This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

Ethics Statement: All these experiments follow the animal care and ethical guidelines of the University of Nottingham.
1. Preparation of Heparin Beads for Grafting
<ol>
	<li>Wash heparin beads thoroughly in PBS before use. Note: Beads can be stored at 4 &#176;C as a slurry in PBS.
	<ol>
		<li>Select beads for grafting by removing them from the stock with a 20 &#181;l pipette into a 1 ml drop of PBS. Then use a micropipette set to 2 &#181;l to transfer beads into a 20 &#181;l drop of PBS in a 3 cm petri dish. Choose beads based on size and, using a stereo dissecting microscope, transfer selected beads with a P2 pipette tip; those around 100 &#181;m diameter are ideal.</li>
	</ol>
	</li>
	<li>Remove the PBS from the beads. Remove most of the liquid with a 20 &#181;l pipette and the residual liquid by capillary action with fine watchmakers forceps. Note: It is important to remove as much as possible so the growth factor is not diluted when it is added to the beads.</li>
	<li>Pipette the growth factor directly onto the beads. For FGF18 add 0.5 &#181;l of recombinant FGF18 reconstituted at 0.5 mg/ml in 0.1% BSA in PBS. Place several drops of water around the edges of the dish to prevent the liquid from evaporating.</li>
	<li>Incubate the beads for 1 hr at RT. Remove the drops of water from the dish with a Pasteur pipette and place on ice ready for grafting.</li>
	<li>If required (for transparent beads) transfer 4-5 beads to 2% phenol red before grafting to help visualize them. Rinse in PBS before transferring to the embryo.</li>
</ol>
2. Preparation of AG 1-X2 Beads for Grafting
<ol>
	<li>Prior to use derivatize beads with 0.2 N formic acid.
	<ol>
		<li>To do this use a spatula to transfer beads to a 1.5 ml microcentrifuge tube. Add 1ml of 0.2 N formic acid and wash on a shaking platform for 1 hr.</li>
		<li>Then remove the formic acid with a p1000 pipette and replace with 1 ml of water. Wash for 1 hr and repeat six times to remove remaining formic acid.</li>
		<li>Remove remaining liquid with a p1000 pipette and store the beads at 4 &#176;C. Note: It is not necessary to remove all the residual water at this stage.</li>
	</ol>
	</li>
	<li>Remove a small pellet of beads of approximately 5-10 &#181;l volume from the stock using a small spatula into a 3 cm petri dish and add 1 ml of DMSO (or whichever solvent the drug is dissolved in). Note: This should provide several hundred beads from which to select those of the right size.</li>
	<li>Then use a P2 pipette set to 2 &#181;l to transfer beads of approximately 100 &#181;m diameter to a 20 &#181;l drop of solvent in another 3 cm petri dish.</li>
	<li>Remove solvent with a 20 &#181;l pipette and any residual solvent with a 2 &#181;l pipette. Replace with 20 &#181;l of the drug to be applied. 
	NOTE: For consistency dissolve the drug at a concentration of 100 &#181;M in DMSO (or which ever solvent is used), aliquot into 20 &#181;l and store at -80 &#176;C as some small molecule inhibitors are unstable and it is best to avoid repeated freeze-thawing. If necessary then dilute the drug to the desired concentration before soaking beads. Effective concentrations may need to be determined empirically for each drug.</li>
	<li>Incubate for 1 hr. Protect from light as many of these molecules are light sensitive.</li>
	<li>Before grafting transfer 4-5 beads with a p2 pipette set to 2 &#181;l into 20 &#181;l of 2% phenol red dissolved in water. Perform this step using a stereo dissecting microscope to visualize the beads. Remove a single bead with fine watchmakers forceps or a wire loop and rinse in PBS before transferring to the embryo. Do not leave beads in 2% phenol red for more than 15 min before use as this can cause loss of activity.</li>
</ol>
3. Preparing Tungsten Needles
<ol>
	<li>Cut fine tungsten wire into 3-4 cm lengths. Insert one piece of wire into the end of a glass Pasteur pipette and melt in a Bunsen burner to fix in position. Prepare up to 10 needles to ensure that there are spares if they are damaged during use.</li>
	<li>Place the end of the wire into a blowtorch flame and hold it there until the tungsten glows white and the tip is sharp (around 2-3 min). Note: During use the needle can be cleaned and re-sharpened in the blowtorch as needed. 
	NOTE: These needles can also be used to make loops for transferring beads. Gently touch the needle to the bench and it will bend to form a loop.</li>
</ol>
4. Preparing Embryos for Bead Grafts
<ol>
	<li>Incubate eggs with the blunt end up until they reach the desired stage (3-5 days for most limb bud manipulations). Using blunt forceps tap on the blunt end to break the shell and then use the forceps to remove the shell and expose the embryo. Ensure the eggshell membrane is also removed. 
	NOTE: 1-2 ml of PBS can be added with a Pasteur pipette to assist with this if the membrane sticks to the yolk. Gently pipette the PBS onto the surface of the yolk, grasp the membrane with the forceps, taking care not to damage the yolk, and gently pull it away from the egg.</li>
	<li>Remove up to 5 ml of albumen from the egg with a 10 ml syringe and a 19 G needle. Take only as much as needed to ensure the embryo does not make contact with the tape used to seal the egg as removal of larger volumes can reduce embryo survival rates.</li>
	<li>To visualize the embryo add a 5-6 drops of artists india ink to 15 ml of PBS containing 100 U penicillin and 0.1 mg streptomycin/ml. Inject 0.5-1 ml directly under the embryo with a 1 ml syringe and a 25 G needle.</li>
	<li>Add 2-3 ml of PBS with 1% fetal calf serum and 100 U penicillin and 0.1 mg streptomycin/ml into the egg to ensure the embryo does not dehydrate. Using sharpened watchmakers forceps remove the vitelline membrane and open the amnion over the developing limb buds. Ensure the embryo does not dry out and, if needed, add more PBS / FCS. 
	NOTE: The limb buds can be clearly seen as paired outgrowths on the flank of the embryo. At these stages the embryo will turn such the right side is normally uppermost. As a result it is normally easier to graft beads into the right limbs and use the left ones as contralateral controls.</li>
	<li>Use a sharpened tungsten wire to make an incision into the limb bud where the bead will be implanted. Avoid cutting all the way through the limb bud where possible although at younger stages this is difficult. 
	NOTE: In older embryos (HH stage 22 and above) it is possible to select specific regions of the limb bud for grafting but at younger stages this is more difficult as the limb bud itself is small. It is also important to remember that the limb bud will grow past the grafted bead so, especially at younger stages, the bead will remain in the proximal limb.</li>
	<li>Pick up a bead from either the growth factor or drug using either extra fine watchmakers forceps or a loop made from a tungsten needle. If the bead has been soaked in either DMSO or phenol red, rinse in PBS before applying to the embryo. Transfer the bead to the embryo with the forceps / wire loop. Then use the sharpened tungsten wire to insert the bead into the incision.</li>
	<li>Add 1-2 ml of PBS/FCS to the egg ensuring that the embryo is kept hydrated but avoid applying directly onto the embryo itself as this can damage the embryos and cause the bead to be dislodged. Seal the egg with tape and return to the incubator.</li>
</ol>

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The authors have nothing to disclose.

The use of bead grafts applied directly to developing tissues in ovo is a powerful tool to dissect the role of growth factor signaling during development giving unparalleled control over the developmental stage at which they are applied and the duration of exposure.
The choice of bead for each type of molecule is important. Small hydrophobic molecules, such as the inhibitors described here and retinoic acid, usually bind well to derivatized AG 1-X2 beads although it is necessary to test the ef...

Avian embryos have provided a powerful tool for the study of development for many years1. One of their most useful characteristics is that they are relatively easy to manipulate. External development makes it possible to open the egg to access the embryo and perform various micromanipulations including examples such as the classic quail-chick chimera system for studying cell fate2,3, the injection of retroviruses for overexpression in specific tissues during development4,5 and explant culture to identify developmental signaling sources6. More recently chimeras generated between unlabeled hosts with grafts from a transgenic chicken line expressing GFP has shown that the combination of classical grafting and genetic modification can provide important insights into development7,8.
The ease with which the avian embryo can be manipulated has made it an excellent model for studying limb development9. Application of specific growth factors to developing limbs in vivo has been instrumental in identifying factors that alter limb patterning10,11 and continues to provide insights into this process12. This approach has also been used to study the factors that regulate muscle development and has uncovered roles for numerous signals such as Wnts13, BMPs14 and HGF15.
Recently this technique has been used to investigate the signals controlling myogenic gene expression in the limb bud and has shown that interactions between FGF18 and retinoic acid can control the timing of MyoD expression16. Using a combination of growth factors and small molecules that can be loaded onto beads and then grafted directly into specific tissues at defined developmental stages gives the opportunity to intervene at almost any time and region during development. This has been used to investigate many processes including somite patterning17,18, neural specification19, neural crest migration20 and axis extension21.
Here we describe a method for grafting beads soaked in either growth factors or inhibitors into developing chicken limbs. This has been used to determine the effects of these signals on myogenesis by analyzing muscle specific gene expression with in situ hybridization. We describe grafts using either heparin soaked beads, which are used for growth factors, or AG 1-X2 beads for small hydrophobic molecules such as retinoic acid or small molecule inhibitors of specific signaling pathways. However other beads are also available which have been used to deliver both FGFs22 and Shh23.

<table>
	<tbody>
		<tr>
			<td>Heparin acrylic beads</td>
			<td>Sigma</td>
			<td>H5263</td>
			<td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td>
		</tr>
		<tr>
			<td>AG 1-X2 beads</td>
			<td>Bio-Rad</td>
			<td>140-1231</td>
			<td></td>
		</tr>
		<tr>
			<td>Affi Gel blue beads</td>
			<td>Bio-Rad</td>
			<td>153-7301; 153-7302</td>
			<td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td>
		</tr>
		<tr>
			<td>FGF18</td>
			<td>Peprotech</td>
			<td>100-28</td>
			<td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&#176;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td>
		</tr>
		<tr>
			<td>U0126</td>
			<td>Cell Signalling</td>
			<td>9903</td>
			<td>Make to 20mM stock in DMSO and store in single use aliquots at -80&#176;C. Protect from light.</td>
		</tr>
		<tr>
			<td>BMS-493</td>
			<td>Tocris Biosciences</td>
			<td>3509</td>
			<td>Resuspend in DMSO and store in single use aliquots at -80&#176;C. Protect from light.</td>
		</tr>
		<tr>
			<td>Black Indian ink</td>
			<td>Windsor and Newton</td>
			<td>5012572003384 (30ml)</td>
			<td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td>
		</tr>
		<tr>
			<td>Tungsten wire, 0.1mm dia. 99.95%</td>
			<td>Alfa Aesar</td>
			<td>10404</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td>Dilute 100X in PBS/ink and PBS/FCS</td>
		</tr>
	</tbody>
</table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

At HH stage 21 MyoD is not expressed in developing limb myoblasts although staining can be seen in the myotome of the developing somites (Figure 1A). Figure 1B shows in situ hybridization for MyoD 6 hr following an FGF18 bead graft. MyoD is induced in myoblasts close to the bead while there is no expression in the contralateral limb. Co-grafting a bead soaked in U0126, a specific inhibitor of MEK, blocks FGF18 induction of MyoD (Figure 1C). Similarly grafting a ...

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

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8.7K Views.  University of Nottingham. By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Video: Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

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

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

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

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

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

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

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 ...