We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

هذا الإجراء هو إجراء 2 يوم. يوم 1 (2 ساعة + تشريح الوقت) 1. العذراء القرص تشريح العين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حدد خادرة للتشريح. ملاحظة: عمر خادرة أن تشريح ستحدده الاحتياجات التجريبية. ومع ذلك، إذا فحص مورفولوجيا الخلية، وهذا كثيرا ما يتم في 42 ساعة بعد تشكيل puparium (APF) عند 25 درجة مئوية، وهو عمر الشرانق يظهر في شريط الفيديو. جمع الشرانق الأبيض (APF اعتبر 0 ساعة) مع الفرشاة المبللة وترتيبها حسب التسلسل الزمني (على أساس جمع الوقت) في جديدة قارورة ذبابة المواد الغذائية حافظت على 25 درجة مئوية. تشريح الشرانق العمر في نفس هذا الترتيب الزمني للحفاظ على فروق التوقيت بين الشرانق في أقرب وقت ممكن. </li><li style=";text-align:right;direction:rtl"> نقل خادرة إلى قطرة من الفوسفات مخزنة المالحة (PBS، ودرجة الحموضة 7.4، انظر المواد للصفة) على لوحة سيليكون تشريح. </li><li style=";text-align:right;direction:rtl"> عقد الجزء العلوي من حالة العذراء مع ملقط وبيرس وفتح لOWER جزء من حالة العذراء مع زوج من ملقط حادة. إزالة خادرة من الحال في حفرة تشكلت حديثا. </li><li style=";text-align:right;direction:rtl"> اجراء خفض قطري مع مقص، منتصف الصدر، في خادرة. إزالة الحطام أو نقل رئيس خادرة إلى انخفاض جديد من برنامج تلفزيوني. نظيف باستخدام ماصة أنبوب منفاخ (انظر المواد والشكل 1 لمزيد من التفاصيل) وإضافة برنامج تلفزيوني إضافية حسب الحاجة. تأكد لتجنب الجفاف. </li><li style=";text-align:right;direction:rtl"> قبضة آمن الجزء العلوي من رأسه إهاب العذراء مع زوج من ملقط. باستخدام غيض من أنبوب منفاخ، ودفع بلطف على حالة العذراء لبثق المخ والأنسجة الأخرى، بما في ذلك أقراص العين، للخروج من حالة العذراء. تأكد من تجنب الاستيلاء على أي نوع من الأنسجة داخل بشرة. </li><li style=";text-align:right;direction:rtl"> باستخدام أنبوب منفاخ مليئة PBS، ضربة بلطف أنسجة الجسم الدهون وغيرها من الحطام للخروج من حالة الرأس لفصل بوضوح والتعرف على أقراص الدماغ والعين. ملاحظة: سيتم الحفاظ على الدماغ تعلق على حالة رئيس العذراء يسمح الباحث لانه يستخدمحالة الإعلان بأنه &quot;مقبض&quot; للتلاعب في المستقبل من الأنسجة دون الإضرار أقراص العين أو الدماغ. ومع ذلك، فإنه من الشائع لإزاحة أقراص الدماغ والعين من الرأس وهذه الحالة ليست ضارة لإجراء الشامل والنتائج. وعادة ما يستغرق عدة محاولات للنفخ ودفع لاستخراج بنجاح الأقراص العين والدماغ من داخل حالة رئيس العذراء وبحيث تكون منفصلة عن الأنسجة المحيطة بوضوح. وأنظف وأكثر واحد منفصل يمكن الحصول على أقراص العين من الأنسجة المحيطة بها سوف تساعد على منع الأنسجة الغريبة من تحديد إلى أقراص العين. </li><li style=";text-align:right;direction:rtl"> بعد التشريح، ونقل على الفور إلى الأنسجة طبق زجاج 3 جيدا على الجليد مع 0.5 مل من محلول فيكس (انظر المواد). تطفو على الأنسجة على السطح من الحل فيكس لبضع دقائق (عادة الوقت الذي يستغرقه لتشريح المجموعة التالية من أقراص العين). ملاحظة: إذا لم يكن هذا أول قرص العين ليتم تشريح في هذه الجولة من تشريح، completelذ غمر القرص العين السابق في حل فيكس. إن القيام بإجراءات تلطيخ الخاصة (على سبيل المثال، أنواع الاكسجين التفاعلية، الجسيمات الحالة، الخ) يجب أن يتم ذلك قبل التثبيت. من أجل القيام بذلك، تخزين أقراص تشريح العين في برنامج تلفزيوني الجليد الباردة قبل الاستمرار مع بروتوكول تلطيخ المناسب. </li><li style=";text-align:right;direction:rtl"> بعد تشريح وغمر جميع أقراص يجري تشريح العين، تحريك الطبق الزجاجي 3 بالإضافة إلى الروك والروك في درجة حرارة الغرفة لمدة 30 دقيقة. </li></ol> 2. المناعية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة بعناية الحل فيكس وإضافة 0.5 مل من برنامج تلفزيوني مع 0.3٪ تريتون X-100 (0.3٪ PBT، انظر المواد) وغسل الأنسجة على الكرسي الهزاز لمدة 10 دقيقة. كرر هذه الخطوة ثلاث مرات أكثر ليصبح المجموع 4 يغسل (10 دقيقة لكل منهما). ملاحظة: إزالة بعناية من الحل من البئر يمكن إجراء تشريح تحت المجهر لمنع تلف الأنسجة أو الخسارة. يمكن أن يستنشق الحل بعناية مع الماصة. نحن نفضل-tippe غرامةد، يمكن التخلص منها نقل الماصة التي تمت زيارتها فتح بالارض لتقليل فرصة الشفط الأنسجة. </li><li style=";text-align:right;direction:rtl"> في نهاية غسل 4، وإزالة غسل وإضافة 0.5 مل من محلول كتلة (انظر المواد) إلى البئر واحتضان لمدة 30 دقيقة. ملاحظة: يمكن تمديد هذا الإطار الزمني وفقا لاحتياجاتك. </li><li style=";text-align:right;direction:rtl"> في نهاية الوقت كتلة 30 دقيقة، الماصة 10 ميكرولتر من الأجسام المضادة الحل الابتدائية (مخففة في حل بلوك) إلى العدد المناسب من الآبار في لوحة microwell. ملاحظة: سيكون أحد بئر من لوحة microwell تستوعب حوالي 4 أزواج من أقراص العين التي تعلق على حالة العذراء أو 8 أزواج منعزلة من أقراص العين مع العقول المرفقة. الأجسام المضادة الأولية المستخدمة في هذا البروتوكول لبيانات تمثيلية هي مكافحة فسفوتيروزين (1: 500 تمييع) ومضادة للقطع (1: 200 تمييع). </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة تشريح في الآبار لوحة microwell مليئة الجسم المضاد الحل الأساسي من الطبق الزجاجي 3 أيضا. </li><li style=";text-align:right;direction:rtl"> مكانلوحة microwell على منشفة ورقية مبللة (لمنع الجفاف) في علبة فارغة طرف الماصة (أو حاوية أخرى مماثلة) وتخزينها في 4 درجات مئوية خلال الليل. </li></ol> يوم 2 (4 + ساعة الوقت التركيب) <ol start="6" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نقل الأنسجة من لوحة microwell إلى 3 جيدا طبق زجاج جديد مع 0.3٪ PBT (≈0.5 مل)، وغسل الأنسجة لمدة 10 دقيقة على الروك. كرر هذه الخطوة ثلاث مرات أكثر ليصبح المجموع 4 يغسل (10 دقيقة لكل منهما) </li><li style=";text-align:right;direction:rtl"> إزالة غسل الماضي وإضافة 0.5 مل من محلول بلوك إلى البئر. منع لمدة 30 دقيقة. ملاحظة: يمكن تمديد هذا الإطار الزمني وفقا لاحتياجاتك. </li><li style=";text-align:right;direction:rtl"> إزالة كتلة الحل وإضافة 0.5 مل من محلول الأجسام المضادة الثانوية (مصنوعة من الأجسام المضادة fluorescently المسمى المناسبة لالضد الخاص الابتدائي والطول الموجي الفلورسنت، مخففة في حل بلوك) إلى البئر وحماية طبق زجاج 3 جيدا من الضوء مع غطاء أو الألومنيوم احباط واحتضان لمدة لا تقل عن 2 ساعة في غرفةدرجة الحرارة. ملاحظة: جميع الخطوات اللاحقة ينبغي حماية بالمثل من التعرض للضوء. هذه الحضانة يمكن تمديدها ليلة وضحاها في 4 درجات مئوية، في الإعداد مماثل للحضانة الأجسام المضادة الأولية، ولكن يمكن أيضا أن يؤديها في طبق زجاج 3 جيدا مغطاة فيلم المختبر، أو لوحة microwell إذا كان المطلوب المحافظة على الأجسام المضادة. </li><li style=";text-align:right;direction:rtl"> في نهاية الحضانة الضد، وإزالة الأجسام المضادة الثانوية الحل وإضافة 0.5 مل من 0.3٪ PBT إلى البئر وتغسل على الروك لمدة 10 دقيقة. كرر هذه الخطوة ليصبح المجموع 4 يغسل (10 دقيقة لكل منهما). ملاحظة: إذا كان المطلوب تلوين دابي، قبل البدء في غسل الأولى، إضافة 0.5 ميكرولتر من محلول المخزون دابي (انظر المواد) إلى 500 ميكرولتر من PBT التي سيتم استخدامها لغسل الأولى (1: 1،000 التخفيف). </li></ol> 3. تركيب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في نهاية غسل الماضي، تركيب أقراص antennal / العين على شريحة باستخدام 70٪ الجلسرين أو برنامج تلفزيوني كوسيلة لإزالة رعين والأنسجة الأخرى غير المرغوب فيها في بركة على جانب واحد من الشريحة. </li><li style=";text-align:right;direction:rtl"> تحرك بحذر الأنسجة باستخدام الضغط الهيدروليكي بين ملقط، أو بلطف شديد مع طرف الملقط، ويصطف على أقراص العين في عمود نحو وسط الشريحة. </li><li style=";text-align:right;direction:rtl"> إزالة جميع الجلسرين دخيلة أو برنامج تلفزيوني من جميع أنحاء الأنسجة (باستخدام إجراءات فتل مختبر مسح أو ملقط لهذا العمل). </li><li style=";text-align:right;direction:rtl"> ضع كمية صغيرة (≈10 ميكرولتر) من تصاعد المتوسطة على طول جانب واحد من العمود قرص العين ووضع ساترة على الأنسجة وذلك لنشر المتوسطة المتزايدة على أقراص العين. </li><li style=";text-align:right;direction:rtl"> ترك الجلوس الشريحة من 1 إلى 2 دقيقة للسماح للأنسجة / متوسطة متزايدة لتنتشر بشكل كامل. </li><li style=";text-align:right;direction:rtl"> يمسح أي وسيلة إضافية متصاعدة خارج من حول حواف ساترة وختم ذلك مع طلاء الأظافر واضحة. </li><li style=";text-align:right;direction:rtl"> التقاط الصور الفلورسنت من أقراص العين باستخدام متحد البؤر المجهري. </li></ol>

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The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

مجالات البيولوجيا التطورية والخلايا قد تأثرت إلى حد كبير من قبل كائن نموذج: ذبابة الفاكهة السوداء البطن. ضمن هذا النموذج، وقد ساهمت الدراسات القرص العين على قدر كبير من المعرفة فيما يتعلق الإشارات، بيولوجيا الخلية وغيرها من المناطق. وقد درس الراحل ثالث اليرقات القرص الطور العين وعلى نطاق واسع هو نموذج قوي للاستفادة، كما أنه يعطي لمحة عن سلسلة من الفترات التنموية، مع كل جزيئاته الإشارات الفريدة الخاصة والعمليات، كما تقدم ثلم المخلق عبر القرص العين 8. ومع ذلك، هناك حاجة إلى توسيع فهمنا للعمليات التنموية في مرحلة العذراء للتنمية. في حين كانت هناك دراسات على القرص عين العذراء 3-7 والمعرفة لدينا لا تقترب من اتساع نطاق العمل الذي تم القيام بها على القرص الثالث الطور العين. ويرجع ذلك، في جزء منه، إلى صعوبة أكبر في تشريح العين القرص العذراء. لذا، عرضا للالأسلوب السليم للتشريح يمكن أن يتوسع كثيرا من البحوث في هذا المجال. في حين أن هناك مراحل في تطوير القرص عين العذراء التي يتم تشريح بسهولة، وخاصة حول فترة منتصف العذراء، وفترات زمنية أخرى هي أكثر تحديا لتشريح. هذا البروتوكول يمثل طريقة واحدة لتشريح أقراص عين العذراء التي يمكن استخدامها عالميا لجميع الأطر العذراء الوقت التنموية. هذا البروتوكول يمكن استخدامها كبديل للبروتوكول آخر 9 يبين طريقة أسهل وأسرع لتشريح أقراص العين من النقاط الزمنية midpupal. تم تصوير هذا البروتوكول أصلا وضعت لتدريب طلاب المرحلة الجامعية المتقدمة في جامعة كاليفورنيا الجامعية اتحاد البحوث في علم الجينوم الوظيفي (URCFG) 10،11 في تقنية العذراء تشريح العين. كانوا قادرين على الاستفادة من هذا الفيديو وطريقة لتعلم هذه التقنية الصعبة العديد من الطلاب الجامعيين.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>80g NaCl, 2g&#160; KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>Caution: Irritant! Wear gloves.</td>
		</tr>
		<tr>
			<td>0.3% PBT</td>
			<td></td>
			<td></td>
			<td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Fisher Scientific</td>
			<td>F75P1GAL</td>
			<td>Caution: Toxic, probable human carcinogen! Wear gloves.&#160;</td>
		</tr>
		<tr>
			<td>Fix Solution</td>
			<td></td>
			<td></td>
			<td>(&#8776;4% Formaldehyde in PBS) 50 &#956;l of 37% Formaldehyde, 450 &#956;l 1XPBS, make fresh before use</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Rockland antibodies &#38; assays</td>
			<td>B304</td>
			<td>Aliquot in 1 ml volumes and store at -80C</td>
		</tr>
		<tr>
			<td>Block Solution</td>
			<td></td>
			<td></td>
			<td>10% NGS in PBT.&#160; This can be made and stored at 4 &#176;C for a few days prior to use.</td>
		</tr>
		<tr>
			<td>DAPI stock solution</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O.</td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For mounting. Prepare 70% dilution with ddH2O.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Nutating mixer</td>
			<td>VWR</td>
			<td>82007-202</td>
			<td>Used to rock tissue in 3 well glass dish</td>
		</tr>
		<tr>
			<td>SylGard 182 Silicone Elastomer Kit</td>
			<td>Krayden</td>
			<td>NC9897184</td>
			<td>Used to make silicone dissection dish</td>
		</tr>
		<tr>
			<td>Silicone dissecting dish</td>
			<td></td>
			<td></td>
			<td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &#176;C incubator.</td>
		</tr>
		<tr>
			<td>3 well glass dish</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td>The 3 well variety of these are no longer available, this is the 9 well product.</td>
		</tr>
		<tr>
			<td>72 well microwell minitray</td>
			<td>Nunc</td>
			<td>438733</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps (Dumont #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td><a name="RANGE!A19">Vannas-type Micro Scissors, Straight, 5mm blade</a></td>
			<td>Ted Pella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td>
		</tr>
		<tr>
			<td>Disposable Transfer Pipets, Fine Tip</td>
			<td>Samco Scientific</td>
			<td>231</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td>
		</tr>
		<tr>
			<td>PVC tubing (1/8 x 3/16 x 1/32)</td>
			<td>Nalgene</td>
			<td>8000-0010</td>
			<td rowspan="3">Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00004</td>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00001</td>
		</tr>
	</tbody>
</table>

dissection and mounting of drosophila pupal eye discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

كمثال لاستخدام هذا البروتوكول، توضح النتائج midpupal (42 ساعة APF عند 25 درجة مئوية) أقراص العين immunostained مع الأجسام المضادة المختلفة ترد في الشكل 2. باستخدام الأجسام المضادة الموجهة ضد بقايا فسفوتيروزين، غشاء الخلايا يمكن أن يكون الملاحظ (الشكل 2A). وهذا يمكن أن تستخد...

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

تشريح وتركيب<em&gt; ذبابة الفاكهة</em&gt; العذراء أقراص العين

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.0K Views.  University of California at Los Angeles (UCLA). The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Video: Dissection and Mounting of Drosophila Pupal Eye Discs

Dissection of Drosophila Ovaries

تشريح المبايض ذبابة الفاكهة

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

تشريح الجهاز العصبي المركزي اليرقات في Melanogaster ذبابة الفاكهة

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

تشريح أقراص تخيلي من يرقات ذبابة الفاكهة طور مرحلي 3

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ذبابة الفاكهة تشريح اليرقات NMJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

تشريح وتلون ذبابة الفاكهة اليرقات المبايض

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

ذبابة الفاكهة المناعية العذراء البطن

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

التصوير من خلال حالة العذراء من<em&gt; ذبابة الفاكهة السوداء البطن</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

تشريح والمناعية من تخيلي أقراص من<em&gt; ذبابة الفاكهة السوداء البطن</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

فيفو السابقين الثقافة من ذبابة الفاكهة العذراء خصية واحدة ذكر الخراجات جرثومة خط: تشريح، التصوير، والمعالجة الدوائية

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

تشريح وشقة-تصاعد للThreespine أبو شوكة الخيشومي الهيكل العظمي

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...