Name: dissection and flat-mounting of the threespine stickleback branchial skeleton
Uploaded: 2016-05-07T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.

모든 물고기 작품은 캘리포니아 버클리 대학의 기관 동물 관리 및 사용위원회 (프로토콜 번호 R330)에 의해 승인되었다. 안락사 0.1 % 중탄산 나트륨 39 % 완충 0.025 Tricaine-S에 침지하여 수행 하였다. 모든 단계는 실온에서 수행된다. 1. 준비 참고 : 단단히 밀봉 할 수 있고 수평으로 배치 할 원뿔 튜브 또는 섬광 유리 병에 단계 1.1-1.5를 수행합니다. 물고기는 지속적으로 동요 될 필요가 조심스럽게 반전 또는 염색 액에 물고기의 모든면을 노출하고 얼룩이 균등하게 조직을 통과 할 수 있도록 튜브 또는 튜브의 랙을 흔들어 가능한 한 자주 솔루션을 혼합하려고하지 않습니다. 액체의 무거운 무게가 통을 깰 것 같은 플랫폼 통에 물고기의 큰 배치를 두지 마십시오. <ol><li> 하룻밤 10 % 중성 완충 포르말린 (NBF)와 에탄올에 저장하거나 갓 안락사 생선이나 생선을 수정합니다. 대안 적으로, 4 % 파라 포름을 사용1X PBS 용액 대신에 10 % NBF에서 알데히드. 주 : DNA를 추출하는 경우에는 에탄올에 고정 및 저장 전에 꼬리 지느러미 또는 가슴의 일부를 클립. </li><li> 2 시간 동안 (즉, ~의 pH 7.0) 화학 후드에서 제대로 수정 폐기 및 수돗물로 교체합니다. 종종 산성 수 있으며 뼈 석회 수있는 탈 이온수를 사용하지 마십시오. </li><li> 24 시간 동안 물에 1 % KOH에 0.008 % 알리자린 레드 S 물과 얼룩 물고기를 제거합니다. 물고기보다 20mm 표준 길이를 들면, 0.004 % 알리자린 레드 S.는 (다음 희석 될 수있다 알리자린 레드 S의 100 배 (0.8 %) 원액을 확인)를 사용합니다. </li><li> 몇 시간 동안 수돗물에 얼룩 (후드에서 적절한 폐기 용기에 넣어)과 장소 물고기를 제거합니다. 물 린스는 대부분 깨끗해질 때까지 필요에 따라 물을 변경합니다. </li><li> 50 % 글리세롤, 가벼운 청소 및 후속 해부 0.25 %의 KOH에 물과 장소 물고기를 제거합니다. 참고 :이 염색 프로토콜은 앞서 설명한 방법 40, 41에서 수정됩니다. </li></ol><p클래스 = &quot;jove_title&quot;&gt; 2. 해부 참고 : 관련 머리 골격 형태의 검토를 위해 그림 1을 참조하십시오. <img alt="그림 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> 그림 1 :. 큰가 머리 골격 형태 알리자린 레드는 로다 민 B 필터 세트에서 형광 이미징 큰가시 고기의 머리를 염색. 유용한 형태는 표지 할 것 : 연산 = opercle, Subop = subopercle, BSRs = branchiostegal 광선, 수술 전 = preopercle, Infraorb 1-3 = 눈꺼풀 1-3 (또한 circumorbitals 또는 suborbitals), 덴트는 = 치골, Premax = premaxilla, 최대 = 상악을 , 나스 = 코, 위도. ethm = 측면 사골, Psph = parasphenoid, 프론트 = 정면 뼈. 큰가시 고기의 머리 골격에 대한 자세한 설명은, (1974) 31하기 Anker를 참조하십시오. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">의 더 큰 버전을 보려면 여기를 클릭하십시오</a>이 그림. <ol><li> 물고기 평면 (그림 2A)을 놓고 눈을 덮고있는 막에 구멍을 위해 ~ 45 ° 각도로 눈의 측면에 날카로운 # 5 시계의 집게를 삽입합니다. </li><li> 거리 요구르트 뚜껑 (도 2b)을 박리 유사한 눈으로부터 막을 벗겨. </li><li> 눈 뒤에 열린 집게를 삽입 눈 뒤에있는 시신경을 붙잡고, 및 눈 (그림 2C)를 제거합니다. 이 멜라닌을 누출로 눈에 구멍을하지 마십시오. 구멍이있는 경우, 멜라닌 이후 단계에서 씻겨 할 수 있습니다. </li><li> 반대편에 반복합니다. </li><li> 후방에서 시작하여, opercle 플랩 아래에 하나의 작은 해부 가위 블레이드를 배치, 드래그 가위 블레이드는 등쪽 opercle 위, 그 다음 눈 소켓 (그림 2D)까지 연부 조직을 잘라. opercle 뼈에 지느러미를 잘라. </li><li> 정면 뼈 (눈 소켓에 지느러미) (그림 2E)을 잘라. </li><li> t 주위의 중간 선 parasphenoid 뼈를 잘라눈 소켓 (그림 2 층)의 그는 센터. </li><li> 반대 측의 반복 opercle 잘라. </li><li> opercle에서 집게를 삽입하고 천천히 여전히 (그림 2G - H)에 연결된 모든 연부 조직을 트리밍, 멀리 몸에서 얼굴을 떼어. 아가미 레이커의 첫 번째 행을 방해하지 않도록주의하십시오. <ol><li> 외부 피부 opercle, preopercle, subopercle 포함한 전체 설골 골격을하고, 핀셋으로 벗겨과 전방 두개 안면 골격 (치골, premaxilla 및 상악 포함한 전체 턱을 분리 할 때 중간 선 basihyal에서 양쪽에 ceratohyals를 분리 branchiostegal 광선 및 기본 지느러미와 복부 연골 요소와 코, 측면 사골을 포함한 두개골의 앞쪽 부분과 눈꺼풀 뼈)도 1 및도 2I를 참조하십시오. </li><li> 골반 쪽은 몸에서 밖으로 접을 수 있으며, 집게 보류 O를 잡아에 대한 핸들 역할을 할 수때 F 본. 등뼈는 제자리에 고정. 잠금을 해제하려면, 부드럽게 직접 거리에 물고기의 몸에서 집게로 척추를 당겨 부드럽게 물고기에 평평 척추를 눌러 뒤쪽으로 구부리십시오. </li></ol></li><li> 삽입 떨어져 아가미 골격 (- K 그림 2J)에 부착 된 나머지 근육과 인대를 괴롭 히고, 전방 및 드래그 집게 (단지 장 관 아래) 아가미 골격에 집게 후방과 복부를 마감했다. </li><li> 전방 방향 (그림 2L)에 후방에서 복부 braincase에 지느러미 아가미 골격을 부착 근육을 멀리 긁어, 닫힌 포셉의 팁을 사용. </li><li> 반대 측에 2.9와 2.10를 반복합니다. </li><li> 장내 튜브의 기반을 잡고 아가미 골격과 창자 관 (그림 2M - N)을 제거하기 위해 전방으로 잡아 당깁니다. </li><li> (다섯 번째 ceratobranchial의 끝에도 2O을 수직 절단 후방을 만들어 장내 튜브를 분리 </strong&gt;). </li><li> 아가미 골격의 등 쪽의 braincase에서 남아있는 뼈 조각을 제거한 후, 지느러미 치아 판의 양측 세트 사이에 지느러미 절단 (후방에 전방 절단) (- D 그림 3A)를 만들기 위해 아가미 바구니에 가위를 삽입합니다. 확인 컷은 지느러미 치아 판의 손상을 방지하기 위해 중앙에. </li><li> 아가미 골격 (그림 3E)를 개방을 지원하기 위해 아가미 골격 (창자 관의 앞쪽 끝)의 후방 끝 부분에있는 고무 장 루멘에서 두 개의 얕은 측면 상처를 확인합니다. </li><li> 물고기와 항아리에 모든 조직의 조각을 놓고 더 이상 청소가 필요하지 않은 경우 부드러운 청산, 또는 100 % 글리세롤을 계속 50 % 글리세롤, 0.25 %의 KOH와 microcentrifuge 관에 아가미 골격을 배치합니다. 고유 식별자와 라벨 항아리와 튜브는 그래서 그들은 추적 할 수 있습니다. 필요한 정화 량은 40m 이상 (대체로 물고기 큰 성어의 크기의 함수 인표준 길이 m)는 일반적으로 추가적인 청소가 필요합니다. </li></ol><img alt="그림 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> 그림 2 :. 큰가 아가미 골격 해부 알리자린 레드 해부에 대한 큰가시 고기 생선 준비 스테인드. 눈은 광범위한 청산에서 탈색된다. 파란색 화살표는 동작 방향을 나타낸다. (A) 큰가 헤드의 측면도, 전방 우측이다. (B) 눈을 덮는 막 제거. 눈의 (C)를 ​​제거. (D) 지느러미는 opercle 위에 잘라. (E) 정면 뼈를 잘라. (F) Parasphenoid 절단. (G - I) 안면 골격의 제거. 복부 아가미 골격 연조직 연결 (J) 제거. (K - L) 등의 아가미 골격 연결 제거. (&lt;강한&gt; M - N) 아가미 골격의 제거. (O) 아가미 골격에서 장 튜브를 분리합니다. 자세한 내용은 단계 2.16 통해 2.1를 참조하십시오. 스케일 바 = 5 mm이다. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 3. 아가미 해골 다시 염색 (필요한 경우) <ol><li> 아가미 골격 어둡거나 명확한 조직을 더 얼룩, 50 % 글리세롤, 0.25 %의 KOH 용액을 제거하고 배 1 % KOH (플랫폼 통에 수평으로 흔들어 섞으면 두 번째 24 시간 세척 한 다음 1 ~ 5 분 세척)으로 씻는다. </li><li> 24 시간 동안 1 % KOH에서 0.008 % 알리자린 레드 S와 1 % KOH 다시 얼룩을 제거합니다. </li><li> 얼룩을 제거하고 24 시간 동안 1 % KOH로 교체합니다. </li><li> KOH 용액을 제거하고 50 % 글리세롤, 0.25 % KOH로 교체합니다. </li></ol> 4. 아가미 해골 장착 <ol><li> 아가미 골격을 제거등쪽면이 22mm X 60mm 유리 커버 슬립 하단 50 % 글리세롤, 0.25 % KOH 또는 100 % 글리세롤과 장소에서 (그림 3 층)이 위를 향. 아가미 골격 위에 50 % 글리세롤, 0.25 %의 KOH 또는 100 % 글리세롤 몇 방울을 추가한다. 미세 원심 관에 50 % 글리세롤, 0.25 % KOH 100 % 글리세롤, 변화 솔루션에서 전환과&gt; 5 분 전에 조직을 평형 설치에 흔들합니다. </li><li> 스페이서의 역할을 커버 슬립의 양쪽 끝에서 모델링 점토와 장소의 두 개의 작은 공을 굴립니다. </li><li> 느슨하게 앞쪽 아가미 골격 (그림 3G)를 평평하게하기에 충분한 압력으로 상단에 두 번째 coverslip에 배치합니다. </li><li> 껍질, 지느러미 치아 플레이트를 포함 왼쪽 지느러미 플랩을 열고 평평하고, 커버 슬립 (그림 3H) 사이에 슬라이드. </li><li> 오른쪽 지느러미 플랩 반복 기술과는 커버 슬립 (그림 3I)의 가장자리에서 떨어져 전체 아가미 골격을 누릅니다. <ol><li> 바꾸다기본적으로, 집게로 열린 두 지느러미 플랩을 잡고 조심스럽게 한 부드러운 움직임의 아가미 골격을 평평하게,에 coverslip에 배치합니다. </li><li> 중력은 아가미 골격가 다시 닫습니다 허용하지 않도록 또는 밖으로 측면 각 지느러미 쪽을 플레잉, 하나의 커버 슬립에 거꾸로 아가미 골격을 탑재합니다. 그런 다음 두 번째 22mm X 60mm 유리 커버 슬립으로 커버하고 준비를 반전. 참고 : 다른 장착 기술이 좋든 나쁘 든 각 개인에 대한 작업을하는 경향이있다. 각 시도하고 가장 편안한 느낌을 확인. </li></ol></li><li> 가볍게 아가미 골격을 유지하기에 충분 점토 공을 평평하게하기 위해 상단 커버 슬립에 평평하게 탑재 눌러하지만, 시료를 분쇄하지 않도록주의. <ol><li> 장착 과정 ceratobranchials는 회전 레이커 행을 가릴 수있다. 커버 슬립 다시 배향 ceratobranchials 또는 전체 아가미 골격 사이에 집게를 밀어이를 해결. </li></ol></li><li> 쇼핑몰 소유주에 슬라이드 트레이에 평평하게 준비합니다톰 온도. 100 % 글리세롤에 장착 준비합니다 적어도 십년 가교 된 커버 사이에 저장 될 수있다. 청소 집게, 이소프로판올, 에탄올 및 커버 팁 가위. </li></ol><img alt="그림 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> 그림 3 :. 플랫 아가미 골격 조작을 장착하고 아가미 골격의 설치가 표시됩니다. 파란색 화살표는 동작 방향을 나타낸다. (A) 아가미 골격 등의 측면입니다. 지느러미 치아 판 사이 - (B D) 회전과 절개. 부드러운 조직 (E) 측면 컷은 또한 창자 관의 기반을 엽니 다. 설치를위한 준비 커버 슬립의 하단에 위치 (F) 아가미 골격. (지느러미 치아 플레이트 위) 아가미 골격의 앞쪽 절반에 위치 (G) 두 번째 커버 슬립. (H - I)지느러미 치아 판 플랩을 열고 두 커버 전표 사이에 밀어 아가미 골격의 평면 장착. 자세한 내용은 단계 4.6을 통해 4.1을 참조하십시오. 스케일 바 = 5 mm이다. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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The authors have nothing to disclose.

The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all...

다양성의 놀라운 양이 특히 물고기들, 척추 동물 중 머리 골격에 존재합니다. 대부분의 경우 이러한 다양성은 다른 공급 전략 1 용이 - 4, 모두 외부 및 내부 두개 안면 패턴에 큰 변화를 포함 할 수있다. 아가미 골격은 물고기의 목에 내부에 위치하여 구강의 대부분을 둘러싸고있다. 아가미 골격은 아가미를 지원하는 전방 네있는 5 직렬로 동종 세그먼트로 구성되어 있습니다. 함께이 다섯 세그먼트는 물고기와 음식 (5) 사이의 인터페이스로 작동합니다. 아가미 레이커, 인두 이빨과 아가미 뼈를 포함하여 특성의 다수의 변화는 다양한 종류의 음식에 효율적 꼴에 기여한다. 큰가시 고기과는 북반구에 걸쳐 민물 호수와 시내 식민지 조상의 해양 양식 후 적응 방사선을받은. 다이어트의 변화민물에서 더 큰 먹이에 바다에서 작은 동물성 플랑크톤에서 여러 두개 안면 특성 (6)에 극적인 영양 변화를 가져왔다. 많은 연구가 큰가시 고기과 7 외부 두개 안면의 차이에 초점을 맞추고 있지만 - 13 중요한 두개 안면의 변화는 내부 아가미 골격에 반복적으로 진화. 형태 학적으로 구별 큰가시 고기 집단 사이의 비옥 한 하이브리드를 만들 수있는 능력은 아가미 골격에 진화 변화의 유전 적 기초를지도 할 수있는 좋은 기회를 제공한다. 생태 학적 의미 중 하나는 영양 특성은 아가미 레이커, 아가미 뼈의 전방 및 후방 얼굴 라인과 먹이 항목을 필터링하는 데 사용되는주기적인 피부 뼈의 패턴입니다. 일반적으로 작은 먹이 항목을 먹고 물고기는 더 이상 더 조밀하게 이격 아가미가 더 큰 먹이 14,15 먹이 물고기에 비해 레이커 경향이있다. 아가미 레이커의 변화가보고 된 적이 모두 within 종 14-19 사이, 그리고 아가미 레이커 패턴의 양상은 영양 틈새 시장 및 피트니스 (16)에 기여한다. 연구의 수십 년은 광범위하게 threespine 큰가시 고기과 (17)의 아가미 레이커 번호와 길이의 변화 문서화 - (21); 그러나, 이러한 연구는 일반적으로 아가미 레이커의 첫 번째 행에 초점을 맞춘다. 최근 작품은 하나 하나의 아가미 레이커가 이해하는 행보다 더 많은 공부의 중요성을 강조 (23)과 길이 (24) 간격 아가미 골격 (22, 23)에서와 아가미 레이커의 단일 행에 걸쳐 아가미 레이커 수의 유전자 제어 모듈을 보여 주었다 아가미 레이커 감소의 발달 유전 적 기초. 모두 생태 및 생물 의학 의미의 두 번째 영양 특성은 인두 치아의 패턴입니다. 물고기에서 치아 인두 이빨 공지 모두 경구 턱과 아가미 골격 중에 위치 할 수있다. 구강 치아 페이지에 주로 사용된다27 - 인두 치아 저작 및 먹이 조작 (25)에 사용하는 동안 레이 캡처. 두 세트는 공유 발달 메커니즘을 통해 형성과 상동 (28) 발달로 간주됩니다. 때때로 이러한 일부 제브라 피쉬, 부족으로 구강 및 인두 등의 치아 (29) 다른 종의 여러 톱니 ceratobranchials, pharyngobranchials있는 동안 같은 종, 그리고 basihyal 톱니와 30 hypobranchials있다 흥미 모듈화가 발생합니다. 큰가시 고기과에, 인두 치아 앞쪽에 다섯 번째 ceratobranchial과 등쪽에 복부 발견하고 후방은 31 pharyngobranchials 있습니다. 큰가시 고기 공급에 운동학은 구강 턱은 주로 먹이 캡처 및 인두 턱에 저작을 떠나 흡입 공급 (9)을 용이하게 사용됩니다 보여줍니다. 시클리드에서 하부 인두 턱 형태 극적 32,33를 가변 적응 및 영양 틈새 (34) 상관 관계가 밝혀졌다. 멀티PLE 담수 큰가시 고기의 인구는 복부 인두 치아 번호 23,35,36에서 극적인 증가를 진화했다. 최근 작업이 진화 치아 이득의 발달 유전 적 근거는 민물 큰가시 고기과 (36)의 두 개의 독립적으로 파생 된 인구에서 크게 구별 있음을 보여 주었다. 포유 동물의 치아와는 달리, 물고기는 성인 생활 (37)를 통해 지속적으로 자신의 치아를 재생. 이러한 전술 높은 치형 민물 인구 모두 재생 (36)의 유전 적 기초를 연구하는 희귀 척추 제공 시스템, 가속 보철물 속도 진화 해왔다. 민물 큰가시 고기과에 반복적으로 진화하고 세 번째 영양 특성은 더 이상 각각 38 epibranchial 및 ceratobranchial 뼈, 상부 및 하부 턱의 아가미 아치 분절 동족체이다. 긴 아가미 뼈는 더 큰 구강을 부여하고 가능성이 더 큰 먹이 항목을 다 할 수 있도록하기위한 적응이다onsumed. 또한, 다른 물고기에, epibranchial 뼈는 등쪽 인두 치아 플레이트 (25)의 우울증 중요하다. 아가미 레이커와 인두 치아처럼, 아가미 뼈는 쉽게 시각화 또는 정량화하기 어려운, 따라서 내부입니다. 여기에서 우리는 해부 자세한 프로토콜을 제시하고 중요한 두개 안면 특성의 다양한 쉽게 시각화 및 정량화를 허용, 아가미 골격을 평면 마운트합니다. 이 프로토콜은 큰가시 고기의 해부를 설명하지만,이 같은 방법은 다른 물고기의 다양한 작동합니다.

<table><tbody><tr><td>Potassium Hydroxide (KOH)</td><td>EMD</td><td>PX1480-1</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893-4L</td><td></td></tr><tr><td>10% Neutral Buffered Formalin (NBF)</td><td>Azer Scientific</td><td>NBF-4-G</td><td></td></tr><tr><td>Alizarin Red S</td><td>EMD</td><td>AX0485-3</td><td></td></tr><tr><td>Microscope Cover Glasses 22 mm x 60 mm</td><td>VWR</td><td>16004-350</td><td></td></tr><tr><td>100 mm x 10 mm Glass Petri Dish</td><td>Kimble Chase</td><td>23064-10010</td><td>To dissect samples on</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>Can be poured into glass or plastic Petri dishes to make dissecting plates</td></tr><tr><td>Modeling Clay</td><td>Sargent Art</td><td>22-4000</td><td>1 lb cream</td></tr><tr><td>Scintillation Vials (case of 500)</td><td>Wheaton</td><td>986586</td><td>Borosilicate Glass with Screw Cap</td></tr><tr><td>Forceps-Dumont #5 Inox (Biologie tip)</td><td>FST</td><td>11252-20</td><td>Dumostars are an alternative</td></tr><tr><td>Dissecting Scissors&amp;nbsp;</td><td>FST</td><td>15003-08</td><td>Alternate sizes are available depending on size of sample</td></tr><tr><td>Dissecting Microscope</td><td>Leica</td><td>S6E with KL300 LED</td><td>Many other models work nicely, having a flat base helps</td></tr><tr><td>Microcentrifuge Tubes 1.7 ml</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Cardboard slide tray</td><td>Fisher</td><td>12-587-10</td><td></td></tr></tbody></table>

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.

<html> 중요한 영양 특성의 다양한 정량화 할 수있는 해부와 평면 탑재 아가미 골격이 프로토콜 결과 (그림 4). 지느러미보기에서 아가미 레이커, 모든 인두 치아 판, 거의 모든 아가미 뼈의 모든 행은 쉽게 시각화, 22 정량화 할 수있다 - 24,35,36,38,42합니다. 알리자린 레드 S는 다른 마커 (예, 유전자 변형 GFP 42) 및 시각화하는 ...

Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

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.

해부와 큰가시 고기 아가미 해골의 평면 장착

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

dissection-flat-mounting-threespine-stickleback-branchial

Research

JoVE Journal

Biology

9.7K 조회수.  University of California, Berkeley. 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. 

비디오: Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

형광 단백질의 발현에<em&gt; 창고 기</em&gt; 미 수정 난자에 mRNA의 주입에 의한

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

연구

발생학

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Threespine 큰가시 고기과에 형질 전환 및 게놈 편집에 대한 미세 주입

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Zebrafish의 턱 역학을 조사하기 위해 유한 요소 모델을 구축

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

다층 물고기 비늘 (의 특성<em&gt; Atractosteus 주걱</em&gt;) 나노 인 덴 테이션, X-선 CT, FTIR, 및 SEM을 사용하여

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

생체공학

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

신경과학

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

해부 및 제브라 피쉬 배아의 측면 장착 : 척수 개발의 분석

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

얼룩말피에서 척추동물 세분화를 연구하는 3-D 꼬리 이식 문화

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

척추동물 축 신장 및 세분화를 연구하기 위한 세 가지 및 네 차원 시각화 및 분석 접근법

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

재생 능력의 비교 분석을 위해 Zebrafish와 Medaka를 사용한 성인 시신경의 찔린 상처 부상 모델

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

전체 마운트하여 제브라 피쉬 배아 시편 스테인드 관찰 및 분석을위한 평면 마운트 준비<em&gt; 현장에서</em&gt; 하이브리드

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

해부와 큰가시 고기 아가미 해골의 평면 장착

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