עבודה זו מומנה על ידי R01-R01 DE018234 ו-DE019638.

1. הכנת רקמות התורם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן התקשורת לתרבות, לחדד סיכות זכוכית, לחדד מחטים טונגסטן. </li><li style=";text-align:right;direction:rtl"> איסוף העובר מן המעטפת, להתרחץ קר כקרח PBS. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעזרת מזרק 10 מ&quot;ל ו - 18 מחט מד להסיר 1.0 מ&quot;ל של אלבומין מהקצה המחודד של מעטפת הביצית. </li><li style=";text-align:right;direction:rtl"> עושים חור קטן בחלק העליון של הקליפה באמצעות נקודת מספריים, ולאחר מכן לחתוך פתח עגול לחשוף את העובר </li></ol></li><li style=";text-align:right;direction:rtl"> הסר את הראש אל המקום DMEM (נסיוב בחינם, בטמפרטורת החדר) </li><li style=";text-align:right;direction:rtl"> לנתח תהליכים Frontonasal מעובר (או לאתר תורם אחרים, כגון תהליך לסתי או mandibular) </li><li style=";text-align:right;direction:rtl"> תקציר ב 2.4U / mL dispase ב PBS על הקרח עבור 20mins </li><li style=";text-align:right;direction:rtl"> כיסוי רקמות עם DMEM עם BSA 1% כדי לעצור העיכול </li><li style=";text-align:right;direction:rtl"> שימוש במחט טונגסטן חידד את האאקטודרם mesenchyme נפרד, ו neuroectoderm </li><li style=";text-align:right;direction:rtl"> חנות רקמת שתל ב BSA DMEM 1% על הקרח עד מארח מוכן להשתלה </li></ol> 2. הכנת מארח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחשוף את העובר <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעזרת מזרק 10 מ&quot;ל ו - 18 מחט מד להסיר 1.0 מ&quot;ל של אלבומין מהקצה המחודד של מעטפת הביצית. </li><li style=";text-align:right;direction:rtl"> עושים חור קטן בחלק העליון של הקליפה באמצעות נקודת מספריים. מניחים נייר דבק על החור, ולאחר מכן לחתוך פתח עגול לחשוף את העובר. </li></ol></li><li style=";text-align:right;direction:rtl"> שימוש 2 זוגות מלקחיים, לתפוס את amnion בעדינות דמעה לעשות חור. </li><li style=";text-align:right;direction:rtl"> סובב את ראשו על ידי הנחת זוג מלקחיים על עין ימין ועל הפעלת לחץ. בעוד מחזיק את הראש יציב, להשתמש במחט טונגסטן להסיר את האאקטודרם לארח כדי להתאים את השתל. </li><li style=";text-align:right;direction:rtl"> מעבירים את השתל לארח בעזרת פיפטה מזכוכית. </li><li style=";text-align:right;direction:rtl"> מיקום השתל להחליף את האאקטודרם הוסרו. הכנס סיכה זכוכית בכל פינה של הרקמה מורכבים להצמיד את השתל במקום. כדי להפוך את סיכות זכוכית, למשוך צינור microcapillary לנקודה קנס על אש אלכוהול. </li><li style=";text-align:right;direction:rtl"> מקום קלטת בחוזקה מעל החור ולהחזיר העובר אל האינקובטור לאורך זמן מתאים לניתוח. </li><li style=";text-align:right;direction:rtl"> ראה גם: 9 </li></ol> 3. נציג תוצאות: כדי להעריך מפלצות, עוברים יש לאסוף ולעבד לניתוח ההתפלגות של המארח רקמות התורם. עבור זיהוי של תאים שליו, אימונוהיסטוכימיה באמצעות נוגדן QcPN (המתואר: 3) הוא מועסק, וכן עבור זיהוי של תאים בעכבר, ב הכלאה באתרו על סעיפים פרפין משמש 6. תאים התורם צריך להיות מוגבל האפיתל, ולא צריך להיות שום עדות של רקמות התורם mesenchymal (איור 1). נוכחות של תאים מתורם בודד mesenchyme מצביע על נוכחות של תאים עצביים לפסגה זיהום אשר לבלבל כל פרשנות מורפולוגיים בשל השפעתם של תאים אלה על היבטים רבים של פיתוח פנים (למשל, 10). לאחר משוכנע כי הטכניקה שתל ללא זיהום mesenchyme, מדדי התוצאה מורפולוגי נוסף או מולקולרית ניתן להשתמש כדי להעריך את מפלצות. לענייננו, זיהינו את נוכחותו של סחוסים ועצמות מחוץ לרחם כי תאמו כפילויות של הלסת העליונה שהיו המושרה על ידי רקמות להשתלה (איור 2), ואת השינויים המולקולריים ברקמות mesenchymal בתגובה האאקטודרם מורכבים (איור 3) 5,6. <img src="/files/ftp_upload/2557/2557fig1.jpg" alt="איור 1" /> באיור 1. הערכת chimerism (א) נגד QcPN מכתים משמש כדי לזהות תאים שלווים שלווים, חומוס מפלצות. אין ראיות של מכתים ב mesenchyme. (ב) הכלאה באתרו משמש כדי לזהות את הביטוי של תעתיקים B2 סינוס ב עכבר חומוס מפלצות. הביטוי מוגבל האאקטודרם המרכיבים את השתל. <img src="/files/ftp_upload/2557/2557fig2.jpg" alt="איור 2" /> איור 2. מכתים Trichrome מראה שכפול של השלד הלסת העליונה הכימרה (א) שליו, חומוס, ו (ב) עכבר חומוס מפלצות. <img src="/files/ftp_upload/2557/2557fig3.jpg" alt="איור 3" /> איור 3. האאקטודרם regultes ביטוי גנים mesenchyme. (א) הביטוי רגיל Bmp7 ב mesenchyme של עובר אפרוח (רדיואקטיביים הכלאה באתרו עם אדום אות חיובי פסאודו בצבע ב Adobe Photoshop). (ב) הוא ביטוי Bmp7 המושרה mesenchyme הסמוך השתל (חץ) בעוברים chimeric.

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	<li>Noden, D. M. <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+the+Neural+Crest+in+Patterning+of+Avian+Cranial+Skeletal,+Connective,+and+Muscle+Tissues.">The Role of the Neural Crest in Patterning of Avian Cranial Skeletal, Connective, and Muscle Tissues.</a> Developmental Biology. 96, 144-144 (1983).</li><li>Bronner-Fraser, M., Stern, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Mesodermal+Tissues+on+Avian+Neural+Crest+Cell+Migration.">Effects of Mesodermal Tissues on Avian Neural Crest Cell Migration.</a> Developmental Biology. 143, 213-213 (1991).</li><li>Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+can+form+cartilages+normally+derived+from+mesoderm+during+development+of+the+avian+head+skeleton.">Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton.</a> Developmental Biology. 208, 441-441 (1999).</li><li>Couly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Hox-negative+cephalic+neural+crest+cells+and+the+foregut+endoderm+in+patterning+the+facial+skeleton+in+the+vertebrate+head.">Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head.</a> Development. 129, 1061-1061 (2002).</li><li>Evans, D. J., Noden, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+repetitive+mouse+B2+element+to+identify+transplanted+mouse+cells+in+mouse-chick+chimeras.">Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.</a> Exp Cell Res. 248, 75-75 (1999).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Eames, B. F., Schneider, R. 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אין ניגודי אינטרסים הכריז.

באמצעות שיטה זו השתלת אפשרה לנו לקבוע כי האאקטודרם מכיל מידע איתות המווסת את הקוטביות dorsoventral והסיומת proximodistal של הלסת העליונה. הדמיון של תוצאות בעת שימוש שליו או האאקטודרם העכבר השימור של אותות מולקולריים ברקמה זו בקרב מינים רבים 6,11 מציין כי מדובר במרכז איתות שמור ביותר בקרב בעלי החוליות. יתר על כן, חוקרים אחרים השתמשו בטכניקות דומות כדי לבדוק את המאפיינים של איתות באזורים שונים של פני השטח האאקטודרם cephalic ו חשפו את נוכחותו של מרכזי איתות מרובים הממוקמים האאקטודרם שתורמים המורפוגנזה הפנים 12. שיטה זו מאפשרת לנו להמשיך את הבנתנו את התפקיד רקמות שונות, כמו גם הבדלים אזוריים בתוך רקמות, לשחק במהלך התפתחות הפנים. לבסוף, על ידי שילוב העוצמה של העכבר הגנטיקה עם כוח של מערכת העופות, הכימרה נוכל להבהיר את המתווכים מולקולרית של אפיתל-mesenchymal אינטראקציות במהלך פיתוח, נוכל לזהות את המנגנונים שבאמצעותם אינטראקציות אלה מווסתים הפיתוח.

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assessing signaling properties of ectodermal epithelia during craniofacial development

הנגישות של עוברי העופות סייע embryologists ניסיוני להבין את גורלם של תאים במהלך ההתפתחות ואת תפקידו של אינטראקציות רקמות המווסתים דפוסים המורפוגנזה של בעלי חוליות (לדוגמה: 1, 2, 3, 4). כאן, אנו ממחישים שיטה זו מנצלת את הנגישות כדי לבדוק את איתות ותכונות דפוסים של רקמות ectodermal במהלך התפתחות הפנים. בניסויים אלו, אנו יוצרים שליו, חומוס 5 או עכבר חומוס 6 מפלצות ידי השתלת האאקטודרם משטח cephalic המכסה את הלסת העליונה של שליו או בעכבר על אזור או באותו אזור או מחוץ לרחם של עוברים חומוס. השימוש שליו כמו רקמות התורם להשתלה לתוך אפרוחים פותחה על מנת לנצל את הסמן nucleolar נוכח שליו אך לא תאים חומוס, ובכך מאפשר לחוקרים להבחין מארח ורקמות תורם 7. בדומה לכך, מרכיב חוזרות נמצא גנום העכבר, והוא הביע בכל מקום בגוף, אשר מאפשר לנו להבחין המארח רקמות התורם אפרוח ועכבר מפלצות 8. השימוש האאקטודרם עכבר כמו רקמות התורם יהיה מאוד להרחיב את ההבנה שלנו של האינטראקציות הללו רקמות, כי זה יאפשר לנו לבחון את המאפיינים של איתות האאקטודרם שמקורם בעוברים מוטנטיים שונים.

Watch this Scientific Journal Video about Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development at JoVE.com

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

מאמר זה מתאר השתלת רקמות טכניקה שנועדה לבדוק את איתות ותכונות של דפוסים האאקטודרם משטח cephalic במהלך הפיתוח craniofacial.

הערכת נכסים איתות של Ectodermal epithelia במהלך פיתוח Craniofacial

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

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9.4K Views.  University of California San Francisco. מאמר זה מתאר השתלת רקמות טכניקה שנועדה לבדוק את איתות ותכונות של דפוסים האאקטודרם משטח cephalic במהלך הפיתוח craniofacial.

Video: Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation של Mesenchyme Craniofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

ניתוח גנטי מהיר של איתות אפיתל-mesenchymal במהלך התחדשות שיער

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

מעקב נייד באמצעות חלבוני Photoconvertible במהלך פיתוח דג זברה

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

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 ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

הרבייה מכאנית מושרה גירוי סיד גל בmonolayers הסלולרי: דוגמה לתאי אנדותל קרני שור

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

ויזואליזציה של Craniofacial פיתוח בsox10: קאאד מהונדס דג הזברה הקו באמצעות זמן לשגות מיקרוסקופיה confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

מטבולית תיוג וממברנה היפוך חלוקה לניתוח השוואתי של proteomic<em&gt; Thaliana ארבידופסיס</em&gt; תא תרבויות השעיה

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

פורסים תרבות השיטה למעקב אחר ההתפתחות של חיידקים בשיני explant תרבות

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

ניתוח Craniofacial המורפוגנזה בדג הזברה שימוש 4D מיקרוסקופיה confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

הערכת תרומות מינים ספציפיות כדי Craniofacial פיתוח באמצעות מפלצות שליו-ברווז

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...