Investigating Molecular Mechanisms of Embryonic Mammary Gland Development by Bead-Implantation in Embryonic Flank Explant Cultures – A Protocol

The involvement of molecular mechanisms in a particular process such as embryonic mammary gland development, can be revealed by modulation of one or several factors that purportedly act in that process. If those factors or their inhibitors are soluble, their function can be tested by loading them onto small inert beads, which are then implanted in cultured explants of the tissue of interest, in this case embryonic flanks. We here describe a protocol for such experiments.


Introduction
Nowadays, genetic modification is a widely used technique to identify the role of a particular gene in a specific process. But if genetically modified animals are not available, too costly, or don't survive until the developmental stage of interest; or if the molecule of interest is not encoded by genes (as is the case for e.g. retinoic acid), alternative methods are available to test the potential roles of these molecules. For example, if the molecule of interest is soluble, it can be loaded onto inert beads, which can be applied to, or implanted in, explant cultures of the tissue or organ of interest. Compared to diluting the soluble factor in the culture medium, this method has the advantage of localized application and reduced consumption of the factor. This technique can be applied to the study of various stages of embryonic mammary gland development. Mammary gland development in mice starts at around embryonic day (E) 10.5, as inferred from the formation of Wnt10b positive mammary streaks on the flanks, in the axillae and in the inguinae. On each lateral side of the body, these streaks soon fuse into one continuous line coincident with the asynchronous formation of five mammary disc-shaped placodes, one in the axilla, one in the inguen, and the other three residing on the flank in between forelimb and hindlimb [1]. In the course of 1 to 2 days, these placodes transform into spherical buds which subsequently sprout into the underlying dermis and branch out into the hypodermal fat pad precursor before birth [2]. Klaus Kratochwil developed a culture method in which mammary rudiments (MRs) from E12.5 mouse embryos onwards can undergo normal morphogenesis, albeit it with a delay of about 1 day. For these cultures, Kratochwil dissected individual mammary buds with a few layers of contiguous mesenchyme. He placed these on a filter resting on a metal grid which itself was hanging over a central depression in a special glass culture dish (Grobstein-design), filled with less than 1 ml medium to just touch the filter [3]. This culture method is based on the principle of a Trowell culture, i.e. organ culture at the medium/gas interface on a thin filter membrane supported by a metal grid [4]. For ex vivo culture of MRs at younger stages, including those prior to the onset of mammary gland formation, one can culture a wide band of the flank encompassing all prospective MRs and the limbs [5]. The presence of the limbs prevents retraction of the ectoderm during culture, but has the disadvantage that only MR2, MR3 and MR4 can be monitored, as MR1 and MR5 are covered by the limbs. This protocol describes the culture of E10.5 and E11.5 flank explants with application or implantation of beads soaked in soluble molecules, to monitor the effect of these molecules on mammary development. In short, beads are loaded with the molecule of interest. Embryos are harvested at ages ranging between E10.5 and E12, and their flanks are dissected for culture as explants. A loaded bead is then grafted underneath the ectoderm [5] or laid on top of it [6]. These explants can be cultured ex vivo for 1-3 days, which is sufficiently long to test the effect of any factor that is loaded onto beads. If culture is extended beyond 3 days, the dermal mesenchyme will stiffen, which interferes with normal growth. For ex vivo experimentation with mammary development from E12.5 onwards, one can use Kratochwil's culture method [3] or its modification as described elsewhere in this issue [7] and apply beads that are soaked in molecules of interest as described here. Approximately at the level of the anterior side of the hindlimb is the boundary between somites #24 and #25 in embryos at around E11.5 (Fig. 1). Count from there downwards to the tip of the tail. Note that relative boundaries of limbs and somites shift slightly in the course of development between E10.5 and E11.5, which may create slight errors in counting when comparing litters at E10.5 and E11.5. Nonetheless, this method allows distinguishing the lesser advanced from the more advanced embryos within one litter. 4 Deeply submerge one embryo at a time in sterile DPBS.

Materials
Use Graefe knifes to remove the head and tail. Lay the trunk ventral side down and place the Graefe knifes in and aligning with the dorsal midline of the neural tube, one in the neck, one in the lumbar area. Slide the knives along each other to bisect the embryo along the dorsal midline, through the visceral organs and as near to the ventral midline as possible. Remove all internal organs as they inhibit development of mammary tissue. 5 The peritoneum should be peeled away, but the dermal mesenchyme and the somites should be retained, as they contain inductive signals for mammogenesis [5]. The heart can serve as a control for culture conditions, and some organs may be useful controls to test activity of the factors loaded onto beads. 5 Set up one of the following three Trowell-type culture dishes according, using forceps: a. place a bent metal grid into a 35 mm petri dish b. hang a flat metal grid over the rim of the central well of an organ culture dish c. hang a Millicell membrane insert in a 35 mm petri dish or 6-well culture plate 6 Using forceps, transfer a washed filter (convex if curled) into the dish with embryonic flanks and push to the bottom of the dish. With closed forceps, gently push a flank onto the center of the filter, mesenchymal side down, ectodermal side facing up. Drag the filter towards the periphery of the dish, and decant the dish so that the explant is no longer submerged. Lift the filter with explant out of the dish and place it on the grid or membrane insert, above the hole and fill the dish with supplemented medium until it just touches the filter. The explant may not be submerged, but has to be situated at the medium/air interface. Avoid air bubbles in the medium and certainly underneath the filter. 7 Place the lid on the dish and the dish in the incubator, and prepare flank explant cultures of the next embryo. 8 In a separate dish, set up a similar Trowell-type culture of a heart, as a positive control for culture conditions. If the heart is not beating at the end of the experiment, culture conditions were not good. 9 In separate dishes, set up similar Trowell-type cultures of an organ that can serve as a positive control for activity of the molecule that is loaded onto beads, e.g. several Fibroblast Growth Factors (most certainly FGF10) will enhance branching morphogenesis of the embryonic lung [14]. 3 Commercially available chick extract (several vendors) may be a practical alternative, but I have not tested these. 4 A movie showing embryo dissection (step 2) is available online at http://www.ijdb.ehu.es/data/11/113424ls/IntJDevBiol-113424ls-SuppMovie1.mp4 [13], and needs to be downloaded (by opting "save link as…") for viewing. Note that for this protocol, cleaning of surfaces with RNase-away is not necessary, and after dissection, embryos should NOT be collected in RNA-later, but in sterile DPBS. 5 A movie showing flank dissection (step 4) is available online at http://www.ijdb.ehu.es/data/11/113424ls/IntJDevBiol-113424ls-SuppMovie2.mp4 [13], and needs to be downloaded (by opting "save link as …") for viewing. Note that embryos in the movie were stored in RNA-later and thus look wrinkled/shriveled compared to the fresh embryos one uses for culture experiments. Once the visceral organs have been removed, the subsequent manipulations in this movie should NOT be followed for culture experiments.  [11] and e.g. retinoic acid or its inhibitor citral [6]. When working with acrylic beads, make sure to coat the inside of Pasteur pipettes by aspirating a small volume of serum and washing with DPBS before aspirating beads, so they will not stick to the pipette. & Bovine Serum Albumin (BSA) (lyophilized, BioReagent; Sigma cat# A9418), as a 'vehicle' for growth factors. Prepare a small stock solution of 1 mg/ml in DPBS and store at 4°C for a few weeks. & (recombinant) Growth factors (e.g. from R&D Systems) or other molecules of interest. For growth factors: Buy in lyophilized state and reconstitute according to manufacturer's recommendations and 10× more concentrated than the final concentration in culture, usually in at least 0.1 % (1 mg/ml) BSA.
Final concentrations for culture must be retrieved from the literature and by experimentation. Store small aliquots at −80°C where they can be kept for at least several months. Dilute on ice to final concentration preferably just prior to the experiment, although single use aliquots (e.g. 10 μl) can be made of excess volume at final concentration and stored for at least several months at −80°C.

Methods
1 Using a 1 ml Stripette, transfer a small volume of bead suspension from the commercial stock to a 1.5 ml microfuge tube. 2 Wash these beads several times with DPBS. 3 Using a 1 ml Stripette, transfer beads to a 60 mm petri dish. 4 Under the stereoscope, select beads with a diameter that is about similar to the width of a somite. 5 Transfer sufficient beads for one experiment (add extra for unforeseen loss during experimentation) with a 2 or 10 μl pipette (or mouth pipette with capillary drawn Pasteur pipette) to a sterile 0.2 ml PCR reaction tube.
Preferably keep the number (e.g. 40) of beads the same for similar experiments, to minimize loading variation among multiple experiments. 6 Under the stereoscope, aspirate the DPBS entirely with 2 or 10 μl pipette or mouth pipette.   When beads are loaded with growth factors or BSA as vehicle control, they need not be replaced. When beads are loaded with relatively unstable factors (e.g. retinoic acid) and placed on top of the explant, beads may be replaced daily. 2 Capture images of all cultures at the end of the culture period (Fig. 1). 3 Fixate and analyze explants by the method of choice, e.g. visualize the mammary line or MRs by LacZ staining if the embryos carry the TOPGAL transgene as a marker for mammary line and rudiments, or use a fluorescence stereoscope if they carry the s-SHIP-GFP transgene, or perform whole mount in situ hybridization for endogenous Wnt10b; or test any other gene of interest.

Conclusion
The use of beads loaded with molecules of interest in explant flank cultures facilitates the analysis of the role of those molecules in the earliest stages of mammary gland development. This technique can also be used to test epistatic cascades without having to crossbreed different mouse mutant strains with each other.
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