We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

Ovarioles 1. 해부 <ol><li> 추가 활성 건조 효모와 신선한 플라이 식품 유리 병에 약 15 ~ 4 일 된 여성 파리와 몇 명의 남성을 전송합니다. 바이알을위한 마개로 솜을 사용하여, 높은 습도를 유지하는면에 몇 방울의 물을 추가한다. </li><li> 14-16 시간 동안 25 ° C의 배양기에서 찾는 바이알 스테이지 8-10 달걀 챔버의 개수를 최대화한다. 참고 : 배양 시간은 온도와 원하는 단계에 따라 달라집니다. </li><li> 다음날에 필요한 해부 미디어를 준비, 0.1M KPO 4 NP40 솔루션 (자료 참조). </li><li> CO 2를 사용하여 파리를 마취하고, 해부 현미경으로 배치합니다. 피펫 유리 우울증 슬라이드의 각 공동으로 해부 미디어를 몇 방울. </li><li> 한 여성 비행 아래로 날개를 가진 테이블 상단과 동양에 약 30 ° 각도로 각 손에 집게를 잡습니다. 당신의 비 지배적 인 손으로, 그녀를 잡고, 집게를 사용하여 여성을 데리러흉부 근처 복부의 nterior 및 우울증 슬라이드 (그림 1A 인세 트)의 해부 미디어에 그녀의 잠수함. </li><li> 포셉의 다른 쌍으로, 복부의 복부 후방에서 외골격을 잡고 관통. 후방 끝에 난소를 잡고, 다음 복부에서 그들을 끌어와 우울증 슬라이드의 반대편에 신선한 미디어에 배치합니다. (그림 1A 인세 트). 장 떨어져 당겨하지 마십시오. 시체를 버리십시오. </li><li> 모든 난소를 해부 할 때까지 다른 여성 파리로 반복합니다. 남성을 폐기하십시오. </li><li> 포셉 한 쌍, (가장 큰 달걀 챔버 근처) 후방 끝에 난소를 잡고 다른 손으로 가장 앞쪽에 계란 챔버 (germarium) 중 하나를 꼬집어 집게 한 쌍을 사용합니다. (도 1a). </li><li> 천천히 그리고 꾸준히, 난소 칼집에서 ovariole 체인을 당깁니다. 원하는 모든 계란 챔버 R 때까지 개별적으로 ovarioles를 해부 계속emoved. </li><li> 반복하여 모든 해부 난소에 대한 1.7-1.8 단계를 반복합니다. </li><li> 완료되면, 플라스틱 전송 피펫을 사용하여 0.6ml 튜브에 ovarioles를 전송할 수 있습니다. </li><li> ovarioles는, 튜브의 바닥에 정착 한 후 고정 및 염색 단계로 진행하자. </li></ol> Ovarioles 2. 면역 형광 (IF) 염색 <ol><li> 조심스럽게 피펫 ovarioles에서 해부 용지를 제거합니다. 어떤 ovarioles 또는 달걀 챔버를 제거하지 마십시오. ovarioles 및 해부 미디어의 약 50 μl를 남겨주세요. </li><li> 튜브에 0.1 M KPO 4 버퍼의 150 μl의 16 % 파라 포름 알데히드의 50 μl를 추가하고이를 해결하기 위해 계란 챔버에 솔루션을 추가합니다. 10 분 동안 흔들 동안 품어. 주의 : 파라 포름 알데히드는 독성이있다. </li><li> 정착액을 제거하고 0.5 ml의 NP40 세척 버퍼로 두 번 씻어. </li><li> RT에서 15 분 각각 두 번 씻으십시오. </li><li> 다음 단계에 대한 표준 IF 프로토콜 15을 참조하십시오. <ol><li> 간단히, 고정 후, 추가적절한 희석에 차 항체와 4 ° C에서 O / N을 품어. (200 희석 1) 다음 이차 항체를 추가, NP40 여러 번에 씻으십시오. </li><li> NP40에서 여러 번 세척 한 후 DAPI (1 : 1,000)를 추가 10 분 동안, 다음 세척을 반복합니다. 프로토콜이 완료되면 일단, 계란 챔버에 PBS에 50 % 글리세롤 200 μl를 추가하고 2 시간 동안 실온에서 앉아 보자. </li><li> , 용액을 제거 PBS에 70 % 글리세롤로 교체하고, 호일 커버. 설치 준비가 될 때까지 4 ᵒC에서 샘플을 놓습니다. 한편, 3 단계를 계속합니다. </li></ol></li></ol> 글리세린 젤리 슬라이드 3. 사전 준비 <ol><li> 글리세린 젤리 주걱을 이용하여 적어도 4 mL로 측정하고 반쯤 마크에 유리 셀 배양 관을 채운다. 30 분 동안 55 ℃에서 수욕 글리세린 젤리 녹아. </li><li> 두 현미경 슬라이드 상 스톡 용액으로부터 글리세롤의 작은 방울, 약 300 μL를 붓는다. 조직을 사용하여 얇은 라 글리세롤 확산yer의 각 슬라이드에 걸쳐. 이 기반을 제공하고 단계 5.6에서 글리세린 젤리 쉽게 제거 할 수 있습니다. </li><li> 필터링 된 1,000 μL 피펫 팁 오프 끝을 잘라. 사용 컷 피펫 팁 천천히 각 현미경 슬라이드에 따뜻한 글리세린 젤리 1,000-1,500 μl를 전송합니다. 거품을 방지하기 위해 필요한주의를 기울입니다. </li><li> 글리세린 젤리 슬라이드 설정 실온에서 5 분 동안 앉아 보자. </li><li> 60mm × 15mm 페트리 접시에 각각의 글리세린 젤리 슬라이드를 삽입하고 플라스틱 포장으로 덮고. 4 ° CO / N에서 장소. 선택적으로, 상점은 4 ° C에서 최대 5 일 동안 젤리 슬라이드 글리세린. </li></ol> 4. 계란 챔버 장착 <ol><li> 단계 2.5, 하나의 글리세린 젤리 슬라이드에 걸쳐 70 % 글리세롤 계란 챔버의 피펫 여러 3 μL 상품에서 샘플을 사용. 모든 계란 챔버이 슬라이드 때까지 피펫 3 μL 상품을 계속합니다. 각각의 강하가 적어도 10 달걀 챔버가 포함되어 있는지 확인합니다. 한편, 55 ° C의 물을 욕조에 글리세린 젤리의 문화 튜브를 가열. </li><li> 해부 현미경으로 계란 챔버와 글리세린 젤리 슬라이드를 놓습니다. 계란 챔버의 한 방울의 시야 (도 1b)에 있도록 배율을 조정한다. </li><li> 원하는 단계 달걀 챔버를 데리러, 숟가락으로 30 게이지 바늘을 사용. 시 칼 등을 이용하여 바늘 ovariole 체인에서 필요한 별도 소망 달걀 챔버. </li><li> 영상을 방해 할 수있는 파편을 피하기 위해, 왼쪽 (그림 1E -1)에 직면 전방에, 두 번째 글리세린 젤리 슬라이드에 계란 챔버를 전송합니다. 적어도 7 계란 챔버 열 (그림 1C)에 줄 지어있을 때까지 반복합니다. </li><li> 원하는 모든 계란 챔버가 제 2 글리세린 젤리 슬라이드로 전송 될 때까지 일곱의 새 열을 형성합니다. </li><li> 조직과 트위스트의 작은 조각을 떼어냅니다. 가볍게 하나 하나를 만져 계란 챔버에서 초과 PBS-글리세롤을 흡수하는 데 사용합니다. 달걀 챔버를 제거하지 않도록주의하십시오. </li><li> 잘라필터링 된 200 μL 피펫 팁의 끝. 계란 챔버의 모든 열을 충당하기 위해 젤리 글리세린의 150 μl를 전송하는 데 사용합니다. 주 : 칼럼을 커버하기 위해 필요한 글리세린 젤리의 양을 알 챔버 단계 및 열 번호에 기초한다. 사용량이 적절히 조절 될 수있다. </li><li> 글리세린 젤리 상부층 완전히 (도 1D) 고화 될 때까지 장착 달걀 챔버 RT에서 5 분 방치. </li><li> 장소 60mm × 15mm 페트리 접시에 장착 된 계란 챔버 슬라이드 및 플라스틱 포장으로 덮고. 4 ° CO / N 스토어는 (photobleaching에 대한 우려가있는 경우 어두운 장소) 글리세린 젤리 층이 함께 응고 허용합니다. </li></ol> 이미징 계란 챔버 5. 준비 <ol><li> 해부 현미경 아래에 장착 계란 챔버의 슬라이드를 놓습니다. 계란 챔버 한 열만이 시야에 있도록 배율을 조정한다. </li><li> 45 ° 각도 소형 메스를 사용하여, straig를 잘라(계란 챔버의 후방에 가까운 측) 달걀 챔버의 첫 번째 열의 우측에 HT 라인. (그림 1D-E) </li><li> 다음에, 열의 마지막 달걀 챔버 맨 먼저 계란 챔버의 상하 절단. 이 시점에서 달걀 챔버 열 삼면 절단 예정. </li><li> 계란 챔버 (도 1E -3)의 전방 측에 최대한 가깝게 microknife 열의 왼쪽의 슬라이스를 이용. </li><li> 절단 된 열을 통해 감기 1X PBT 100 μl를 피펫. </li><li> 슬라이드에 나머지를 떠나, 계란 챔버 및 폐기의 절단 열 세 가지 측면 주위에 여분의 글리세린 젤리를 제거하기 위해 둥근 가장자리 주걱을 사용합니다. </li><li> 계란 챔버의 전방 측에 만든 컷 주걱을 삽입하고 기본 글리세린 젤리 블록에서 열을 구분합니다. </li><li> 샘플 중 하나를 반전 (5.8.1) 또는 수직 (5.8.2) 현미경을 준비합니다. <ol><li> 거꾸로 현미경의 경우 : 전송 t계란 챔버의 앞쪽면이 아래로 향하게하여 커버 슬립에 계란 챔버 그는 열입니다. 글리세린 젤리의 모든 열 제거 및 장착 될 때까지 반복 5.2-5.8 단계를 반복합니다. 마르지 않도록 달걀 챔버의 각 블록에 50 % PBS-글리세롤의 몇 방울을 추가합니다. 직접 커버 슬립에 이미지 계란 챔버. </li><li> 직립 현미경의 경우 : 전방면이 위로 향하게하여 현미경 슬라이드에 계란 챔버의 열을 전송합니다. 글리세린 젤리의 모든 열을 제거하고 현미경 슬라이드에 장착 될 때까지 반복 5.2-5.9 단계를 반복합니다. # 1 ½ coverslip에 계란 챔버 커버 블록의 각 블록에 50 % PBS-글리세롤의 몇 방울을 추가합니다. </li></ol></li></ol>

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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+strategies+for+protein+trapping+in+Drosophila.">Exploring strategies for protein trapping in Drosophila.</a> Genetics. 175 (3), 1089-1104 (2007).</li><li>Buszczak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carnegie+protein+trap+library:+a+versatile+tool+for+Drosophila+developmental+studies.">The carnegie protein trap library: a versatile tool for Drosophila developmental studies.</a> Genetics. 175 (3), 1505-1531 (2007).</li><li>Belu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upright+imaging+of+Drosophila+embryos.">Upright imaging of Drosophila embryos.</a> J Vis Exp. (43), (2010).</li><li>Witzberger, M. 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The authors have no competing financial interests.

여기서 우리는 말에 관점에서 계란 챔버를 개발, 탑재하는 방법을 설명하고 이미지 작은. 영상 계란 챔버의 일반적인 기술은 측면 플레이에 최적화 된 형광 항체 염색을 할​​ 때 주로 메디 오 - 측면 모낭 세포의 정확한 시각화를 허용하고 있습니다. 여러 초점면을 볼 수있는 Z-스택 또는 3 차원 재구성 보조기구의 사용,하지만 여전히 타원형의 달걀 실 (그림 2D)의 극의 서브 세포 해?...

초파리의 연구 현상의 넓은 범위의 유전자 조절에 큰 통찰력을 제공하고 있습니다. oogenesis 티슈 패터닝 셀 극성의 변화, 세포주기 스위칭, 및 병진 규제 1,2,3,4- 등 다양한 발달 과정을 조사하기 취급 용이 한 방법을 제공하기 때문에 특히, 계란 개발에 대한 광범위한 연구가있어왔다. oogenesis 때 중요한 형태 발생 이벤트 (5 검토) 테두리 세포라는 세포의 집합의 명세서, 운동성의 취득 및 마이그레이션이다. 세포 이동하기 때문에 동물 형태 발생의 중요한 특징이며,이 프로세스의 유전자 조절이 잘 보존되어 있기 때문에, 파리 결정 메커니즘은 다른 상황에서 중요 할 것으로 보인다. 따라서, 우리는 경계 세포 이동의 분자 제어를 조사하고있다. 우리의 연구를 위해, 우리는 계란을 개발하는 전방 극을 관찰 할 수있는 새로운 방법을 개발했다,경계 셀이 발생하는 경우, 그들이 어떻게 발달 상세히 검토한다. 난소 내에서 다양한 발달 단계에서 계란 챔버는 얇은 외피 (그림 1A)에 싸여있다 ovarioles라는 체인을 따라 존재한다. 각 달걀 챔버는 한 계란을 형성에 갈 것입니다. oogenesis 동안 여러 종류의 세포 조화롭게 개발해야한다. D. 달걀 melanogaster의 챔버는 단일 모낭 상피 층 (6)에 의해 둘러싸인 하나의 난자를 포함한 16 생식계 세포로 구성된다. 극성 세포라는 특수한하는 소수의 세포는 상피의 전후방 극에서 발생한다. 6-8 테두리 세포 극성 세포에 의해 유도 된 5,7- 달걀 챔버의 전방 상피에서 발생. 중간 oogenesis (단계 9)에서, 경계 셀들이 이웃으로부터 분리하고 계란 5,7- 챔버의 후방에 도달하는 난자 세포 간호사 사이에 이동한다. 이 운동은 달성해야테두리 세포 집단이 세포 이동의 유형 만드는 두 운동성 극성 주변 세포 클러스터에 남아있다. 경계 셀 클러스터의 성공적인 마이그레이션 수정에 필요한 달걀 껍질의 micropyle의 적절한 개발을 보장합니다. 전방 극성 세포 신호 전달 캐스케이드를 활성화하여 경계 셀 운명 지시. 폴라 난자 세포의 발달 8,9 스테이지 (8) 중에 모낭 인접 셀에, 경막 수용체 Domeless (DOME)에 결합하는 사이토킨으로 독립 (UPD)를 분비한다. UPD의 결합은 야누스 티로신 키나제 (JAK)의 전사 (STAT) 8,10,11의 신호 변환기 및 활성제를 인산화됩니다. STAT는 전사를 활성화하기 위해 핵으로 이동합니다. 느린 테두리 세포 (SLBO)는 STAT의 직접 전사 대상이며, 또한 테두리 세포 이동 (12)에 요구되는 전사 인자이다. 계란 챔버의 측면보기는 t을 나타냅니다모자 STAT 활동은 전방 상피 8,11,13에 걸쳐 그라데이션으로 조절된다. 극성 세포에 가장 가까운 여포 세포 따라서 그들이 경계 셀과 인접되어 생식계 조직 침입, STAT 활성화의 가장 높은 수준을 갖는다. 국경 세포 내에서 지정 및 상피 세포에서 분리하는 방법을 이해하기 위해, 우리는 조직이 어떻게 구성되어 있는지 관찰해야합니다. 우리가 전방-에 관점에서 계란 챔버를 보면, 우리는 극성 세포를 둘러싸고있는 여포 세포에서 STAT 활동의 반경 대칭을 기대. 최종에서보기는 더 정확하게 이전에 다른 초점면에 세포를 비교하는 것보다 분리하는 동안 막 단백질과 세포 - 세포 인터페이스의 차이를 보여 것입니다. 달걀 챔버 직사각형과 줄기 세포에 의해 서로 연결되어 있기 때문에 어려운 전방 구조를 관찰하기 위해, 측 방향 슬라이드 상에 정착. 따라서, 달걀 챔버의 극의 세포에 대한 많은 정보가 있습니다측면 뷰에서 유추 된. 일부 정보는 photobleaching에 광학 부, 광 산란의 알고리즘 3-D 재구성 및 Z 축에서의 해상도 불량한 한계를 통해 얻어 질 수 있지만, 초 - 해상도 현미경 (14)과 같은 고가의 기술이없는 경우에이 정보는 이하 상세하고 신뢰성을 . 섹션 기반 이미징의 다른 종류 (예를 들어, 전자 현미경 또는 마이크로톰 절편)는 아티팩트 가능성을 증가, 탈수 포함한 조직의 광범위한 조작을 필요로한다. 따라서, 우리는 이미지 D.에 새로운 방법을 개발 똑바로 동안 melanogaster의 달걀 챔버. 이 방법은 이미 (검토중인 대표 결과와 매닝 등을 참조) 운동성이있는 세포가 운명하는 방법을 아는데 도움이 검증 된, 그리고 더 넓게 가치 oogenesis의 다른 측면의 연구 될 가능성이있다.

<table border="0" cellpadding="0" cellspacing="0" width="1027">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4&#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:348px;">Glass culture tube</td>
			<td style="width:279px;">VWR</td>
			<td>47729-576</td>
			<td>14mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
			<td style="width:279px;">VWR</td>
			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerin Jelly&#160;</td>
			<td style="width:279px;">Electron Microsocpy Sciences</td>
			<td>17998-10</td>
			<td>Mounting media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Glycerol</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB15760</td>
			<td>70% in PBS</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>For processing confocal Z-stacks</td>
		</tr>
	</tbody>
</table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

똑바로 이미징 방법은 우리가 단 8시에 전방 모낭 상피 세포의 직접 조직을 볼 수 여포 세포 운명의 일반적인 마커, 눈에 결석 (EYA) 단백질뿐만 아니라 핵 DNA 마커 DAPI, 심지어 표현을 보여 주었다 세포의이 들판을 가로 질러, 모든 세포가 비슷한 염색 강도 (그림 2B &quot;)로 볼 수 있다는 것을 보여 주었다. 사이토킨 UPD에 응답하여 조절 단백질 그러나, 가변 패턴 및 발현 수준을 보였다. ...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

의 수직 이미지<em&gt; 초파리</em&gt; 계란 챔버

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.5K 조회수.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

비디오: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

의 라이브 영상<em&gt; 초파리</em&gt; 번데기 눈

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

연구

발생학

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

에서 이미징 칼슘<em&gt; 초파리</em&gt; 계란 활성화에

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

장기의 라이브 영상<em&gt; 초파리</em&gt; 아이 디스크

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

배양 된 초파리 계란 챔버의 시간 경과 영화를 사용하여 로컬 세포 및 조직 저울에서 Actomyosin 역학분석

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

초파리 배아에서 근육 수축의 라이브 이미징 및 분석

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

녹고, 경작, 그리고 초파리 셀 라인 Cryopreserving

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

인간 계란 성숙도 평가 및 임상 적용

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

초파리 날개 디스크에서 이미징 Dpp 릴리스

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.