Culture of Mouse Embryonic Foregut Explants

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1189)

Abstract

The ability to culture embryonic organ rudiments and follow their development ex vivo has helped to understand how tissues are constructed and what cellular and biological events are important in this process. Here we outline a technique for isolation and ex vivo growth of foregut explants from E8.5 mouse embryos. This technique serves as a reliable tool for the analysis of the morphogenetic processes and signaling networks during early development of foregut derivatives, such as the lungs.

Key words

Organ explant Foregut culture Development Organogenesis Mouse Lung development 

1 Introduction

The emergence of tissue engineering as a discipline requires a good understanding of tissue formation during embryonic development [1]. The ability to culture embryonic organ rudiments ex vivo and follow their development has emerged as an important tool to understand how tissues are constructed and what cellular and biological events are important in this process. Organ culture protocols developed over the past five decades allowed exploration of complex tissu interactions and provided invaluable insights into mechanisms that control morphogenesis and differentiation. These techniques have been widely used in the study of kidney, salivary gland, and gut-derived organs, such as the pancreas and lung [2, 3, 4, 5, 6, 7].

Early in organogenesis, the gut endoderm is specified into distinct organ domains along the anterior-posterior axis of the gut tube, which then is roughly divided into three regions: the foregut (the most anterior or cranial), midgut, and hindgut (the most posterior or caudal). The foregut gives rise to the thyroid, lung, stomach, liver, and pancreas. In mice, thyroid and liver progenitors are specified as early as embryonic day E8.0 (approximately 4–6-somite stage). However in organs, such as the lungs, specification occurs later around E9.0 and primordial buds form at E9.5 (20–25-somite stage) [8, 9, 10].

This protocol describes how to isolate and culture mouse embryonic foregut explants and follow the initial stages of organogenesis of some of its derivatives, with a particular focus on the lung. We have previously shown that the initial stages of lung development in foregut cultures recapitulate key early events observed in vivo [11, 12, 13, 14]. The response of the foregut to agonists or inhibitors added directly to the culture medium or locally applied in the tissue using heparin beads can be studied with relative ease using this model. Mechanistic insights into the role of developmental pathways and how they interact can be readily obtained in many cases without the cost and time to generate genetically engineered animals. Moreover, this culture system allows studies of the development of the foregut derivatives in genetic models that result in early embryonic lethality shortly after E8.5 [14]. The combination of genetic and pharmacological manipulations using this system represents a robust approach to address developmental processes. In summary, ex vivo culture of the foregut is a powerful tool to dissect some of the early events that regulate organogenesis of foregut-derived structures.

2 Materials

All solutions, tools, and equipment used for dissection and culture should be sterilized to prevent contamination. Culture media should be aseptically prepared.

2.1 Isolation of Foregut from Embryonic Mice

  1. 1.

    Pregnant female mouse, gestational age day 8.5 (embryos approximately 6–18 somites).

     
  2. 2.

    Absorbent papers.

     
  3. 3.

    70 % ethanol in a spray bottle.

     
  4. 4.

    Surgical scissors.

     
  5. 5.

    Forceps, Dumont #5, two pairs.

     
  6. 6.

    Petri dishes, 60 and 100 mm diameters.

     
  7. 7.

    Cold PBS, 1×, with calcium and magnesium.

     
  8. 8.

    Ice.

     
  9. 9.

    Laminar flow hood equipped with a dissecting microscope, magnification ranging from 0.8× to 5×.

     
  10. 10.

    Tungsten needles, 0.25 mm diameter, secured in pin holders (Fine Science Tools).

     

2.2 Culture of Foregut Explants

  1. 1.

    Foregut culture medium: BGJb medium (Life Technologies) containing 0.02 % l-ascorbic acid, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 % fetal calf serum. Pass the medium through a 0.22 μm disposable filter. Store at 4 °C for up to 1 week.

     
  2. 2.

    Micropipetter with tips (1,000 μl).

     
  3. 3.

    6-Well plate and collagen-coated transwell inserts, 0.4 μm pore size (Fisher Scientific #07-200-558).

     
  4. 4.

    Medicine dropper with rubber bulb, 2 ml (glass to avoid sticking to the tissue).

     
  5. 5.

    Incubator preset to 37 °C, 5 % CO2, humidified.

     

3 Methods

3.1 Isolation of Foregut Explants

Euthanize the pregnant mouse by cervical dislocation (seeNote1).
  1. 1.

    Lay the mouse on its back on a few pieces of absorbent paper and soak its abdomen thoroughly with 70 % ethanol from a spray bottle (seeNote2).

     
  2. 2.

    Make a longitudinal cut in the abdomen and reflect the skin using surgical scissors and forceps (seeNote2). Cut the abdominal wall through the peritoneum to reveal the abdominal contents. Push the gut out of the way and locate the two horns of the uterus. Remove the uterus with the embryos by holding it at one end of the uterine horns and cutting along the mesometrium and the blood vessel connections on the dorsal side of the uterus.

     
  3. 3.

    Place the dissected uterus in a Petri dish on ice containing cold 1× PBS and transfer the Petri dish to a dissecting microscope in a biological laminar flow hood.

     
  4. 4.

    Insert the forceps at the nonvascular end of the uterus to create an opening and remove the muscular layer. Dissect the decidua to reveal the embryos.

     
  5. 5.

    Separate the placenta and yolk sac from each embryo (seeNote3).

     
  6. 6.

    Using a medicine dropper, move each embryo to an individual 60 mm Petri dish containing approximately 5 ml of cold PBS (seeNotes4 and 5).

     
  7. 7.
    Place the embryo on the dish with its dorsal side facing right. Identify the heart, foregut, somites, spinal cord, and the otic vesicles (see Fig. 1a). Separate the somites with the spinal cord from the foregut by doing a longitudinal incision using the Tungsten needles (see Fig. 1b, Note6). Transect the embryo slightly caudal to the inferior margin of the heart (see Fig. 1c), and again at the level of the otic vesicle (see Fig. 1d). The heart (on the left) remains attached to the foregut (seeNote7). The explant is now ready to be cultured (see Fig. 1e).
    Fig. 1

    Dissection and culture of mouse embryonic foreguts. (a) E8.5 embryo; dashed line representing somite (so) and spinal cord (sc) regions; ov otic vesicle, fg foregut, ht heart. (bf) Stepwise isolation of the foregut explant; red arrows represent surgical incisions. (g) Culture of the foregut explant at the air–liquid interface in transwell filters

     

3.2 Culture of Foregut Explants

  1. 1.

    Prewarm the foregut culture medium to 37 °C.

     
  2. 2.

    Moisten the transwell membrane insets with a minimal amount of culture medium.

     
  3. 3.

    Fill each well of the culture plates with 1.5 ml of culture medium and place the moistened transwell into the well.

     
  4. 4.

    Transfer the foregut explant onto the moistened transwell inset using the medicine dropper. Using Tungsten needles, place the explant with the heart lying on the left side of the foregut, not on top of it (seeNote8). Excess medium should be removed with a medicine dropper (see Fig. 1f, Notes9 and 10).

     
  5. 5.
    Explants can be cultured for up to 5 days in a humidified 37 °C incubator with 5 % CO2. Change the medium at the bottom of the well every 24 h and monitor the explant’s growth under a dissecting or an inverted microscope daily. During the initial 24 h in culture the explant flattens and primordial buds form in the lung and pancreatic fields. At this time these structures are still difficult to visualize in live specimens. However by days 2–3 most of the organ primordia, including the stomach, can be promptly distinguished by morphology. By days 4–5 primary lung buds have elongated and in some cases secondary buds have started to form (usually in explants initially older than 8-somite stage). Both thyroid and pancreas (ventral and dorsal) primordia are prominent and the liver grows broadly at the posterior region of explant. At this time, strong rhythmic peristaltic movements are seen throughout the foregut, particularly prominent in the lung buds and the stomach. All organs can be recognized not only by morphology in live specimens but also by expression of characteristic markers of differentiation, when analyzed by in situ hybridization (ISH) or immunohistochemistry (see Fig. 2, Note11).
    Fig. 2

    Foregut organogenesis in vitro. At the center, whole-mount view of a live foregut explant in culture at around days 3–4. Th thyroid, Lu lungs, St stomach, Gb gall bladder, Pv ventral pancreas, Pd dorsal pancreas, Li liver, g prospective gallbladder. Lateral panels, whole-mount ISH of markers of cell fate markers in day-3 foregut cultures. Pax8 (Th), Albumin (Li), Nkx2-1 (Th, Lu), Pdx1 (Pv, Pd). Modified from ref. 11

     

4 Notes

  1. 1.

    Cervical dislocation can be performed by breaking the mouse’s neck by applying firm pressure at the base of the skull while at the same time pulling the tail backward. Some animal facilities require the animal be anesthetized prior to cervical dislocation, so consult your institutional animal facility for guidelines.

     
  2. 2.

    These steps markedly reduce the risk of contaminating the dissection with bacteria or fungi from the mouse’s skin and hair.

     
  3. 3.

    We find that it is much easier to separate the yolk sac from the embryo using Tungsten needles than forceps.

     
  4. 4.

    The level of PBS should be just enough to cover the embryo to prevent adherence of tissue to the Tungsten needles and also minimize embryo motion during dissection.

     
  5. 5.

    While foregut explant from most E8.5 embryos can be cultured successfully, we find that those that are older than the 8-somite stage grow the best in culture. Explants from embryos younger than the 6-somite stage generally grow poorly in culture. Thus, the system is not optimized to culture foreguts prior to the specification of the thyroid and liver progenitors.

     
  6. 6.

    It is essential to dissect out and exclude the neighbor somite tissue as thoroughly as possible as it will overgrow in culture and obscure the foregut.

     
  7. 7.

    It is important to leave the heart attached to the foregut for the culture; explants tend to grow poorly or break apart during culture without the heart attached.

     
  8. 8.

    The mechanical forces generated by the pulsation of the heart can break the foreguts if the heart is placed on top of the foregut.

     
  9. 9.

    The explant should not be covered by medium; it grows best at the air–liquid interface, not when submerged.

     
  10. 10.

    When multiple explants are placed in the same filter, allow enough space for each explant to grow; otherwise they will fuse in culture. Up to nine foreguts can be cultured on each transwell filter.

     
  11. 11.

    Cultured explants at the desired time points can be harvested and stored for protein/RNA analysis (quick freeze or RNAlater [QIAGEN kit]) or processed for immunohistochemistry and in situ hybridization in whole-mount or histological sections (fixation with 4 % paraformaldehyde solution overnight at 4 °C). Methods for gene or protein analysis of these cultures are reported elsewhere [13, 14].

     

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Pulmonary Center – Boston University School of MedicineBostonUSA
  2. 2.Department of MedicineColumbia University Medical Center and Columbia Center for Human DevelopmentNew YorkUSA

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