עבודה זו נתמכה על ידי מ &#39;מרגרט Alkek קרן אל RHF.

חלק 1: תיקון העוברים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מלאו צלחת דיסקציה עם קרח קר depc-PBS. שמור על הקרח. פתח את הביצה על ידי הקשה על פגז עם מלקחיים ולהסיר חתיכות הקליפה. </li><li style=";text-align:right;direction:rtl"> הסר עבה אלבומין עם מלקחיים. הטיה שק חלמון עם מלקחיים גס כל כך העובר הפונה כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים לחתוך ריבוע של שק חלמון סביב העובר. </li><li style=";text-align:right;direction:rtl"> בעזרת כף, להסיר העובר מן החלמון ומניחים על קרח קר depc-PBS. </li><li style=";text-align:right;direction:rtl"> מתחת למיקרוסקופ, להסיר ממברנות חלמון ולהעביר לצלחת קיבעון. </li><li style=";text-align:right;direction:rtl"> הצמד מטה, להסיר depc-PBS. החלף PFA 4% ב-PBS depc ולתקן O / N ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> הסר מקבע. החלף עם קרח קר depc-PBS. באמצעות מחט סוף בוטה microcapillary או סכין microdissection, לחרר את מערכת העצבים ואת החללים בלב, כדי למנוע השמנה של בדיקה. </li><li style=";text-align:right;direction:rtl"> לשטוף 2x 10 mn depc-PBS עם 0.1% Tween-20 (PBT). מייבשים 10 mn כל אחד 25%, 50%, 75% מתנול PBT. מייבשים עוברים פעמיים מתנול 100%. חנות ב -20 מעלות צלזיוס מתנול. </li></ol> חלק 2: Prehybridization <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> רעננותם עוברים 10 mn כל אחד 75%, 50% מתנול 25% PBT. לשטוף 2x 10 mn ב PBT. </li><li style=";text-align:right;direction:rtl"> בינתיים, להכין מקבע קרים (paraformaldehyde 4% PBT). החלף PBT עם פתרון K proteinase (3 מ&quot;ל / בקבוקון; [? 5 גרם / מ&quot;ל] הסופי PBT). ודא את הבקבוקון כולו חשוף הפתרון K proteinase ידי בעדינות מגלגלים את הבקבוקון, ראה פתרון בעיות עבור זמני חשיפה. </li><li style=";text-align:right;direction:rtl"> באמצעות RNase חינם פיפטה פסטר להסיר proteinase K ולהוסיף מקבע (2 מ&quot;ל). מיד מקום על קרח בדיוק 20 mn. </li><li style=";text-align:right;direction:rtl"> שוטף כל RT מבוצעות על nutator. לשטוף 2x 10 mn ב PBT, ו 1x 10mn ב PBT המכילות חיץ הכלאה של 50%. </li><li style=";text-align:right;direction:rtl"> לשטוף 1x במאגר הכלאה. לדגור על 65 מעלות צלזיוס חוצץ 2 מ&quot;ל הכלאה של 4-5 שעות. </li></ol> חלק 3: עוברים להכליא עם בדיקות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחפור FITC סינתזה בשילוב בדיקה מתואר בנספח. החללית צפויה לתת איתות חזק, צריך להיות עם synhesized FITC המכילים נוקלאוטידים; בדיקה חלש צריך להיות מסונתז עם עם DIG המכילים נוקלאוטידים. </li><li style=";text-align:right;direction:rtl"> בדיקות Prewarm במאגר הכלאה (500 ng / ml כ&quot;א) באמצעות בקבוקון 2 מ&quot;ל. מיד להחליף את החיץ הכלאה עם פתרון בדיקה. אל תתנו עוברים יבש. דגירה של 16 שעות ב 65 ° C. </li><li style=";text-align:right;direction:rtl"> החלף בדיקה עם חיץ הכלאה, 2 שוטף, כל 30 mn ב 65 ° C. שטפי 15 mn במאגר הכלאה / MABT (1 / 1 v / v /) בשעה 65 ° C. </li></ol> חלק 4: הדגירה DIG נוגדן וצבע תגובה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשטוף 2x20 mn ב MABT. שטפי 1 שעות ב 2% BBR / MABT. לשטוף 5 שעות ב -2% BBR/20% היס / MABT (חסימת חיץ). דגירה של נוגדנים DIG 1:2000 מדולל חיץ חסימה, O / N ב 4 ° C על nutator. </li><li style=";text-align:right;direction:rtl"> לשטוף 3x 10 mn כל MABT, ואת שעות 3x 1 כל MABT. </li><li style=";text-align:right;direction:rtl"> לשטוף 2x 20 mn ב NTMT. הוסף NBT / BCIP המצע 200ul כדי NTMT 10 מ&quot;ל. מיד להסיר NTMT מבקבוקון ולהחליף חיץ 1-2 NBT / BCIP מ&quot;ל. מקום חשוך. צג התגובה צבע אחרי 20 mn, ובו 20 מרווחי mn לאחר מכן. </li><li style=";text-align:right;direction:rtl"> בעקבות התגובה צבע (אמור להופיע כחול), להתרחץ PBS עבור 10 mn, להצמיד על צלחת קיבוע מלא PBS, ולהחליף פתרון עם PFA 4% ב-PBS. תקן O / N ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> העברה בקבוקון scintillation חדש. שטפי ב PBST (PBS עם 1% Tween-20) 2x10 mn. שטפי ב MABT 2x 10 mn. </li><li style=";text-align:right;direction:rtl"> מדגירים את MABT 30 mn ב 63 ° C. </li></ol> חלק 5: הדגירה נוגדן FITC וצבע תגובה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשטוף 2x 10 mn ב MABT, 1 שעות ב 2% BBR / MABT, 5 שעות במאגר חסימת </li><li style=";text-align:right;direction:rtl"> דגירה של אלקליין phosphatase מצמידים אנטי FITC-נוגדן (1:500) O / N ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> לשטוף 3x 10 mn כל MABT, ואת שעות 3x 1 כל MABT. </li><li style=";text-align:right;direction:rtl"> שטפי ב 2x 20 mn ב TBS pH 8.45% עם 0.1 Tween-20 (TBST). </li><li style=";text-align:right;direction:rtl"> הכן וקטור האדום פתרון עובד (5 מ&quot;ל) ב TBST באמצעות ריאגנטים 1,2 ו 3 המסופק בערכה. מיד להסיר TBST מבקבוקון ולהחליף חיץ האדום וקטור. מקום חשוך. צג התגובה צבע אחרי 40 mn, ובו 30 מרווחי mn לאחר מכן. </li><li style=";text-align:right;direction:rtl"> צבע בעקבות התגובה (אמור להופיע אדום), עוברי העברה PBS. </li></ol> חלק 6: קיבוע ועיבוד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העברה צלחת קיבעון המכיל PBS, ולתקן PFA ב -4% במשך 20 mn ב RT או O / N ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> שטפי ב-PBS, 2x 10 mn. כדי תהליך הצילום, להעביר גליצרול 20% PBS (זו יהפכו את העוברים שקוף). כדי ליצור חדר, לחתוך 2 רצועות של סרט PVC, להרכיב אותם בשקופית. גזור ריבוע במרכז באמצעות להב. הסר את מלקחיים באמצעות מרובע. </li><li style=";text-align:right;direction:rtl"> כדי תהליך חתך שעווה, להעביר בחזרה PBS, לשטוף 2x 10 mn, מייבשים בסדרה מדורגת של מתנול (25%, 50%, 75% ו 100% ב-PBS, 10 mn כל אחד). </li><li style=";text-align:right;direction:rtl"> החלף propanol, 3mn. החלף 1,2,3,4-tetrahydronaphthalene, ואחריו 20 mn ידי Histoclear (2x 1 שעות). החלף histoclear / שעווה 01/01 (v / v) 60 ° C עבור 1 שעה. החלף בשעווה תהליךעבור היסטולוגיה. </li></ol> חלק 7: פתרונות חיץ הכלאה, בכל חנות -20 מעלות צלזיוס פלקון, 50 מ&quot;ל: 25 מ&quot;ל לפוראמיד; 3.25 מ&quot;ל (20xSSC); 0.5 מ&quot;ל (EDTA 0.5m); 1 מ&quot;ל (10% Tween-20); 2.5 מ&quot;ל (1% SDS) ; 125 ul (20mg/ml tRNA). 100ul (50 מ&quot;ג / מ&quot;ל ​​הפרין) MABT, להכין חומצה maleic 100mm; pH עד 7.5 עם NaOH כדורי. הוסף 150mm NaCl ו - 0.1% Tween-20. NTMT (עשה רק לפני השימוש), 50 מ&quot;ל: 1ml (NaCl 5M); 2.5 מ&quot;ל (טריס-2M HCl pH9.5); 1.25 מ&quot;ל (2M MgCl 2); 5 מ&quot;ל (10% Tween-20). TBST: טריס-HCl 0.1M, 0.15M NaCl, pH 8.45, 0.1% Tween-20. חלק 8: פתרון בעיות <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> בעיה </th><th> לגרום </th><th> תרופה </th></tr><tr><td valign="top"> העובר מכורבלת </td><td valign="top"> התייבשות התייבשות מספיק / </td><td valign="top"> שמור על 10 מרווחי mn במהלך התייבשות / התייבשות צעדים רצופים </td></tr><tr><td valign="top"> אין אות בדיקה </td><td valign="top"> בדיקה נפגעת זיהום RNase בבקבוקון </td><td valign="top"> הפעל בדיקה על 1% agarose ג&#39;ל ודא את הבקבוקון כולו חשוף הפתרון K proteinase בשלב 2 </td></tr><tr><td valign="top"> בדיקה היא תוויות חללים הלב בצינור העצבי </td><td valign="top"> בדיקה לכוד </td><td valign="top"> בשלב 2, ודא חללים כל microdissection מחורר באמצעות סכין או microcapillary </td></tr><tr><td valign="top"> העובר התפרקה הכלאה הבא (שלב 3) </td><td valign="top"> צעד proteinase K שמאל טמפרטורה הכלאה זמן רב מדי הוא גבוה מדי </td><td valign="top"> Proteinase K אסור להשאיר יותר 1mn (שלבים 3 + ו 4), 2 mn (שלב 5 עד 7), 3 mn (שלב 80-10), 4 mn (שלב 11-13) אל להכליא ב T o גבוה מ 65 מעלות צלסיוס </td></tr></tbody></table> חלק 9: תוצאות נציג עוברים נציג מוצגים להלן: (א) העובר (בשלב HH7) התווית בדיקה DIG-סוקס (כחול) לבין FITC-delta-1 בדיקה (אדום). (ב) העובר (בשלב HH8) התווית בדיקה DIG-Pax6 (כחול) בדיקה FITC-ששש (אדום). NF, עצביים לקפל, ANP, צלחת עצביים PSM הקדמי, והמזודרם presomitic, n, notochord, nt, בצינור העצבי). בדיקה מתנות במעבדות של בני הזוג. ג תבין, מ Goulding, ד אנריקה, וג&#39; בריסקו. <img src="/files/ftp_upload/old/904_2008_10_20_1/904_Figure-01.png" alt="" width="303" height="300" />

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

The authors have nothing to disclose.

הר 2 צבעים כולו בשיטת הכלאה באתרו משמש כדי לקבוע הן תבניות במרחב ובזמן של ביטוי גנים, תוך שימוש באחד או שני גנים של עניין. היישומים כוללים הערכת דפוסי ביטוי גנים של גנים הרומן (למשל 1,2, כמו גם שינויים עלבון גן הביטוי הבא (מניפולציות עובריים 3, 4 חרוזים, ו electroporation של רנ&quot;א או דנ&quot;א בונה 5).

<table><tbody><tr><td>Eggs</td><td>Animal</td><td>Charles River Laboratories</td><td>Premium Fertile</td><td></td></tr><tr><td>Stereomicroscope</td><td>Microscope</td><td>Leica Microsystems</td><td>MZ9.5</td><td></td></tr><tr><td>Marsh Automatic Incubator</td><td>Tool</td><td>Lyon</td><td>RX</td><td></td></tr><tr><td>Hybridization Incubator</td><td>Tool</td><td>Hybaid</td><td>Micro-4</td><td></td></tr><tr><td>Adams Nutator orbital mixer</td><td>Tool</td><td>Fisher Scientific</td><td>14-062</td><td></td></tr><tr><td>Curved Forceps (1)</td><td>Tool</td><td>Electron Microscopy Sciences</td><td>72991-4C</td><td></td></tr><tr><td>Fine scissors</td><td>Tool</td><td>Fine Science Tools</td><td>14161-10</td><td></td></tr><tr><td>Spoon</td><td>Tool</td><td>Fine Science Tools</td><td>10370-18</td><td></td></tr><tr><td>Pasteur Capillary Pipette</td><td></td><td>Electron Microscopy Sciences</td><td>70950-12</td><td></td></tr><tr><td>Microcapillary tube</td><td></td><td>Sigma-Aldrich</td><td>P1049-1PAK</td><td>Pull using vertical micropipette puller; blunt end with fine forceps</td></tr><tr><td>Microdissecting knife</td><td></td><td>Fine Science Tools</td><td>10056-12</td><td>Use to puncture cavities prior to in situ hybridization</td></tr><tr><td>Minuten pins 0.2mm diam</td><td></td><td>Fine Science Tools</td><td>26002-20</td><td>Keep pins together using a magnetic stirrer;</td></tr><tr><td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td><td>Reagent</td><td>Dow Corning</td><td>0001986475</td><td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td></tr><tr><td>Diethylpyrocarbonate (depc)</td><td>Reagent</td><td>Acros Organics</td><td>10025025</td><td>Add 1ml depc to 1l PBS; shake; autoclave</td></tr><tr><td>16% PFA</td><td>Reagent</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Tween-20</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P1379-100ML</td><td></td></tr><tr><td>Proteinase K</td><td></td><td>Sigma-Aldrich</td><td>P2308-5MG</td><td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td></tr><tr><td>Formamide, deionized</td><td>Reagent</td><td>Ambion</td><td>9342</td><td></td></tr><tr><td>Yeast tRNA-25 mg</td><td>Reagent</td><td>Invitrogen</td><td>15401-011</td><td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td></tr><tr><td>Heparin Sodium salt</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4784-250MG</td><td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td></tr><tr><td>SSC x20 pH5.5</td><td>Reagent</td><td>Fisher Scientific</td><td>PR-V4261</td><td></td></tr><tr><td>EDTA (0.5M)</td><td>PR-V4231</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>SDS</td><td>Reagent</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>Maleic acid</td><td>Reagent</td><td>Fluka</td><td>63189</td><td></td></tr><tr><td>B&amp;#8211;hringer Blocking Reagent</td><td>Reagent</td><td>Roche Group</td><td>11096176001</td><td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td></tr><tr><td>Heat inactivated sheep serum</td><td>Reagent</td><td>Jackson ImmunoResearch</td><td>013-000-121</td><td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Reagent</td><td>Roche Group</td><td>11093274910</td><td></td></tr><tr><td>NBT/BCIP stock solution</td><td>Reagent</td><td>Roche Group</td><td>11681451001</td><td></td></tr><tr><td>Anti-Fluorescein-AP, Fab fragments</td><td>Reagent</td><td>Roche Group</td><td>11426338910</td><td></td></tr><tr><td>2M Tris</td><td>BP1759-500</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Vector Red Alkaline Phosphatase Substrate Kit</td><td>Reagent</td><td>Vector Laboratories</td><td>SK-5100</td><td></td></tr><tr><td>1,2,3,4-tetrahydronaphthalene</td><td>Reagent</td><td>Sigma-Aldrich</td><td>429325-100ML</td><td>Under hood</td></tr></tbody></table>

double whole mount in situ hybridization of early chick embryos

עובר אפרוח הוא כלי רב ערך במחקר ההתפתחות העוברית המוקדמת. שקיפות, שלה נגישות וקלות מניפולציה, הופכים אותו לכלי אידיאלי ללימוד ביטוי גנים במוח, בצינור העצבי, somite היווצרות primordia לב. סרט הווידאו הזה מדגים את השלבים השונים בהר 2 צבעים שלם הכלאה באתרו, ראשית, העובר גזור מן הביצה ואת קבוע paraformaldehyde. שנית, העובר עיבוד עבור prehybridization. העובר היא הכלאה אז עם שתי בדיקות שונות, אחת יחד לחפור, ואף אחד מצמידים את FITC. בעקבות הכלאה בין לילה, העובר הוא מודגרות עם נוגדנים בשילוב לחפור. תגובת צבע על מצע DIG מבוצע, ואת האזור של עניין נראה כחול. העובר הוא מודגרות אז בשילוב עם נוגדנים FITC. העובר מעובד לתגובת צבע עם FITC, ואת האזור של עניין נראה אדום. לבסוף, העובר מקובע ומעובד phtograph ואת חתך. מדריך לפתרון בעיות מוצג גם.

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

סרט הווידאו הזה מדגים 2 צבעים הר שלם הכלאה באתרו, שיטה שבאמצעותה דפוס ביטוי במרחב ובזמן של 2 גנים שונים יכולים להיות דמיינו בעוברים אפרוח צעיר. שיטה זו הוצגה לראשונה על ידי דייוויד וילקינסון, דומינגוס אנריקה, פיל אינגהם ודוד איש-Horowicz.

הר שלם כפול באתרו הכלאה של עוברים צ&#39;יק מוקדם

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

double-whole-mount-in-situ-hybridization-of-early-chick-embryos

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K Views.  Institute of Biosciences and Technology - Texas A&M Health Science Center. סרט הווידאו הזה מדגים 2 צבעים הר שלם הכלאה באתרו, שיטה שבאמצעותה דפוס ביטוי במרחב ובזמן של 2 גנים שונים יכולים להיות דמיינו בעוברים אפרוח צעיר. שיטה זו הוצגה לראשונה על ידי דייוויד וילקינסון, דומינגוס אנריקה, פיל אינגהם ודוד איש-Horowicz.

Video: Double Whole Mount in situ Hybridization of Early Chick Embryos

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 ...

Developmental Biology

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

רב-יעד Chromogenic כל הר<em&gt; באתר</em&gt; הכלאה להשוואת תחומים ביטוי גנים ב<em&gt; דרוזופילה</em&gt; עוברים

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

שיטת מכתים נוגדן שלם הר ב צ&#39;יק

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

ניתוח של הגירה קרסט בידול עצבית ועל ידי חוצה מינים להשתלות

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

Wholemount ב באתרו הכלאה עבור עוברי Astyanax

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

יצירת כימרות במוח הקדמי של העופות להערכת התפתחות הפנים

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

הר שלם<em&gt; באתר</em&gt; הכלאה של E8.5 לעוברי E11.5 עכבר

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

מעקב אחר דפורמציות המוךפו&quot;גנטי שהולך רקמות בעובר צ&#39;יק מוקדם

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

הר כולו RNA פלורסנט<em&gt; באתר</em&gt; הכלאה של<em&gt; דרוזופילה</em&gt; עוברים

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

הר שלם ברזולוציה גבוהה<em&gt; באתרו</em&gt; הכלאה בתוך עוברי דג הזברה לחקר ביטוי גנים ותפקוד

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

הר שלם כפול באתרו הכלאה של עוברים צ'יק מוקדם

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הבנת organogenesis המוקדם שימוש פשוטה

רב-יעד Chromogenic כל הר

שיטת מכתים נוגדן שלם הר ב צ'יק

ניתוח של הגירה קרסט בידול עצבית ועל ידי חוצה מינים להשתלות

Wholemount ב באתרו הכלאה עבור עוברי Astyanax

יצירת כימרות במוח הקדמי של העופות להערכת התפתחות הפנים

הר שלם

מעקב אחר דפורמציות המוךפו"גנטי שהולך רקמות בעובר צ'יק מוקדם

הר כולו RNA פלורסנט

הר שלם ברזולוציה גבוהה