) וIroquois-3 (irx3) - רכישת שני מאפיינים של תא ZLI: הסמכות ל להביע את ששש, ואת היכולת להפריד מתאי צלחת עצביים קדמי. כמערכת מודל, האינדוקציה של גורל ZLI לתאי neuroepithelial יוצג 8. פרוטוקולים אלה מכוונים למתן וכלים פשוטים, זולים, יעילים לביולוגים התפתחותיים וחוקרים אחרים לחקור את MEC הבסיסיhanisms של התנהגויות תא עצביות מרכזיות. פרוטוקולים אלה הם מגוונים מאוד ומאפשרים החקירה של מגוון גדול של רמזי נחישות עצביים חיצוניים ופנימיים. זה מאפשר לטווח ארוך בניתוח vivo של מחויבות עצביות שושלת, אינטראקציות אינדוקטיביים והתנהגויות תא.

<table><tbody><tr><td>Paraformaldehyde</td><td>VWR</td><td>&amp;nbsp;20909.290</td><td>Toxic</td></tr><tr><td>anion exchange resin beads</td><td>Biorad</td><td>140- 1231</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>SIGMA</td><td>A-7888</td><td>For culture of animal cappH 7.6</td></tr><tr><td>Gentamycine&amp;nbsp;</td><td>GIBCO</td><td>15751-045&amp;nbsp;</td><td>antibiotic</td></tr><tr><td>Bovine Serum Albumin</td><td>SIGMA</td><td>A7906</td><td>&amp;nbsp;for bead preparation</td></tr></tbody></table>

stem cell-like xenopus embryonic explants to study early neural developmental features in vitro and in vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

בהתבסס על שיקולים מורפולוגיים במינים שונים, מניפולציות עובריות, ואת דפוס הביטוי של גני רגולציה, מודל רעיוני גורס כי הצלחת העצבית מחולקת למגזרים רוחביים ואורך המגדירים רשת התפתחותית יצירת שדות histogenic מובחנים. בצלחת העצבית, primordia של המוח הקדמי, המוח ה?...

Watch this Scientific Journal Video about Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo at JoVE.com

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

כמו תא גזע<em&gt; Xenopus</em&gt; Explants העובריים לחקר תכונות המוקדמות עצבי התפתחותית<em&gt; במבחנה</em&gt; ו<em&gt; In vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

stem-cell-like-xenopus-embryonic-explants-to-study-early-neural

Research

JoVE Journal

Developmental Biology

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

7.9K Views.  Institut Curie. In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. 

Video: Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

הבנת organogenesis המוקדם שימוש פשוטה<em&gt; באתר</em&gt; הכלאת פרוטוקול ב<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

ומניפולציה<em&gt; במבחנה</em&gt; ההבשלה של<em&gt; Laevis Xenopus</em&gt; ביציות, ואחריו הזרקת Intracytoplasmic זרע, לחקר התפתחות העוברית

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

שיטות לימוד המורפוגנזה העצבית:<em&gt; Ex vivo</em&gt; RNAi electroporation בקליפת עובריים מוחין Murine

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Neuroscience

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

ברזולוציה גבוהה הדמיה חיה של התנהגות התא ב Neuroepithelium פיתוח

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

התרחבות של תאי גזע עצביים עובריים ומבוגרים על ידי<em&gt; ברחם</em&gt; Electroporation או הזרקת Stereotaxic ויראלי

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

תרבויות Explant עצביות מן<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

סידן פלואורסצנטי הדמיה ובעקבות היברידיזציה באתרו לאפיון עצבי בלתי מבשר בקספוס זריזה

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

דור של מסלקי בלסטודרם נאיביים מעוברי דגי זברה

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

הערכת Neurogenesis יסודי<em&gt; Xenopus</em&gt; עוברים באמצעות Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

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הבנת organogenesis המוקדם שימוש פשוטה

ומניפולציה

שיטות לימוד המורפוגנזה העצבית:

ברזולוציה גבוהה הדמיה חיה של התנהגות התא ב Neuroepithelium פיתוח

התרחבות של תאי גזע עצביים עובריים ומבוגרים על ידי

תרבויות Explant עצביות מן

סידן פלואורסצנטי הדמיה ובעקבות היברידיזציה באתרו לאפיון עצבי בלתי מבשר בקספוס זריזה

דור של מסלקי בלסטודרם נאיביים מעוברי דגי זברה

הערכת Neurogenesis יסודי

Dissection של