אנו מודים ג&#39;יי צ&#39;ן מתן בכלוב והעמסת-dextran פרוטוקול סינתזה. מחקר זה נתמך על ידי מצעד הפרוטות פרס 5-78-FY09, איגוד הלב האמריקאי הפרס 09BGIA2090075, ואת הפרס NIH / NHLBI HL107369 R01-01 אס תודה מיוחדת ג Closson לסיוע במיקרוסקופ שלו.

<ol>
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אין ניגודי אינטרסים הכריז.

<html> מאמר זה מתאר שיטה צדדי למיפוי תא בודד גורל בהתבסס על photoactivation של בכלוב והעמסת-dextran. משחררות רפרוף של בכלוב והעמסת-dextran בתאים בודדים מושגת באמצעות שני פוטונים הקליטה של ​​לייזר femtosecond מאי טאי מצמידים את המיקרוסקופ LSM 510 META Zeiss confocal. שברחה מכלוב והעמסת-dextran בתאים photoactivated הוא מדמיין במהירות באמצעות הליך פשוט אימונוהיסטוכימיה. בפרוטוקול זה הגל שני הפוטונים קליטה של ​​photoactivation בכלוב והעמסת-dextran הוא 740 ננומטר. אורך גל זה נקבע באופן ניסיוני על ידי photoactivating בכלוב והעמסת-dextran עוברים wildtype מוזרק. גל עירור הלייזר היתה מגוונת 600-800 ננומטר, ועל נוכחות או העדר והעמסת הקרינה שלאחר ההפעלה נקבע איכותית. מצאנו כי 740 ננומטר המיוצר ההפעלה החזקה והעמסת האות הבאה. באופן אידיאלי, 740 ננומטר צריך לעבוד עבור כל מערכות המודל, אך אנו מייעצים בדיקות מגוון רחב של אורכי גל כדי לקבוע את אורך הגל האופטימלי שני הפוטונים עירור למדגם שלך. בשנת עוברים בכלוב והעמסת-dextran wildtype מוזרק, עבור גל שני הפוטונים כל עירור נבדק גם אנחנו מגוונים את הכוח לייזר הממוצע. עבור גל עירור של 740 ננומטר, כוח לייזר ממוצע של 47 mW המיוצר אות והעמסת חזקה על האזור הפעיל בהשוואה נמוך סמכויות לייזר הממוצע. גבוהה סמכויות לייזר הממוצע לא לייצר אותות והעמסת חזק, אבל אבלציה המושרה של הרקמה. מאז לייזר פולסים femtosecond יעילים ב ablating רקמות ביולוגיות 15, אנו ממליצים על קביעת סף ממוצע לייזר כוח photoactivation כי ימנע אבלציה רקמות. שני הפוטונים הקליטה מתרחשת בתוך אינטראקציה נפח קטן. כדי להבטיח הפעלה ראויה של בכלוב והעמסת-dextran, התא חייב להיות מובא אל המוקד הנכון. בפרוטוקול לעיל, תאים GFP חיובית היו צילמו בו זמנית תחת אור לבן כדי לאשר להתמקד התא. לכן, רכישת אור לבן, בנוסף לערוצים אחרים מומלץ. לאחר אישור להתמקד התא, פרוטוקול שלנו מציע מגדלת התא הפעיל. גורם זום של 50-70 הוא הציע, עם זאת, ערך זה תלוי בגודל של התא הנבחר. הגדלת זום מבודד את התא הפעיל של תאים סמוכים, ומגביל את אזור הסריקה להפעלת שני הפוטונים. באופן אידיאלי, לסרוק את האזור צריך לכסות שטח גדול של התא. איתור של תאים מופעל יחיד דורש כי מכתים רקע להיות מינימלית. בפרוטוקול immunodetection לעיל חשוב כי המדגם הוא חסום חוסם חיץ במשך 5 עד 6 שעות (שלב 4.13). בעת פיתוח עם NBT / BCIP, שברחה מכלוב והעמסת-dextran צריך להיות גלוי ב 10 עד 20 דקות. אם מכתים רקע חזק, אנו ממליצים זמן ארוך יותר חסימת ושוטף נוספת כדי להסיר את הנוגדן (שלב 4.17). איורים 1 ו -2 לספק תוצאות נציג photoactivation ו immunodetection. באיור 1, מוקדם 1 - ל 2 תאים בשלב עוברי wildtype הוזרקו בכלוב והעמסת-dextran. עוברים הועלו על somitogenesis באמצע ועלה נמוך ההיתוך agarose בכיוון לרוחב. איור 1 (a, b) מתארים brightfield ותמונות הניאון של העובר לפני photoactivation. ראיות כי העובר נשאר שברחה מכלוב באה לידי ביטוי בהעדר והעמסת הקרינה באיור 1 (ב). אזור קטן בתוך somite הופעל עם אורך גל של 740 ננומטר במשך 31 שניות באמצעות כוח לייזר ממוצע של 47 mW, איור 1 (ג; החץ). לאחר ההפעלה, שני הפוטונים משחררות רפרוף של והעמסת-dextran התרחשו כפי שמוצג על ידי הקרינה מאזור מופעל, איור 1 (ד; החץ). הפעלה לא לוותה אבלציה לייזר, כמו רקמת somitic איור נשאר שלם, 1 (ג). איור 2 מדגים את immunodetection שברחה מכלוב של והעמסת-dextran. אזור קטן והמזודרם את הצלחת רוחבי של העובר 10-somite שלב היה photoactivated באמצעות פרמטרים לייזר זהה כאמור באיור 1. העובר גדל ומעובד באמצעות צעדים 4.1 עד 4.20. החץ באיור 2 מאתרת באזור הפעיל, כפי שצוין על ידי הצטברות של משקע כחול. רק המיקום מופעל מזוהה בבירור בלי להתערב מכתים רקע. ההכנה של פתרונות: 1 X PBT (1 L) 100 מ&quot;ל 10 X PBS 2 גרם BSA 10 מ&quot;ל 20% Tween 10 X PBS (1 L) 80 גרם NaCl 2 גרם KCl 6.1 גרם Na 2 HPO 4 1.9 גרם KH 2 PO 4 DDH 2 O ל L 1 עד 7.3 pH Paraformaldehyde 4% (160 מ&quot;ל) Paraformaldehyde 32% (שני בקבוקונים 10 מ&quot;ל) 8 X 20 מ&quot;ל PBS 132 מ&quot;ל DDH 2 O &quot;jove_content&gt; AP חוצץ (50 מ&quot;ל) 1 מ&quot;ל 5 M NaCl 2.5 מ&quot;ל 1 M MgCl 2 5 מ&quot;ל 1 M טריס pH 9.5 250 Tween 20% μL 41.25 מ&quot;ל DDH 2 O פתרון נוגדן (למדגם 1) 5.5 אצטון μL אבקת 40 μL PBT 0.8 LS μL 0.1 μL נוגדן Anti-והעמסת-AP LS 2% (360 μL למדגם 1) 7.2 LS 100% μL 352.8 μL PBT 1 X Danieau מדיה (1 L) ‡ 11.6 mL 5 M NaCl 700 μL 1 M KCl 400 μL 1 M MgSO 4 600 μL 1 M Ca (NO 3) 2 5 מ&quot;ל 1 M HEPES DDH 2 O ל L 1 pH להסתגל 7.6 ‡ Danieau הוא פתרון אידיאלי עבור מלח גידול עוברי דג הזברה dechorionated. מדיה אחרים עשויים לשמש, בתנאי שהם פתרונות איזוטוני עם osmolarity כראוי (~ 300 mOsm עבור דג הזברה) NBT מניות (1 מ&quot;ל) 50 מ&quot;ג Nitro כחול Tetrazolium 0.7 מ&quot;ל אנהידריד לפוראמיד דימתיל 0.3 מ&quot;ל DDH 2 O BCIP מניות (1 מ&quot;ל) 50 מ&quot;ג 5-bromo-4-כלורו-3-indolyl פוספט 1 מ&quot;ל אנהידריד לפוראמיד דימתיל NBT / BCIP בפיתוח פתרון 1 מ&quot;ל AP חיץ 4.5 NBT μL מניות 3.5 BCIP μL מניות אצטון אבקת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר 20 דגים בוגרים להקפיא אותם בחנקן נוזלי. </li><li style=";text-align:right;direction:rtl"> טוחנים את הדג הקפוא עם במכתש ועלי לאבקה. </li><li style=";text-align:right;direction:rtl"> מעבירים את אבקת דגים לתוך צינור חרוטי 50 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> מלאו את צינור חרוטי עם אצטון 100%. וורטקס את הצינורית. </li><li style=";text-align:right;direction:rtl"> ספין התחתית מקסימום סל&quot;ד במשך 10 דקות. בטל supernatant. </li><li style=";text-align:right;direction:rtl"> שטפו את גלולה אחרת 3 פעמים אצטון 100% ספין התחתית מקסימום סל&quot;ד במשך 10 דקות. עד לשטוף האחרון supernatant אמור להופיע ברור. </li><li style=";text-align:right;direction:rtl"> הוסף אצטון 100% עד שוב את הכדור ולתת הצינור לעמוד בטמפרטורת החדר. החלקיקים צפופים יותר יהיה להסתפק, ואילו חלקיקי עדין יישארו ההשעיה. </li><li style=";text-align:right;direction:rtl"> למזוג חלקיקים לתוך צינור חדש. Aliquot אבקת פתרון לתוך צינורות נפרדים ולאחסן אותם ב -20 ° C. </li></ol>

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th></tr><tr><td> CMNB-בכלוב והעמסת SE </td><td> Invitrogen </td><td> C20050 </td></tr><tr><td> 10 kDa Aminodextran </td><td> Invitrogen </td><td> D1860 </td></tr><tr><td> זבה Desalt טור ספין </td><td> לנקב </td><td> 89889 </td></tr><tr><td> התכה נמוכה agarose </td><td> Amresco </td><td> 0815-100 גרם </td></tr><tr><td> נתרן borate </td><td> Amresco </td><td> 1131117-100 גרם </td></tr><tr><td> Tricaine Methylsulfonate / MS222 </td><td> מחקר אורגניקס </td><td> 1347A </td></tr><tr><td> Anti-והעמסת-AP </td><td> רוש </td><td> 11376622 </td></tr><tr><td> חסימת חיץ </td><td> רוש יישומית למדעים </td><td> 11 096 176 001 </td></tr><tr><td> Maleic חומצה </td><td> סיגמא </td><td> MO375-500 גרם </td></tr><tr><td> Paraformaldehyde </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 15714 </td></tr><tr><td> NBT </td><td> סיגמא </td><td> טבליות של 250-I8377 </td></tr><tr><td> BCIP </td><td> פישר סיינטיפיק </td><td> PI-34040 </td></tr><tr><td> Microinjector </td><td> הרווארד Apparatus </td><td> PLI-2000 </td></tr><tr><td> LabTek החדרים coverglass שקופיות </td><td> Nalge Nunc הבינלאומי </td><td> 155380 </td></tr><tr><td> Proteinase K </td><td> Invitrogen </td><td> AM2544 </td></tr><tr><td> כבש בסרום </td><td> Invitrogen </td><td> 16070096 </td></tr><tr><td> LSM 510 META NLO </td><td> Zeiss </td><td></td></tr><tr><td> 20X 0.8 NA האוויר apochromatic אובייקטיבי </td><td> Zeiss </td><td></td></tr><tr><td> מסנן צהוב שקוף </td><td> תיאטרון בית בע&quot;מ </td><td> Roscolux מסנן צבע סדין: 312 </td></tr></tbody></table>

single cell fate mapping in zebrafish

היכולת דיפרנציאלי תווית תאים בודדים יש השלכות חשובות בביולוגיה התפתחותית. למשל, לקבוע כיצד היווצרות הדם, הלימפה, כלי הדם שושלות להתעורר עוברים לפתח מחייב מיפוי גורל השושלת העתקה של תאים מבשר מובחן. לאחרונה, חלבונים photoactivatable הכוללים: Eos 1, 2, 3 PAmCherry, קאךה 4-7, pKindling 8, KikGR 9, 10 קיבלו אינטרס רחב כמו בדיקות מעקב התא. ספקטרום הקרינה של החלבונים האלה רגישים ניתן להמיר בקלות עם עירור UV, המאפשר אוכלוסייה של תאים כדי להבחין בין אלה סמוכים. עם זאת, photoefficiency של החלבון הפעיל עלול להגביל בטווח הארוך מעקב תא 11. כחלופה חלבונים photoactivatable, בכלוב והעמסת-dextran כבר בשימוש נרחב במודל מערכות העובר 7, 12-14. באופן מסורתי, כדי uncage והעמסת-dextran, עירור UV מבית המנורה הקרינה או לייזר יחיד UV פוטון נעשה שימוש, אולם מקורות כגון להגביל את הרזולוציה המרחבית של photoactivation. כאן אנו מדווחים פרוטוקול מפת גורל, זכר השושלת, וכדי לזהות תאים תווית אחת. תאים יחיד בעוברים הזריקו בכלוב והעמסת-dextran הם photoactivated עם קרוב אינפרא אדום פעימות לייזר המופק לייזר femtosecond טיטניום ספיר. לייזר זה נהוג בכל שני הפוטונים מיקרוסקופים confocal כמו מיקרוסקופ 510 LSM NLO META בשימוש בנייר זה. מאז רקמות ביולוגיות שקופה קרינה אינפרא אדום הקרוב 15, קטניות הלייזר יכול להיות ממוקד עמוק ללא משחררות רפרוף תאים מעל או מתחת למישור המוקד נבחרים בתוך העובר. לכן, שאינו ליניארי שני הפוטונים קליטה מושרה רק במוקד גיאומטריים uncage והעמסת-dextran בתא בודד. כדי לזהות את התא המכיל שברחה מכלוב והעמסת-dextran, אנו מתארים פרוטוקול פשוט אימונוהיסטוכימיה 16 במהירות כדי להמחיש את התא הפעיל. פרוטוקול הפעלה וזיהוי המוצג במאמר זה הוא צדדי יכול להיות מיושם על כל מערכת מודל. הערה: חומרים כימיים המשמשים פרוטוקול זה ניתן למצוא בטבלה המצורפת בסוף המאמר.

Watch this Scientific Journal Video about Single Cell Fate Mapping in Zebrafish at JoVE.com

Single Cell Fate Mapping in Zebrafish

השיטה מתוארת על photoactivate תאים בודדים המכיל חלבון פלואורסצנטי בכלוב באמצעות שני פוטונים קליטה מתוך טי: לייזר ספיר מתנד femtosecond. כדי למפות גורל התא photoactivated, אימונוהיסטוכימיה משמשת. טכניקה זו יכולה להיות מיושמת לכל סוג תא.

תא בודד הגורל מיפוי של דג הזברה

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Research

JoVE Journal

Biology

13.3K Views.  Cincinnati Children's Hospital Medical Center. השיטה מתוארת על photoactivate תאים בודדים המכיל חלבון פלואורסצנטי בכלוב באמצעות שני פוטונים קליטה מתוך טי: לייזר ספיר מתנד femtosecond. כדי למפות גורל התא photoactivated, אימונוהיסטוכימיה משמשת. טכניקה זו יכולה להיות מיושמת לכל סוג תא.

Video: Single Cell Fate Mapping in Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

השתלה של מח כליות שלמים דג הזברה למבוגרים

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

תא בודד יניקה הקלטות של photoreceptors קון עכבר

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

גישה מעשית מיפוי גנטי ועין מתנהלת הגורל: מדריך חזותי כדי מארק עקוב אחר תאים In vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

הגורל מיפוי בתאי גזע עובריים אנושיים על ידי גיבוש תפלצת

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

ב electroporation vivo של Morpholinos לתוך הרשתית דג הזברה למבוגרים

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

ב electroporation vivo של Morpholinos לתוך למבוגרים התחדשות זנב סנפיר דג הזברה

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Explants Blastomere לבדיקת מחויבות גורל תא במהלך התפתחות עוברית

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

דור של תרביות תאים המפוזרים Presomitic המזודרם להדמיה של דג הזברה פילוח השעון בתאים בודדים

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

זיהוי של Myod Interactome שימוש טיהור טנדם זיקה מצמידים ספקטרומטריית מסה

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

ניתוח קוד פתוח Single-חלקיק למיקרוסקופיה סופר-רזולוציה עם VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...