עבודה זו נתמכה על איגוד כירורגי ילדים אמריקאי פרס קרן (AG) והמכון הלאומי לבריאות K08DK098271 (AG) bythe.

אוניברסיטת ויסקונסין טיפול בבעלי חיים ושימוש הוועדה אישרה את כל הנהלים. 1. הכנה של פתרונות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש פוספט 1x שנאגרו (PBS) כחיץ לנתיחה ופתרון שטיפה. </li><li style=";text-align:right;direction:rtl"> הכן סוכרוז 30% על ידי במשקל 30 גרם של סוכרוז ומקום לבקבוק. להוסיף 99 מיליליטר של 1x PBS ולהוסיף 1 מיליליטר 10% אזיד הנתרן. מערבבים היטב עד שכל סוכרוז הוא נמס. עד נדרשת חנות ב 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> הכן חסימת פתרון על ידי ערבוב 1x PBS, 3% אלבומין בסרום שור (BSA) ושל 0.1% Triton-X-100. מערבבים היטב ולאחסן במקרר עד צורך. </li><li style=";text-align:right;direction:rtl"> הכן 8% paraformaldehyde פתרון (PFA) ב1x PBS על ידי שקילת הכמות המתאימה של PFA לPBS 1x ולאחר מכן לדגור על 65 מעלות צלזיוס עד שהוא נמס לגמרי. חנות ב -25 aliquots מיליליטר ב-20 ° C במקפיא עד צורך. לדלל את PFA 4% ב- ​​PBS 1x ביום שימוש. </li></ol> 2. העובר וגוט לנתחיון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בהתאם למוסדי טיפול בבעלי חיים ושימוש הוועדה אישר פרוטוקולים, להרדים עכבר בהריון בעיתוי ולהעביר את הרחם לקרח קר המכיל 60 מ&quot;מ צלחת פטרי 1x PBS. </li><li style=";text-align:right;direction:rtl"> תחת מיקרוסקופ לנתיחה באמצעות מספריים חדים, לחתוך את דופן הרחם הפתוחה לחשוף את העוברים. הסר את העוברים מן הרחם והמקום ל-60 מ&quot;מ צלחת פטרי המכילה נקייה קר כקרח 1x PBS. </li><li style=";text-align:right;direction:rtl"> להרדים כל עובר על ידי עריפת הראש ב1x קר כקרח PBS. אם אתה משתמש בעכברים מהונדסים המכילים חלבוני ניאון, תחת תאורת הקרינה, לזהות את העוברים מהונדסים החיוביים. </li><li style=";text-align:right;direction:rtl"> לנתח את מערכת עיכול מכל עובר באמצעות מיקרוסקופ לנתיחה. בעזרת מלקחיים בסדר, לכוון את העובר כך שצד השמאל פונה כלפי מעלה וצד ימין הוא נגד החלק התחתון של צלחת פטרי. הסר את קיר הגוף העליון מהעובר כדי לחשוף את האיברים הפנימיים. הכנס מלקחיים עדינים בין קיר גוף גב והאיברים הפנימיים. CrOSS מלקחיים אחד נגד השני בפעולת חיתוך כמו מספריים כדי להסיר את האיברים הפנימיים של העובר. </li><li style=";text-align:right;direction:rtl"> בהמשך תת-לנתח את מערכת העיכול מהאיברים שמסביב ואז למקם את כל מערכת עיכול לתוך צינור microcentrifuge 1.5 מיליליטר המכיל קר כקרח 1x PBS. </li></ol> 3. קיבוע של קטעי GI <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יש לשטוף את כל מערכת עיכול 3 פעמים עם 1x קר כקרח PBS ולאחר מכן להחליף עם PFA 4%. תקן את שטחי GI בצינורות 1.5 מיליליטר microcentrifuge על פלטפורמת נדנדה ב RT תמורת 1.5 שעות. יש לשטוף את שטחי GI 3 פעמים במשך 5 דקות ב RT ולאחר מכן במשך שעה 1 על פלטפורמת הנדנדה. בשלב זה, לאחסן קטעי GI על 4 מעלות צלזיוס בסוכרוז 30% ב 1x PBS המכיל 0.1% אזיד הנתרן עד צורך. הערה: לחלופין, לאחסן את העוברים בסוכרוז 30% לשנה עד אחת ללא כל השפעה על שלמות הרקמות. אחסון של הדגימות בסוכרוז 30% מאפשר עיבוד מאוחר יותר של הדגימות או לimmunostaining או לאוקטובר לcryo-מדור שng. לחלופין, להמשיך בפרוטוקול immunostaining מפורט להלן. </li></ol> 4. Immunostaining פרוטוקול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם דגימות אוחסנו ב -30% סוכרוז, לשטוף אותם 3 פעמים במשך 20 דקות ב1x PBS על פלטפורמת נדנדה. </li><li style=";text-align:right;direction:rtl"> מניחים את שטחי GI לפתרון חסימה על פלטפורמת נדנדה 1h ב RT. </li><li style=";text-align:right;direction:rtl"> הסר את פתרון חסימת דגירה שטחי GI עם הכמות המתאימה של נוגדנים עיקריים מדולל חסימת פתרון גם עבור 4 שעות ב RT או O / N ב 4 ° C על פלטפורמת נדנדה. השתמש 1: 1,000 דילול של נוגדן אנושי אנטי-הו (סרום המתקבל ממטופל), 1: 1,000 דילול של נוגדן עוף אנטי-ירוק חלבון פלואורסצנטי (GFP) ו -1: של נוגדן אנטי צ&#39;אט עז 100 דילול. הערה: אנו מנצלים נוגדן אנטי-הו אנושי שהושג באופן מקומי ממטופל, לעומת זאת, נוגדנים נגד הו זמינים מסחרי, למשל, עכבר אנטי-הו, (שימוש ב1: 500 דילול). </li><li style=";text-align:right;direction:rtl"> יש לשטוף את שטחי GI ב1x PBS3 פעמים במשך 5 דקות ולאחר מכן עבור שעה 1 ב RT על פלטפורמת נדנדה. </li><li style=";text-align:right;direction:rtl"> החלף את PBS 1x עם נוגדנים משני בדילול 1: 500 בחסימת פתרון על פלטפורמת נדנדה לאו 4 שעות על RT או O / N ב 4 מעלות צלזיוס. השתמש צבע אמין-reactive אנטי אנושי חמור כגון DyLight, Cy2 אנטי-עוף חמור וCy5 אנטי-העז החמור. אם נוגדן אנטי-הו העכבר באמצעות לאחר מכן להשתמש דילול 1: 500 של צבע אמין-reactive החמור אנטי עכבר 405. הערה: עוצמת GFP אנדוגני וביטוי tdTomato והבהירות של immunostaining עלייה עם גיל התפתחותי. אנחנו בדרך כלל immunostain אומץ עוברי בנפרד ב0.2 מיליליטר צינורות עם 150 μl של פתרון מכתים במטרה לצמצם את הנפח של נוגדן המשמש וגם כדי להבטיח צביעה יעילה של הרקמות. </li><li style=";text-align:right;direction:rtl"> יש לשטוף את שטחי GI בPBS 3 פעמים 1x למשך 5 דקות ולאחר מכן עבור שעה 1 ב RT על פלטפורמת נדנדה. </li><li style=";text-align:right;direction:rtl"> מניחים כמה טיפות של מדיום הרכבה הקרינה עם DAPI על גבי זכוכית. לטבול את tra GICT להרכבה בינוני הקרינה ולהוסיף מכסה זכוכית ישירות על גבי הרקמה. הקרינה ההרכבה -G הבינוני היא תרכובת מסיס במים המספקת חותם חצי קבוע. הערה: הרכבה בינונית הקרינה השימוש כגון Vectashield שהוא גליצרול מבוסס הרכבה בינונית שמונע דהייה וphotobleaching של נוגדנים. שני מוצרים אלה מאפשרים רקמות להיות מאוחסנות במשך תקופות ארוכות של זמן על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> צלם תמונות של כל אחד מfluorophore באמצעות מיקרוסקופ confocal. השתמש באורכי גל עירור לfluorophores ומסננים המועסקים מוצגת בלוח 3. </li><li style=";text-align:right;direction:rtl"> לבצע ניתוח תמונה בעזרת מחשב באמצעות תוכנה מתאימה, בהתאם לסוג של confocal משמש ללכידת תמונות. </li></ol>

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The authors have nothing to disclose.

המעבדה ואחרים שלנו הראו כי ליקויים במערכת העיכול בHSCR אינם מוגבלים למעי גס aganglionic אבל הרחיבו proximally, אפילו למעי הדק ganglionated 5,15,16. שינויים אלה כוללים שינויים בפנוטיפ צפיפות והעצבי העצבי ENS ועשויים להסביר dysmotility שנצפה בחולים עם HSCR. יש לנו מנוצלים הטכניקות לעיל במאמצי...

מערכת תפקוד מעי עצבים (ENS), השולטת בתנועתיות, מזין קליטה, וזרימת דם מקומית, היא חיונית לחיים 1. ENS נוצר על ידי תאים עצביים פסגה (NCC) שמתרבים, להעביר וליישב את הבטן, שבו הם להתמיין הנוירונים הגרעינים המכילים ותאי גלייה. מחלת הירשפרונג (תורשה של מנדל HSCR, באינטרנט באיש), הפרעה מולדת multigeneic עם שכיחות של 1 ב4,000 לידה חי, יכולה להיחשב המחלה אבטיפוס ללימוד היווצרות ENS שיבש. בHSCR, NCC לא מצליח להעביר ולליישב אורכים משתנה של המעי האחורי דיסטלי 2. בנוסף, במערכת העיכול נפוצה אחרת (GI) פגמים התפתחותיים באוכלוסיית הילדים, כגון מומים אנו-רקטליות, atresias מעיים, והפרעות תנועתיות קשורות עם הפרעות בתפקודים בסיסיים ENS, וצפויה הקשורים בשינויים קלים, אינם מוערכים, אנטומיים ושינויים תפקודיים ב3-6 ENS. לכן, טכניקות שתאפשרנה לנו להבין את הגורמים התפתחותיים של היווצרות ENS עשויות לשפוך אור על הפתוגנזה וטיפול פוטנציאלי של הפרעות בדרכי עיכול בילדים. בעקבות הגירה והתישבות, NCC מבדיל לתוך הנוירונים עם סמנים ספציפיים לפנוטיפ העצבי שלהם. נוירונים כולינרגית מהווים כ 60% מתאי עצב מעיים 7, ויכולים להיות מזוהים על ידי מכתים לacetyltransferase כולין (צ&#39;אט), אנזים הסינתזה להנוירוטרנסמיטר אצטילכולין מעורר. מבחינה הסטורית, מנסה לדמיין נוירונים כולינרגית היו מבולבל על ידי שונה סגוליות אנטיגן של נוגדנים המכוונים נגד מערכת עצבים מרכזית (CNS) צ&#39;אט לעומת מערכת עצבים היקפית (PNS) צ&#39;אט 8-10. עם זאת, נוגדנים המכוונים נגד צ&#39;אט שליה להכיר שני צ&#39;אט המרכזי והיקפי 11-13, ויש לנו טכניקות מתוארות לאחרונה המאפשרות לvisualization של נוירונים כולינרגית ENS עם רגישות גבוהה מוקדם יותר בפיתוח מאשר הושג עם קווי כתב צ&#39;אט 14. כאן, אנו מציגים טכניקה לנתח, לתקן וimmunostaining של מערכת העיכול עוברי עכברית לדמיין ביטוי עצבי ENS בתאי עצב. למחקרים אלה, שנוצלנו עכברי הצ&#39;אט-Cre הזדווגו עם R26R: בעלי חיים tdTomato לייצר צ&#39;אט-Cre; R26R: floxSTOP עכברי tdTomato (המוגדרים כצ&#39;אט-Cre tdTomato ברחבי כתב היד): floxSTOP. בעלי חיים אלה ולאחר מכן היו הזדווגו עם עכברי כתב צ&#39;אט-GFP הומוזיגוטים, להשיג עכברים להביע שני כתבי ניאון המזהים ביטוי צ&#39;אט 14. שני בעלי חיים אלה הם כתב על רקע C57BL / 6J והם זמינים מסחריים (ג&#39;קסון מעבדות, בר הרבור, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining to visualize murine enteric nervous system development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

שתארנו בעבר את הדור של עכברים המבטאים את שני כתבי ניאון GFP וtdTomato המזהים ביטוי צ&#39;אט 14. בקצרה, עכברי צ&#39;אט-Cre זווגו עם R26R: floxSTOP: בעלי חיים tdTomato לייצר צ&#39;אט-Cre; R26R: עכברי tdTomato (נקראים צ&#39;אט-Cre tdTomato): floxSTOP. בעלי חיים אלה ולאחר מכן היו הזדוו?...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

צביעה חיסונית כדי להמחיש את התפתחות מערכת העצבים האנטרית של ה- Murine | ביולוגיה התפתחותית | וִידֵאוֹ

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining לדמיין פיתוח מערכת עצבים Murine מעיים

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

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11.3K Views.  University of Wisconsin School of Medicine and Public Health. The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Video: Immunostaining to Visualize Murine Enteric Nervous System Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

מותאם באופן מיטבי<em&gt; Ex-אובו</em&gt; Culturing של Chick עוברים לשלבים מתקדמים של פיתוח

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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 ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

רומן חדש פאט שומן טכניקת השתלת לדמיין את כלי השיט הדור של כלי הדם בתאי גזע אנדותל

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

פוטון רב זמן לשגות הדמיה כדי להמחיש את התפתחות בזמן אמת: ויזואליזציה של העברת תאי הרכס העצבי עוברי דג זברה

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

שיטת Organotypic תרבות ללמוד על התפתחות עובריים עוף רקמות

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

ניתוח חישובית של Germline Caenorhabditis elegans ללמוד הפצה של גרעינים, חלבונים ו את שלד התא

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

תרביות רקמה השחלות כדי לחזות תופעות העכבר השחלה

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

דמיינו דרוזופילה הרגל מוטור נוירון אקסונים דרך הקוטיקולה למבוגרים

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

3-מימדי אנגיוגנזה שיטת מערכת באמצעות Spheroids שיתוף תרבות נוצר על ידי ויוצרים מושבת המושבה תאים ותאי גזע Mesenchymal

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

הר שלם בתוך היברידיזציה ב-Zebrafish עוברים ושיטת היווצרות צינור ב-iPSC-ECs ללמוד את התפקיד של אנגלין בפיתוח כלי דם

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...