Name: reverse dissection and dicect reveal otherwise hidden data in the evolution of the primate face
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face at JoVE.com

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...

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	<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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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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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 ...

This method can help answer key questions in evolutionary biology, such as the evolution of social communication. The main advantages of this technique are that it provides direct evidence for form and presence of mimetic musculature. It eliminates guesswork in the dissection process, and it preserves muscle layers.Combined with DiceCT, reverse dissection is the new gold standard for mimetic muscle investigation. To begin, follow the accompanying text protocol for specimen fixation and preparation. Then, transfer the specimen to the fume hood.Then, rinse the specimen for several hours in running water. Once the specimen has been rinsed, pat it dry with paper towels, and transfer it to a work station. With the brain and overlying calvaria removed, make one incision near the glabella region of the skull, and another along the caudal aspect of the skull, where the cranial bone is intact.Palpate for the external occipital protuberance at the caudal edge of the skull. This marks the area where the skull meets the spinal column. Then use a scalpel and make a midline incision, beginning at the external occipital protuberance.Cut the specimen rostrally over the parietal and frontal region of the skull. Continue and pass over the orbital region in between the eyes, down the nasal region, all the way through the external nasal area between the nares. Next, make a cut on the mandible between the lower and central incisors, and move the scalpel caudally toward the clavicle.At this point, choose one side of the face mask, and release it from the skull, leaving the other side in place. 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 and laterally. 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 and cartilaginous skull, attachments can be preserved and recorded at a later time. Once the external ear is hit, locate the auricular muscles. Cut through these muscles, so that a small portion of each muscle remains with the skull.Then, cut through the elastic cartilage attaching the external ear to the skull. Continue to pull the mask rostrally, and release each mimetic muscle from the skull, leaving a small portion of each muscle behind on the skull. Once there is an intact facial mask from one side of the skull, allow it to sit out with the musculature exposed to the air, for one to three hours, depending on the size of the specimen.This will desiccate the connective tissue on the face mask, and increase the color contrast between muscle and connective tissue. With the contrast between muscle and connective tissue now enhanced, use the number three scalpel, forceps, and micro scissors to remove sufficient connective tissue, in order to visualize the musculature. The superficial layer of mimetic musculature in this reverse dissection, gently lift and separate the deep layer of musculature from the superficial layer, and remove the connective tissues surrounding the superficial musculature.When finished, return the resulting face mask back into formalin. The procedure may be paused here, before moving on to further dissection, or before continuing on to staining. At each intervening step, allow sufficient drying time of the facial mask, so that the connective tissue can be easily discerned from the muscle tissue.In order for the mimetic musculature to be visualized using CT scanning, stain the masks using an iodine solution by placing the mask into Lugol's staining solution, as described in the accompanying text protocol. The staining process generally takes about two weeks, in 1.75%Lugol's solution. However, this timing varies, depending on several factors.Intermittent scanning with accompanied staining adjustment is recommended. For CT scanning, first prepare face mask by using wooden toothpicks to mount the face mask onto a low density material, such as floral foam. This will eliminate any wrinkles in the mask, and limit its movement as it dries out slightly during the scan.Next, place the specimen in a low density container, and secure it into place. Once secure, place the specimen into the scanner, close the door, and turn on the X-rays. Then, set up the CT scan parameters for a high resolution X-ray computed tomography scan.Using inter-slice spacing and inter pixel distance of around 0.05 millimeters for these scans, as the small fascicles will be obscured at lower resolution. If fascicles are unable to be fully visualized, due to overstaining, place face mask into a de-stain solution such as 10%formalin, or 5%sodium thiosulfate to remove some of the stain. After adjusting the staining, scan the face mask again, and continue to make the necessary adjustments, until all fascicles are able to be fully visualized.By using reverse dissection to create a facial mask, a fuller representation of mimetic muscle can sometimes be seen, than in traditional dissection methodology. This method works on a range of body sizes, from the tiny, small body of the Marmoset, to large-bodied primates such as the chimpanzee. In small-bodied primates that have gracil facial muscles, some of the facial musculature may be indistinguishable from the surrounding connective tissue, and may be lost during dissection.An iodine stain, such as Lugol's, may improve the contrast and help to resolve both individual mimetic muscles, as well as individual muscle fascicles, and for the first time, obtain whole muscle volumes of these gracil muscles. As shown here, some of the very small muscles, associated with the external ear, are clearly visible in the DiceCT scans. It is not uncommon for these muscles to be missed in some reverse dissection procedures, perhaps due to their small size.This three dimensional volume rendering allows for basic volume thresholding and color mapping, to visualize the musculature more easily, than with cross-examination alone. Here you can see that the individual muscle fascicles are clearly visible in cross section, making digital dissection feasible. While attempting this procedure, it's important to keep in mind, that you should move slowly, cautiously, and with deliberation, as this method is destructive in nature.Following this procedure, other methods, such as histological processing, may be used to answer additional questions, such as fiber diameter, or myosin fiber typing. After its development, this technique paved the way for researchers in the field of evolutionary biology, to explore the evolution of facial expression and visual communication in non-human mammals, and in humans. Don't forget, that working with formalin, and a loaded scalpel can be extremely hazardous.Precautions, such as rinsing your specimen, and knowing where your scalpel is at all times, should be taken while performing this procedure.

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Biology

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 ...

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 ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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 ...

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...