The authors wish to acknowledge Yerkes National Primate Research Center for access to chimpanzee and rhesus macaque specimens, and Chris Vinyard (Northeast Ohio Medical University) for access to common marmoset specimens. We thank Marissa Boettcher, Kaitlyn Leonard, and Antonia Meza at the University of North Carolina for assistance with the scanning process. This work was performed in part at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (Grant ECCS-1542015) as part of the National Nanotechnology Coordinated Infrastructure (NNCI). This is Duke Lemur Center publication number 1405.

Because these procedures use animals that died from natural causes at zoos or were sacrificed in research labs where they were part of unrelated studies, these protocols do not require IACUC approvals.
1. Reverse Dissection
Note: The protocol for reverse dissection is effective for very small mammals, such as laboratory mice, all the way to large land mammals, such as the domestic horse. The mimetic muscles are often best preserved and best visualized when left with the overlying dermis instead of leaving them behind on the skull. See Figure&#160;1-3.
<ol>
	<li>Disarticulate head from cervical part of the vertebral column prior to dissection procedures. If the cadaver is frozen, allow it to thaw completely before proceeding through this step.
	<ol>
		<li>Regardless of the state of preservation, palpate the occipital region of the skull and the spinous process of the second cervical vertebra (C2). Note that the first cervical vertebra (C1) lacks a spinous process, so palpating C2 and the occipital region of the skull will give an approximate location of C1.</li>
		<li>Using a scalpel, make horizontal cuts between the occipital region of the skull and C2 in order to begin separating the skull from the vertebral column at C1. Continue to make cuts until the skull is disarticulated from the vertebral column.</li>
		<li>If the specimen is fresh and unfixed, steps 1.1.2-1.2 need to be executed in a relatively short time frame. Additionally, if the brain remains intact inside the cranial cavity, necrosis of the neural tissue may proceed rapidly, so it is highly recommended that the brain be removed from the cadaver if the specimen is to remain unfixed. 
		&#8203;NOTE: As an optional step, the specimen can be immersed in 10% buffered formalin for 24 hours in order to fix the specimen. This will allow for an unhurried data collection procedure.</li>
		<li>If the specimen is already fixed with ethyl alcohol or 10% buffered formalin, rinse for several hours with fresh water.</li>
	</ol>
	</li>
	<li>Make incisions to release facial mask from skull. If the brain and overlying calvaria have been removed during necropsy, make one incision near the glabellar region of the skull and another along the caudal aspect of the skull where the cranial bone is intact.
	<ol>
		<li>Once the specimen has been rinsed for several hours, pat dry with paper towels and transfer to work station.</li>
		<li>Palpate for the external occipital protuberance at the caudal (or rear) edge of the skull. This marks the area where the skull meets the spinal column. Using a #4 scalpel for large specimens or a #3 scalpel for small specimens, make a mid-line incision beginning at the external occipital protuberance and coming rostrally (or forward) over the parietal and frontal region of the skull, over the orbital region and between the eyes, down the nasal region, all the way through the external nasal area between the nares.</li>
		<li>Make a cut on the mandible between the lower, central incisors and move caudally (or backwards) toward the clavicle.</li>
		<li>Choose one side of the face mask to remove and leave the other side in place.</li>
	</ol>
	</li>
	<li>Release one side of the facial mask from the skull.
	<ol>
		<li>Starting in the occipital region, use the scalpel to cut the facial mask, including mimetic musculature, away from the occipital bone of the skull, pulling the mask rostrally (or forward) and laterally (to the side). Cut the occipitalis muscle away from the skull, leaving a small amount of the muscle behind on the skull so that its attachment can still be visualized once the facial mask has been removed. By leaving some portion of the muscle behind on the bony/cartilaginous skull, attachments can be preserved for recording at a later time.</li>
		<li>Once the external ear is hit, locate the auricular muscles. Cut through these so that a small portion of each remains with the skull. Cut through the elastic cartilage attaching the external ear to the skull.</li>
		<li>Continue to pull the mask rostrally and release each mimetic muscle from the skull by leaving a small portion of each muscle behind on the skull.</li>
		<li>Once there is an intact facial mask from one side of the skull, allow it to sit out, exposed to the air, for an hour (for small specimens like a lab rat) up to three hours (for a large specimen like a domestic horse), with the musculature side facing up. This step desiccates some of the connective tissue on the face mask and increases color contrast between muscle and connective tissue.</li>
	</ol>
	</li>
	<li>Remove sufficient connective tissue to visualize musculature.
	<ol>
		<li>Using a #3 scalpel, forceps, and microscissors, pluck away the connective tissue that separates the mimetic musculature from one another.</li>
		<li>To access the superficial layer of mimetic musculature in this &#34;reverse dissection&#34;, gently lift and separate the deep layer of musculature from the superficial layer and remove the connective tissue surrounding the superficial musculature. The resulting face mask can be returned to formalin, and the procedure can be paused here before moving on to further dissection or to staining (Step 2). At each intervening step in further dissection, allow the facial mask to dry enough so that connective tissue can be easily discerned from muscle tissue.</li>
	</ol>
	</li>
</ol>
2. Staining Process for DiceCT
NOTE: Specimens must be fixed in 10% buffered formalin if DiceCT is to be used in order to preserve the tissue during the lengthy staining procedure. If the specimen has not yet been fixed, place it in a container with enough 10% buffered formalin to submerge all tissue and leave it for 48 hours.
<ol>
	<li>Make 1.75% (m/v) Lugol&#39;s iodine solution (I2KI) for staining face mask. 
	&#8203;NOTE: Make enough I2KI solution to fully submerge the face mask within the chosen container. Make more or less solution by scaling the values of both the solutes and the solvent equally in steps 2.1.1 and 2.1.2.
	<ol>
		<li>Under a fume hood, add 3.50 g potassium iodide (KI) to 200 mL distilled water in a large glass beaker. Stir using glass stirring rod until fully dissolved. 
		CAUTION: Potassium iodide (KI) can cause eye, skin, and respiratory irritation or damage. Wear protective eyewear and handle under a fume hood.</li>
		<li>Slowly add 1.75 g of iodine crystals (I2), stirring until fully dissolved. This will take several minutes. 
		CAUTION: Iodine crystals (I2) can cause eye, skin, and respiratory irritation or damage. Wear eye and skin protection and work under a fume hood while handling this chemical.</li>
		<li>Filter solution into amber glass bottle using filter paper with 11 micron pore size to remove any undissolved crystals that could produce inconsistent staining. 
		CAUTION: This solution is highly toxic to aquatic life. Do not dispose of solution down drain. In case of spill, neutralize aqueous triiodide (I3-) into iodide (I-) with 5% (m/v) sodium thiosulfate (Na2S2O3). Check institutional waste disposal policies for iodide and dispose of this waste accordingly.</li>
	</ol>
	</li>
	<li>Stain the face mask.
	<ol>
		<li>Place the face mask cut side down in an amber glass or blacked out container (to keep out light) large enough to allow the face mask to rest in a natural shape and to be fully submerged.</li>
		<li>Pour the Lugol&#39;s iodine solution over the face mask until it is completely submerged. A weight may be needed to keep the face mask fully submerged and prevent it from floating to the top of the solution. Seal the container to prevent evaporation and keep out light.</li>
		<li>Agitate the container continually on an electric rocker or regularly by hand to facilitate even staining and prevent crystals from settling for at least two weeks. If a longer time is needed to adequately stain the muscle fascicles, replace the Lugol&#39;s solution with fresh solution.</li>
		<li>After two weeks, remove the face mask from the solution. Pour a small amount of distilled water over both surfaces of the face mask to rinse. Wrap the face mask in damp paper towels and place in a sealed plastic bag for at least 24 hours to allow for slight drying before moving on to the next step. 
		NOTE: The protocol can be paused for up to a week. Ensure that the face mask remains slightly damp during this period.</li>
	</ol>
	</li>
</ol>
3. DiceCT scanning
<ol>
	<li>Prepare face mask for CT scanning.
	<ol>
		<li>Mount the face masks onto a low-density material, such as floral foam, to eliminate any wrinkles in the mask and to limit movement of the mask as the mask dries out slightly during scanning. Secure the face mask to the foam with wooden toothpicks.</li>
	</ol>
	</li>
	<li>Setting the CT scan parameters and scanning.
	<ol>
		<li>Use a high resolution for these scans, as the small fascicles will be obscured at lower resolution. Use an inter-slice spacing and inter pixel distance around 0.05 mm.</li>
		<li>Scan the mounted face mask on a High-Resolution X-ray Computed Tomography Scanner.</li>
		<li>If fascicles are unable to be fully visualized, due to over-staining, place face mask into 10% formalin solution or into 5% sodium thiosulfate to remove some of the stain. If fascicles are not sufficiently stained, return to Lugol&#39;s iodine solution. After adjusting the staining, scan the face mask again. Continue to make necessary adjustments and scan until fascicles are able to be fully visualized.</li>
	</ol>
	</li>
</ol>
4. Prepare the face mask for long-term preservation.
<ol>
	<li>Chemically neutralize the iodine stain in the face mask, removing discoloration from the staining process.
	<ol>
		<li>Place stained face mask into 5% (m/v) sodium thiosulfate solution to chemically neutralize specimen. This may take several hours or days. Agitate regularly during destaining.</li>
		<li>Return destained face mask to 10% formalin solution for preservation. If stain continues to leach out of specimen into solution, replace discolored formalin with fresh formalin until it remains clear for long-term preservation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gregory, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Our face from fish to man. , (1929).</li><li>Burrows, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+facial+expression+musculature+in+primates+and+its+evolutionary+significance.">The facial expression musculature in primates and its evolutionary significance.</a> BioEssays. 30, 212-215 (2008).</li><li>Santana, S. E., Dobson, S. D., Diogo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plain+faces+are+more+expressive:+comparative+study+of+facial+colour,+mobility+and+in+primates.">Plain faces are more expressive: comparative study of facial colour, mobility and in primates.</a> Biology Letters. , (2014).</li><li>Burrows, A. M., Cohn, J. F., Li, S. Z., Jain, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+of+the+face.">Comparative anatomy of the face.</a> Encyclopedia of Biometrics. , (2014).</li><li>Liebal, K., Waller, B. M., Burrows, A. M., Slocombe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Communication. , (2013).</li><li>Rowe, N., Myers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All the World's Primates. , (2016).</li><li>Burrows, A. M., Waller, B. M., Micheletta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimetic+muscles+in+a+despotic+macaque+(Macaca+mulatta)+differ+from+those+in+a+closely+related+tolerant+macaque+(M.+nigra).">Mimetic muscles in a despotic macaque (Macaca mulatta) differ from those in a closely related tolerant macaque (M. nigra).</a> Anatomical Record. 299, 1317-1324 (2016).</li><li>Burrows, A. M., Parr, L. A., Durham, E. L., Matthews, L. C., Smith, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+faces+are+slower+than+chimpanzee+faces.">Human faces are slower than chimpanzee faces.</a> PLoS One. 9, 0110523 (2014).</li><li>Burrows, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+mimetic+musculature+in+primates:+how+social+variables+and+body+size+stack+up+to+phylogeny.">Functional morphology of mimetic musculature in primates: how social variables and body size stack up to phylogeny.</a> Anatomical Record. 301, 202-215 (2018).</li><li>Burrows, A. M., Smith, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+of+facial+expression+in+Otolemur,+with+a+comparison+to+Lemuroidea.">Muscles of facial expression in Otolemur, with a comparison to Lemuroidea.</a> Anatomical Record. 274, 827-836 (2003).</li><li>Burrows, A. M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+inside+tarsier+faces.">What's inside tarsier faces.</a> Yearbook of Physical Anthropology. 60, 96 (2015).</li><li>Dickinson, E., Stark, H., Kupczik, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-destructive+determination+of+muscle+architectural+variables+through+the+use+of+DiceCT.">Non-destructive determination of muscle architectural variables through the use of DiceCT.</a> Anatomical Record. 301, 363-377 (2018).</li><li>March, D., Hartstone-Rose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+and+behavioral+correlates+to+postcranial+musculature.">Functional morphology and behavioral correlates to postcranial musculature.</a> Anatomical Record. 301, 419-423 (2018).</li><li>Santana, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+of+bat+jaw+musculature+via+diffusible+iodine-based+contrast-enhanced+computed+tomography.">Comparative anatomy of bat jaw musculature via diffusible iodine-based contrast-enhanced computed tomography.</a> Anatomical Record. 301, 267-278 (2018).</li><li>Burrows, A. M., Waller, B. M., Parr, L. A., Bonar, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+of+facial+expression+in+the+chimpanzee+(Pan+troglodytes):+descriptive,+comparative+and+phylogenetic+contexts.">Muscles of facial expression in the chimpanzee (Pan troglodytes): descriptive, comparative and phylogenetic contexts.</a> Journal of Anatomy. 208, 153-167 (2006).</li><li>Burrows, A. M., Waller, B. M., Parr, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+musculature+in+the+rhesus+macaque+(Macaca+mulatta):+evolutionary+and+functional+contexts+with+comparisons+to+chimpanzees+and+humans.">Facial musculature in the rhesus macaque (Macaca mulatta): evolutionary and functional contexts with comparisons to chimpanzees and humans.</a> Journal of Anatomy. 215, 320-334 (2009).</li><li>Cox, P. G., Jeffery, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reviewing+the+Morphology+of+the+Jaw-Closing+Musculature+in+Squirrels,+Rats,+and+Guinea+Pigs+with+Contrast-Enhanced+MicroCt.">Reviewing the Morphology of the Jaw-Closing Musculature in Squirrels, Rats, and Guinea Pigs with Contrast-Enhanced MicroCt.</a> Anatomical Record. 294, 915-928 (2011).</li><li>Gignac, P. M., Kley, N. J., Clarke, J. A., Colbert, M. 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The authors have no disclosures to report.

Following the steps for the &#34;reverse dissection&#34; protocol typically produces a facial mask that can be slowly and methodically dissected to reveal mimetic musculature, regardless of the size of the head. It is especially important to move slowly and continuously assess whether the muscles have been completely cut through inadvertently, especially in smaller bodied species.
In order to determine where the musculature is located, it is especially critical to allow the developing mask to ...

Mimetic musculature, or facial expression musculature, is skeletal muscle and is found throughout Mammalia1. While most mammalian skeletal muscle attaches to discrete bony landmarks, mimetic musculature is unique in its attachments primarily into the skin of the face, scalp, and the ventral aspect of the neck1,2,3,4. Mimetic musculature contraction deforms the &#34;facial mask&#34; into expressions or facial displays of social and emotional intent, changes the size and shape of the sphincters of the eye, nasal cavity, and oral cavity used in feeding, respiration, and in vocalizations, and is part of the overall close-proximity visual communication mechanism found among most mammals2,3,4,5. Across Mammalia, the facial displays generated by mimetic muscles assist in regulating and maintaining territorial boundaries, social bonds, and the social group by cuing conspecifics on the emotional and behavioral intentions of the sender2,5.
Among mammals, primates are characterized in part as employing a high level of social behavior throughout the life cycle with all species living in a social group2,5. While some taxa, such as the nocturnal galagos and lorises, may live in groups consisting only of a mother and offspring, other taxa, such as the diurnal macaques and baboons, may live in groups of over 100 individuals6. No matter the size of the social group, primates often use stereotyped social behaviors associated with rank and territoriality and these behaviors typically include a facial display component. Facial displays are part of the process of maintaining bonds among member of social groups, dominance hierarchies, reproduction, and the communication that is part of daily life, especially in diurnal species2,5,7. While it has been clear for some time that facial musculature is used to create these facial displays, it has only recently become apparent that facial musculature form and physiology are associated with the functional demands of social variables2,8. Previous studies on phylogenetically and behaviorally diverse ranges of primates have shown that diurnal species living in large, complex social groups tend to have a high number of discrete facial displays that focus on movement of the lips, eyebrows, and eyelids with a high number of facial muscles clustered around the lips and orbital region9. In contrast, there have been few studies on nocturnal species living in small groups, but these species have a high number of discrete facial muscles with attachments around the external ear and lips, which may be associated with movements of the ear and lips (which have been documented in some nocturnal species in agonistic encounters with conspecifics and locating sounds)2,9,10,11. In addition, humans have a relatively higher percentage of slow-twitch myosin fibers in mimetic musculature than either rhesus macaques or chimpanzees, which may be related to the &#34;slowing down&#34; in contraction of human mimetic musculature around the lips used during production of speech sounds or to general fatigue-resistance capability of the muscles8.
Humans are, arguably, the most social of all primates, and have developed language as one component of social communication. Still, though, humans use facial expression as a means of visual communication and have the greatest known facial display repertoire among primates. In an effort to more completely understand the variables surrounding the evolution of human and general primate social behavior, an increased understanding of the morphology and physiology of primate mimetic musculature is highly desirable. Because mimetic musculature is attached to the skin itself and may, in some species, be exceptionally thin and difficult to visualize, we have developed a unique method of visualizing this musculature for both the processes of recording gross presence/absence and attachments as well as sampling for microanatomical processing.
&#34;Reverse dissection&#34; is a method for preserving the mimetic musculature by removing the entire facial mask from the head and increasing visibility of even small muscles. Because reverse dissection is a destructive process, rare and valuable specimens may not always be available for this methodology. DiceCT is an effective method that can visualize many of the mimetic muscles in even tiny species12,13,14. This method can be used in concert with reverse dissection or in cases where rare, valuable specimens may not be dissected and can provide much information without having to remove the facial mask in &#34;reverse dissection&#34;12,13,14. The present protocol describes a set of methods for combining reverse dissection with DiceCT in order to examine primate mimetic musculature.

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			<td height="21" style="height:21px;width:216px;">Nikon XTH 225 ST</td>
			<td style="width:71px;">Nikon</td>
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			<td style="width:167px;">no catalog numbers</td>
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			<td height="42" style="height:42px;width:216px;">10% buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SF98-4</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Iodine, ACS Grade</td>
			<td style="width:71px;">Lab Chem, Inc.</td>
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			<td height="42" style="height:42px;width:216px;">Sodium thiosulfate</td>
			<td style="width:71px;">Acros Organics</td>
			<td style="width:119px;">AC450620010</td>
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reverse dissection and dicect reveal otherwise hidden data in the evolution of the primate face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

This section presents examples of results on facial musculature form that can be achieved by using &#34;reverse dissection&#34; in concert with DiceCT scanning. By using &#34;reverse dissection&#34; to create a facial mask, a fuller representation of mimetic (facial) muscle can sometimes be seen than in traditional dissection methodology. This method works on a range of body sizes from the tiny, small-bodied primates, for example the common marmoset Callithrix jacchus (<strong cl...

Watch this Scientific Journal Video about Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face at JoVE.com

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

reverse-dissection-dicect-reveal-otherwise-hidden-data-evolution

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.8K Views.  Duquesne University. Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Video: Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

This article describes the procedure for the identification and characterization of a gene family in grapevine applied to the family of Arabidopsis T&#243;xicos in Levadura (ATL) E3 ubiquitin ligases.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

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Chapters in this video

Molecular Evolution of the Tre Recombinase

Dissecting the Non-human Primate Brain in Stereotaxic Space

The Gateway to the Brain: Dissecting the Primate Eye

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

Dissection of Organs from the Adult Zebrafish

Dissection of the Adult Zebrafish Kidney

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Dissection of the Endolymphatic Sac from Mice