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, 141 (22), 4231-42

Symmetry Breaking, Germ Layer Specification and Axial Organisation in Aggregates of Mouse Embryonic Stem Cells

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Symmetry Breaking, Germ Layer Specification and Axial Organisation in Aggregates of Mouse Embryonic Stem Cells

Susanne C van den Brink et al. Development.

Abstract

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.

Keywords: Axial elongation; Endoderm; Gastrulation; Live cell imaging; Mesoderm; Mouse; Neural ectoderm; Pattern formation; Polarisation; Self-organisation; Symmetry breaking.

Figures

Fig. 1.
Fig. 1.
Comparative analysis of the effect of exposure time and signalling on aggregate formation. (A) The stimulation protocol. The vertical black dotted lines indicate medium changes, and the red vertical line corresponds to the beginning of day 2. Aggregates were cultured in N2B27 (grey shading) continuously (P0), or treated with continuous (P2) or 24 h pulses (P1,3,4,5) of Act, Chi or Act/Chi (blue shading) before being returned to N2B27. Data for P1 not shown. (B) Cartoon renderings (see Materials and Methods; unprocessed images are shown in supplementary material Fig. S2) of typical aggregate morphologies on day 5 following the conditions shown in A; images are not to scale. Maximum elongation was observed following pulsed treatment within the day 2-3 time frame (P3). (C,C′) Comparison of aggregate morphologies following a 24 h pulse on days 2-3 of Act, Chi, Act/Chi and BMP4 for three different cell types: (a) Sox17::GFP, (b) TBX6::EYFP and (c) wild-type E14-Tg2A. (C) Aggregates were scored based on whether they were spherical (white), contained a single outgrowth (ovoid, grey), showed overt elongation (green) or had multiple protrusions (blue). Examples of these aggregate morphologies are shown in the form of cartoon renderings, processed as described above. (C′) These data are also represented as ratios between the indicated morphologies and the proportion of aggregates with ovoid appearance. Note how transient exposure to Chi results in a much higher ratio of elongated to ovoid morphologies. The number of Sox17::GFP, Tbx6::EYFP and E14-Tg2A aggregates for each condition (C,C′) are as follows (respectively): Act: 71, 27, 52; Chi: 60, 32, 48; Act/Chi: 69, 17, 46; BMP4: 64, 24, 38.
Fig. 2.
Fig. 2.
Effect of initial cell density on the elongation of aggregates. (A) Aggregates formed from increasing numbers of cells (200-1600 cells) as indicated were exposed to Chi for the duration of the experiment (at least six aggregates per condition). (B) Aggregates with an initial size of between 400 and 800 cells showed elongation. (C) Aggregates with 800 cells tended to exhibit multiple elongations (arrowheads).
Fig. 3.
Fig. 3.
Polarisation, patterning and gene expression in aggregates. (A,A′) Two single sections through GPI-GFP mESC aggregates exposed to N2B27 for 5 days with a 24 h pulse of either Act (n=10), Chi (n=5), or Act/Chi (n=14) between 48 and 72 h and imaged by confocal microscopy (GPI-GFP channel not shown). The expression of the indicated markers on the surface of the aggregates is shown in A′, with the corresponding orthogonal view through the aggregate in A″. The arrows in A″ indicate the z-section shown in A and A′. Note how the expression of Sox17 is localised to the surface of the aggregate. (B,B′) A representative aggregate from GPI-GFP mESCs exposed to Act between 48 and 72 h was imaged at the end of the treatment after being fixed and stained for E-cadherin and Sox17 (B); the boxed region is enlarged to show E-cadherin (B′). Note the depressions that are associated with Sox17 expression and high levels of E-cadherin. (C) Section through an E7.5 embryo stained for Sox17 and with DAPI.
Fig. 4.
Fig. 4.
Polarised expression of Bra in response to Wnt signalling. (A) Bra::GFP cells exposed to a 24 h pulse of Act or Chi between 48 and 72 h and imaged at 120 h (n>3). (B) Aggregates of a Sox17::GFP cell line treated with sustained Act/Chi and stained on day 4 for either Bra (left, n>10) or FoxA2 (right, n=13). (C,D) The β-catenin transcriptional reporter line TCF/LEF::mCherry treated with (C) Chi in culture (as in A, n=11) and compared with its expression in the PS of an E6.5 embryo (D), both stained for Bra. These conditions not only produce elongations of the aggregates, but also result in polarised gene expression.
Fig. 5.
Fig. 5.
Emergence of polarised gene expression in aggregates. (A) Stills from live cell imaging of mESCs in suspension in N2B27 showing aggregate formation within the first 8 h. (B,C) Emergence and progression of Sox17::GFP following addition of secondary Act/Chi medium. (D) Early stages of Sox17::GFP and Bra expression. Initially, Bra and Sox17::GFP are heterogeneously expressed before polarisation occurs. (E) Live imaging of Bra::GFP mESCs following addition of Act/Chi. Every cell initially expresses Bra before downregulation in regions that will not form the elongation. A, B, C and E are from supplementary material Movies 1-4, respectively. Data are representative from at least two experiments.
Fig. 6.
Fig. 6.
Qualitative summary of the tissue-specific response of aggregates to different signalling environments. Reporter lines were used for neural (Sox1::GFP), endoderm (Sox17::GFP), mesendoderm (Bra::GFP) and paraxial mesoderm (TBX6::EYFP) as well as for Wnt signalling (TCF/LEF::mCherry). Aggregates from the different lines were treated as indicated and the results in terms of expression levels within the population as a whole are summarised by the colour intensity within each square. Representative examples are shown in Fig. 7. ND, not determined.
Fig. 7.
Fig. 7.
Gene and tissue-specific response of aggregates to different signalling environments. (A-E) Representative examples from the summary in Fig. 6. Sox1::GFP (A, n=16, 14, 16, 24, 16 per labelled condition, respectively), TBX6::EYFP (B, n=11 or 10 per labelled condition, respectively), TCF/LEF::mCherry (C, n=4 per condition), Bra::GFP (D, n=9, 11 or 11 per labelled condition, respectively) and Sox17::GFP (E, n>3) mESCs were treated as indicated. The boxed region in the Chi pulse image (A) is enlarged section to the right to show a region within the tip of the aggregate that is negative for Sox1::GFP. Compare with the expression pattern of Bra and Sox17::GFP and Wnt activity from Figs 3 and 4. Note that the expression of the reporters is associated with specific morphogenetic events; two examples of each are given. The colour coding of each treatment label corresponds to that used in Fig. 6. (F,G) Aggregates of Sox1::GFP mESCs following a pulse of Chi on day 3 were stained for GFP (Sox1), Sox2 and Sox17. The boxed region is magnified to the right and also shows orthogonal views. Sox1 at the tip of the aggregate is co-expressed with high levels of Sox2, whereas Sox2 levels decrease in regions high for the endoderm marker Sox17. (G) Section through a 12-somite stage embryo stained for Sox2 and with DAPI. Note how Sox2 is expressed in the neural tissue (n) and in the gut (g), similar to the expression pattern seen in F.
Fig. 8.
Fig. 8.
Gastrulation-like movements in aggregates. (A-C″) Cell extrusion and intra-aggregate movement in (A,A′) Sox17::GFP, (B,B′) Bra::GFP and (C-C″) TBX6::EYFP. Aggregates were treated as indicated. Images correspond to the indicated time points from the associated movies (see supplementary material Movies 5-8). Arrows in C″ indicate a single extruded cell from a second aggregate between 102 and 103 h (see supplementary material Movie 8); curved arrows in A′ indicate the direction of movement of the cells leaving the aggregate. (D,D′) Live imaging of GPI-GFP mESCs following treatment with Act/Chi, showing membrane blebbing at the elongated region of the aggregate. Two different z planes are shown. Arrows indicate the blebbing region (see supplementary material Movie 10).
Fig. 9.
Fig. 9.
Comparison of events in embryos and aggregates. (Top) Timeline of embryogenesis, with the illustrated stages acting as landmarks. (Bottom) A representation of the behaviour of aggregates exposed to different signalling environments over the indicated periods of differentiation, as inferred from our experiments labelled here as a, b and c. We propose that the third day of differentiation of the aggregates is equivalent to the E5.5-6.0 postimplantation epiblast. DD, day of aggregate differentiation. The dark blue shading indicates anterior Sox1 expression.

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