Name: a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse
Uploaded: 2017-10-24T13:00:00
Description: Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

We very much appreciate the assistance of Rong Zhao, Jie Li, and Yan Ying.

These studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine under protocol number AN-4203.
1. Preparation of the Pregnant Mouse for Ultrasound
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	<li>Timed mating
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		<li>Place the dam with a proven fertile male mouse overnight beginning after 1700. Separate dam an...

<ol>
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E., Hildebrandt, T. B., Kuhl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reprod Biol Endocrinol. 12, 38 (2014).</li><li>Nguyen, T. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+High-Frequency+Ultrasonography+for+Early+Detection+and+Characterization+of+Embryo+Implantation+Site+Development+in+the+Mouse.">Three-Dimensional High-Frequency Ultrasonography for Early Detection and Characterization of Embryo Implantation Site Development in the Mouse.</a> PLoS One. 12 (1), e0169312 (2017).</li><li>Song, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+phospholipase+A2alpha+is+crucial+[correction+of+A2alpha+deficiency+is+crucial]+for+'on-time'+embryo+implantation+that+directs+subsequent+development.">Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for 'on-time' embryo implantation that directs subsequent development.</a> Development. 129 (12), 2879-2889 (2002).</li><li>Nallasamy, S., Li, Q., Bagchi, M. 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This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. However it should be noted that histological methods would still be considered more optimal when characterization at a more magnified and more cellular level is ...

Recurrent early pregnancy loss is one of the most common complications after conception and affects approximately 1% of couples trying to conceive1,2. The underlying mechanisms of early pregnancy loss are varied: from intrinsic embryonic abnormalities and maternal comorbidities to defects in endometrial receptivity1,3,4. Because of their genetic tractability, mouse models have been widely utilized for investigations of early embryo implantation and pregnancy. Furthermore, the short gestational time of the mouse and the ability to perform large-scale studies have ensured the growing utility of the mouse in addressing key clinical questions in human reproductive medicine5. That said, the vast majority of murine experimental designs still require numerous dams to be euthanized on sequential gestational days to quantify and analyze implantation site location, number, size, and spacing patterns during pregnancy6,7,8, thereby precluding longitudinal studies on the same animal.
In the clinic, ultrasound is a reliable and invaluable tool to monitor human fetal viability and development in a non-invasive manner9,10,11. More recently, high-frequency ultrasound (HFUS) has begun to find limited applications in the mouse as a method for monitoring fetal viability and growth during pregnancy12,13,14. The recent technological advances in ultrasound imaging have permitted the application of three-dimensional (3-D) data for visual reconstruction of animal organs and subsequent monitoring of pathologies15,16,17. Use of this advanced imaging technology has markedly improved the power to detect smaller volume fluctuations, to reduce inter-animal variability, and to monitor the progression of a pathology or the efficacy of a therapeutic intervention17. While the primary utility of this technology has been to monitor malignancy progression in oncomouse models15,16, 3-D HFUS imaging has only recently been used to quantitate and monitor the active growth of embryo implantation and fetal development in the mouse uterus18.
Here, we demonstrate how to perform HFUS imaging to produce 2-D and 3-D data to generate reconstructions of the early pregnant mouse uterus. We demonstrate the utility of this novel method to detect these early embryonic implantation events without the need for pregnancy termination, allowing researchers to collect data in a non-invasive manner.

<table>
	<tbody>
		<tr>
			<td>VisualSonics Vevo 2100 Ultrasound Imaging Platform/Machine</td>
			<td>VisualSonics, inc.</td>
			<td>VS-11945</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Imaging Station</td>
			<td>VisualSonics, inc.</td>
			<td>SA-11982</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasonic 100 Ultrasound Transmission Gel</td>
			<td>Parker</td>
			<td>#SKU PLI 01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (IsoThesia) 100mL bottle</td>
			<td>Henry Shein</td>
			<td>#29404</td>
			<td></td>
		</tr>
		<tr>
			<td>PuraLubenAnimal Ophthalmic Ointment</td>
			<td>Dechra</td>
			<td>#12920060</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

As demonstrated in Figure 1, the high frequency ultrasound can detect implantation site development beginning as early as the 5.5 d.p.c time point. Using the lighter hyperechoic decidualized endometrium as a marker of implantation sites at 6.5 d.p.c allows the number of implantation sites and spacing of these sites to be quantified. As the pregnancy progresses to 7.5 d.p.c., a darker hypoechoic gestational sac and fetal pole are also easily identifiable.
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Watch this Scientific Journal Video about A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse at JoVE.com

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

The overall goal of this ultrasound imaging procedure is to detect, monitor and characterize early pregnancy in the mouse. This method can help answer key questions in the field of murine reproductive biology and establishing the growth, development and viability of early mouse pregnancies. The main advantage of this technique is that these pregnancies can be characterized and phenotyped in vivo, without interruption of the mouse pregnancy, allowing for longitudinal collection of data.Generally, individuals new to this method will struggle with getting all the images in one plane. This is due to the small incremental movements of the ultrasound probe that is necessary. Begin with preparing the ultrasound machine.Turn on the transducer and then attach the 3-D motor stage to the transducer. Next, attach the probe to the transducer clamp. Now, position the mouse, supine, on the monitoring platform.Provide it anesthetic via nose cone and gently tape all of its paws to the heart rate monitoring pads on the platform. Next, rub about one or two milliliters of ultrasound transmission gel onto the abdomen. Then position the ultrasound probe on the lower abdomen using the manual motor stage.Using the ultrasound, locate the bladder, which should appear as a food-filled dark circle just cephalad to the vaginal opening. Once the bladder is located, move the probe very slowly cephalad to visualize the pregnant uterus, which should appear as a cylindrical shape with round areas at pregnancy sites. This can be described as looking like pearls on a chain.Now, proceed with 2-D imaging. Once the pregnant uterus has been identified, begin at the pregnancy site closest to the bladder and slowly and sequentially move cephalad to determine location of all the pregnancy sites. Save the image.If the kidney, spleen or liver can be seen, then the scan is too far cephalad. Reposition the probe closer to the bladder and save the image. Next, image the contralateral uterine horn in the same manner.Freeze the image when the ultrasound frame is at the center of the pregnancy or implantation site and save the image for analysis. Now or later, use the saved images to measure implantation positions using the hyperechoic decidualization reaction as a marker. Manually click on the measure tool and click on the location of an implantation site.Then, drag the cursor to the next implantation site and click again to draw a line. The software then reports the length of the line. After all the implantation sites are measured, use the same technique and proceed to measure the gestational sac size and the fetal pole size.After the 2-D imaging, position the probe in an area that is at the approximate mid-point of the desired 3-D image. For example, if one were imaging an apple in 3-D, position the probe at the core. Once the probe is properly positioned, obtain a 3-D acquisition.The 3-D motor stage will travel this distance across the uterus in a series of steps or frames. As the scan is obtained, ensure that the intended structures are completely captured in the scan. After collecting the scan, load it up for image processing.Choose the parallel and rotational methods for processing, which will load all the 3-D image frames into one 3-D box. Next, choose the step size of 0.08 millimeters, then begin at one end of the image block and scroll through all the frames to become familiar with the data. Next, start at one end of the stack and manually trace the outline of an object of interest.Then, proceed to the next frame and manually trace the outline of the object again. Continue this process until all frames have been manually annotated. Once complete, click finish to obtain a 3-D image and a calculation of the object's volume.For the image, choose between a mesh or a solid overlay. Then, choose to keep or remove the surrounding image information for clarity. The high frequency ultrasound can detect the development of an implantation site as early as the 5.5 days post-conception.Using the lighter hyperechoic decidualized endometrium as a marker of implantation sites, the number of implantation sites and spacing of these sites, to be quantified at 6.5 days post-conception. As the pregnancy progresses to 7.5 days post-conception, a darker, hypoechoic gestational sac and fetal pole are both easily identifiable. Three-dimensional reconstructions of the mouse uterus at 6.5 days post-conception, were made in both mesh and solid overlay formats.At 7.5 days post-conception, the 3-D scan was just as revealing. After watching this video, you should have a good understanding of how to obtain accurate ultrasound images in both 2-D and 3-D, to characterize a pregnancy without interruption. The total time needed for an ultrasound acquisition, in both 2-D and 3-D, is approximately 10 to 20 minutes in the hands of an experienced user.While attempting this procedure, it's important to remember to keep your movements small and incremental. This technique paved the way for researchers to explore normal and abnormal pregnancy phenotypes in the mouse.

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JoVE Journal

Developmental Biology

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...