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Multi-Axial Self-Organization Properties of Mouse Embryonic Stem Cells Into Gastruloids Leonardo Beccari1,6, Naomi Moris2,6, Mehmet Girgin3,6, David A

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Multi-Axial Self-Organization Properties of Mouse Embryonic Stem Cells Into Gastruloids Leonardo Beccari1,6, Naomi Moris2,6, Mehmet Girgin3,6, David A LETTER https://doi.org/10.1038/s41586-018-0578-0 Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids Leonardo Beccari1,6, Naomi Moris2,6, Mehmet Girgin3,6, David A. Turner2, Peter Baillie-Johnson2,5, Anne-Catherine Cossy4, Matthias P. Lutolf3, Denis Duboule1,4,7* & Alfonso Martinez Arias2,7* The emergence of multiple axes is an essential element in the Recent work on organoids derived from stem cells has revealed a sur- establishment of the mammalian body plan. This process takes prising autonomy in the development of particular tissues and organs4,5. place shortly after implantation of the embryo within the uterus When around 250 ESCs are aggregated, given a pulse of the Wnt agonist and relies on the activity of gene regulatory networks that coordinate CHIR99021 (Chi) between 48 and 72 h after the start of culture, and transcription in space and time. Whereas genetic approaches have returned to N2B27 medium (Fig. 1a), a pole of Bra (brachyury, also revealed important aspects of these processes1, a mechanistic known as T) expression emerges reproducibly6 (Fig. 1b, Extended Data understanding is hampered by the poor experimental accessibility of Fig. 1), resembling the elongating embryonic tail bud. The aggregates early post-implantation stages. Here we show that small aggregates continue to elongate up to 120 h after aggregation (AA), when they of mouse embryonic stem cells (ESCs), when stimulated to undergo display a ‘rostral’ cell-dense region and a polar extension towards a gastrulation-like events and elongation in vitro, can organize a ‘caudal’ extremity, reaching up to 500 µm in size (Fig. 1b). Shaking post-occipital pattern of neural, mesodermal and endodermal the culture enables aggregates to grow to 850–1,000 µm in length at derivatives that mimic embryonic spatial and temporal gene 168 h AA (Fig. 1c, d). At 120 h AA, a Gata6-positive domain is visible expression. The establishment of the three major body axes in opposite to a Bra and Cdx2-expressing region, probably corresponding these ‘gastruloids’2,3 suggests that the mechanisms involved are to the cardiac crescent, which delimits the embryonic post-occipital interdependent. Specifically, gastruloids display the hallmarks of region7 (Fig. 1b–d, Extended Data Fig. 1, Supplementary Videos 1, 2). axial gene regulatory systems as exemplified by the implementation By contrast, at 120–168 h AA Sox1/Sox2-positive cells are localized of collinear Hox transcriptional patterns along an extending antero- centrally, with the exception of those at the rostral extremity (Fig. 1c, d). posterior axis. These results reveal an unanticipated self-organizing To characterize the transcriptional programmes of these gastru- capacity of aggregated ESCs and suggest that gastruloids could be loids, we carried out RNA-sequencing (RNA-seq) analysis on duplicate used as a complementary system to study early developmental events pools of gastruloids and compared their profiles with those of devel- in the mammalian embryo. oping mouse embryos from E6.5 to E9.5. Because gastruloids display 120 h z = 27 μm a e c 0 h 24 h 48 h 72 h 96 h 120 h 144 h 168 h b Chi 24 h Suspension Shaking E6.5 a 48 h a b 50 c 48 h 72 h96 h 72 h b E7.8 b 25 μm 25 μm 96 h 50 μm 50 μm a BRA Gata6 SOX2 a 0 120 h a 168 h 168 h b c d E8.5 b DAPI SOX2 SOX1 BRA DAPI CDX2 a SOX2 SOX1 PC2 (26.5% of explained variance) a b a BRA SOX1 b b a 144 h a b b b a –50 –50 0 50 E9.5 168 h PC1 (34.4% of explained variance) Fig. 1 | Elongation of gastruloids. a, Schematic of the culture protocol: proteins. Scale bar, 150 µm. Each image is representative of an experiment 200–300 ESCs were allowed to aggregate; the Wnt agonist CHIR99201 performed in 20 biological replicates. The reported expression pattern was (Chi) was added between 48 h and 72 h AA; organoids were cultured in observed in at least 80% of the cases. e, PCA analysis of RNA-seq datasets suspension until 120 h AA (grey rectangle) and transferred into shaking using time-pooled gastruloids from 24 to 168 h AA (n = 2 replicates per cultures until 168 h AA. b, Three-dimensional renderings and confocal time point) and pooled mouse embryos at E6.5 (n = 3), E7.8 (n = 3), sections of gastruloids at different times showing the elongation and E8.5 (12–14 somites, n = 2 replicates) and E9.5 (~24 somites, n = 2 expression of BRA, SOX2 and Gata6H2B-Venus (green). The right-most panel replicates). Each replicate was derived from an independent sample. For is a confocal section of the 3D rendering of the neighbouring 120 h AA E7.8 embryos, only their posterior half was used. For E8.5 and E9.5, the gastruloid. Scale bars, 25 µm (48 h, 72 h), 50 µm (96 h, 120 h). Each image post-occipital embryonic domain was dissected. In all cases, the portion is representative of an experiment with seven biological replicates showing used for RNA extraction is coloured in pale green. All autosomal genes the same expression pattern. c, d, Three-dimensional rendering (c, left) were considered for this analysis. PC1 shows a strong temporal component and confocal sections (c, centre, right and d, tail region) of gastruloids whereas PC2 discriminates between gastruloids or embryonic samples. at 168 h AA, showing the localization of CDX2, SOX2, SOX1 and BRA 1Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland. 2Department of Genetics, University of Cambridge, Cambridge, UK. 3School of Life Sciences, Federal Institute of Technology EPFL, Lausanne, Switzerland. 4Swiss Cancer Research Institute (ISREC), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. 5Present address: Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. 6These authors contributed equally: Leonardo Beccari, Naomi Moris, 7 Mehmet Girgin. These authors jointly supervised this work: Denis Duboule, Alfonso Martinez Arias. *e-mail: [email protected]; [email protected] NATURE| www.nature.com/nature © 2018 Springer Nature Limited. All rights reserved. RESEARCH LETTER a 24 h 48 h 72 h 96 h 120 h 144 h 168 h PCA E6.0 E7.8 E8.5 E9.5 PCA determinant determinant –3 –2 –1 0 1 2 3 –1 0 1 50 Msgn1 Cdx1 Pltp Bra 1 Cdx2 Nkx1-2 2 20 Prl3d1 Wnt3aTbx6 Hoxb1 H2-Ke6 Pcdhga8 Tdgf1Cts7 Emcn 25 Hes7 Hoxb9 Prl3d2 Hoxa4 Myl3 Nr2f1 Hoxa1 Hoxb8 Hoxa7 Mt4 Copg2 Mt3 1 10 Olig2 0.5 Hoxc8 Hoxb2 Itga7 Pax1 Hoxb6 Hoxc6 Arg1 Hoxb3 Pou3f2 Hoxd4 Elf5 Meox1 Pax6 Myl4 0 Hoxd9 Dnmt3l Meis2 0 0 Foxd1 Platr10 Hoxc10 Meis1 Hoxc4 Hoxc6 0 Hoxc10 Eomes Utf1 Copg2 Hoxb9 Hoxd4 Hbb-y Ydjc Gata4 Nanog Tnni1 Platr10 L1td1 Nr2f1 Hoxc8 –1 –10 Mesp1 Hoxa2 –25 Rpl39l Ydjc Hoxb3 Dlk1 Hoxa3 Foxd3 Spp1 Hbb-y Meox1 Hba-x PC2 (25.1% explained variance) Tnni1 PC2 (19.4% explained variance) Hoxb2 Utf1 Dnmt3l Actc1 Arg1 –0.5 Tceb2 Lgals2 Hes3 –2 Hbb-bh1 Tnnt2 Hba-x –20 Pdzk1 Hoxb1 Pfkp Myl4 Hbb-bh1 Nkx1-2 Msgn1 –50 –50 0 50 –20 0 20 PC1 (53.7% explained variance) PC1 (55.9% explained variance) b Gastruloids Embryos log2(FPKM + 1) 01 23 Afp Cubn Apoc2 Amn Extraembryonic Pou5f1 Otx2 Fgf4 Zfp42 Pluripotency Nanog Six3 B Hesx1 Dmbx1 En1 FB/MB/H En2 Nkx6-1 Sox2 Pax6 eural tube Foxa2 Irx3 Mixl1 Wnt3 Eomes Gsc Gastrulation Chrd Nkx1-2 mN Cdx2 Meox1 Gata6 Tcf15 Mesendoder abab ababababab a b cab cab ab 24 h 48 h 72 h 96 h 120 h 144 h 168 h E6.5 E7.8 E8.5 E9.5 Fig. 2 | Temporal patterns of gene expression in gastruloids. a, PCA also known as Elob. b, Heat map of scaled expression of genes associated of either pooled gastruloids during temporal progression from 24 h to with development of different embryonic structures in pooled gastruloids 168 h AA (left) or mouse embryos from E6.5 to E9.5 (right). The 100 top and embryos over time. The replicates represented in these graphs were contributing genes to the first two principal components are labelled, with derived from biologically independent samples, as in Fig. 1e. FB, forebrain; those common to both gastruloid and embryo datasets in red. Tceb2 is MB, midbrain; HB, hindbrain. hallmarks of post-occipital embryos6 (Fig. 1b–d) we excluded the ante- around 48 h AA (Fig. 2b, Extended Data Fig. 2b), suggesting that at rior portion of E7.5–E9.5 embryos (Fig. 1e, left). Principal component this stage the gastruloid transcriptome resembles that of mouse epi- analysis (PCA) demonstrated reproducibility between samples and a blast at the onset of gastrulation. By 72 h AA, we observed an increase clear clustering along principal component 1 (PC1) corresponding to in the complexity of gene-expression profiles, with the appearance of their temporal order (Fig. 1e), whereas embryo samples segregated markers for different embryonic lineages, including mesendoderm and from gastruloids along PC2 only. The main (top 100) clustering deter- neuroectoderm, and the transcription of Hox gene clusters (Fig. 2a, minants of gastruloid samples included several pluripotency-related b, Extended Data Fig. 2a, b; see below). Genes associated with either genes, epiblast markers and genes involved in gastrulation. They also extra-embryonic structures or anterior neural plate derivatives were comprised different Hox genes and other transcription factors usually not (or were poorly) expressed in gastruloids (Fig. 2b, Supplementary expressed in post-occipital structures of the developing mouse embryo Information datasets 1, 2).
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