The author thanks Doug for his constant encouragement and unwavering support, as well as the members of the Melton lab for their helpful feedback and comments on the manuscript. The author would also like to thank the Harvard Office of Animal Resources (OAR) for their dedicated animal care.

All animal experiments were performed in accordance with IACUC (Protocol #: 11-32) and AAALAC guidelines at Harvard University.
1. Preparing solutions and setup for anesthesia and recovery
<ol>
	<li>Prepare fresh 0.1% tricaine solution for anesthesia and 0.5% sulfamerazine sodium salt solution for recovery. Make the solutions using water suitable for axolotl husbandry37 according to approved IACUC protocols at the relevant research institution (modified Holtfreter&#8217;s solution, for instance). Make sure that the solutions are well-mixed and that enough volume is prepared in order to submerge the entire axolotl.
	<ol>
		<li>To prepare 0.1% tricaine solution, mix 1 g of tricaine and 1 g of sodium bicarbonate with 1 L of water. The solution can be scaled up according to this recipe.</li>
		<li>To prepare 0.5% sulfamerazine sodium salt solution, mix 5 g of sulfamerazine sodium salt with 1 L of water. The solution can be scaled up according to this recipe. Sulfamerazine solution is an anti-biotic that will prevent bacterial infection during surgical recovery.</li>
	</ol>
	</li>
	<li>Sanitize the surgical area by spraying it down with Clidox-S or 70% ethanol. Sterilize surgical tools (forceps, dissecting scissors, spring scissors) by autoclaving. If performing multiple surgeries, make sure to sterilize the surgical tools with a hot bead sterilizer between animals.</li>
	<li>To set up the recovery area, place a 15 cm Petri dish or any container that will fit the axolotl on top of a bucket filled with wet ice. Fill the Petri dish with a low level of 0.5% sulfamerazine sodium salt solution, enough such that the axolotl would not be fully submerged. The recovery on ice post-surgery will slow the animal&#8217;s movement while it awakens from anesthesia, allowing the sutured area to heal relatively undisturbed. 
	NOTE: This setup can be customized by researchers depending on what materials they have available.</li>
</ol>
2. Performing the full skin flap surgical procedure
<ol>
	<li>Anesthetize the axolotl by submersing it in a container of 0.1% tricaine solution. This will take approximately 15-20 minutes. Ensure that the axolotl is indeed fully anesthetized by performing a tail pinch. If there is no response from the axolotl, proceed with the surgery. 
	NOTE: Use older, larger axolotls for this surgery (at least 15 cm in size). Make sure that the axolotl remains well hydrated throughout the surgery by wetting the skin periodically with axolotl system water using a plastic pipette. Make sure to aseptically prepare the surgical site by irrigating the area with sterile PBS prior to surgery. The animal should also be placed on a sterile surgical drape for the procedure.</li>
	<li>Perform a limb amputation at the distal end of the zeugopodial skeletal elements using the dissecting scissors (Figure 1.1).</li>
	<li>Using the spring scissors, make a small incision (approximately 2 mm) on the ventral portion of the skin (Figure 1.2).</li>
	<li>Using the forceps, carefully peel back the skin to approximately the midline of the zeugopodial skeletal elements, exposing the underlying limb tissues (muscle, bone, etc.) (Figure 1.3). Make sure not to damage the skin. See note after step 2.8.</li>
	<li>Amputate the exposed underlying limb tissues at the midline of the zeugopod using surgical scissors (Figure 1.4).</li>
	<li>Push back the muscle tissue with the surgical scissors and trim the exposed bone. 
	NOTE: This is necessary to ensure improved healing and also to increase the success of the surgery as protruding bone can be jagged against the sutured flap and disrupt the integrity of an intact skin flap later on.</li>
	<li>Using the forceps, carefully pull the extra full thickness skin over the amputation plane to cover the exposed underlying tissues and suture in place by connecting with the ventral full thickness skin (Figure 1.5).</li>
	<li>Suture the remaining right and left sides of the skin flap into the underlying ventral portions of intact skin. This can be done by either suturing the sides of the flap in a &#8220;criss-cross&#8221; manner (recommended) (Figure 1.6-1.9), or simply suturing straight into the ventral skin. Use the forceps and curved spring scissors for suturing. Make sure no exposed underlying tissues can be seen and that sutures are tied tight (knotted at least three times). 
	NOTE: It is critical that the intact skin is not damaged in steps 2.4, 2.7-2.8. We have found that damage to the full thickness skin has been correlated with unsuccessful surgeries, as the areas of damage may still form a small wound epidermis. If possible, try to use a duller pair of forceps when handing the full thickness skin flap.</li>
	<li>Perform an amputation on the contralateral limb (optional internal animal control) by amputating it at mid-zeugopod level with surgical scissors. Push back the muscle tissue with surgical scissors and trim the exposed bone. 
	NOTE: An internal contralateral limb control can be done to better assess the success of the surgery during step 4 in the same animal. However, amputation of the same limb in a separate animal can also be used to serve as a control.</li>
</ol>
3. Post-operative recovery and care
<ol>
	<li>Once the surgery is complete, place a Kimwipe or sterile paper towel at the bottom of the container or Petri dish to wet it. Place the animal into the container on wet ice and gently wrap the exposed ends of the Kimwipe or paper towel around the top of the animal to keep it well-hydrated with sulfamerazine solution. Leave on wet ice for 30 minutes to 1 hour to ensure minimal movement during recovery from anesthesia.</li>
	<li>Place the animal into a static housing container with 0.5% sulfamerazine solution. Axolotls must remain in this solution for the first 24 hours in order to prevent infection.</li>
	<li>Place the axolotl into normal system water and monitor health daily. Ensure that no sutures fall out each day as this can result in a small wound epidermis forming which will confound results. 
	NOTE: Make sure that the housing container has ample room for the axolotl to move around and minimize the chances that the sutured limb on the axolotl can come into contact with the sides of the container. This will help to ensure that the sutures stay in place, especially during the first week post-surgery.</li>
</ol>
4. Assessing success of the surgery under a stereo microscope
NOTE: We recommend checking animals under a stereo microscope at least once a week to assess the integrity of the full skin flap and success of the surgery.
<ol>
	<li>Anesthetize the axolotl in 0.1% tricaine as in Step 2.1. Make sure there is ample room in the container for the axolotl to move around.</li>
	<li>If inspecting during the first two weeks post-surgery, inspect the sutured limb using a stereomicroscope to make sure no sutures have popped out and that a clear thin wound epidermis is not visible anywhere. If inspecting on the third week post-surgery or later, make sure a blastema has not formed and compare with how the normal control amputated limb (either from the same animal or a different animal) has progressed during regeneration (i.e., whether a blastema has formed).</li>
	<li>When done, return the axolotl to normal system water and husbandry conditions.</li>
</ol>

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The authors have nothing to disclose.

This article describes a protocol for performing full skin flap surgeries in axolotl limbs to inhibit wound epidermis formation. While this surgery is relatively simple and technically reproducible compared to other methods of inhibiting wound epidermis formation, there are several critical steps that can impact the success of the surgery. First, when pulling the intact full skin flap over the exposed underlying tissues, it is paramount that the full thickness skin not be damaged in any way. Damage to the skin flap can still lead to the formation of a small wound epidermis, which can result in a small blastema-like outgrowth. Second, ensure sutures do not fall out during post-operative care as this can also lead to the formation of a small wound epidermis. To this point, minimizing the potential contact between the sutured limb and any surfaces is important, especially during the first week post-surgery. Several ways to prevent this entail housing and anesthetizing the axolotl in a large enough container such that the axolotl has plenty of room to move around post-surgery.
This surgery also has several limitations. Perhaps the most notable is that the success of surgeries can only be assessed in two ways: using the dissecting scope during the first two weeks of surgery to search for an absence of a wound epidermis and/or checking whether a blastema forms within 3 weeks. While these methods are effective, they are relatively low throughput. The development of future transgenic reporter axolotls for wound epidermis-specific markers may aid in quicker screening for successful versus unsuccessful surgeries. Furthermore, this surgery is more difficult to perform on younger animals as the intact skin is more fragile. Using sub-adult or adult axolotls is thus recommended.
While this surgery was originally developed in N. viridiscens19, it has been easily adapted for axolotls25,39 and can likely be applied to other salamander species as well. In sum, applying this technique to future limb regenerative studies will empower researchers to both develop more tools to address wound epidermis biology and identify the underlying mechanisms driving its function in initiating blastema formation.

Since Lazzaro Spallanzani reported it in 17681, salamander limb regeneration has been one of the most well-studied natural regenerative phenomenon that has enamored biologists for centuries. Successful limb regeneration hinges on the formation, outgrowth, and subsequent patterning of an undifferentiated cellular structure known as the blastema. Researchers have made significant strides in understanding the cellular composition of the blastema as well as which supportive tissues and cell types are necessary for its formation2,3,4,5,6,7,8,9,10,11,12,13. Yet, the coordinated signaling mechanisms between different tissues and cell types that lead to the initiation of blastema formation remain poorly understood.
A key requirement for successful blastema formation and regeneration is the wound epidermis, a transient and specialized epithelium that covers the amputation plane within 12 hours post-amputation10. Following amputation, epithelial cells from the intact skin proximal to the injury rapidly migrate over the amputation plane to form a thin wound epithelium14. As the blastema forms in the following weeks, the early wound epidermis develops into a thicker epithelial signaling structure called the apical epithelial cap (AEC)15. While normal full thickness skin contains both an epithelial and dermal layer separated by a basal lamina, the wound epidermis/AEC consists only of an epithelial layer and lacks a basal lamina16,17. The absence of the basal lamina and dermis allows for direct contact between the wound epithelial cells and the underlying tissues, which facilitates bi-directional signaling between the two compartments that is critical for both blastema formation and maintenance17,18.
Classical experimental studies devised various innovative surgical methods to probe wound epidermis/AEC function and necessity via inhibiting its formation. These methods included suturing19 or grafting full thickness skin20,21 over the amputation plane, immediately suturing the amputated limb into the body cavity22, and continuous daily removal or irradiation of the early wound epidermis and AEC23,24. Altogether, these experiments not only established the importance of the wound epidermis/AEC, but also further determined its roles in early tissue histolysis as well as maintaining progenitor cell proliferation and blastemal outgrowth13 throughout regeneration.
However, these previous studies were largely limited to histological staining as well as tritiated thymidine pulses to track cell proliferation. In fact, revisiting these classical experiments with modern sequencing technologies and functional techniques in salamanders has only recently been done and has led to the discovery of additional roles for the wound epidermis in modulating inflammation and ECM degradation/deposition during early stages of regeneration25. With the release of various salamander genome and transcriptome sequences26,27,28,29,30,31,32,33,34, as well as the burgeoning number of functional methods available in salamander species11,35,36,37,38, researchers are now well-positioned to begin to unravel the molecular mechanisms driving wound epidermis formation, function, and AEC development.
Unfortunately, several of these classical methods used to inhibit wound epidermis formation are technically challenging, presenting difficulties for reproducibility between biological replicates in the same experiment. For instance, maintaining skin grafts can be challenging as grafts may eventually fall off the host limb and removal of the wound epidermis/AEC daily is difficult without damaging the underlying tissues. Furthermore, suturing the amputated limb into the body cavity is challenging and also requires additional injury at the site of insertion. On the other hand, suturing full thickness skin immediately over the amputation plane is relatively simple, technically reproducible, and introduces minimal tissue damage. This full skin flap (FSF) surgical method was previously developed by Anthony Mescher in 1976 in adult newts (Notophthalmus viridiscens). He demonstrated that the FSF surgery inhibited wound epidermis formation and function by prohibiting both epithelial cell migration over the amputation plane and direct contact between epithelial cells and the underlying tissues.
Here, this surgical procedure is shown step-by-step using the axolotl limb. Coupled with modern day molecular and sequencing technologies, this technique may prove to be very helpful for researchers to deepen our understanding of wound epidermis/AEC formation and function during limb regeneration.

<table>
	<tbody>
		<tr>
			<td>Curved spring scissors</td>
			<td>Fine Scientific Tools</td>
			<td>15009-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>886-86-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific Tools</td>
			<td>11252-40</td>
			<td>Need two pairs</td>
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		<tr>
			<td>Nylon monofilament sutures (9-0)</td>
			<td>Roboz</td>
			<td>SUT-1000-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfamerazine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>127-58-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
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inhibition of wound epidermis formation via full skin flap surgery during axolotl limb regeneration

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling structure that forms rapidly post-amputation and is required for limb regeneration. However, methods to study its precise function at the molecular level over the last decades have been limited due to a paucity of precise functional techniques and genomic information available in salamander model systems. Excitingly, the recent plethora of sequencing technologies coupled with the release of various salamander genomes and the advent of functional genetic testing methods, including CRISPR, makes it possible to re-visit these foundational experiments at unprecedented molecular resolution. Here, I describe how to perform the classically developed full skin flap (FSF) surgery in adult axolotls in order to inhibit wound epidermis formation immediately following amputation. The wound epidermis normally forms via distal migration of epithelial cells in the skin proximal to the amputation plane to seal off the wound from the outside environment. The surgery entails immediately suturing full thickness skin (which includes both epidermal and dermal layers) over the amputation plane to hinder epithelial cell migration and contact with the underlying damaged mesenchymal tissues. Successful surgeries result in the inhibition of blastema formation and limb regeneration. By combining this surgery method with contemporary downstream molecular and functional analyses, researchers can begin to uncover the molecular underpinnings of wound epidermis function and biology during limb regeneration.

This surgical protocol will allow for the complete inhibition of wound epidermis formation (Figure 1) and ultimately, limb regeneration. A successful surgery results in no blastema formation in approximately 2-3 weeks depending on the size of the animal, while control regenerating limbs should form a blastema normally.
Researchers should inspect the sutured limb by naked eye every 2-3 days to make sure that the sutures have not popped out and that a blastema is not forming. If one or more of the sutures pop out, a wound epidermis can still form resulting in either a small or large blastema and an unsuccessful surgery (Figure 2). Additionally, researchers should inspect the sutured limb at least once every week under a stereomicroscope to make sure that a thin wound epidermis is not evident anywhere on the amputation surface. For comparison, researchers should also examine the control regenerating limb which should have a wound epidermis over the amputation plane and form a blastema over 2-3 weeks. The wound epidermis will appear thin and clear, while the normal skin will appear more opaque and pale pink (almost white), light yellow, or dark green in leucistic, albino, or wildtype axolotls, respectively.
If researchers wish to collect tissue prior to the blastema formation stages at 2-3 weeks, they should inspect the sutured limbs prior to sample collection to make sure the sutures remained in place and that a small wound epidermis did not form. Additionally, sectioning sagitally through the sutured limb tissue and performing histological analyses at any time point can also verify the presence of the dermis from the full skin flap encircling the entire amputation plane and the absence of a wound epidermis (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61522/61522fig01.jpg" /> 
Figure 1: Schematic of the steps of the full skin flap surgery. 
The steps of the protocol are numbered and diagrammed here. The dotted lines denote the planes of amputation at steps 1 and 3 of the protocol. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61522/61522fig02.jpg" /> 
Figure 2: Examples of successful and unsuccessful full skin flap surgeries. 
Representative brightfield image of a limb that has undergone a successful surgery (left), an unsuccessful surgery (right), and a control regenerating limb (no surgery) at 25 days post-amputation (dpa). The successful surgery has a flat amputation plane where the full skin flap was sutured over, whereas the unsuccessful surgery has a small blastema developing. Arrowheads denote the amputation plane and white dotted lines are there to aid visualization of the absence of a blastema in the successful surgery and presence of blastemas in the unsuccessful surgery and control regenerating limbs. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61522/61522fig03.jpg" /> 
Figure 3: Histological staining of normal regenerating and FSF sutured limbs. 
(A-B&#8217;) Representative brightfield images of picro-mallory stained sections from regenerating (A-A&#8217;) and sutured axolotl limbs (B-B&#8217;) at 7 dpa. Insets in A and B are shown in A&#8217; and B&#8217;, respectively. The collagen-heavy dermal layer lines and covers the entire amputation plane in sutured limbs. Amputation plane is denoted by arrowheads in A-B. Scale bars represent 500 &#181;m. This figure was adapted from Tsai et al.25. <a href="https://www.jove.com/files/ftp_upload/61522/61522fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration at JoVE.com

Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Classical experiments in salamander regenerative biology over the last century have long established that the wound epidermis is a crucial signaling ...

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3.0K Views.  Harvard University. This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration.

Video: Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...