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.
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