VCU에서 A. 디킨슨 시동 돈이 작업을 지원했다. 저자는 개략적 인 그림을 만드는 그의 예술적 재능 댄 Nacu을 인정하고 싶습니다.

<ol>
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저자가 공개하는 게 없다.

아프리카 발톱 개구리는 laevis의 발달 기전 구강 안면 개발을 해부하는 데 유용한 도구가되었다; 그러나, 개구리에서이 지역의 크기와 모양 변화를 기술 한 프로토콜은 현재 존재하지 않는다. 여기에 설명 된 방법은 제노 푸스 다른 척추 동물에 구강 안면 표현형보다 엄격한 정량 허용함으로써 구강 안면 개발 분야에 크게 기여할 것이다. 제대로이 프로토콜을 ?...

인간의 출생 결함의 가장 흔하고 치명적인 유형 중 같은 구강 안면 쪼개진 조각 1로 입과 얼굴에 영향을 미치는 것들이다. 잘못된 구강 안면 구조와 아이들은 평생 동안 여러 수술을 받아야 얼굴 disfigurements, 언어, 청각 먹는 문제를 투쟁. 따라서, cranio- 및 구강 안면 개발의 새로운 연구를 촉진하는 것은 인간의 출생 결함의 이러한 유형의 예방 및 치료에 중요합니다. 아프리카 발톱 개구리 laevis의는 두개 안면 개발을 관리하는 메커니즘을 해부를위한 새로운 도구로 떠오르고있다 (몇 가지 예 2,3,4 포함 -11). 따라서,이 종류의 헤드의 개발 및 얼굴 중 크기 및 형상 변화를 정량적으로 분석하는 방법은 세 매우 강력 할 수있다. 여기서, 우리는 이러한 방법을 제시한다; Xenopus의 연구 (12)에서 적응 기하학적 morphometrics 전통적인 크기 측정을 결합 </인간의 얼굴 형태 13-15을 분석하는 연구의 SUP&gt;를 찾을 수있다. 이 프로토콜의 목표는 연구자가 정상 및 비정상 개발하는 동안 다른 구강 안면 표현형을 구별하는 얼굴 크기와 모양을 정량화 할 수 있도록하는 것입니다. 이 분석은 유전자 및 / 또는 환경 적 요인의 시너지 효과에서 발생하는 것과 같은 미묘한 두개 안면 결함 사이의 더 나은 차별화 수 있습니다. 또한, 이러한 정량화 방법은 또한 구강 안면 결함에도 약간 개선 또는 구조를 밝힐 수있다. 이것은 따라서 잠재적 인 치료제 분석에 유용한 가이드한다. 여기 제시 안면 측정 및 기하학적 morphometrics의 조합은 대체로 하나 또는 다른 15-18 활용 크기 및 현재의 프로토콜보다 구강 안면 영역의 형상 모두 포괄적 통계 분석을 허용한다. 또한, 우리는 내측과 외측의 양쪽면을 평가할 수있는 간단한 방법을 제시현재 연구 13, 19에서 사용 된 복잡한 3 차원 화상 장비 없이도 얼굴. 우리는 아프리카 발톱 개구리에이 프로토콜은 비정상적인 구강 안면 개발 및 중간 구개열 2,3를 유도하는 레티노 산 수용체 억제제로 치료 배아를 laevis의 보여줍니다. 이러한 배아의 구강 안면 영역의 크기와 모양의 정량화 비슷한 구개 쪼개진 조각 및 마우스 모델 (20, 21)와 인간과 유사하다 midface의 변화를 공개했다. 그러나이 프로토콜은 예컨대 성장 인자와 같은 천연 물질, 제초제, 또는 단백질과 같은 구강 안면 발달에 다른 화합물의 효과를 평가하는데 이용 될 수있다. 또한, 손실 또는 함수 실험의 이득을 통해 유전자 발현을 교란으로부터 발생하는 구강 안면 크기 및 형태의 변화 (안티센스 morpholinos 또는 Crispers를 사용하여이 / Talens)도이 프로토콜을 이용하여 정량화 할 수있다. 마지막으로, 우리는이 방법의 사양보다 개발에서야 Xenopus의 형태를 평가하기 위해; 그러나, 용이하게 임의의 척추 동물의 분석을 위해 수정된다. 다른 응용 프로그램도 진화 또는 생태 연구에 밀접하게 관련 종을 비교하는이 프로토콜을 사용하여 포함 할 수있다. 우리가 여기에서 제공하는 예는 구강 안면 영역의 분석을 설명하는이 프로토콜을 사용하지만, 쉽게 다른 지역, 기관, 또는 구조의 분석을 위해 수정할 수 있습니다. 이 구강 안면 정량화 프로토콜은 연구 지역 사회를위한 귀중한 자원뿐만 아니라, 비디오 데모와 같은 학부 학생들을위한 훌륭한 교육 도구가 될 것이다.

<table><tbody><tr><td>Dissecting microscope</td><td>Zeiss</td><td></td><td>fitted with AxioCamICC1 camera</td></tr><tr><td>Dumont #5 Inox forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Sterile, disposable scalpel</td><td>Sklar</td><td>06-2015</td><td></td></tr><tr><td>24-well plate</td><td>Fisher Scientific</td><td>087721</td><td></td></tr><tr><td>Standard Disposable transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>150 mm x 15 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>Incubators</td><td>Ectotherm</td><td></td><td>set to 15 &amp;deg;C or 20 &amp;deg;C</td></tr><tr><td>Modeling Clay</td><td>Premo, or other non-toxic modeling clay</td><td></td><td>in black or white</td></tr><tr><td>Straight teasing needle</td><td>Thermo Scientific</td><td>19010</td><td></td></tr><tr><td>Capillary Tubing (for needles)</td><td>FHC</td><td>30-30-1</td><td>Borosil 1.0 mm OD x 0.5 mm ID/Fiber, 100 mm each</td></tr><tr><td>Needle Puller, Model P-97</td><td>Sutter Instrument Co.</td><td>Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B</td><td>For filaments, use Sutter 3.00 mm square box filaments, 3.0 mm wide.</td></tr><tr><td>Pipettemen</td><td>Gilson</td><td>F144802, F123600, F123602</td><td></td></tr><tr><td>BMS-453</td><td>Tocris</td><td>3409</td><td></td></tr><tr><td>DMSO</td><td>American Bioanalytical</td><td>AB00435-01000</td><td></td></tr><tr><td>Cysteine</td><td>Sigma-Aldrich</td><td>52-90-4</td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Petri dishes</td><td>Falcom</td><td>353003, 351058</td><td>100 mm diameter and 150 mm in diameter</td></tr><tr><td>100% Ethanol</td><td>VWR</td><td>89125-170</td><td></td></tr></tbody></table>

quantification of orofacial phenotypes in xenopus

Xenopus의 개발은 두개 안면 결함을 관리하는 메커니즘을 해부위한 중요한 도구가되었다. 구강 안면 현상을 정량화하는 방법은 유전자 또는 분자 유전자 발현 또는 단백질의 기능을 조작 할 수있는 물질과 폐기시 구강 안면 표현형보다 엄격한 분석을 허용 할 것이다. 배아 머리의 두 차원 이미지를 사용하여, 기존 사이즈의 치수를-같은 구강 안면 폭, 높이 등 및 지역 - 측정한다. 또한, 배아 입 개구의 진원도 측정 입의 형상을 설명하는 데 사용된다. 이러한 이차원 영상의 기하학적 morphometrics 또한 구강 안면 영역의 형상의 변화에​​보다 정교한 뷰를 제공하기 위해 수행된다. 랜드 마크는 구강 안면 영역의 특정 지점에 할당 및 좌표가 생성됩니다. 주성분 분석은 치료를 판별 원리 성분 표식 좌표를 줄이기 위해 사용되는그룹. 이러한 결과는 비슷한 구강 안면 형태와 개인이 함께 클러스터링하는 산포도로 표시됩니다. 또한 통계적 두 치료군 간의 랜드 마크의 위치를​​ 비교 판별 함수 분석을 수행하는 것이 유용하다. 이 분석은 랜드 마크의 위치 변화가 벡터로 볼 수 있습니다 변환 그리드에 표시됩니다. 그리드는 왜곡 패턴이 중요한 랜드 마크 위치가 변경된 위치를 표시하는 표시되도록이 벡터에 놓이게됩니다. 판별 함수 분석 형상 변화는 통계적 측정치에 기초하며, 따라서 p- 값에 의해 평가 될 수있다. 이 분석은 입체경 프리웨어 소프트웨어를 필요로하는, 단순하며, 따라서 귀중한 연구 및 교육 자료가 될 것이다.

여기서, 구강 안면 크기 및 형상의 정량 분석​​ 미처리 컨트롤 레티노 산 수용체 억제제 (RAR 억제제)로 처리 배아를 비교하는 것으로 입증되었다. 배아, 30 (26 ~ 35 HPF)로 단계 24에서이 화학 물질 억제제의 1 μM 농도로 처리 세척하고, 단계 (42) (82 HPF)로 고정되었다. 그런 다음 처리 프로토콜에 기술 된 바와 같이 분석 하였다. 결과는 원본 데이터,하지만 이전의 출판물 2,3 관측과 일치한다. ?...

Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Quantification of Orofacial Phenotypes in Xenopus

아프리카 발톱 개구리 laevis의 배아의 구강 안면 크기 및 형상을 정량화하는 방법이 개발되었다. 이 프로토콜에서, 기존의 크기 측정은 구강 안면 현상 결함의보다 정교한 분석을 허용하기 위해 기하학적 morphometrics과 결합된다.

구강 안면 표현형의 정량화에<em&gt; Xenopus의</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

quantification-of-orofacial-phenotypes-in-xenopus

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K 조회수.  Virginia Commonwealth University.  아프리카 발톱 개구리 laevis의 배아의 구강 안면 크기 및 형상을 정량화하는 방법이 개발되었다. 이 프로토콜에서, 기존의 크기 측정은 구강 안면 현상 결함의보다 정교한 분석을 허용하기 위해 기하학적 morphometrics과 결합된다. 

비디오: Quantification of Orofacial Phenotypes in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

이해 간체를 사용하여 초기 형성기<em&gt; 현장에서</em에서&gt; 하이브리드 프로토콜<em&gt; 아프리카 발톱 개구리</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

연구

발생학

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

의 배아 혈관 신생에 대한 시각화 및 정량 분석<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

제노푸스 laevis 배아에서 눈부안으로 DNA의 미세 주입 및 GFP의 이미징 그대로 광학 축색 아버를 표현, 살아있는 제노푸스 올챙이

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

생물학

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

에서 차 신경 발생 평가<em&gt; Xenopus의</em&gt; 배아 사용하여 면역 염색

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

신경과학

Electroporation of Craniofacial Mesenchyme

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

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

Craniofacial Mesenchyme의 Electroporation

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

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

생활 Xenopus Tadpoles에 후 각 통로의 기능 평가

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

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

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

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

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

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

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Zebrafish를 사용하여 색소 침착 조절자를 식별하기 위한 역 유전적 접근

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

구강 안면 표현형의 정량화에

요약

더 많은 비디오 탐색

이 비디오의 챕터

이해 간체를 사용하여 초기 형성기

의 배아 혈관 신생에 대한 시각화 및 정량 분석

제노푸스 laevis 배아에서 눈부안으로 DNA의 미세 주입 및 GFP의 이미징 그대로 광학 축색 아버를 표현, 살아있는 제노푸스 올챙이

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis

에서 차 신경 발생 평가

Craniofacial Mesenchyme의 Electroporation

생활 Xenopus Tadpoles에 후 각 통로의 기능 평가

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

의 얼굴 이식

Zebrafish를 사용하여 색소 침착 조절자를 식별하기 위한 역 유전적 접근