The research described in this study was supported by a National Science Foundation grant (DEB-0805818) to W.A.F., A.W.S and I.D. This study was also supported by grants IOS-0845847 to A.W.S., IOS-0919855 to A.W.S. and I.D. and IOS-0920720 to W.A.F.

1. Rearing and manipulation of diet to produce variation in body size and wing size
Rational and Overview. The expression of scaling relationships ultimately hinges on the developmental regulatory processes that produce variation in final organ and body size. These processes are best described from work on fully metamorphic (i.e., holometabolous) insects such as D. melanogaster, where organs grow as undifferentiated 'imaginal discs' within the grub-like larva11,15-17. In Drosophila, final organ and body size are regulated by growth during the larval stages; larvae grow through the first and second larval instar until they reach a minimal viable weight for eclosion (MVWE) towards the beginning of the third larval instar18. Starvation before attainment of MVWE prevents adult eclosion. However, larvae that are starved at MVWE generate viable adults, albeit at a very small size. This is because reduced nutrition during development reduces final body and organ size, not just in flies but in almost all animals19-21. By manipulating food availability after MVWE is attained, but before pupation occurs, it is possible to induce extreme phenotypic plasticity in body size that greatly outweighs other sources of variation (e.g., individual genetic variation).
<ol>
	<li>Eggs are collected from the population to be phenotyped, divided into lots of 50 and placed in vials containing 10ml of standard fly food. Because oviposition occurs continuously, eggs are collected every 24h for three days, producing three age cohorts.</li>
	<li>When the oldest cohort reaches the wandering stage or has just begun to pupate, the diet manipulation is applied. At this point, the oldest cohort is well beyond the MVWE, the youngest cohort is very close to the MVWE and the remaining cohort is somewhere in between these extremes. Thus, removal of food at this point will produce very large adults from the oldest cohort, very small adults from the youngest cohort and intermediate-sized adults from the remaining cohort. To remove the larvae from the vials, approximately 5ml of 40% sucrose solution is added to all vials. To free the larvae, the food is agitated for 15-20 minutes on a shaker table set to an intermediate speed.</li>
	<li>Floating larvae are removed from the sucrose solution with a fine (00) paintbrush, placed into vials containing a wet cotton plug and allowed to pupate. Alternatively, larvae could be placed on a non-digestible food additive such as methyl cellulose22.</li>
</ol>
2. Estimation of body size
Rational and Overview. Previous studies indicate that thorax length is a less than ideal proxy for overall body size22. Instead, we use pupal size as a measure of body size. Maximum body size is fixed at the cessation of feeding that precedes pupation. As a result, there is a tight correlation between pupal size and adult body size23.
<ol>
	<li>Pupae are arranged ventral side up on glass microscope slides and imaged using a digital camera attached to a dissecting microscope. Each image is captured, assigned a unique identifying code and transferred onto a computer via a live feed connecting the digital camera to the computer.</li>
	<li>Imaged pupae are moved to individual 2 ml epitubes containing 1ml of fly food, labeled with the unique code for that individual, and punctured with airholes to allow gas exchange.</li>
	<li>Pupal size is estimated as the number of pixels of the pupal silhouette. Other size metrics could also be used.</li>
</ol>
3. Trait (wing) size estimation
Rational and Overview. To image wings, we use a 'wing grabber'24, which pulls and holds the wing of a live, anesthetized fly perpendicular to its body. Pressed between two pieces of glass, this allows the wing to be imaged as a two dimensional object.
<ol>
	<li>Flies are anesthetized lightly with CO2 and placed in position in the wing grabber on a dissecting microscope fitted with a digital camera that is connected to a computer.</li>
	<li>The wing is imaged and saved under the unique code for that individual.</li>
	<li>Wing size can be estimated using the minimum convex polygon that circumscribes marginal fixed landmarks or as the pixel count of the wing silhouette.</li>
</ol>
4. Scaling relationship estimation
<ol>
	<li>Data are pooled across cohorts but separated by sex and log-transformed.</li>
	<li>Scaling relationships are fit to the size data using Type II (i.e., Reduced Major Axis) regression (see review in 12) and the parameters estimated for each group.</li>
</ol>
5. Representative Results:
Our diet manipulation will produce a broad range of body sizes (Figure 1) and trait sizes (Figure 2). Plotting the log transformed traits against each other reveals their pattern of covariation within a given biological group and permits fitting the scaling relationship to the group (Figure 3). Extraction of the scaling relationship parameters that describe how shape scales with body size allows comparison of how shape covaries with size across groups.
<img alt="Figure 1" src="/files/ftp_upload/3162/3162fig1.jpg" /> 
Figure 1. Pupa from extremes of the size distribution produced via diet manipulation after individuals achieve the MVWE. Actual pupal images are shown at left with their corresponding silhouettes for the measurement of pupal size at right. Scale bar is 1mm.
<img alt="Figure 2" src="/files/ftp_upload/3162/3162fig2.jpg" /> 
Figure 2. Wings taken from extremes of the body size distribution produced via diet manipulation after individuals achieve the MVWE. Actual wing images are shown on the left with their corresponding silhouettes for the measurement of wing size shown on the right. Scale bar is 1mm.
<img alt="Figure 3" src="/files/ftp_upload/3162/3162fig3.jpg" /> 
Figure 3. Wing-body size scaling for Drosophila melanogaster by sex. Log-transformed data for individuals of each sex that were fully-fed (dark circles), starved one (grey circles) or two days (open circles) as larvae. Type II regression line is shown for each sex. Between each age cohort, declines in average wing area of ~0.1mm (~7%) and ~0.2mm (~8%) occurs, increasing the total range of phenotypes observed. The regressions reveal that females are slightly hypoallometric for this scaling relationship (slope of 0.96) whereas males are slightly hyperallometric (slope 1.06).

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No conflicts of interest declared.

Morphological scaling has been the subject of intense study in evolutionary biology for nearly a century. More recently, the proximate basis of the regulation of growth and scaling has become increasingly important in development, physiology and genetics (e.g., see reviews in 12,15,23-25). Our methodology permits estimation of morphological scaling relationships across the entire range of trait and body sizes. This can be important, as trait size may not scale linearly with body size (e.g., 13,14). However, this may not be apparent if animals are reared only under ideal lab conditions.
Although our protocol was developed explicitly to allow estimation of the wing-body size scaling relationship in Drosophila, it can be used with trait-specific modification to estimate the scaling relationship for any morphological trait in flies. Similarly, the diet manipulation can likely be used to produce the full range of trait and body sizes in other fully metamorphic insects; such applications simply require knowing when the MVWE is achieved and applying the manipulation at that point in ontogeny.

experimental manipulation of body size to estimate morphological scaling relationships in drosophila

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4.
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bx&alpha;, where x and y are the size of the two traits, such as organ and body size8,9. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + &alpha;log(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of &alpha;, called the 'allometric coefficient'9,10. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and &alpha;) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12).
Not all traits scale linearly with each other or with body size (e.g., 13,14) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila, our methodology should be applicable to nearly any fully metamorphic insect.

Watch this Scientific Journal Video about Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila at JoVE.com

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

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13.7K Views.  University of Houston. Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.

Video: Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Experimental Approaches to Tissue Engineering

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...