The authors would like to kindly thank Dr. J. Luther and K. Ubieta for preparing the data and Dr. B. Gr&#246;tsch for proofreading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (BO3811/1-1-Emmy Noether).

<ol>
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The authors have nothing to disclose.

Adipocytes are characterized phenotypically by their size, numbers and area, revealing adipocyte hyperplasia and hypertrophy, induced by excessive energy storage due to over-nutrition 5. These events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms, which is also referred to as &#34;healthy&#34; fat expansion. For example, Kusminski et al. 21 showed that mice with massive fat expansion remain metabolically healthy, suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes. The adipose tissue (AT) plays a pivotal role in the regulation of body metabolism. AT is the biggest endocrine organ that could influence dyslipidemia, atherosclerosis, hyperinsulinemia and hyperglycemia 3. Evaluating AT homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders. Therefore, unravelling the mechanisms regulating adipocyte differentiation, adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders. Using in vivo and in vitro methods, it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity. To determine the AT homeostasis, determining the balance between adipocyte differentiation, proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response 22-24.
Expression profiling analyses of genes involved in adipogenesis, lipogenesis, lipolysis, fatty acid uptake, hypoxia, apoptosis and proliferation in primary adipocytes and visceral AT is a high throughput method for obtaining an overview on adipocyte homeostasis and their possible dysfunction. Interesting candidates should be further analyzed at the protein level by western blot or immunohistological staining. To obtain optimal results through real-time PCR system using unsymmetrical cyanine dyes, the concentrations of cDNA ranging from 1 to 10 ng and the optimal primer concentrations ranging from 50 to 900 nM should be tested to minimize nonspecific amplification. The critical components are the primers; for each run, the melting curves need to be strictly controlled to ensure the specificity and to exclude the formation of primer dimers. Therefore, using a negative control with H2O instead of cDNA is recommended. Furthermore, commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye (such as ROX) to provide an internal reference signal. The cDNA signal is normalized during data analysis to the ROX signals to correct well-to-well signal fluctuations. Another point to be considered in order to establish a good qPCR system is the choice of the housekeeping gene. For each condition, several housekeeping genes are used, e.g., HPRT, &#946;-actin, GAPDH, &#946;-2-MG or HSP90.
HIF proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival, but also metabolic changes, such as glucose, insulin tolerance and lipid metabolism 11,25,26. To ascertain hypoxic areas in the fat pad, HIF-1&#945; is determined in AT sections by immunohistochemistry. Since HIF proteins are rapidly degraded within 5 to 10 min under normoxic conditions, the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time. Therefore, to ensure hypoxia not only through HIF staining, pimonidazole is used to determine hypoxic areas in the AT. Pimonidazole is able to distribute into tissues, as it was already shown in bones 27, and effectively mark hypoxic areas by binding to thiol-containing proteins specifically in hypoxic cells, which is further detected in histological sections by specific antibody binding 28. However, other markers and methods can be used to analyze the hypoxia pathway. For example, the involvement of the prolyl hydroxylase (PHD) enzyme, which induce hydroxylation of proline residues under normoxia, as well as the von Hippel-Lindau (VHL) protein, which recognize hydroxylated prolines and induce the poly-ubiquitination to mediate proteasomal HIF degradation, need to be analyzed for a full overview of the pathway 29,30. Moreover, ubiquitous detection of HIFs would also determine the protein stability and degradation that can be alter 31,32.
Furthermore, proliferation by Ki67 and apoptosis by TUNEL staining are determined in vivo through staining of AT histological sections. Quantification of proliferation by Ki67 and apoptosis by TUNEL or Annexin V staining through flow cytometry analysis is also carried out 33. Proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle, which is not addressed by the measurement of Ki67 positive cells. Moreover, apoptosis study by TUNEL can be completed by FACS analyses of Annexin V and TOP-PR-3 which will determine the levels of necrosis versus the apoptosis cell death process. Apoptosis is a fundamental process for the program of cell death, which is important for AT homeostasis. Indeed, dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy 34. Moreover, in 2011, Keuper et al. linked adipose tissue inflammation to adipocyte apoptosis. They showed that macrophages induced apoptosis in preadipocytes and adipocytes, which in turn attract macrophages. The recruitment of macrophages accelerates inflammation, which contributes to metabolic syndromes such as glucose and insulin tolerance 35. However, adipocyte apoptosis is still a poorly studied phenomenon, despite the hypothesis that induced adipocyte apoptosis could lead to decreased weight.
The present protocol uses immunohistochemical approaches to study different phenomenon such as proliferation, apoptosis and hypoxia in vivo. Therefore, the tissue was fixed with 4% formaldehyde, which is a critical step. An extended tissue fixation time leads to change of the epitopes, which become non-accessible for the antibody. In contrast, a short fixation time increases the sensitivity of epitopes to reagents. The recommended optimal time of fixation is 24 hr. Moreover, the thickness of the sections also influences the antibodies binding to their epitopes; optimal thickness is between 2 and 5 &#181;m. Sections thicker than 5 &#181;m will give false positive results due to increased binding sites. In contrast, sections thinner than 2 &#181;m contain less binding sites and positive areas are not well defined. Further critical factors are the antibody itself, incubation time, concentration and even temperature, which influence the quality of the specific binding to the epitopes. Therefore, validating antibody concentration and incubation time is necessary for each condition.
To complete the study, we provide an in vitro adipocyte differentiation protocol, which could be extended by different treatments, stimulation or co-cultures. By using in vitro adipocyte cultures, it is feasible to determine defects in adipocyte differentiation and functions. To obtain reliable results, as for all primary cells, the healthy behavior and appearance of the ADSCs and adipocytes is quite important. The granularity, cytoplasmic vacuolations and/or detachment are signs of deterioration, indicating inadequate medium, microbial contamination or senescence of the primary cells. This protocol is using isolated adipocytes from the fat pad tissue, whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols 36. The latest includes stromal progenitor cells, which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation, this might be missed in our current protocol. Moreover, ADSCs can be expanded rapidly (more than 10 times within one week), and long-term cultured ADSCs after some passages still retain their mesenchymal pluripotency 37,38. Another advantage using ADSCs is that one can easily switch to human, since ADSCs can be harvested from patients by liposuction which is a simple and minimally invasive method.
As AT influences several other organs in an endocrine manner, the protocol should be extended to adipokines. Adipokines, such as leptin, adiponectin, tumor necrosis factor-&#945; (TNF) and resistin, secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism, energy homeostasis and insulin sensitivity 39. Therefore, serum and adipocyte secretome analyses should be performed. In the case of AT dysfunction, adipokines and pro-inflammatory cytokines, such as IL-6, can lead to dysregulation of organs such as the liver and pancreas, and of muscle function 4. In order to exclude systemic organ dysfunction, animal models or cell cultures could be tested for their response to glucose stimulation and uptake.
Here we provide a protocol for analyzing the basic state of the AT and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality.

According to the 2014 report from the World Health Organization, 39% of the world&#39;s adult population is overweight, and 13% is obese 1. In the near future, overweight people will comprise a significant proportion of the elderly population. An important feature of obesity and aging is dysregulation of fat in relation to morbidity and mortality 2. Adipokines, proteins secreted by the adipose tissue (AT), can trigger metabolic syndromes such as obesity and type 2 diabetes 3. Metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes, which results in AT expansion 4. It is therefore of interest to determine the causes and the molecular mechanisms of AT expansion in order to find opportunities to control it.
Over-nutrition leads to AT expansion, which is regulated by two events: excessive energy storage into the lipid droplets of adipocytes, a process leading to hypertrophy (increase in adipocyte size), and increased adipogenesis, also known as adipocyte hyperplasia 5. Adipogenesis is a process of differentiation of multipotent mesenchymal stem cells (MSC) into adipocytes. Firstly, MSCs develop into preadipocytes during the commitment phase. Secondly, preadipocytes further differentiate to acquire the features of mature and functional adipocytes 6. Several transcription factors have been identified as master regulators for preadipocyte determination, such as zinc finger protein 423 (Zfp423) and early B cell factor 1 (Ebf1). Whereas Zfp423 induces early commitment, Ebf1 is required for the generation of adipocyte progenitors 6. Terminal differentiation is tightly controlled by a transcriptional cascade, whereby peroxisome proliferator-activated receptor &#947; (PPAR&#947;) is the essential transcription factor 7. Further key transcriptional factors are the CCAAT/enhancer-binding protein (C/EBP) family members (i.e., C/EBP&#945;, C/EBP&#946;, and C/EBP&#948;), kruppel-like factors (KLFs), cAMP responsive element binding protein (CREB) and early growth response 20 (Krox20) 6.
Recently, it has been shown that the activator protein-1 (AP-1) family is involved in the adipocyte differentiation process 8,9. The AP-1 family is formed by a dimeric protein complex, composed of Fos, Jun and/or activating transcription factor (ATF) members. Fos-related antigen 1 and 2 (Fra-1 and Fra-2) are able to regulate adipocyte differentiation. Fra-1 impairs adipocyte differentiation by inhibiting C/EBP&#945; 8, whereas Fra-2 controls adipocyte turnover 9. Fra-2 thereby not only decreases the adipocyte number by repressing PPAR&#947;2 expression during adipocyte differentiation, but also decreases adipocyte apoptosis through direct repression of hypoxia-inducible factors (HIFs) expression. The HIF family is a heterodimeric transcription factor complex, composed of HIF-1&#945;, HIF-2&#945; and HIF-1&#946;. The heterodimers consist of an oxygen-sensitive HIF-&#945; protein (HIF-1&#945; or HIF-2&#945;) and the oxygen-insensitive HIF-1&#946; subunit 10. During normoxia, HIF-&#945; proteins are poly-ubiquitinylated and are finally degraded by proteasomes 11. Under hypoxic conditions, occurring in AT during expansion, HIF-&#945; proteins are no longer hydroxylated. They therefore become stabilized and form dimers with the constitutively expressed HIF-1&#946;. Transcriptional activation of genes controlled by the HIF response elements is involved in the regulation of angiogenesis, metabolism, and inflammation 12. Indeed, HIF-1&#945; promotes AT dysfunction by inducing glucose tolerance, inhibiting energy expenditure and peripheral use of lipid, as well as by increasing leptin level and HFD-induced hepatic steatosis 13. Moreover, HIF-1&#945; regulates adipocyte apoptosis in vivo and in vitro9.
The present protocol describes methods for studying AT status to unravel the molecular characteristics of adipocyte homeostasis in adult mice. It shows how apoptosis, proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia. To do so, we use mice with adipocyte specific deletion of Fra-2 generated by crossing mice carrying the Fra-2 floxed alleles with Fabp4-CreERT mice 9. By using Fabp4-Cre ERT mice, the deletion is adipocyte specific and inducible by tamoxifen injection 14. For the adult model, intra peritoneal injections of tamoxifen are performed over 5 consecutive days starting at the age of 6 weeks. Thus, the mice are subjected to a normal diet or high-fat diet for 6 weeks before the analysis is done. The mice used in this study were male based on a C57Bl6 background to avoid female hormones, such as estrogens, shown to regulate the body fat distribution 15. Using another genetic background might also alter the metabolic phenotype, due to strain-related differences in lipid management 16.
This protocol demonstrates how to analyze AT under hypoxia using histology and how to quantify adipocyte apoptosis, proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses. The study is completed by in vitro experiments, showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia.

<table>
	<tbody>
		<tr>
			<td>RNAlater solution</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>RNA stabilization solution</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td rowspan="2">Applied Biosystems</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Select Master Mix</td>
			<td>4472908</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human Ki-67&#160;&#160;</td>
			<td rowspan="2">BD Biosciences</td>
			<td>550609</td>
			<td>Clone &#160;B56&#160;(RUO)</td>
		</tr>
		<tr>
			<td>Purified Mouse Anti-Human Ki-67&#160; Clone &#160;B56&#160;(RUO)</td>
			<td>550609</td>
			<td>Proliferation marker</td>
		</tr>
		<tr>
			<td>FITC Annexin V</td>
			<td>BioLegend</td>
			<td>640906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 Rabbit mAb</td>
			<td rowspan="2">Cell signalling</td>
			<td>9664S</td>
			<td>&#160;Clone 5A1E</td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td>
			<td>9664</td>
			<td>Apoptosis marker</td>
		</tr>
		<tr>
			<td>Hypoxyprobe-1 Plus Kit</td>
			<td>Hypoxyprobe</td>
			<td>HP2-200Kit</td>
			<td>Hypoxia marker, Solid pimonidazole HCl (Hypoxyprobe-1), FITC conjugated to mouse IgG&#8321; monoclonal antibody (FITC-MAb1), Rabbit anti-FITC conjugated with horseradish peroxidase</td>
		</tr>
		<tr>
			<td>Lipofectamine2000 Reagent&#160;</td>
			<td rowspan="2">Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>TO-PRO-3 Iodide</td>
			<td>T3605</td>
			<td>Nuclear counterstain, Monomeric cyanine nucleic acid stain, Excitation&#8260;Emission: 642&#8260;661 nm</td>
		</tr>
		<tr>
			<td>Mayer&#8217;s hemalum</td>
			<td>Merck</td>
			<td>109249</td>
			<td>hematoxylin</td>
		</tr>
		<tr>
			<td>pegGOLD TriFast</td>
			<td rowspan="3">Peqlab</td>
			<td>30-2030</td>
			<td>TRIzol, single-phase solution of guanidinisothiocyanat and phenol</td>
		</tr>
		<tr>
			<td>Percellys Ceramic Kit 1.4 mm</td>
			<td>91-PCS-CK14</td>
			<td>tubes containing ceramic beads (1.4 mm)</td>
		</tr>
		<tr>
			<td>Precellys 24</td>
			<td>91-PCS24</td>
			<td>homogenizer</td>
		</tr>
		<tr>
			<td>HIF-1 alpha Antibody</td>
			<td rowspan="2">Pierce</td>
			<td>PA1-16601</td>
			<td></td>
		</tr>
		<tr>
			<td>HIF-1 alpha Antibody, 16H4L13</td>
			<td>700505</td>
			<td>Hypoxia marker</td>
		</tr>
		<tr>
			<td>In Situ Cell Death Detection Kit, Fluorescein</td>
			<td>Roche</td>
			<td>11 684 795 001</td>
			<td>TdT-mediated dUTP-biotin nick end labeling (TUNEL)&#160;</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma</td>
			<td>318906</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I Solution (1 unit/&#181;L)</td>
			<td>Thermo Scientific</td>
			<td>EN0525</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated anti mouse IgG (H+L)</td>
			<td rowspan="4">Vector Laboratories</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated anti mouse IgG (H+L)</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectastain ABC Kit</td>
			<td>PK-4000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Medium with DAPI</td>
			<td>H-1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanism of regulation of adipocyte numbers in adult organisms through differentiation and apoptosis homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

We show how to determine adipocyte homeostasis in vivo and in vitro using the example of Fra-2fl/fl Fabp4-CreERT mice compared to wild-type littermates. Our protocol defines how increased HIF expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis.
Increased adipocyte size and area in high-fat diet (HFD) treated mice
Over-nutrition, among other factors, results in adipocyte hypertrophy, caused by excessive energy storage in the lipid droplets. The adipocyte size and area is an indicator of hypertrophy. Sections of the fat pad from normal (ND; Figure 6a) and high-fat diet (HFD; Figure 6b) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of HFD, which is indicated by increased adipocyte size in the HFD treated mice (Figure 6c and d).
Increased hypoxia in the adipose tissue (AT) of adult Fra-2fl/fl Fabp4-CreERT mice leads to increased HIF-1&#945; level and adipocyte apoptosis
To determine the in vivo status of hypoxia in AT, Fra-2fl/fl Fabp4-CreERT mice are analyzed 6 weeks after Fra-2 deletion at the age of 12 weeks and compared with wild-type littermates. Pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker; it is nontoxic and able to distribute into the AT. Hypoxic adipocytes in the AT in vivo are defined by immunohistochemical antibody staining (Figure 7a). Furthermore, the increased hypoxic status of the AT in mice is accompanied by increased HIF-1&#945; positive adipocytes as indicated by the immunohistochemical staining Figure 7b), which is confirmed by quantification of HIF-1&#945; expression levels and its targets genes 9. Additionally, TUNEL staining of AT sections from Fra-2fl/fl Fabp4-CreERT mice and control littermates (Figure 7c) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and HIF-1&#945; expression.
Increased HIF-1&#945; expression in primary adipocytes through hypoxia induced adipocyte apoptosis
To analyze adipocyte apoptosis in vitro, we use adipocytes generated from subcutaneous fat pads as described elsewhere 20. As expected, the HIF-1&#945; expression in adipocytes is increased after 24 hr of hypoxia (Figure 8a). To further analyze HIF-1&#945; activities, the RNA levels of HIF target genes such as Inos (inducible nitric oxide synthase) are quantified by qPCR. Figure 8b shows that the increased expression of HIF-1&#945; under hypoxic conditions leads to increased Inos mRNA level. Since we have already shown in vivo (Figure 8) that increased HIF-1&#945; expression in adipocytes correlates with increased adipocyte apoptosis, apoptosis is also quantified in in vitro cultures by Annexin V staining under hypoxic conditions. Consistent with the in vivo data (Figure 7), an increased HIF-1&#945; level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions (Figure 8b). Moreover, to prove that hypoxic-induced apoptosis is HIF-dependent; HIF-1&#945; or HIF-2&#945; is silenced by RNA interference in adipocytes derived from wild-type or Fra-2 deficient mice. The increased adipocyte apoptosis is restored by silencing HIF-1&#945; or HIF-2&#945; as shown by Annexin V staining in Figure 8b, proving that the hypoxia sensor HIF-&#945; regulates the adipocyte apoptosis.
<img alt="Figure 6" src="/files/ftp_upload/53822/53822fig6.jpg" /> 
Figure 6: Increased adipocyte size and area in high-fat diet (HFD) mice. (a, b) H&#38;E staining of sections from the perigonadal fat pad of male wild-type mice fed with normal diet (ND) (a) or high-fat diet (HFD) (b) for 6 weeks. Bars represent 500 &#956;m. (c, d) Quantifications of adipocyte size (c) and area (d) from the perigonadal fat pad of wild-type mice fed with ND (a) or HFD (b) for 6 weeks. n = 10. Data are shown as mean values &#177; SEM. Statistical analysis was performed using Student&#180;s t-test. ***p &#60;0.0001.<a href="https://www.jove.com/files/ftp_upload/53822/53822fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53822/53822fig7.jpg" /> 
Figure 7: Increased HIF-1&#945; level and apoptosis in adipocytes of adult Fra-2fl/fl Fabp4-CreERT mice. Hypoxia (a) and HIF-1&#945; (b) staining in the AT of male Fra-2fl/fl Fabp4-creERT mice and male control littermates 6 weeks after tamoxifen injection. Magnification 20X, insert 40X. Black arrows indicate hypoxic areas and HIF-1&#945; positive cells. (c) TUNEL staining in Fra-2fl/fl Fabp4-CreERT mice and control littermates AT 6 weeks after tamoxifen injection. Magnification 20X, insert 40X. Black arrows indicate TUNEL positive cells. This figure has been modified from Luther et al. 9. <a href="https://www.jove.com/files/ftp_upload/53822/53822fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53822/53822fig8.jpg" /> 
Figure 8: Increased HIF-1&#945; expression and adipocyte apoptosis in primary adipocytes induced by hypoxia. (a) Real-time PCR analysis of HIF-1&#945; and HIFs target Inos mRNA levels in primary adipocytes placed in hypoxic chambers analyzed at the indicated time points. (b) Quantification of apoptosis by Annexin V FACS staining in primary adipocytes isolated from Fra-2fl/fl Fabp4-CreERT mice or wild-type controls transfected with sh control or sh plasmid against HIF-1&#945; or HIF-2&#945; and placed under hypoxia (1% O2) for 24 hr. This figure has been modified from Luther et al. 9. Data are shown as mean values &#177; S.D. Statistical analysis was performed using Student&#39;s t-test. *P &#60;0.05 and **P &#60;0.01 were accepted as significant. <a href="https://www.jove.com/files/ftp_upload/53822/53822fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis at JoVE.com

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

mechanism-regulation-adipocyte-numbers-adult-organisms-through

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JoVE Journal

Developmental Biology

14.9K Views.  Universitätsklinikum Erlangen. Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

Video: Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

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Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

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Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

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Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

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Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

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Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

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Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.