Yusuke Marikawa, Dana Ann A. Tamashiro, Toko C. Fujita, Vernadeth B. Alarcón
In this paper (1), Marikawa et al. use P19 embryonal carcinoma (EC) cells grown as 3D aggregates to study the properties of early axial development, specifically mesendodermal formation and axial elongation. Although this paper is now 10 years old, it is in my opinion, an important piece of work that demonstrates how useful 3D tissue-culture techniques are as model systems in exploring the signalling requirements of axial specification during embryo development.
Here, Marikawa et al. build on previous observations that mesendodermal derivatives can be generated when EC cells (P19) are cultured in high density and as embryoid bodies (2). The authors grow small numbers of cells as hanging drops in medium containing DMSO, previously shown to be an good inducer of mesendodermal derivatives from P19 EC cells (2, 3). When cultured in this manner, over time and up to 6 days after aggregation, pooled aggregates showed an increase in the level of expression for genes associated with the primitive streak (~embryonic day 6.5-7.5 such as Brachyury and Fgf8), mesoderm (Wnt3a, Wnt5a), and definitive endoderm/posterior neural tube (Cdx2), with concurrent down-regulation of pluripotency-related genes (e.g. Oct). Interestingly, the authors note that the temporal progression of these gene expression patterns happens on a similar time-scale to that of the embryo.
However, one of the most striking observations from this work is the subsequent change in the morphology which had not been seen in previous studies, possibly (as the authors themselves mention) due to the low seeding density of cells compared to other work (~200 cells vs <5000). As they point out, this number was chosen to be a close approximation to the numbers of cells expected in the epiblast just prior to gastrulation. As time progressed, the aggregates began to elongate and become thinner, in a manner similar to the convergent extension (CE) that is observed in the posterior of embryos. Correlating the emergence of this CE-like morphological development to the expression of genes that are expected to be expressed in the posterior of the embryo during early gastrulation (e.g. Brachyury, Cdx2, Fgf8 and Wnt3A) showed polarisation and spatial confinement of a number of these genes to one pole of the elongating aggregate (as assessed by in situ hybridisation). By comparison, a contemporaneous paper by ten Berge et al. (2008) used a similar approach to generate aggregates of mouse embryonic stem cells, but seeded 2,000 cells as hanging drops (4), an order of magnitude of cells more than Marikawa and colleagues, and transferred individual aggregates to 96-well plates for further culture. Although ten Berge et al. were able to show polarised expression of Brachyury and a Wnt/β-Catenin in a selection of embryoid bodies, they were not able to generate such elongated structures, suggesting that the size of the aggregate (e.g. cell number), and therefore the number of the cells in the embryo by extrapolation, may be an important determinant for proper axial development.
The authors’ experiments were terminated following 6 days in culture, mainly due to the lack of subsequent changes in morphology and, as they report, possible degeneration of the aggregates manifested by emergence of loose cells coming away from the aggregate. Because there are no data in the paper showing the state of these emerging cells in these post-day 6 aggregates, it is tempting to hypothesise that these cells may not in fact be indicating the degeneration of the aggregate, but rather an attempt at gastrulation (the appearance of a Brachyury positive region in one region of the aggregate may be suggestive of a primitive streak-like population, which marks the site of gastrulation (5-7)).
Although beyond the scope of the study, these experiments would be well complemented by taking a time-lapse microscopy approach, assessing the temporal progression of fluorescent reporters for genes associated with anterior or posterior embryonic regions (i.e. GATA6 and Brachyury), providing a correlation between acquisition of an elongated phenotype and the associated gene expression. Additionally, testing the time-periods when the aggregates are most sensitive to particular stimuli/inhibitors would add further dimension which would feed back to generate hypotheses into the temporal requirement of specific factors during embryo development.
However, the hanging-drop method employed by the authors might not be best suited for these experiments; for example, it is difficult to keep hanging drops separate from one another, and to accurately apply different signalling regimes throughout the time-course. One method that might have been tried is the SFEBq (serum-free culture of embryoid body-like aggregates; quick reaggregation) culture developed a year earlier by Sasai and colleagues in their studies on neuroectodermal differentiation (8). With this SFEBq culture, individual aggregates could be cultured in individual 96-well plates, making it much easier to precisely control the application and removal of stimuli at specific temporal intervals, as well as facilitating basic single-aggregate imaging. Indeed, it was a combination of this Marikawa paper and the Sasai SFEBq protocol that lead to the development of the gastruloid model system some time later, using small aggregates of mouse embryonic stem cells to probe axial elongation, cell fate specification, and the development of the three embryonic axes during development (9-11), in a similar vein to this work by Marikawa and colleagues.
In summary, this paper shows how many of the properties of early axial mesendodermal development can be recapitulated in vitro, highlighting the inherent self-organisational ability of these cells, providing that they are exposed to the correct signalling conditions and cultured appropriately. This therefore provides an interesting and useful model system to study events similar to those that occur during development, but in a way that doesn’t require the use of animals, is much cheaper, and if combined with SFEBq culture provides a way to precisely manipulate signalling conditions and perform experiments that are difficult or otherwise impossible to accomplish in the embryo.
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