המחברים מבקשים להודות לג&#39;פרי פארל, ג&#39;יימס גניון, וג&#39;ניפר ברגמן להערות על כתב היד. KWR נתמכה על ידי קרן מדע בוגר תכנית עמיתי המחקר הלאומית במהלך הפיתוח של assay FDAP. עבודה זו נתמכה על ידי מענקי מ- NIH לAFS ועל ידי מענקים מקרן המחקר הגרמנית (Emmy תכנית נטר), מקס פלאנק החברה, והתכנית לאדם Frontier המדע (פיתוח קריירה פרס) לראש הממשלה.

אי לנו קונפליקטים לחשוף.

ההצלחה של ניסוי FDAP מסתמכת על הדור של חלבון היתוך photoconvertible פונקציונלי. תיוג חלבון יכול להשפיע על פעילותה הביולוגית ו / או נכסי biophysical, כוללים הלוקליזציה שלה, מסיסות, ויציבות 36-41. להיות מוכן לבחון את הפעילות של כמה מבני היתוך שונים כדי למצוא אחד שהוא פעיל. מצאנו כי ש...

הרמות של חלבונים בתאים ואורגניזמים נקבעות על ידי תעריפי ייצור שלהם ואישור. זמן מחצית חיים של חלבון יכולים לנוע בין דקות לימים 1-4. בהרבה מערכות ביולוגיות, יש הייצוב או אישור של חלבוני מפתח השפעות חשובות על פעילות הסלולר. אפנון של יציבות חלבון תאית נדרש להתקדמות מחזור התא 5,6, איתות התפתחותית 7-9, אפופטוזיס 10, ותפקוד תקין ותחזוקה של נוירונים 11,12. יציבות חלבון תאית משפיעה על ההתפלגות וזמינות של חלבונים מופרשים, כגון morphogens 13,14, בתוך רקמה. במהלך העשורים האחרונים, יציבות חלבון בעיקר הוערכה בתרבית תאים באמצעות דופק תיוג הרדיואקטיבי או ניסויי מרדף cycloheximide 15. בניסויי דופק מרדף כזה, תאים או זמניים נחשפו ל&quot; דופק &quot;של אמין רדיואקטיבייםמבשרי חומצה שמשולבים בחלבונים מסונתזים חדש, או שהם נחשפים לcycloheximide, אשר מעכב את סינתזת חלבון. תאים בתרבית ולאחר מכן נאספו בנקודות זמן שונות, וגם immunoprecipitation אחרי autoradiography (בניסויי דופק מרדף רדיואקטיבי) או מערבית סופג (בניסויי cycloheximide) משמשת לכמת את האישור של חלבון לאורך זמן. יש מבחני יציבות חלבון קונבנציונליים כמה חסרונות. ראשית, חלבונים במבחנים אלה הם בדרך כלל לא באו לידי ביטוי בסביבות אנדוגני, אלא בתרבית רקמה ולעתים בתאים ממינים שונים. לחלבוני יציבותה תלוי קשר, גישה זו היא בעייתית. שנית, לא ניתן לעקוב אחרי שחרור מחלבון בתאים או אורגניזמים בודדים לאורך זמן, והנתונים ממבחנים אלה משקפים ממוצע של אוכלוסיות שונות של תאים בנקודות זמן שונים. מאחר ותאים בודדים אולי התחילועם כמויות שונות של חלבון, אולי לקח את התווית או cycloheximide רדיואקטיביים בזמנים שונים, או שיש לי קינטיקה אישור שונה, נתונים מצרפיים כזה יכולים להיות מטעים. לבסוף, במקרה של ניסויי מרדף cycloheximide, תוספת של מעכב הסינתזה של החלבונים עלולה להיות השפעות פיסיולוגיות בטעות שיכולות לשנות באופן מלאכותי יציבות חלבון 16-18. ניתן להימנע מחסרונות אלה באמצעות הקרינה רדיואקטיבית לאחר photoconversion (FDAP), טכניקה שמשתמשת בחלבוני photoconvertible למדוד אישור חלבון באופן דינמי באורגניזמים החיים 19-25 (ראה דיון למגבלות של טכניקת FDAP). חלבוני photoconvertible הם חלבוני ניאון עירור פליטה שהתכונות ולשנות לאחר חשיפה לאורכי גל מסוים של אור 26. גרסה הנפוצה אחת היא Dendra2, חלבון &quot;ירוק-עד-אדום&quot; photoconvertible שתחילה יש לשעברמאפייני ציטוט ופליטה דומים לחלבוני ניאון ירוקים, אבל אחרי חשיפה ל&quot; photoconversion &quot;בהיר UV -its עירור / מאפייני פליטה להיות דומים לאלה של חלבוני ניאון אדומים 23,27. חשוב לציין, החלבון חדש שיוצר לאחר photoconversion לא יהיה לי מאפייני עירור / פליטה זהות לחלבון photoconverted, המאפשר ניתוק של ייצור ואישור על photoconversion והתבוננות רק בריכה של חלבון photoconverted. תיוג חלבוני עניין עם חלבוני photoconvertible כך מספק דרך נוחה לדופק תווית חלבונים באורגניזמים שלמים, אופטי נגישים חיים. במבחני FDAP (איור 1 א), חלבון של העניין מתויג עם חלבון photoconvertible ובא לידי ביטוי באורגניזם חי (איור 1). חלבון ההיתוך הוא photoconverted, והירידה באות photoconverted לאורך הזמן מנוטרת על ידי fluorescenמיקרוסקופיה ce (איור 1 ג). הנתונים אז מצויד במודל מתאים כדי לקבוע את זמן מחצית החיים של חלבון ההיתוך (1D איור). Assay FDAP המתואר כאן נועד לקבוע את זמן מחצית החיים תאיים של חלבוני איתות מופרשים בעוברי דג הזברה בעובר מוקדם 19. עם זאת, גישה זו יכולה להיות מותאמת לכל אורגניזם מודל שקוף שסובל הדמיה לחיות, ויכול לשמש כדי לפקח על הפינוי של כל חלבון תאיים או תאי taggable. וריאציות של הטכניקה המתוארת כאן בוצעו בתאים בתרבית 20,23 ותסיסנית 22 ועכבר 21 עוברים.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PyFDAP (download from the following website: http://people.tuebingen.mpg.de/mueller-lab)</td>
			<td></td>
			<td></td>
			<td>Install and operate using the instructions provided on the PyFDAP website; PyFDAP is compatible with Linux, Mac, and Windows operating systems.</td>
		</tr>
		<tr>
			<td>mMessage mMachine Sp6 Transcription Kit (Life Technologies, AM1340)</td>
			<td></td>
			<td></td>
			<td>To generate capped mRNA for injection into embryos</td>
		</tr>
		<tr>
			<td>Alexa488-dextran conjugate, 3 kDa (Life Technologies, D34682)</td>
			<td></td>
			<td></td>
			<td>Co-inject with mRNA to create intracellular and extracellular masks</td>
		</tr>
		<tr>
			<td>6-well plastic dish (BD Falcon)</td>
			<td></td>
			<td></td>
			<td>Incubate embryos in agarose-coated wells until ready for mounting</td>
		</tr>
		<tr>
			<td>Embryo medium</td>
			<td></td>
			<td></td>
			<td>250 mg/l Instant Ocean salt, 1 mg/l methylene blue in reverse osmosis water adjusted to pH 7 with NaHCO3</td>
		</tr>
		<tr>
			<td>Protease from Streptomyces griseus (Sigma, P5147)</td>
			<td></td>
			<td></td>
			<td>Make a 5 mg/ml stock and use at 1 mg/ml to dechorionate embryos at the one-cell stage</td>
		</tr>
		<tr>
			<td>5 cm diameter glass petri dish</td>
			<td></td>
			<td></td>
			<td>For embryo dechorionation</td>
		</tr>
		<tr>
			<td>200 ml glass beaker</td>
			<td></td>
			<td></td>
			<td>For embryo dechorionation</td>
		</tr>
		<tr>
			<td>Microinjection apparatus</td>
			<td></td>
			<td></td>
			<td>For injection of mRNA and dye into embryos at the one-cell stage</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td></td>
			<td></td>
			<td>For injecting and mounting embryos</td>
		</tr>
		<tr>
			<td>1x Danieau&#39;s medium</td>
			<td></td>
			<td></td>
			<td>Dilute low melting point agarose and perform imaging in this medium; recipe: 0.2 mm filtered solution of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.3 mM CaCl2, 5 mM HEPES pH 7.2</td>
		</tr>
		<tr>
			<td>UltraPure low melting point agarose (Invitrogen, 16520-100)</td>
			<td></td>
			<td></td>
			<td>For mounting embryos; use at a concentration of 1% in Danieau&#39;s medium: add 200 mg to 20 ml Danieau&#39;s medium, microwave until dissolved, then aliquot 1 ml into microcentrifuge tubes; aliquots can be stored at 4 &#176;C, re-melted at 70 &#176;C, and cooled to 40 - 42 &#176;C when ready to use</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette (Kimble Chase (via Fisher), 63A53WT)</td>
			<td></td>
			<td></td>
			<td>For mounting embryos; flame the tip to prevent jagged edges from injuring embryos</td>
		</tr>
		<tr>
			<td>Metal probe</td>
			<td></td>
			<td></td>
			<td>For positioning embryos during mounting</td>
		</tr>
		<tr>
			<td>Glass bottom dishes (MatTek, P35G-1.5-14-C)</td>
			<td></td>
			<td></td>
			<td>Use the appropriate cover glass thickness for your objective; part number listed here is for cover glass No. 1.5</td>
		</tr>
		<tr>
			<td>15 ml tube (BD Falcon) filled with ~5 ml embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>For rinsing residual agarose from the Pasteur pipette</td>
		</tr>
		<tr>
			<td>Inverted laser scanning confocal microscope</td>
			<td></td>
			<td></td>
			<td>A mercury arc lamp, 488 nm laser, 543 nm laser, and the appropriate filter sets are required</td>
		</tr>
		<tr>
			<td>Heated stage</td>
			<td></td>
			<td></td>
			<td>To maintain embryos at the optimal temperature of 28 &#176;C during the experiment</td>
		</tr>
		<tr>
			<td>Confocal software capable of time-lapse imaging</td>
			<td></td>
			<td></td>
			<td>Must be able to define multiple positions and automatically image them at defined intervals&#160;</td>
		</tr>
		<tr>
			<td>25x or 40x water objective</td>
			<td></td>
			<td></td>
			<td>Objective for imaging</td>
		</tr>
		<tr>
			<td>10x air objective</td>
			<td></td>
			<td></td>
			<td>Objective for photoconversion</td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td></td>
			<td></td>
			<td>Immersion oil with the same refractive index as water</td>
		</tr>
	</tbody>
</table>

measuring protein stability in living zebrafish embryos using fluorescence decay after photoconversion (fdap)

יציבות חלבון משפיעה על היבטים רבים של ביולוגיה, ומדידת קינטיקה השחרור של חלבונים יכולה לספק תובנות חשובות במערכות ביולוגיות. בניסויי FDAP, האישור של חלבונים בתוך אורגניזמים חיים ניתן למדוד. חלבון של העניין מתויג עם חלבון פלואורסצנטי photoconvertible, בא לידי ביטוי בvivo וphotoconverted, והירידה באות photoconverted לאורך הזמן היא פיקוח. הנתונים אז מצויד במודל פינוי מתאים כדי לקבוע את חלבון מחצית החיים. חשוב לציין, קינטיקה פינוי אוכלוסיות חלבון בתאים שונים של האורגניזם יכולה להיבחן בנפרד על ידי יישום מסכות compartmental. גישה זו נעשתה שימוש כדי לקבוע את זמן מחצית החיים התוך וחוץ-תאיים של חלבוני איתות מופרשים במהלך פיתוח דג הזברה. כאן, אנו מתארים פרוטוקול לניסויי FDAP בעוברי דג הזברה. זה צריך להיות אפשרי להשתמש FDAP לקבוע קינטיקה סליקתכל חלבון taggable בכל אורגניזם אופטי נגיש.

FDAP נעשה שימוש כדי לקבוע את זמן מחצית החיים של חלבוני איתות תאיים בעוברי דג הזברה 19. אחד מחלבונים אלו, פזילה, גורמת ביטוי של גני mesendodermal במהלך embryogenesis 34. לפזול-Dendra2 מפעיל ביטוי של גני mesendodermal ברמות דומות לפזילה לא מתויגת, כפי שהודגם על ידי qRT-PCR ובמבחני הכל?...

Watch this Scientific Journal Video about Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP at JoVE.com

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

רמות חלבון בתאים וברקמות לעתים קרובות מוסדרות היטב על ידי האיזון של ייצור חלבון ופינוי. שימוש בקרינה רדיואקטיבית לאחר photoconversion (FDAP), קינטיקה האישור של חלבונים ניתן למדוד באופן ניסיוני בvivo.

מדידת יציבות חלבון בחיים עוברי דג הזברה שימוש הקרינה רדיואקטיבית לאחר photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

measuring-protein-stability-living-zebrafish-embryos-using

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

11.1K Views.  Harvard University.  רמות חלבון בתאים וברקמות לעתים קרובות מוסדרות היטב על ידי האיזון של ייצור חלבון ופינוי. שימוש בקרינה רדיואקטיבית לאחר photoconversion (FDAP), קינטיקה האישור של חלבונים ניתן למדוד באופן ניסיוני בvivo. 

Video: Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

פגיעה כלי מכאנית בעוברי דג הזברה

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

הדמית מבני subcellular בעובר דג הזברה החייה

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

ניתוח<em&gt; In vivo</em&gt; נדידת תאים באמצעות השתלות תא והדמיה זמן לשגות עוברי דג הזברה

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

באמצעות מיקרוסקופ פלואורסצנטי גיליון אור פיתוח תמונה דג הזברה העין

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Biosensing מוטורית נוירון פוטנציאל ממברנה עוברי דג הזברה חיים

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

גיליון מבוסס אור מיקרוסקופ פלואורסצנטי של חיים או קבוע מוכתמת<em&gt; Tribolium castaneum</em&gt; עובר

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

אינדוקציה של היפוקסיה צפרדע חיה דג הזברה עוברי

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion תא בודד בהמחיה שלם דג זברה

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

הפרקים הבאים רקמה Endocardial ויה Photoconversion תאים של העובר דג זברה

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

כל הר אימונוהיסטוכימיה בזיבפיש עוברים וזחלים

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...