A vertebrate embryo undergoes gastrulation, wherein it reorganises into a structure with three germ layers and three distinct body axes. Here, Vikas Trivedi and colleagues investigate spatial patterning of germ layers in the absence of external cues. To find out more about their work, we spoke to the first author, Kerim Anlaş, and the corresponding author, Vikas Trivedi, Research Group Leader and Co-Chair of Theory Transversal Theme at European Molecular Biology Laboratory (EMBL), Barcelona, Spain.
Kerim Anlas (left) and Vikas Trivedi (right)
Vikas, what questions are your lab trying to answer?
VT: Our long-term research vision is to decipher the complexity in biology and generate an understanding that can be harnessed for regenerative medicine and translational research. What makes this challenge so difficult, yet fascinating, is the unique property of biological tissues to combine chemistry, materials science and thermodynamics. A central theme in the lab is to decipher how a collection of cells (e.g. an embryo) generates a system of body axes to organize patterns reproducibly and robustly. Our unique approach to this recurrent theme in biology is to integrate the traditionally segregated fields of cell signalling, tissue mechanics and metabolism with comparative study of embryos and embryonic organoids across species.
Kerim, how did you come to work in the lab and what drives your research today?
KA: I first met Vikas when he had just obtained a group leader position at EMBL Barcelona, but the lab was not yet set up at that time. I would say we got along very well from the beginning and I ended up becoming his first PhD student, since I was also very excited about his research vision for the group. Beyond the science itself, it was special (and very instructive) to experience the lab grow and develop so much over the years. I am thankful and appreciated Vikas' mentorship a lot – as well as my time there in general. These days, I am aiming to explore evolutionary questions related to the conservation of anteroposterior polarisation mechanisms, for which, I believe, we contributed to laying the groundwork in this research article by studying the earliest patterning processes in our model system of choice (gastruloids).
Tell us about the background of the field that inspired your work
KA & VT: The idea of taking embryonic stem cell (ESC)-like populations outside their native embryonic context and (re-)aggregating them in vitro is not new, as this has been performed in starfish and Hydra about 30-40 years ago. More recently though, there has been a renewed focus on mammalian ESC-based embryo-like structures, particularly in the context of the organoid and embryoid body field(s). Therefore, we believe there is now an increased appreciation of insights from such in vitro systems, in the sense that these can be used to uncover developmental principles that may be more difficult or perhaps even impossible to study in the native embryo. A natural question to ask is to what extent can the cells achieve robust fate-decisions in the absence of external cues (morphogens) typically present in an embryo? This was our motivation to use mouse gastruloids to study the true self-organizing capacity of mouse ESCs during anterior-posterior (AP) axis development in the absence of external Wnt stimulation. This also gave us an opportunity to ask whether cells achieve similar cell fates in vivo and in vitro in the same way or if they can access the same fate via different developmental trajectories in vivo and in vitro.
Can you give us the key results of the paper in a paragraph?
KA & VT: When it comes to the establishment of an AP axis in gastruloids, autonomous polarization of the transcription factor brachyury (or T) at the posterior end has been interpreted to be the first tissue-level symmetry-breaking event. However, the initial mouse ESC culture conditions vary across labs that grow cells either in 2i or serum-LIF (ESL) conditions. In the latter case, spontaneous polarization of T/Bra has been observed without external Wnt stimulation. We wanted to investigate this self-organizing process and the extent of concomitant tissue patterning of other cell types before external Wnt stimulation. Our studies show that this process is robust to modifications in aggregate size. Furthermore, we were also curious about whether the emergence of in vivo-like meso- and endodermal cell types in gastruloids mimics the process in the embryo or whether this can be achieved via different developmental trajectories. We found that gastruloids develop (transcriptionally) similar mesendodermal cell types, despite initial differences in their primed pluripotent populations, which adopt a more mesenchymal state in lieu of an epiblast-like transcriptome. Such ‘alternative developmental modes’ (Anlas and Trivedi, 2021) have been observed, for example, in re-aggregated Nematostella gastrulae (Kirillova et al., 2018), and we hope that this work will make us all think about our strict notions of developmental trajectories, particularly in mammalian development.
Kerim, when doing the research, did you have any particular result or eureka moment that has stuck with you?
KA: When Nicola, the other first author of the paper to whom I would like to give immense credit and express my appreciation, first performed integrations or alignments of our gastruloid early development single cell atlas with a corresponding mouse embryo dataset, we observed that some of the populations did not overlap well. This was a bit unexpected and particularly exciting, implanting and/or reaffirming the idea of alternative (transcriptional) developmental trajectories between the native embryo and the in vitro system.
Representative gastruloids at 72 h post aggregation, generated from 50 (top), 100 (middle) or 300 (bottom) initial mouse embryonic stem cells. Samples were stained (hybridisation chain reaction in situ) for the key germ layer and anteroposterior axial markers Bra/T (green), Eomes (yellow), Aldh1a2 (cyan), Sox2 (magenta) and Foxa2 (white). Image courtesy of Laura Salamó Palau.
Representative gastruloids at 72 h post aggregation, generated from 50 (top), 100 (middle) or 300 (bottom) initial mouse embryonic stem cells. Samples were stained (hybridisation chain reaction in situ) for the key germ layer and anteroposterior axial markers Bra/T (green), Eomes (yellow), Aldh1a2 (cyan), Sox2 (magenta) and Foxa2 (white). Image courtesy of Laura Salamó Palau.
Kerim, and what about the flipside: any moments of frustration or despair?
KA: Now, I cannot think of a big, singularly frustrating moment, although there have certainly been slumps, experiments not working out and periods of lack of clear perspective (but I want to highlight here that Vikas has been an excellent supervisor!). However, I believe that this is somewhat normal for the research process (at least in my experience) – though we should certainly not normalise suffering or prolonged states of despair in the scientific community! In the case of this paper, I felt that it was particularly difficult for me to find the right structure, to choose what to include and what to omit in order to best convey and highlight our key findings.
Vikas, why did you choose to submit this paper to Development?
VT: The core work of our paper is about understanding the dynamics of symmetry breaking in embryonic organoids (gastruloids) and at the earliest stages of development, i.e. how do gastruloids, which are made of mouse ESCs, compare to the mouse embryo? Development was thus the natural choice. We are hoping that the groundwork we have done can be taken up by the community that makes up the readership of Development.
Kerim, what is next for you after this paper?
KA: As alluded to above, I would like to address questions related to the degree of evolutionary conservation of early embryonic patterning mechanisms that we can observe in mouse gastruloids, perhaps by focusing on less established model species, particularly early branching metazoans.
We are hoping that the groundwork we have done can be taken up by the community that makes up the readership of Development
Vikas, where will this story take your lab next?
VT: This work has allowed us to understand the early patterning of gastruloids in the absence of any external stimulus. This is quite powerful because now we can perform experiments where we can understand cellular interactions (chemical, mechanical and metabolic) without being confounded by external factors. We have a manuscript (soon to be out) that investigates this inherent signalling dynamics within ESC aggregates, linking it with non-invasive measurements of tissue rheology based on the methodological framework that we developed in a previous work (Oriola et al., 2022). The early patterning is also allowing us to investigate the metabolic control of germ layer proportions through regulation of signalling (Stapornwongkul et al., 2023). We are looking into how the initial culture conditions of the cells influences later differentiation trajectories, something that we have also touched upon in the current work. Beyond addressing fundamental questions, we are also interested in technology and methodology development that can allow us to ask questions that we couldn't have asked before. In this context, we have benefitted from ARISE program and we are keen to do more in that direction. We are working with three other labs within our ERC Synergy consortium called BREAKDANCE to investigate questions related to control mechanisms and robustness of multicellular symmetry breaking (https://hpscreg.eu/browse/project/1544). So, in a nutshell, as you can see this work has raised many questions and possibilities that we are deeply interested to pursue, and we are always looking out for talented researchers who want to join hands!
Kerim, finally, let's move outside the lab – what do you like to do in your spare time?
KA: I really like basketball, both following the NBA and playing myself. Apart from that, I try to keep up with global politics and to educate myself on socio-political and economic theories.
K.A. & V.T.: 1EMBL Barcelona, 08003 Barcelona, Spain.
V.T.: EMBL, Heidelberg, Developmental Biology Unit, 69117 Heidelberg, Germany.
E-mail: [email protected]
