Gastruloid

An example of a Gastruloid formed from Brachyury::GFP mouse ESCs,[1] treated with a pulse of the Wnt/β-Catenin agonist CHIR99021[2] between 48 and 72h and imaged by wide-field fluorescence microscopy at 120h. Notice the polarised expression of Brachyury::GFP (Bra) at the elongating tip of the Gastruloid. Image from van den Brink et al. (2014), used with CC-BY licence.

Gastruloids are three dimensional aggregates of embryonic stem cells (ESCs) that, under appropriate culture conditions develop an embryo-like organization with three orthogonal axes and a precise distribution of the primordia for multiple derivatives of the three germ layers in the absence of extraembryonic tissues. Significantly, they lack fore- mid- and hindbrain. They are a model system for the study of mammalian development and disease[3]. They are a model system for the study of mammalian development and disease[3][4][5][6][7][8].

Background

The Gastruloid model system draws its origins from work by Marikawa et al.[9][10]. In those studies, small numbers P19 embryonal carcinoma (EC) cells were aggregated as embryoid bodies (EBs) and used to investigate the processes involved in mesoderm formation.[9] The EBs were able to organise themselves into structures similar to the gastrulating embryo with polarised gene expression, axial elongation/organisation and up-regulation of posterior mesodermal markers. This was in stark contrast to work using EBs from mouse ESCs, where this level of organisation had not been reported, even though progenitors of axial populations can be generated in culture[11][12].

Following this study, the Martinez Arias laboratory in the Department of Genetics at the University of Cambridge demonstrated how aggregates of mouse ESCs were able to generate structures that exhibited collective behaviours with striking similarity to those during early development such as symmetry-breaking (in terms of gene expression), axial elongation and germ-layer specification[3][7]. To quote from the original paper: "Altogether, these observations further emphasize the similarity between the processes that we have uncovered here and the events in the embryo. The movements are related to those of cells in gastrulating embryos and for this reason we term these aggregates ‘gastruloids’". As noted by the authors of this protocol, a crucial difference between this culture method and previous work with mouse EBs was the use of small numbers of cells which may be important for generating the correct length scale for patterning.[3][4][13]

Brachyury (T/Bra), a gene which marks the primitive streak and the site of gastrulation, is up-regulated in the Gastruloids following a pulse of the Wnt/β-Catenin agonist CHIR99021[2] (Chi; other factors have also been tested[3]) and becomes regionalised to the elongating tip of the Gastruloid. From or near the region expressing T/Bra, cells expressing the mesodermal marker tbx6 are extruded from the similar to cells in the gastrulating embryo; it is for this reason that these structures are called Gastruloids[3].

Further studies revealed that the events that specify T/Bra expression in gastruloids mimic those in the embryo[7]. Modifications in the protocol[14] and extended culture show that after seven days gastruloids exhibit an organization very similar to a midgestation embryo with spatially organized primordia for all mesodermal (axial, paraxial, intermediate, cardiac, cranial and hematopoietic) and endodermal derivatives as well as the spinal cord[8]. They also implement Hox gene expression with the spatiotemporal coordinates as the embryo[8].

A feature of gastruloids is a disconnect between the transcriptional programs and outlines and the morphogenesis.

Further reading

Martinez Arias lab website article containing up-to-date information on the history, methods and current protocols on the culture of Gatruloids and their uses.

The term Gastruloid has been expanded to include self-organised human embryonic stem cell arrangements on patterned (micro patterns) that mimic early patterning events in development.[15][16]

References

  1. ↑ Fehling, Hans Jörg; Lacaud, Georges; Kubo, Atsushi; Kennedy, Marion; Robertson, Scott; Keller, Gordon; Kouskoff, Valerie (2003-09-01). "Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation". Development. 130 (17): 4217–4227. doi:10.1242/dev.00589. ISSN 0950-1991. PMID 12874139.
  2. 1 2 Ring, David B.; Johnson, Kirk W.; Henriksen, Erik J.; Nuss, John M.; Goff, Dane; Kinnick, Tyson R.; Ma, Sylvia T.; Reeder, John W.; Samuels, Isa (2003-03-01). "Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo". Diabetes. 52 (3): 588–595. doi:10.2337/diabetes.52.3.588. ISSN 0012-1797. PMID 12606497.
  3. 1 2 3 4 5 6 Brink, Susanne C. van den; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A.; Arias, Alfonso Martinez (2014-11-15). "Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells". Development. 141 (22): 4231–4242. doi:10.1242/dev.113001. ISSN 0950-1991. PMC 4302915. PMID 25371360.
  4. 1 2 Turner, David A.; Baillie-Johnson, Peter; Martinez Arias, Alfonso (2016-02-01). "Organoids and the genetically encoded self-assembly of embryonic stem cells". BioEssays. 38 (2): 181–191. doi:10.1002/bies.201500111. ISSN 1521-1878. PMC 4737349. PMID 26666846.
  5. ↑ Turner, David Andrew; Glodowski, Cherise R.; Luz, Alonso-Crisostomo; Baillie-Johnson, Peter; Hayward, Penny C.; Collignon, Jérôme; Gustavsen, Carsten; Serup, Palle; Schröter, Christian (2016-05-13). "Interactions between Nodal and Wnt signalling Drive Robust Symmetry Breaking and Axial Organisation in Gastruloids (Embryonic Organoids)". bioRxiv 051722.
  6. ↑ Turner, David; Alonso-Crisostomo, Luz; Girgin, Mehmet; Baillie-Johnson, Peter; Glodowski, Cherise R.; Hayward, Penelope C.; Collignon, Jérôme; Gustavsen, Carsten; Serup, Palle (2017-01-31). "Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling". bioRxiv 104539.
  7. 1 2 3 Turner, David A.; Girgin, Mehmet; Alonso-Crisostomo, Luz; Trivedi, Vikas; Baillie-Johnson, Peter; Glodowski, Cherise R.; Hayward, Penelope C.; Collignon, Jérôme; Gustavsen, Carsten (2017-11-01). "Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids". Development. 144 (21): 3894–3906. doi:10.1242/dev.150391. ISSN 0950-1991. PMC 5702072. PMID 28951435.
  8. 1 2 3 Beccari, Leonardo; Moris, Naomi; Girgin, Mehmet; Turner, David A.; Baillie-Johnson, Peter; Cossy, Anne-Catherine; Lutolf, Matthias P.; Duboule, Denis; Arias, Alfonso Martinez (October 2018). "Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids". Nature. 562 (7726): 272–276. doi:10.1038/s41586-018-0578-0. ISSN 0028-0836.
  9. 1 2 Marikawa, Yusuke; Tamashiro, Dana Ann A.; Fujita, Toko C.; Alarcón, Vernadeth B. (2009-02-01). "Aggregated P19 mouse embryonal carcinoma cells as a simple in vitro model to study the molecular regulations of mesoderm formation and axial elongation morphogenesis". genesis. 47 (2): 93–106. doi:10.1002/dvg.20473. ISSN 1526-968X. PMC 3419260. PMID 19115346.
  10. ↑ Li, Aileen S. W.; Marikawa, Yusuke (2015-12-01). "An in vitro gastrulation model recapitulates the morphogenetic impact of pharmacological inhibitors of developmental signaling pathways". Molecular Reproduction and Development. 82 (12): 1015–1036. doi:10.1002/mrd.22585. ISSN 1098-2795.
  11. ↑ Gadue, Paul; Huber, Tara L.; Paddison, Patrick J.; Keller, Gordon M. (2006-11-07). "Wnt and TGF-β signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells". Proceedings of the National Academy of Sciences. 103 (45): 16806–16811. doi:10.1073/pnas.0603916103. ISSN 0027-8424. PMC 1636536. PMID 17077151.
  12. ↑ Kouskoff, Valerie; Lacaud, Georges; Schwantz, Staci; Fehling, Hans Jöerg; Keller, Gordon (2005-09-13). "Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation". Proceedings of the National Academy of Sciences of the United States of America. 102 (37): 13170–13175. doi:10.1073/pnas.0501672102. ISSN 0027-8424. PMC 1201570. PMID 16141334.
  13. ↑ Baillie-Johnson, Peter; Brink, Susanne Carina van den; Balayo, Tina; Turner, David Andrew; Arias, Alfonso Martinez (2015). "Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro". Journal of Visualized Experiments (105). doi:10.3791/53252. PMC 4692741. PMID 26650833.
  14. ↑ Girgin, Mehmet; Turner, David Andrew; Baillie-Johnson, Peter; Cossy, Anne-Catherine; Beccari, Leonardo; Moris, Naomi; Lutolf, Matthias; Duboule, Denis; Martinez Arias, Alfonso (2018-10-12). "Generating Gastruloids from Mouse Embryonic Stem Cells". Protocol Exchange. doi:10.1038/protex.2018.094. ISSN 2043-0116.
  15. ↑ Etoc, Fred; Metzger, Jakob; Ruzo, Albert; Kirst, Christoph; Yoney, Anna; Ozair, M. Zeeshan; Brivanlou, Ali H.; Siggia, Eric D. (2016). "A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization". Developmental Cell. 39 (3): 302–315. doi:10.1016/j.devcel.2016.09.016. PMC 5113147. PMID 27746044.
  16. ↑ Warmflash, Aryeh; Sorre, Benoit; Etoc, Fred; Siggia, Eric D; Brivanlou, Ali H (2014). "A method to recapitulate early embryonic spatial patterning in human embryonic stem cells". Nature Methods. 11 (8): 847–854. doi:10.1038/nmeth.3016. PMC 4341966. PMID 24973948.
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