Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/β-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.

Widefield, single-time point images of Gastruloids were acquired using a Zeiss AxioObserver.Z1 (Carl Zeiss, UK) in a humidified CO 2 incubator (5% CO 2 , 37°C) with a 20x LD Plan-Neofluar 0.4 NA Ph2 objective with the correction collar set to image through plastic. Illumination was provided by an LED white-light system (Laser2000, Kettering, UK) in combination with filter cubes GFP-1828A-ZHE (Semrock, NY, USA), YFP-2427B-ZHE (Semrock, NY, USA) and Filter Set 45 (Carl Zeiss Microscopy Ltd. Cambridge, UK) used for GFP, YFP and RFP respectively, and emitted light recorded using a back-illuminated iXon888 Ultra EMCCD (Andor, UK). Images were analysed using FIJI (Schindelin et al., 2012) and plugins therein as previously described  and when required, images were stitched using the 'Pairwise Stitching' plugin in FIJI (Preibisch et al., 2009). Briefly, the fluorescence intensity was measured by a line of interest (LOI) drawn from the posterior to anterior region of the Gastruloid with the LOI width set to half the diameter of a typical Gastruloid at 48h (100px with the 20x objective). The background for each position was measured and subtracted from the fluorescence for each Gastruloid. Shapedescriptors were generated by converting brightfield images of Gastruloids to binary images and measuring them by particle detection in FIJI.
Fluorescence levels were normalised to the maximum obtained in following Chi stimulation, and the maximum length of each Gastruloid was rescaled 1 unit. Average fluorescence traces of Gastruloids ±S.D. are shown in the main figures, and the raw data and individual traces in the supplemental data. For live imaging experiments, each well of a 96-well plate containing individual Gastruloids were imaged as described above using both the 20x (24-72h) and the 10x (72-96h) objectives, and images captured every 20 min for a maximum of 96h (120h AA). All images were analysed in FIJI (Schindelin et al., 2012) using the LOI interpolator (Soroldoni et al., 2014) with the LOI set as described above.
We define a measure of assessing differences between intensity profiles, of a Gastruloid in a given condition and the control Gastruloids, similar to the root-mean-square deviation used to measure differences between values of an estimator and the values observed. We call this measure the Normalised Root Square Distance ( ) and for the !! Gastruloid in condition it is defined as follows: where denotes the maximum number of points (typically 100) defining the normalised length of the Gastruloid. As a physical interpretation of this measure, it can be seen that !,! = 0 means that the signal intensity profile for the !! Gastruloid in condition is identical to the mean intensity profile of the control Gastruloids. !,! ≤ 1 means that the signal intensity profile for the !! Gastruloid in condition is within the standard deviation around the mean intensity profile of the control Gastruloids, thereby implying that the Gastruloid in condition is similar to the control. Significance between treatments within each time-point was determined using a nonpaired Student's t-test.

Development • Supplementary information
In experiments which required the addition of specific factors to Gastruloids on the second day of aggregation (24-48h), 20µl medium was carefully removed with a multichannel pipette, and 20µl of N2B27 containing twice the concentration of the required factors was added. This method was preferable to the addition of smaller volumes containing higher concentrations of agonist/antagonists, as the data from these experiments showed more variation between Gastruloids (DAT, PB-J, AMA unpublished). Control experiments showed that replacement of half the medium at this stage did not significantly alter the ability of Gastruloids to respond to signals on the third day (DAT, PB-J, AMA unpublished). The next day, 150µl fresh N2B27 was added to each of the wells with a multichannel pipette and left for no more than 30 min to wash the Gastruloids; a time delay ensured that sample loss was prevented. Following washing, 150µl N2B27 containing the required factors was then applied. The small molecules used in this study and their concentrations are described in table S4.

Movie 1. T/Bra::GFP expression in Gastruloids following DMSO treatment (48-72h AA).
Gastruloids made from T/Bra::GFP mESCs stimulated with a mock pulse of DMSO and imaged by wide-field microscopy from 24h to 120h AA every 20 min. The 20x objective was used between 24 and 72h, followed by the 10x objective from 72h to the end of the experiment. Quantification of both the length and fluorescence as a function of time can be seen in Fig. 3D (top). (48-72h AA). Gastruloids made from T/Bra::GFP mESCs stimulated with a pulse of Chi and imaged by wide-field microscopy from 24h to 120h AA every 20 min. The 20x objective was used between 24 and 72h, followed by the 10x objective from 72h to the end of the experiment. Quantification of both the length and fluorescence as a function of time can be seen in Fig. 3D (bottom).  Tables   Tables S1-S5   Table S1. Expression phenotype of T/Bra::GFP mESCs. The proportion of T/Bra::GFP Gastruloids not expressing the reporter (No Expression) or displaying either Polarised or Ubiquitous expression at 24, 48 and 72h AA followed by a pulse of DMSO or Chi (72h). The standard deviation is shown in brackets and the number of Gastruloids analysed are shown.         Mann-Whitney U test followed by Bonferroni adjustment, comparing selected columns. Asterisk indicates p < 0.05.