Stem cell-derived three-dimensional (3D) gastruloids show a remarkable capacity of self-organisation and recapitulate many aspects of gastrulation stage mammalian development. Gastruloids can be rapidly generated and offer several experimental advantages, such as scalability, observability and accessibility for manipulation. Here, we present approaches to further expand the experimental potency of murine 3D gastruloids by using functional genetics in mouse embryonic stem cells (mESCs) to generate chimeric gastruloids. In chimeric gastruloids, fluorescently labelled cells of different genotypes harbouring inducible gene expression or loss-of-function alleles are combined with wild-type cells. We showcase this experimental approach in chimeric gastruloids of mESCs carrying homozygous deletions of the Tbx transcription factor brachyury or inducible expression of Eomes. Resulting chimeric gastruloids recapitulate reported Eomes and brachyury functions, such as instructing cardiac fate and promoting posterior axial extension, respectively. Additionally, chimeric gastruloids revealed previously unrecognised phenotypes, such as the tissue sorting preference of brachyury deficient cells to endoderm and the cell non-autonomous effects of brachyury deficiency on Wnt3a patterning along the embryonic axis, demonstrating some of the advantages of chimeric gastruloids as an efficient tool for studies of mammalian gastrulation.

The versatile experimental opportunities offered by functional genetics available in the mouse as a main mammalian model system have greatly enhanced our understanding of embryonic development. However, studies of mammalian embryogenesis are hampered by the relative inaccessibility of the embryo due to intra-uterine growth. Ex vivo embryo cultures partially overcome some of the experimental restrictions but rely on the isolation of often limiting numbers of embryos. More recently, novel approaches have been developed to generate stem cell derived, three-dimensional (3D) embryoids reflecting different stages of mammalian development from the formation of the blastocyst (Kagawa et al., 2022; Li et al., 2019; Yu et al., 2021) to peri-implantation embryos (Amadei et al., 2021; Harrison et al., 2017, 2018) and post-implantation stages recapitulating early organogenesis (Beccari et al., 2018; Moris et al., 2020; Turner et al., 2017; van den Brink et al., 2014, 2020; Veenvliet et al., 2020). These embryoid models demonstrate the remarkable self-organisation and robustness of embryonic programs that guide the processes of morphogenesis, growth, cell lineage specification and differentiation in the embryo as well as in in vitro models.

Among widely used embryoid model systems are 3D gastruloids, which offer a simple experimental procedure to reproducibly generate embryoids that recapitulate development stages from early gastrulation to somitogenesis and onset of organogenesis [comparable with embryonic day 6.5 (E6.5) to E9.0] (reviewed by van den Brink and van Oudenaarden, 2021; Veenvliet et al., 2021). 3D gastruloids are formed by the aggregation of 100-300 mouse or human pluripotent embryonic stem cells (m or hESCs) that are treated with a 24 h pulse of the GSK3β-inhibitor CHIR to activate the canonical Wnt cascade. This signalling stimulus induces an anterior-posterior asymmetry in the aggregate (van den Brink et al., 2014), indicated by the one-sided expression of the Tbx factor brachyury (BRA; encoded by the gene from the T locus) that, in the embryo, marks the site of primitive streak formation at the onset of gastrulation (Rivera-Perez and Magnuson, 2005; reviewed by Arnold and Robertson, 2009). The brachyury-expressing posterior pole of 3D gastruloids elongates over several days, thereby forming different tissues resembling paraxial mesoderm, neural tube and primitive gut tube. 3D gastruloids show a remarkable self-organising capacity as the different tissues are generated in proper spatial organisation and arranged according to the embryonic axes (Beccari et al., 2018). 3D gastruloids thus can be used as model systems for studies of various gastrulation-associated morphogenetic processes, as exemplified for somitogenesis (van den Brink et al., 2020; Veenvliet et al., 2020). However, some tissues of gastrulation stage embryos are less well represented in 3D organoids, including anterior mesoderm derivatives, such as the cardiogenic mesoderm, and cranial structures, such as cranial neural tissues (van den Brink et al., 2020; Veenvliet et al., 2020). This under representation of anterior/cranial tissues most likely results from the initial induction of ESC aggregates with CHIR, which imposes a strong signal for tissue identities of the posterior/caudal primitive streak (Dunty et al., 2008). However, this and other aberrations of 3D gastruloids compared with embryos can also be used to investigate which additional regulatory requirements need to be met for proper morphogenesis of tissues and organ anlagen to more closely recapitulate the embryo (Veenvliet et al., 2021). Owing to the scalability of this ESC-derived embryoid culture system, multiple different environmental cues can be readily tested. It is thus expected that further refinements of current protocols will lead to the generation of gastruloids that more closely resemble the full spectrum of gastrulation and subsequent stages of embryogenesis.

Cell specification to mesoderm and definitive endoderm (DE) cell lineages is regulated by two Tbx transcription factors: eomesodermin (Eomes) and brachyury (T; hereafter referred to as Bra). The functions of both factors were previously extensively studied in mouse, establishing crucial roles for Eomes in cell lineage specification of definitive endoderm (DE) (Arnold et al., 2008; Teo et al., 2011) and anterior mesoderm (Costello et al., 2011), and functions of Bra for the generation of posterior mesoderm derivatives and notochord (Wymeersch et al., 2021). The compound genetic deletion of Eomes and Bra completely abrogates specification of any ME during differentiation of pluripotent cells (Tosic et al., 2019). Although the functions of these Tbx factors in specification of cell fate are well described, their roles in tissue-wide morphogenetic processes are less well defined. For example, the genetic deletion of Eomes in the epiblast abrogates formation of the mesoderm (and DE) cell layer, hindering studies of morphogenetic functions of Eomes-regulated programs (Arnold et al., 2008). Similarly, studies of the cell-intrinsic roles of Bra in posterior body axis extension are compromised by cell non-autonomous functions of Bra in feed-forward regulatory loops to maintain caudal Wnt signals that could independently act on cell specification and morphogenetic programs (Arnold et al., 2000; Dunty et al., 2008; Martin and Kimelman, 2008; Turner et al., 2014; Yamaguchi et al., 1999). These constraints in distinguishing the cell-autonomous effects of disturbed gene function from secondary effects, such as the general disturbance of morphogenesis, can be experimentally addressed by the generation of embryonic chimeras. Here, cell-autonomous gene functions can be studied when limited numbers of genetically altered cells are placed in an otherwise wild-type cellular environment. Chimeric approaches have previously been reported for studies of Bra functions, e.g. in zebrafish (Halpern et al., 1993) and mouse (Wilson et al., 1993, 1995; Wilson and Beddington, 1997), as well as for Eomes functions (Arnold et al., 2008), indicating regulatory roles of both Tbx factors in cell migration and morphogenesis. However, embryonic chimera analyses are highly labour and resource intensive, especially in mouse, and thus are only infrequently employed.

In this report, we demonstrate an approach to further increase the experimental potency of murine 3D gastruloids by expanding it to chimeric analyses. We use genetically engineered traceable mESCs to generate chimeric 3D gastruloids that are composed of cells with different genotypes. To showcase the advantages of this approach and to test experimental feasibility, we generated fluorescently labelled mESC lines with homozygous loss-of-function or inducible expression cassettes for the two Tbx transcription factors Bra and Eomes. These are used to generate chimeric gastruloids, e.g. by mixing defined ratios of inducible Tbx-expressing or Tbx-deficient and wild-type cells to generate chimeric situations with defined cell contribution that are normally difficult to achieve by conventional genetic tools or by cell injections into embryos. In addition to demonstrating the efficiency and feasibility of this experimental approach of chimeric gastruloids, this study also provides some previously unrecognised insights into the morphogenetic functions of Eomes and Bra.

To expand the experimental versatility of the 3D-gastruloid model system, we combined it with the use of genetically modified mESCs and fluorescent imaging. We generated chimeric gastruloids composed of cells with different genetic backgrounds that can be readily traced by different fluorescent membrane labels (Fig. 1A,B). Wild-type mESCs are permanently labelled by monoallelic knock-in of membrane GFP (mG) into the Rosa26 genomic locus (Fig. 1C), while membrane Tomato (mT) labelling was used for genetically manipulated mESCs lines (Fig. 1C). Genetic alterations used in presented experiments were the homozygous deletion of the Tbx transcription factor brachyury (Bra−/−) and the ICE-mediated (induced cassette exchange) targeted integration of Eomes 3′ to a monoallelic Tet-responsive element (TRE) in A2lox (E14-based) mESCs (Iacovino et al., 2014), resulting in TRE.EomesGFP (short TRE.Eo). These cell lines were first tested for their abilities to form regular gastruloids. This demonstrated that fluorescent membrane labels do not have an impact on gastruloid formation, while Bra−/− and doxycycline (DOX)-induced TRE.Eo cells show a failure to form regularly extended gastruloids at 120 h (Fig. S1). To generate chimeric gastruloids, we followed two approaches (Fig. 1A,D,E), either mixing of different cells during the aggregation of mESCs at the beginning of gastruloid culture (Fig. 1E) or merging of two preformed mESCs aggregates composed of different cells 24 h after the initial aggregation (Fig. 1B,D). Such merged ESC aggregates rapidly fuse and stably adhere after placing them together in 96-well plates (Fig. 1D, Movie 1) or in hanging drops (not shown).

Fig. 1.

Experimental approaches for the generation of chimeric gastruloids. (A) Schematic of two experimental approaches to generate chimeric gastruloids from fluorescently labelled embryonic stem cells (mESCs). Wild-type (WT) mESCs are marked by membrane-GFP (mG) and combined with membrane-Tomato (mT)-labelled genetically modified mESCs. The genetic modifications of mESCs comprise homozygous gene deletions (GeneX) or inducible gene-expression by the targeting of cDNAs into a fully controllable pre-engineered locus containing a tetracycline responsive element (TRE) for DOX-dependent induction of gene expression (GeneY). Chimeric gastruloids are generated by either mixing mESCs at the beginning of gastruloid culture or by merging preformed mESC aggregates preceding the induction of gastruloids by CHIR. Chimeric gastruloids can be used as versatile novel model system for various types of studies and embryonic research questions, as indicated. (B) Schematic of the protocol used to form chimeric gastruloids, indicating timepoints for either mixing of cells or merging of aggregates to create chimeric gastruloids. (C) Membrane-GFP (mG) labelling of wild-type mESCs and membrane-Tomato (mT) labelling for genetically modified mESCs allows different cell types in chimeric gastruloids to be distinguished. Scale bars: 10 µm. (D) Time-lapse imaging of the merging process of pre-formed ESC aggregates at indicated time points. 150 mG and mT mESCs were aggregated for 24 h before merging by placing them together in 96-well plates. After 1 h, two aggregates spontaneously aggregated and formed stable contacts. Scale bar: 100 µm. See Movie 1 for 12 h time-lapse footage. (E) Examples of mixed ESC aggregates 24 h after aggregation of 300 mESCs by mixing mG wild-type mESCs and mT Bra−/− mESCs at the indicated ratios. Scale bar: 100 µm.

Fig. 1.

Experimental approaches for the generation of chimeric gastruloids. (A) Schematic of two experimental approaches to generate chimeric gastruloids from fluorescently labelled embryonic stem cells (mESCs). Wild-type (WT) mESCs are marked by membrane-GFP (mG) and combined with membrane-Tomato (mT)-labelled genetically modified mESCs. The genetic modifications of mESCs comprise homozygous gene deletions (GeneX) or inducible gene-expression by the targeting of cDNAs into a fully controllable pre-engineered locus containing a tetracycline responsive element (TRE) for DOX-dependent induction of gene expression (GeneY). Chimeric gastruloids are generated by either mixing mESCs at the beginning of gastruloid culture or by merging preformed mESC aggregates preceding the induction of gastruloids by CHIR. Chimeric gastruloids can be used as versatile novel model system for various types of studies and embryonic research questions, as indicated. (B) Schematic of the protocol used to form chimeric gastruloids, indicating timepoints for either mixing of cells or merging of aggregates to create chimeric gastruloids. (C) Membrane-GFP (mG) labelling of wild-type mESCs and membrane-Tomato (mT) labelling for genetically modified mESCs allows different cell types in chimeric gastruloids to be distinguished. Scale bars: 10 µm. (D) Time-lapse imaging of the merging process of pre-formed ESC aggregates at indicated time points. 150 mG and mT mESCs were aggregated for 24 h before merging by placing them together in 96-well plates. After 1 h, two aggregates spontaneously aggregated and formed stable contacts. Scale bar: 100 µm. See Movie 1 for 12 h time-lapse footage. (E) Examples of mixed ESC aggregates 24 h after aggregation of 300 mESCs by mixing mG wild-type mESCs and mT Bra−/− mESCs at the indicated ratios. Scale bar: 100 µm.

The mixing of cells at different ratios can be used to evaluate cell-autonomous versus cell non-autonomous gene functions, by testing how different levels of cell contribution affect tissue behaviour, e.g. when using cells with loss of gene function. Merging of cell aggregates can be applied when the behaviour of coherent groups of cells is analysed, as in studies of inductive tissue interactions. A similar approach was recently reported where a small aggregate of 50 cells treated with BMP4 was used to induce organiser activities instead of CHIR treatment for gastruloid formation (Xu et al., 2021). Thus, chimeric gastruloids offer multiple experimental opportunities for studying gastrulation development (Fig. 1A) as demonstrated in following experiments.

First, we tested whether chimeric gastruloids are a suitable model for studying instructive gene functions during lineage specification. We therefore generated chimeric gastruloids by merging preformed aggregates of wild-type mESCs (mG-label) and mESCs containing the DOX-inducible EomesGFP expression cassette (TRE.Eo; mT-label) (Fig. 2A,B). Eomes is crucially required for lineage specification towards definitive endoderm (Arnold et al., 2008; Teo et al., 2011) and anterior mesoderm, including the cardiac lineage (Costello et al., 2011; Probst et al., 2021). We tested whether forced Eomes expression in a group of cells (TRE.Eo) within a gastruloid would be sufficient to induce a coherent heart-like domain, which forms only inconsistently in gastruloids composed of wild-type mESCs (Rossi et al., 2021; van den Brink and van Oudenaarden, 2021). Indeed, DOX-induced EomesGFP-expressing cells form a domain of beating cardiomyocytes (Fig. 2C, Movie 2) in chimeric gastruloids in 33.7±3.0% (mean±s.d.) of cases, whereas similar beating areas are only observed in 1.5±2.5% in uninduced chimeric gastruloids (Fig. 2D) or using only A2lox wild-type mESCs (not shown). To correlate induced EomesGFP expression with the cardiogenic domain, we performed in situ hybridisation analyses for early cardiac markers Mlc2a and Nkx2.5, which are expressed in mT-marked TRE.Eo cells that are forming a coherent domain on one side of gastruloids (Fig. 2E,F). To analyse the position of TRE.Eo cell-derived domains along the AP axis of merged chimeric gastruloids, we carried out immunostaining against BRA and CDX2, which indicate the posterior pole of extending gastruloids (Fig. 2G,H). The domains of TRE.Eo cells are predominantly found on one side along the AP axis of the gastruloids and oriented towards the anterior pole (Fig. 2G,H). Of note, these coherent domains of cells are not found in gastruloids when two preformed aggregates of wild-type cells are merged where cells undergo increased mixing (Fig. S2A). Interestingly, TRE.Eo cell containing gastruloids consistently showed a reduction in axis extension, so that the anterior-posterior (AP) axis of resulting gastruloids is less obvious in comparison with only wild-type-derived gastruloids (Fig. S2B,C). In short, these experiments show that induced expression of Eomes suffices to cell-autonomously generate coherent cardiogenic domains oriented to the anterior-lateral side of developing gastruloids in a highly increased proportion of gastruloids compared with uninduced gastruloids.

Fig. 2.

Instructive functions of Eomes for cardiac lineage specification in merged chimeric gastruloids. (A) Schematic illustrating the generation and culture of chimeric gastruloids by merging preformed aggregates of mG-labelled wild-type mESCs and mT-labelled mESCs harbouring EomesGFP in the doxycycline (DOX)-inducible gene locus (TRE.EomesGFP, short TRE.Eo). (B) Fluorescent microscopy of TRE.Eo mESCs showing nuclear expression of DOX-induced (+DOX) EOMESGFP after 24 h of administration that is absent in −DOX conditions. Scale bars: 10 µm. (C) Bright-field (left) and fluorescent (right) images of a chimeric gastruloid at 168 h after induced Eomes expression showing the region of beating cardiomyocytes within the gastruloid that is mostly derived from mT-labelled TRE.Eo cells. The dashed line indicates the domain of beating cells. See also Movie 2 for corresponding time-lapse footage. (D) A comparison of the percentage of beating gastruloids at 168 h in gastruloids with forced Eomes expression +DOX (33.7±3.0%) and uninduced controls −DOX (1.5±2.5%) in n=3 independent experiments that are colour-coded in orange, blue and purple. Data are mean±s.d. The P value for the differences between mean percentages of +DOX and -DOX beating gastruloids was calculated using a two-tailed, unpaired t-test (*P<0.0001). (E,F) Fluorescent microscopy (left) and whole-mount in situ hybridisation (right) of the same chimeric gastruloids after induced Eomes expression (+DOX) showing instructive functions of forced Eomes expression in TRE.Eo cells for the induction of cardiac progenitors, marked by expression of (E) Mlc2a (n=12) and (F) Nkx2.5 (n=10). (G,H) Fluorescent microscopy (left) and immunofluorescence staining (right) showing positioning of TRE.Eo cells at the opposite pole of posterior (G) BRA (n=24) and (H) CDX2 (n=10) immunostaining. Scale bars: 100 µm in C,E-H.

Fig. 2.

Instructive functions of Eomes for cardiac lineage specification in merged chimeric gastruloids. (A) Schematic illustrating the generation and culture of chimeric gastruloids by merging preformed aggregates of mG-labelled wild-type mESCs and mT-labelled mESCs harbouring EomesGFP in the doxycycline (DOX)-inducible gene locus (TRE.EomesGFP, short TRE.Eo). (B) Fluorescent microscopy of TRE.Eo mESCs showing nuclear expression of DOX-induced (+DOX) EOMESGFP after 24 h of administration that is absent in −DOX conditions. Scale bars: 10 µm. (C) Bright-field (left) and fluorescent (right) images of a chimeric gastruloid at 168 h after induced Eomes expression showing the region of beating cardiomyocytes within the gastruloid that is mostly derived from mT-labelled TRE.Eo cells. The dashed line indicates the domain of beating cells. See also Movie 2 for corresponding time-lapse footage. (D) A comparison of the percentage of beating gastruloids at 168 h in gastruloids with forced Eomes expression +DOX (33.7±3.0%) and uninduced controls −DOX (1.5±2.5%) in n=3 independent experiments that are colour-coded in orange, blue and purple. Data are mean±s.d. The P value for the differences between mean percentages of +DOX and -DOX beating gastruloids was calculated using a two-tailed, unpaired t-test (*P<0.0001). (E,F) Fluorescent microscopy (left) and whole-mount in situ hybridisation (right) of the same chimeric gastruloids after induced Eomes expression (+DOX) showing instructive functions of forced Eomes expression in TRE.Eo cells for the induction of cardiac progenitors, marked by expression of (E) Mlc2a (n=12) and (F) Nkx2.5 (n=10). (G,H) Fluorescent microscopy (left) and immunofluorescence staining (right) showing positioning of TRE.Eo cells at the opposite pole of posterior (G) BRA (n=24) and (H) CDX2 (n=10) immunostaining. Scale bars: 100 µm in C,E-H.

Next, we aimed to test the feasibility of using chimeric gastruloids for the assessment of cell-autonomous versus cell non-autonomous functions of Bra during posterior elongation, as the shortening of the posterior body axis is the most prominent phenotype of Bra-mutant embryos (Fig. 3A) (Wymeersch et al., 2021). Gastruloids entirely generated from mT-labelled Bra−/− mESCs present with a phenotype of impaired elongation, reminiscent of the failure of tailbud elongation observed in Bra-mutant embryos (Fig. 3B,E; Figs S1, S3B) (Inman and Downs, 2006). Increasing the proportional contribution of wild-type cells (mG) to the gastruloid by intermixing wild-type with Bra−/− mESCs during the initial aggregation of 300 mESCs leads to the gradual extension and increase in overall tissue mass of the posterior region of the mixed gastruloids (Fig. 3B). We analysed the presence of BRA in the wild-type cells in the posterior regions of chimeric gastruloids with high (80%) and equal (50%) contribution of Bra−/− cells at 24 h time intervals. The posterior region of chimeric gastruloids with an 80% contribution of Bra−/− cells shows less confined and reduced α-BRA staining (Fig. 3C) compared with chimeric gastruloids where Bra−/− cell contribution is 50%. Here, α-BRA staining is found robustly but with decreased intensity in the posterior pole when compared with wild-type gastruloids (Fig. 3D and Fig. S3A).

Fig. 3.

Brachyury functions during axis elongation in chimeric gastruloids generated by cell mixing with high Bra−/− cell contribution. (A) Schematic illustrating the generation of chimeric gastruloids by mixing mG-labelled wild-type mESCs and mT-labelled brachyury-deficient (Bra−/−) mESCs. (B) Chimeric gastruloids at 120 h generated by mixing Bra−/− (mT) and wild-type (mG) mESCs at indicated ratios of cell numbers. Gastruloids with a contribution of Bra−/− cells above 80% show reduced axial elongation. Gastruloids entirely generated from Bra−/− cells fail to axially extend beyond an oval shape. When the wild-type (mG) cell contribution exceeds 50%, axial elongation is similar to that in wild-type gastruloids but occasionally shows a thinning of the posterior region. Across all experiments, Bra−/− (mT) cells preferentially contribute to the posterior region of mixed gastruloids. Anterior is oriented towards the top and posterior towards the bottom in all images. (C,D) Immunofluorescence staining against brachyury (α-BRA) in gastruloids generated by cell mixing of Bra−/− and wild-type cells in ratios of 80:20 (C) and 50:50 (D) at indicated timepoints, showing less confined and reduced Bra expression in 80:20 chimeras at 96 and 120 h in comparison with posteriorly localised, robust Bra expression in 50:50 chimeras. Images are representatives of n=14 (72 h), n=9 (96 h) and n=14 (120 h) in C, and n=16 (72 h), n=25 (96 h) and n=18 (120 h) gastruloids in D. (E) Whole-mount in situ hybridisation for Wnt3a and Rspo3 of wild-type, Bra−/− and chimeric gastruloids of indicated cell ratios at 72 h, 96 h and 120 h showing mislocalised and reduced expression of Wnt3a and Rspo3 in Bra-deficient and chimeric wild-type:Bra−/− mixed gastruloids. The frequencies of representative staining patterns per number of examined gastruloids are indicated below each image. Scale bars: 100 µm in B-E.

Fig. 3.

Brachyury functions during axis elongation in chimeric gastruloids generated by cell mixing with high Bra−/− cell contribution. (A) Schematic illustrating the generation of chimeric gastruloids by mixing mG-labelled wild-type mESCs and mT-labelled brachyury-deficient (Bra−/−) mESCs. (B) Chimeric gastruloids at 120 h generated by mixing Bra−/− (mT) and wild-type (mG) mESCs at indicated ratios of cell numbers. Gastruloids with a contribution of Bra−/− cells above 80% show reduced axial elongation. Gastruloids entirely generated from Bra−/− cells fail to axially extend beyond an oval shape. When the wild-type (mG) cell contribution exceeds 50%, axial elongation is similar to that in wild-type gastruloids but occasionally shows a thinning of the posterior region. Across all experiments, Bra−/− (mT) cells preferentially contribute to the posterior region of mixed gastruloids. Anterior is oriented towards the top and posterior towards the bottom in all images. (C,D) Immunofluorescence staining against brachyury (α-BRA) in gastruloids generated by cell mixing of Bra−/− and wild-type cells in ratios of 80:20 (C) and 50:50 (D) at indicated timepoints, showing less confined and reduced Bra expression in 80:20 chimeras at 96 and 120 h in comparison with posteriorly localised, robust Bra expression in 50:50 chimeras. Images are representatives of n=14 (72 h), n=9 (96 h) and n=14 (120 h) in C, and n=16 (72 h), n=25 (96 h) and n=18 (120 h) gastruloids in D. (E) Whole-mount in situ hybridisation for Wnt3a and Rspo3 of wild-type, Bra−/− and chimeric gastruloids of indicated cell ratios at 72 h, 96 h and 120 h showing mislocalised and reduced expression of Wnt3a and Rspo3 in Bra-deficient and chimeric wild-type:Bra−/− mixed gastruloids. The frequencies of representative staining patterns per number of examined gastruloids are indicated below each image. Scale bars: 100 µm in B-E.

The absence of Bra expression from wild-type cells in gastruloids with a high contribution of Bra−/− cells suggests cell non-autonomous effects of Bra−/− cells. These are most likely explained by disturbances of the previously described feed-forward regulation of Wnt3a and Bra in the tailbud region (Arnold et al., 2000; Dunty et al., 2008; Martin and Kimelman, 2008; Turner et al., 2014; Yamaguchi et al., 1999). To test this, we performed in situ hybridisation to analyse expression of Wnt3a and of the putative Bra target gene and Wnt co-ligand Rspo3 (Koch et al., 2017) in wild-type gastruloids, in gastruloids entirely composed of Bra−/− cells and in mixed gastruloids with 80% and 50% Bra−/− cell contribution at 72, 96 and 120 h (Fig. 3E). This analysis demonstrates the requirement of BRA for the sustained expression of Wnt3a, which is absent from Bra−/− gastruloids at 96 h and greatly reduced and mislocalised in mixed gastruloids at 120 h. Interestingly, Wnt3a expression, which normally is confined to the posterior pole, is found at intermediate levels of mixed gastruloids, most likely resulting from unequal cell distribution of wild-type and Bra−/− cells, which accumulate at the posterior of mixed Bra−/− and wild-type gastruloids (see also Fig. 4 and discussion below). Analysis of Rspo3 expression recapitulates the lack of Bra expression in Bra−/− gastruloids and the prematurely exhausted BRA expression in Bra−/− and wild-type mixed gastruloids (Fig. 3C-E).

Fig. 4.

Brachyury functions in cell lineage specification and tissue sorting in chimeric gastruloids with low Bra−/− cell contribution. (A-D) Chimeric gastruloids generated by mixing of 10% Bra−/− (mT) and 90% wild-type (mG) ESCs at (A) 72 h and 96 h, and (B) three representative gastruloids at 120 h showing the spectrum of distribution of Bra−/− cells between the posterior pole and the midline of chimeric gastruloids. (C,D) Control chimeric gastruloids generated by mixing 10% wild-type (mT) and 90% wild-type (mG) ESCs at (C) 72 h and 96 h, and (D) three replicates at 120 h do not show distinct patterns of cell distribution. (E-G) Immunofluorescence staining against (E) FOXA2 (n=6) and (F) SOX2 (n=8) individually, and (G) double immunofluorescence staining showing the tissue contribution of Bra−/− cells to FOXA2+ endoderm and SOX2+ posterior cells (n=14). (H) Bra−/− cells contribute to the forming primitive gut tubes (arrows) and are positive for the epithelial marker CDH1 (E-cadherin) (n=3). (I) Bra−/− cells are mostly excluded from a mesodermal domain that is positive for FOXC2 (n=7). Scale bars: 100 µm.

Fig. 4.

Brachyury functions in cell lineage specification and tissue sorting in chimeric gastruloids with low Bra−/− cell contribution. (A-D) Chimeric gastruloids generated by mixing of 10% Bra−/− (mT) and 90% wild-type (mG) ESCs at (A) 72 h and 96 h, and (B) three representative gastruloids at 120 h showing the spectrum of distribution of Bra−/− cells between the posterior pole and the midline of chimeric gastruloids. (C,D) Control chimeric gastruloids generated by mixing 10% wild-type (mT) and 90% wild-type (mG) ESCs at (C) 72 h and 96 h, and (D) three replicates at 120 h do not show distinct patterns of cell distribution. (E-G) Immunofluorescence staining against (E) FOXA2 (n=6) and (F) SOX2 (n=8) individually, and (G) double immunofluorescence staining showing the tissue contribution of Bra−/− cells to FOXA2+ endoderm and SOX2+ posterior cells (n=14). (H) Bra−/− cells contribute to the forming primitive gut tubes (arrows) and are positive for the epithelial marker CDH1 (E-cadherin) (n=3). (I) Bra−/− cells are mostly excluded from a mesodermal domain that is positive for FOXC2 (n=7). Scale bars: 100 µm.

Next, in addition to the tissue-wide partially cell non-autonomous phenotype of Bra deficiency in gastruloids with a high contribution of Bra−/− cells, we analysed the cell-autonomous effects of Bra deficiency under conditions with a low contribution of mutant cells (Fig. 4). We generated chimeric gastruloids by mixing cells in a 10:90 ratio of Bra−/− and wild-type mESCs (Fig. 4A,B) and compared resulting gastruloids with controls where 10% mT-labelled wild-type cells were used (Fig. 4C,D). Further controls were included where cells were mixed at 50:50 ratios (Fig. S4A,B). Wild-type cells randomly disperse, while Bra−/− exhibit a tissue-sorting behaviour (Fig. 4A-D, Fig. S4A,B). At 120 h of culture, Bra−/− cells are predominantly found along the midline in the interior of the gastruloids and in the posterior pole (Fig. 4B). To determine tissue identity of these regions, we used immunofluorescence (IF) staining against FOXA2 and CDH1 (E-cadherin) to label the DE (Viotti et al., 2014), SOX2 to label neural tube progenitors (Wood and Episkopou, 1999), and FOXC2 to label mesoderm, and we combined IF staining with the fluorescent labelling of wild-type (mG) and Bra−/− (mT) cells (Fig. 4E-I). FOXC2 and CDH1 staining indicates that Bra−/− cells are biased towards the DE and favour the formation of a gut tube-like structure in the midline of gastruloids (Fig. 4E,G,H). This finding not only demonstrates that brachyury is dispensable for DE lineage specification from pluripotent cells, but suggests that brachyury actually counteracts DE specification programs and thus may not be a suitable marker for early DE-forming cells, as previously suggested (Kubo et al., 2004). In addition to their contribution to endoderm structures of chimeric gastruloids, Bra−/− cells are predominantly found in the posterior region of chimeric gastruloids. Here, Bra−/− cells show SOX2 staining, marking them as neural tube progenitors (Fig. 4F,G). Furthermore, Bra−/− cells are instead excluded from the FOXC2-positive mesoderm-forming domain (Fig. 4I), in accordance with previous findings about NE-repressive and mesoderm-promoting functions of Bra (Koch et al., 2017; Tosic et al., 2019).

In conclusion, this study illustrates the feasibility and experimental potency of using functional genetics in mESCs to generate chimeric 3D gastruloids. These allow the rapid and reproducible analysis of gain and loss of gene functions, which may reveal different phenotypic outcomes according to the degree of cellular contribution to chimeric gastruloids. Close attention should be paid to controlling the general variability in mESC morphogenetic and differentiation behaviour, which might not strictly depend on the genetic manipulation of each cell line. We did not experience these problems in the presented experiments, as they were all based on the use of mESCs lines originating from the same parental clone (A2lox; Iacovino et al., 2014).

Here, we used the inducible expression of the Tbx transcription factor Eomes to demonstrate how limitations of 3D gastruloids can be overcome by genetically providing regulatory cues that are missing or under-represented in CHIR-only treated gastruloids, i.e. the induced formation of anterior mesoderm derivatives including heart tissue. The analysis of Bra-deficient cells in chimeric gastruloids remarkably reflects aspects of previous studies of embryonic chimeras (Wilson et al., 1993, 1995; Wilson and Beddington, 1997). Here, Bra−/− cells progressively accumulate in the tailbud region, most likely as a result of BRA functions in the regulation of mesoderm cell migration, leading to passive replacement of mutant cells in the most posterior embryonic regions (Wilson et al., 1995; Wilson and Beddington, 1997). It thus will be interesting to follow morphogenetic cell movements of Bra−/− cells in chimeric gastruloids. In addition, the analysis of Bra-deficient cells in chimeras with low cell contribution showed a cell-autonomous bias towards the DE, a cell lineage that strictly depends on Eomes functions (Arnold et al., 2008; Teo et al., 2011). As Bra and Eomes are co-expressed in cells of the early primitive streak in early gastrulating embryos (Tosic et al., 2019), this raises the question of their reciprocal regulatory and functional interactions.

In summary, this study demonstrates that chimeric gastruloids represent a powerful experimental tool for the analysis of gastrulation stage embryogenesis. Gastruloids already exhibit a high degree of experimental accessibility, observability and scalability. The additional implementation of chimeric gastruloids allows the analysis of gene functions at the cellular level without affecting the general gastruloid architecture and might thus enhance our understanding of cell-signal interactions during gastrulation morphogenesis.

Cell lines

A2lox mESCs (Iacovino et al., 2014) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, 1× non-essential amino acids (NEAA), 1 mM sodium-pyruvate, 1× penicillin/streptomycin (all from Gibco), 100 µM β-mercaptoethanol (Sigma), leukaemia inhibitory factor (ESGRO LIF, Merck Millipore, 1000 U/ml) and 2i (two inhibitors): CHIR99021 (Axon Medchem, 3 µM) and PD0325901 (Axon Medchem, 1 µM) on 0.1% gelatine-coated dishes. The medium was changed daily and mESCs were passaged every other day. The generation of homozygous Bra−/− mESCs and of A2lox mESCs harbouring a monoallelic DOX-inducible expression cassette for EomesGFP have been described previously (Tosic et al., 2019). A2lox cells with membrane-tagged fluorescent labels (membrane-Tomato, mT; membrane-GFP, mG) were generated by targeted integration of a mT/mG targeting vector (Muzumdar et al., 2007; Addgene 17787) into the Rosa26 locus. 1×106 A2lox mESCs (wild type and Bra−/−) were transfected with 2.5 µg linearised vector using the Nucleofector ESC kit (Lonza) and G418 selected (350 µg/ml) on a monolayer of MitoC (Sigma) mitotically inactivated STO feeder cells. mT-expressing ESC clones were picked on day 9 of selection. To convert the expression of the membrane-Tomato (mT) to membrane-GFP (mG) in wild-type A2lox mESCs, cells were treated for 24 h with 5 µg/ml doxycyclin (DOX; Sigma, D9891) for induced expression of the Cre-recombinase from the DOX-inducible locus of A2lox wild-type cells to excise the loxP-flanked mT expression cassette and bring the mG expression cassette under the transcriptional control of the Rosa26 gene locus. After Cre-excision, mG-expressing wild-type A2lox mESCs underwent one round of clonal selection by minimal dilution of 500 cells onto a 10 cm cell culture dish.

Generation of chimeric gastruloids

Gastruloids were generated using published protocols (van den Brink et al., 2020) with some modifications, as outlined below. Gastruloid formation was performed in ESGRO Complete Basal Medium (Merck Millipore) in the absence of Matrigel. To generate chimeric gastruloids using different mESCs by merging preformed aggregates, 150 cells of each ESC line were aggregated in 40 µl of ESGRO basal medium in 96-well format (Greiner ultra-low attachment plates, 650970) for 24 h before merging by combining two aggregates into the same well. 48 h after first aggregate formation, fused gastruloids were induced by administration of 3 µM CHIR and, if indicated, with DOX (1 µg/µl, Sigma) for 24 h. In the course of gastruloid culture, the medium was changed daily at 72, 96, 120 and 144 h. For the generation of gastruloids by mixing of different ESC lines, aggregates were formed from a total of 300 mESCs at various ratios between the two ESC lines, and further gastruloid formation was followed using previously published protocols (van den Brink et al., 2020).

Whole-mount in situ hybridisation

Whole-mount in situ hybridisation was performed according to standard protocols using standard probes for Mlc2a, Nkx2.5, Wnt3a and Rspo3 (details can be requested from the authors). In brief, gastruloids were fixed in 4% PFA/PBS overnight at 4°C, dehydrated via a methanol series and stored in methanol at −20°C. After rehydration, gastruloids were bleached in 6% H2O2 for 5 min, digested with 1.6 μg/ml Proteinase K in PBT for 2 min, and postfixed in 4% PFA/0.2% glutaraldehyde for 20 min before pre-hybridisation for 2 h and hybridisation overnight according to standard protocols. DIG-labelled RNA probe was detected using anti-Digoxigenin-AP Fab fragments (Roche) in 1% sheep serum, 2% BBR in MAB [0.1 M maleic acid, 0.3 M NaCl, NaOH, 1% Tween-20 in H2O (pH 7.5)] and incubation at 4°C overnight. Antibody was washed out by extensive washes in MAB (>24 h, room temperature) and colour reaction performed in BM purple staining solution (Roche) for 2-6 h at room temperature.

Whole-mount immunofluorescence staining

Gastruloids were fixed in 4% PFA/PBS for 1 h at 4°C, permeabilised (0.3% Triton X-100/ PBT, 30 min) and blocked in 1% BSA/PBT for 1 h at room temperature. Primary antibody incubation was performed at 4°C overnight in 1% BSA/PBT, gastruloids washed four times in PBT before secondary fluorescence-conjugated antibody incubation for 3 h followed by DAPI staining for 30 min at room temperature. Primary antibodies used were anti-brachyury (R&D Systems; AF2085, 1:500), anti-Cdx2 (Biogenex; MU392A, 1:200), anti-Foxa2 (Cell Signaling; 8186S, 1:500), anti-E-cadherin (BD Transduction Laboratories; 610182, 1:500), anti-Sox2 (R&D Systems; AF2018, 1:500) and anti-Foxc2 (R&D Systems; AF6989, 1:200). Secondary anti-goat (A-21447), anti-rabbit (A-31573), anti-mouse (A32787) and anti-sheep (A-21448) Alexa Fluor 647-conjugated antibodies (all from Thermo Fisher) and anti-goat CF405 M (Biotium, 20398) were used at 1:1000.

Histological sections of gastruloids

Fixed gastruloids were processed through 15% and 30% sucrose/PBS, and incubated for at least 1 h in embedding medium (15% sucrose and 7.5% gelatin in PBS) before cryo-embedding. Embedded gastruloids were cut into 8 µm sections, mounted with ProLong Diamond Antifade Mountant (Life Technologies, P36970) and imaged as described below.

Imaging

Images were acquired on a Leica DMi8 Thunder Imager System or a Leica M165FC stereo microscope. Images were processed using the Leica LASX software and Affinity Photo. During time-lapse imaging, gastruloids were maintained under constant conditions at 37°C in 5% CO2.

Data acquisition and statistics

The percentage of beating gastruloids (Fig. 2D) was determined by manually counting during live imaging in three individual experiments. The following numbers of gastruloids were evaluated: 23 (+DOX) and 21 (−DOX) for n1; 61(+DOX) and 57 (−DOX) for n2; and 91 (+DOX) and 90 (−DOX) for n3.

The length of the anterior-posterior axis of gastruloids (Fig. 2J) was measured using LASX software (Leica) for n=34 (wild-type mG-wild-type mT) and n=36 (wild-type mG-TRE.EomT) gastruloids. Graphs (scatter plot; data are mean±s.d.) and column statistics were acquired with GraphPad Prism software (Version 5.04).

We thank T. Bass for excellent technical assistance and Michael Kyba for the A2lox ESC line.

Author contributions

Conceptualization: A.E.W., S.P., S.J.A.; Methodology: A.E.W., K.M.S., A.C., C.M.S., S.P., S.J.A.; Investigation: A.E.W., S.J.A.; Resources: A.E.W., K.M.S., A.C., C.M.S., S.P., S.J.A.; Writing - original draft: A.E.W., S.J.A.; Writing - review & editing: A.E.W., S.P., S.J.A.; Visualization: A.E.W., S.J.A.; Project administration: S.J.A.; Funding acquisition: S.J.A.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft through the Heisenberg Program (AR732/3-1, AR732/2-1, 24678173), Germany's Excellence Strategy (390939984), Sonderforschungsbereiche (89986987 and 431984000) to S.J.A., and by the Else-Kröner-Fresenius-Stiftung (MOTI-VATE program of the Freiburg Medical Faculty to A.C).

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Competing interests

The authors declare no competing or financial interests.

Supplementary information