Transuterine Fetal Tracheal Occlusion Model in Mice.

Fetal tracheal occlusion (TO), an established treatment modality, promotes fetal lung growth and survival in severe congenital diaphragmatic hernia (CDH). Following TO, retention of the secreted epithelial fluid increases luminal pressure and induces lung growth. Various animal models have been defined to understand the pathophysiology of CDH and TO. All have their own advantages and disadvantages such as the difficulty of the technique, the size of the animal, cost, high mortality rates, and the availability of genetic tools. Herein, a novel transuterine model of murine fetal TO is described. Pregnant mice were anesthetized, and the uterus exposed via a midline laparotomy. The trachea of selected fetuses were ligated with a single transuterine suture placed behind the trachea, one carotid artery, and one jugular vein. The dam was closed and allowed to recover. Fetuses were collected just before parturition. Lung to body weight ratio in TO fetuses was higher than that in control fetuses. This model provides researchers with a new tool to study the impact of both TO and increased luminal pressure on lung development.

Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices.

Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment. For complete details on the use and execution of this protocol, please refer to Even et al. (2019).

Fetal tracheal occlusion in mice: a novel transuterine method.

BACKGROUND: Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. MATERIALS AND METHODS: Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. RESULTS: Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. CONCLUSIONS: Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment.

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized α4ß1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions - the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary. The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. OBJECTIVE: Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. METHODS: A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. RESULTS: A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. CONCLUSION: Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. SIGNIFICANCE: LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Loss of Pancreas upon Activated Wnt Signaling Is Concomitant with Emergence of Gastrointestinal Identity.

Organ formation is achieved through the complex interplay between signaling pathways and transcriptional cascades. The canonical Wnt signaling pathway plays multiple roles during embryonic development including patterning, proliferation and differentiation in distinct tissues. Previous studies have established the importance of this pathway at multiple stages of pancreas formation as well as in postnatal organ function and homeostasis. In mice, gain-of-function experiments have demonstrated that activation of the canonical Wnt pathway results in pancreatic hypoplasia, a phenomenon whose underlying mechanisms remains to be elucidated. Here, we show that ectopic activation of epithelial canonical Wnt signaling causes aberrant induction of gastric and intestinal markers both in the pancreatic epithelium and mesenchyme, leading to the development of gut-like features. Furthermore, we provide evidence that ß -catenin-induced impairment of pancreas formation depends on Hedgehog signaling. Together, our data emphasize the developmental plasticity of pancreatic progenitors and further underscore the key role of precise regulation of signaling pathways to maintain appropriate organ boundaries.

Embryo developmental potential of microsurgically corrected human three-pronuclear zygotes.

We explored the embryo development potential of human three-pronuclear (3PN) zygotes reduced to two-pronuclear (2PN) zygotes (3 → 2PN zygotes) by micropuncture. In this study, there were three groups, the 3 → 2PN group (338 zygotes), the non-corrected 3PN group (381 zygotes), and the normal 2PN group (359 zygotes). The first cleavage mode (2-cell cleavage or 3-cell cleavage), 6-8 cell embryogenesis rate, high-quality embryogenesis rate and Day 5/Day 6 blastulation rate were compared between the three groups. The success rate of enucleation was 92.9%. The 2-cell cleavage rate was significantly higher in the 3 → 2PN group (74.3%) than in the 3PN group (36.4%) (P < 0.05), but had no statistical difference compared with the 2PN group (86.0%) (P > 0.05). The 6-8 cell embryogenesis rate was significantly higher in the 3 → 2PN group (91.1%) as compared to the 2PN group (85.6%) (P < 0.05), but had no statistical difference compared with the 3PN group (95.0%) (P > 0.05). Total blastulation rate was significantly higher in the 2PN group (58.8%) as compared to the 3PN group (21.5%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (5.6%) (P < 0.01). Also D5 blastulation rate was significantly higher in the 2PN group (53.7%) as compared to the 3 → 2PN group (8.9%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (1.9%) (P < 0.01). In 3 → 2PN zygotes, the first cleavage mode is mainly 2 cells which is significantly higher than that in 3PN zygotes. Compared with 3PN zygotes, the embryo developmental potential of 3 → 2PN zygotes is improved, but still is lower than that in 2PN zygotes.

An ex vivo culture system to study thyroid development.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the "angio-follicular" units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

Separation of mouse embryonic facial ectoderm and mesenchyme.

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide. Orofacial clefting is caused by abnormal facial development. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments. Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

In vitro induction of nephrogenesis in mouse metanephric mesenchyme with lithium introduction and ureteric bud recombination.

The organ culture setup of embryonic kidney has served as a model of nephrogenesis for several decades. In vitro culture of the mouse metanephric mesenchyme enables easy manipulation and analysis of the tissue and provides information of cellular interactions, morphogenesis, cell differentiation, and molecular biology of the developmental process. The advantages of the tissue culture method include enhanced representativeness of situation in living organism compared to cell culture assays and less demanding and time-consuming possibilities to experimental work compared with in vivo research.

Live imaging of the developing mouse mesonephros.

Embryonic development is a highly dynamic process involving complex tissue interactions and movements. Recent progress in cell labeling, image acquisition, and image processing technologies has brought the study of embryo morphogenesis to another level. It is now possible to visualize in real time the dynamic morphogenetic changes occurring in vivo and to reconstitute and quantify them in 4D rendering. However, extended live embryo imaging remains challenging in terms of embryo survival and minimization of phototoxicity. Here, we describe a procedure to image the developing mesonephros for up to 16 h in intact mouse embryos. This method can easily be adapted to the imaging of other structures at similar developmental stages.

Analysis of migration in primary ureteric bud epithelial cells.

Kidney development has been widely used as a model system to study molecular control of inductive tissue interactions and mechanisms through which branching organs form. Due to lacking or poor methods, less focus has been in understanding details of cellular events that accomplish example ureteric bud (UB) branching. In order to form a branch point, cells need to proliferate, move in relation to each other, and change their shape as well as adhesive properties. In this chapter, detailed description is given how to set up primary UB epithelial cell cultures and study cell motility in these cells.

Use of whole embryo culture for studying heart development.

Congenital heart defects occur in approximately 1% of newborns and are a major cause of morbidity and mortality in infants and children. Many adult cardiac diseases also have developmental basis, such as heart valve malformations, among others. Therefore, dissecting the developmental and molecular mechanisms underlying such defects in embryos is of great importance in prevention and developing therapeutics for heart diseases that manifest in infants or later in adults. Whole embryo culture is a valuable tool to study cardiac development in midgestation embryos, in which ventricular chambers are specified and expand, and the myocardium and endocardium interact to form various cardiac structures including heart valves and trabecular myocardium (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008). This technique is essentially growing a midgestation embryo ex utero in a test tube. One of the strengths of embryo culture is that it allows an investigator to easily manipulate or add drugs/chemicals directly to the embryos to test specific hypotheses in situations that are otherwise very difficult to perform for embryos in utero. For instance, embryo culture permits pharmacological rescue experiments to be performed in place of genetic rescue experiments which may require generation of specific mouse strains and crosses. Furthermore, because embryos are grown externally, drugs are directly acting on the cultured embryos rather than being degraded through maternal circulation or excluded from the embryos by the placenta. Drug dosage and kinetics are therefore easier to control with embryo culture. Conversely, drugs that compromise the placental function and are thus unusable for in utero experiments are applicable in cultured embryos since placental function is not required in whole embryo culture. The applications of whole embryo culture in the studies of molecular pathways involved in heart valve formation, myocardial growth, differentiation, and morphogenesis are demonstrated previously (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008; Nature 446: 62-67, 2010). Here we describe a method of embryo culture in a common laboratory setting without using special equipments.

Stem cell transplantation methods.

Just a few short years ago, we still used to think that we were born with a finite number of irreplaceable neurons. However, in recent years, there has been increasingly persuasive evidence that suggests that neural stem cell (NSC) maintenance and differentiation continue to take ace throughout the mammal's lifetime. Studies suggest that neural stem cells not only persist to mammalian adulthood, but also play a continuous role in brain tissue repair throughout the organism's lifespan. These preliminary results further imply that NSC transplantation strategies might have therapeutic promise in treating neurodegenerative diseases often characterized by isolated or global neuronal and glialloss. The destruction of neural circuitry in neuropathologies such as stroke, Parkinson's disease, MS, SCI prevents signals from being sent throughout the body effectively and is devastating and necessitates a cure. NSC transplantation is among one of the foremost researched fields because it offers promising therapeutic value for regenerative therapy central nervous system (CNS) diseases. Both chemotropic and exogenous cell graft mechanisms ofCNS repair are under review for their therapeutic value and it is hoped that one day, these findings will be applied to human neurodegenerative disorders. The potential applications for NSC transplantations to treat both isolated and global neurodysfunction in humans are innumerable; these prospects include inherited pediatric neurodegenerative and metabolic disorders such as lysosomal storage diseases including leukodystrophies, Sandhoff disease, hypoxic-ischemic encephalopathy and adult CNS disorders characterized by neuronal or glial cell loss such as Parkinson's disease, multiple sclerosis, stroke and spinal cord injury.

Robotic patch-stabilizer using wire driven mechanism for minimally invasive fetal surgery.

The clinical target of this study is intrauterine patch coverage of fetal myelomeningocele. We propose a new surgical robotic system for intrauterine fetal surgery with patch-stabilizer and laser manipulator. The target disease of the fetal surgery is spina bifida or myelomeningocele, which is incomplete closure in the spinal column and one of the common fetal diseases. In the fetal surgery, the collagen patch is supposed to be stabilized onto the fragile fetal tissue during the laser fixation process. In this study, a prototype of the patch-stabilizer using wire driven mechanism has been developed for precise force control on the patch without damaging fetal tissue. The diameter of the patch-stabilizer's shaft is 2.4 mm. The patch-stabilizer including one ball joint and wire driven mechanism is able to bend through 40 degrees. The stabilizing part holds collagen patch with diamond shape mechanism using wire driven. In this paper, we showed that the patch-stabilizer is developed with the stabilizing force control using the tension control of wires. Results of the experiment showed that the tension of driven wires was controlled at 0.3 N to stabilize the collagen patch onto the lesion surface without the damages of fetal tissues and the influence by the amnion liquid.

Animal models for schizophrenia via in utero gene transfer: understanding roles for genetic susceptibility factors in brain development.

Genetic disturbances of brain development may underlie the pathophysiology of schizophrenia. Recent advances in molecular neurobiology suggest that some genetic risk factors for schizophrenia have multiple roles in various brain regions depending on the developmental stage. Furthermore, these factors are likely to act synergistically or epistatically in common molecular pathways, possibly contributing to disease pathology. Thus, a technique that can manipulate the expression of more than one gene simultaneously in animal models is necessary to address such molecular pathways. To produce such animal models, in utero gene transfer technique is one useful method. Given that plasmid-based cell-type-specific and inducible gene expression systems are now available, combining these technologies and in utero gene transfer opens a new window to examine the functional role of genetic risk factors for schizophrenia by conducting multiple-gene targeting in a spatial and temporal manner. The utility of animal models produced by in utero gene transfer will also be expected to be evaluated in terms of functional and behavioral outcomes after puberty, which may be associated with schizophrenia pathology.

Prospects and developments in cell and embryo laser nanosurgery.

Recently, there has been increasing interest in the application of femtosecond (fs) laser pulses to the study of cells, tissues and embryos. This review explores the developments that have occurred within the last several years in the fields of cell and embryo nanosurgery. Each of the individual studies presented in this review clearly demonstrates the nondestructiveness of fs laser pulses, which are used to alter both cellular and subcellular sites within simple cells and more complicated multicompartmental embryos. The ability to manipulate these model systems noninvasively makes applied fs laser pulses an invaluable tool for developmental biologists, geneticists, cryobiologists, and zoologists. We are beginning to see the integration of this tool into life sciences, establishing its status among molecular and genetic cell manipulation methods. More importantly, several studies demonstrating the versatility of applied fs laser pulses have established new collaborations among physicists, engineers, and biologists with the common intent of solving biological problems.

Fetus-supporting flexible manipulator with balloon-type stabilizer for endoscopic intrauterine surgery.

BACKGROUND: Minimally invasive endoscopic fetal surgery enables intrauterine intervention with reduced risk to the mother and fetus. A novel surgical manipulator is described for stabilizing the fetus and restraining it from floating free during endoscopic intrauterine surgery. METHODS: We designed and fabricated a prototype fetus-supporting manipulator equipped with flexible joint and bending mechanisms and a soft balloon stabilizer. The flexible joint and bending mechanisms enable the stabilizer to reach the target sites within the confined space of the uterus under the guidance of an ultrasound device. The balloon stabilizer could be inserted into the uterus through a small incision. RESULTS: The accuracy evaluation showed that the maximum error of the bending mechanism was as small as 7 mm and the standard deviation of the joint mechanism was just 1.6 degrees. In the experiments using a fetus model, the manipulator could be well controlled with guidance from ultrasound images and its bending mechanism with the balloon stabilizer could be clearly visualized while stabilizing the fetus model. CONCLUSIONS: The manipulator has the potential to be used in minimally invasive intrauterine surgery, although further improvements and experiments remain to be carried out.