This Work was supported by NIH R03 HL140264 (Y.-J. N) and Gilead Sciences Research Scholar Program (Y.-J. N).

All animal procedures were performed with the approval of the Vanderbilt University Medical Center Institutional Animal Care and Use Committee.
1. Mouse embryo collection and dissection
<ol>
	<li>Mate 8-10 week old female MLC-2v-tdTomato+/- mice with 8-10 week old male MLC-2v-tdTomato+/- mice to obtain MLC-2v-tdTomato+/+, MLC-2v-tdTomato+/- and MLC-2v-tdTomato-/- embryos.</li>
	<li>Check the dams for vaginal plugs every morning. Noon on the day of vaginal plug detection is considered as E0.5. 
	NOTE: Vaginal examination for detecting a vaginal plug should be performed in the morning (within 8-24 h after sexual activity), since a vaginal plug can be lost throughout the day.</li>
	<li>Euthanize the pregnant dams at different days post coitum (e.g., E8.5, E10.5, and E12.5) using CO2 inhalation followed by cervical dislocation.</li>
	<li>Lay the mice supine and spray 70% ethanol on the abdomen of mice to avoid mouse hair contamination during dissection.</li>
	<li>Open the abdominal cavity by incision of both the skin and abdominal wall using sharp surgical scissors and a forcep.</li>
	<li>Locate bilateral uterine horns in the dorsal part of the abdominal cavity.</li>
	<li>Separate the entire uterus by carefully cutting above the oviducts on both sides using sharp surgical scissors and a forcep.</li>
	<li>Place the entire dissected uterus in a 10 cm Petri dish with ice-cold PBS and carefully separate each amniotic sac along the uterine horn using sharp surgical scissors and a forcep.</li>
	<li>Transfer each embryo into individual wells of 6 well plate filled with ice-cold PBS using a transfer pipette.</li>
	<li>Under a dissecting microscope, open up an amniotic sac and expose each embryo by cutting off the umbilical cord using sharp surgical scissors and a forcep in an individual well of a 6 well plate with ice-cold PBS.</li>
	<li>Trim out extra-embryonic tissues as much as possible without damaging the embryo using sharp surgical scissors and a forcep. 
	NOTE: Whole mount epifluorescent imaging of whole mouse embryo is usually performed before dissecting the developing heart as described below.</li>
	<li>Cut the embryo head using sharp surgical scissors and a forcep and transfer to a 1.5 mL tube with 100 &#181;L of buffer A (25 mM NaOH and 0.2 mM EDTA) for genotyping to correlate with the results of epifluorescent imaging.</li>
	<li>Open the chest of the embryo using fine forceps, remove the heart away from the lungs and vasculature using sharp surgical scissors and forceps, and transfer the dissected embryonic heart into a well of a 12 well plate with PBS using a transfer pipette. All dissection procedures are completed under a dissecting microscope with a fiber optic microscope illuminator. 
	NOTE: It was technically difficult to dissect out mouse embryonic hearts at E8.0 or E8.5, because of their extremely small size and fragile structure. Early embryonic hearts (i.e. E8.0 and E8.5) can be examined within the whole mount embryo without dissection.</li>
</ol>
2. Whole-mount epifluorescence imaging
<ol>
	<li>Place the 12 well plate with mouse embryonic hearts under an epifluorescent dissecting microscope.</li>
	<li>Under an epifluorescent dissecting microscope using fine forceps, position the embryonic heart such that developing ventricles are located close to the examiner.</li>
	<li>Adjust focusing of the image using a 0.63x objective (zoom range between 3.15x and 18.9x) in bright field mode.</li>
	<li>Take bright field exposures and capture multiple images. The images were usually obtained by one second exposure. However, exposure times may vary depending on illumination and camera specificationss and need to be optimized for each setup.</li>
	<li>Turn off a fiber optic microscope illuminator, and set the filter for red fluorescence (Ex545 nm/Em 605 nm) to visualize tdTomato expression.</li>
	<li>Re-adjust focusing of the image if necessary.</li>
	<li>Adjust brightness and contrast, take red fluorescent exposures, and capture multiple images. 
	NOTE: The following image setting was usually used: 1 s exposure time, 2x gain, 1.0 saturation, and 1.0 gamma correction. The optimal setting needs to be optimized for each experiment. Once the optimal setting is determined, the same setting needs to be used for an entire experiment.</li>
</ol>
3. Genotyping
<ol>
	<li>Boil the samples from step 1.12 for 1 h at 100 &#176;C.</li>
	<li>Centrifuge for 2 min at 11,360 x g, transfer 20 &#181;L of supernatant into a new 1.5 mL tube with 20 &#181;L of buffer B (40 mM Tris HCl, pH 5.5) and mix them.</li>
	<li>Take 4.5 &#181;L of the mixed supernatant from step 3.2 as a DNA template, combine it with 0.5 &#181;L of each of the specific forward and reverse primers (10 &#181;M), 10 &#181;L of pre-mixed polymerase and reaction buffer (2x) (see Table of Materials), and then add water to a total volume of 20 &#181;L. Primer sequences are as below. 
	F1: 5&#8217;-TACCCACGGAGAAGAGAAGGACT-3&#8217; 
	R1: 5&#8217;-TGGACTTCTTGGAACTGACTCTGT-3&#8217; 
	F2: 5&#8217;-ACGGCACGCTGATCTACAAGGT-3&#8217; 
	R2: 5&#8217;-TTTGCGCACAGCCCTGGGAT-3&#8217;</li>
	<li>Run a polymerase chain reaction (PCR) with the following PCR program (Table 1 and Table 2).</li>
	<li>Run PCR samples and DNA ladder on a 1% agarose gel at 140 V in 1x TAE (Tris-acetate-EDTA) buffer (40 mM Tris-acetate and 1 mM EDTA) for 25 min. Use a 100 bp DNA ladder to estimate the size of the PCR bands.</li>
	<li>Place the DNA gel on a UV transilluminator to identify the DNA bands and turn on the UV light.</li>
</ol>

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The authors have nothing to disclose.

The method described here is relatively simple to examine ventricular chamber development, without performing labor-intensive experiments to label ventricular or cardiac-specific structural genes or proteins. Thus, this method minimizes technical variabilities that were often found in immunostained heart sections.
There are two critical steps for successfully performing this method including precise estimation of the embryonic age of mice and dissection of embryonic hearts. We practically esti...

Chamber formation during heart development is a complex process transitioning through several morphologically distinct embryonic stages1,2. The crescent shape of cardiac progenitor population cells forms a linear heart tube and then undergoes elongation and looping to form the spiral shape of the developing heart. After its septation process, the developing heart is transformed into the four-chambered heart. Interruption of any of these processes results in developmental heart defects. Thus, it is important to understand the molecular mechanisms underlying chamber formation during heart development. Despite numerous previous studies on heart development, our understanding of this complex process remains limited.
In situ hybridization, immunohistochemistry, and beta-galactosidase staining using LacZ reporter mice have been widely used to study chamber formation during mouse heart development by labeling cardiac specific or chamber specific structural genes or proteins (e.g., Nppa, Coup-TFII, Irx4, MLC-2a and MLC-2v)3,4,5,6,7,8,9,10. However, these experiments using mouse embryos require significant time and expertise, because several different experimental steps have to be performed sequentially11. Here, we describe a simple whole mount epifluorescent microscopy method to visualize the developing ventricles using embryos dissected from MLC-2v-tdTomato reporter knock-in mice12. The advantage of this method compared to previously used methods is to avoid complex experimental steps which may often create experimental variations. The main purpose of this protocol is to describe how to dissect mouse embryos and developing hearts and to examine each stage of mouse cardiac chamber development without tedious histochemical experiments. This method can be easily applied to assess heart development using various other transgenic mouse lines labeling early cardiac markers (e.g., Mesp1Cre: Rosa26EYFP13, Isl1Cre: Rosa26EYFP13, Hcn4H2BGFP14, Hcn4Cre: Rosa mT/mG14, Nkx2-5Cre: Rosa mT/mG14, Hcn4-eGFP15, Isl1Cre: Rosa mT/mG14, Nkx2.5Cre: Rosa26tdTomato15, and TgMef2c-AHF-GFP16 mice).

<table border="0" cellpadding="0" cellspacing="0" style="width:647px;" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">MZ125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DNA ladder (100 bp)</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">G2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">epifluorescence dissecting microscope</td>
			<td style="width:83px;">Leica</td>
			<td style="width:119px;">M165 FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">GoTaq Green master Mix</td>
			<td style="width:83px;">Promega</td>
			<td style="width:119px;">M712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">PCR machine (master cycler)</td>
			<td style="width:83px;">Eppendorf</td>
			<td style="width:119px;">6336000023</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of cardiac chamber development during mouse embryogenesis using whole mount epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

During heart development, MLC-2v is considered to be the earliest marker for ventricular chamber specification17. As depicted in Figure 1, we dissected out mouse whole embryos and embryonic hearts from MLC-2v-tdTomato reporter knock-in mice and examined MLC-2v-tdTomato reporter expression during heart development. In MLC-2v-tdTomato reporter knock-in mice, constitutive tdTomato expression in the developing heart is visualized via epifl...

Watch this Scientific Journal Video about Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence at JoVE.com

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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7.5K Views.  Vanderbilt University Medical Center. We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Video: Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...