Brachyury expression levels predict lineage potential and axis-forming ability of in vitro derived neuromesodermal progenitors
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Abstract
Neuromesodermal progenitors (NMPs) produce the spinal cord and musculoskeleton in the elongating anterior-posterior axis. In vivo , NMPs possess dual potency, coinciding with regions coexpressing SOX2 and Brachyury (TBXT). In vitro , SOX2/TBXT co-expressing cells can be produced from pluripotent cells and, like their in vivo counterparts, can produce neural tube and somitic mesoderm. However, the functional characteristics of in vitro SOX2/TBXT co-expressing cells remain unclear, confounding comparisons with in vivo data. To address this, we developed a dual Sox2/Tbxt reporter mouse ESC line. SOX2/TBXT reporter-positive cells emerge in vitro from pluripotent populations with dynamics that mirror their appearance in the embryo. Purified SOX2/TBXT co-expressing populations can differentiate towards neurectoderm or mesoderm, including lateral mesoderm upon BMP stimulation. In gastruloids, quantitative live imaging shows that WNT or NOTCH inhibition rapidly leads to downregulation of TBXT expression and diminished axial extension. We show that clonally plated SOX2/TBXT co-expressing cells are bipotent NMPs that can also self-propagate. By combining clonal analysis with mathematical modelling, we identify two thresholds of SOX2/TBXT expression, switching clonal output from neural- to mesoderm-biased, and from mesoderm-biased to mesoderm-specified. Media and substrate composition alter the lineage outcomes of in vitro derived mouse NMPs. Thus, this Sox2/Tbxt double reporter cell line provides support for unsuspected heterogeneity in NMPs, together with evidence for a role of these transcription factors in directing cell fate to drive axis elongation.
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Referee #3
Evidence, reproducibility and clarity
Summary
The authors report the construction and validation of a novel SOX2-TBXT dual-reporter mouse embryonic stem cell (mESC) line, a tool enabling the simultaneous, live visualization of SOX2 and TBXT expression. Using this line, they established an in vitro differentiation system that generates populations of SOX2/TBXT co-expressing cells which mimic neuromesodermal progenitors (NMPs). The authors combined clonal analysis to assess the fate potential of these cells and applied gastruloid models to dissect the functional consequences of gene expression dynamics on axis elongation. The central finding is that the level of TBXT …
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Referee #3
Evidence, reproducibility and clarity
Summary
The authors report the construction and validation of a novel SOX2-TBXT dual-reporter mouse embryonic stem cell (mESC) line, a tool enabling the simultaneous, live visualization of SOX2 and TBXT expression. Using this line, they established an in vitro differentiation system that generates populations of SOX2/TBXT co-expressing cells which mimic neuromesodermal progenitors (NMPs). The authors combined clonal analysis to assess the fate potential of these cells and applied gastruloid models to dissect the functional consequences of gene expression dynamics on axis elongation. The central finding is that the level of TBXT expression predicts and directs lineage potential. This work identifies specific expression thresholds that bias progenitors toward a mesodermal fate.
Major comments
The study's conclusions would be substantially strengthened by more direct validation in an embryonic context. Could the authors quantify endogenous SOX2 and TBXT protein levels of the NMPs in the mouse embryos with immunofluorescence? This would test whether a similar heterogeneity in TBXT expression exists in vivo and whether it correlates with the cells' spatial position.
The role of SOX2 in the quantitative model seems underdeveloped. To justify the authors' claim, could the authors analyze their existing imaging data in more detail to disentangle if the absolute TBXT level is essential rather than the ratio of Sox2 to Tbxt is the driving factor for determining NMP fate?
It seems that SOX2 expression also appears heterogeneous from both in vitro differentiation and gastruloid models. Quantifications, and a discussion of this heterogeneity and whether it influences the fate-decision process would be helpful.
To support the authors' claim that the reporter line recapitulates endogenous protein expression in vivo (lines 121-128), please include a control immunostaining of wild-type embryo for SOX2 and TBXT to compare expression patterns side-by-side with those embryos shown in Figure 1B, Suppl. Fig. 1C, D. To substantiate the claims regarding cellular expression patterns within the embryo (line 125-128), the use of higher-resolution imaging, such as confocal microscopy, is recommended.
The differentiation trajectory described culminates in a double-negative (Sox2-mCherry-negative, Tbxt-GFP-negative) population (lines 246-247). To provide a more complete picture of this fate progression, could the authors perform qPCR for relevant lineage markers to validate the molecular identity of this terminal population?
Minor comments
Scale bars for micrographs are missing in Figure 1B, Suppl. Fig. 1C, and D. The claims regarding the dynamics of TBXT and SOX2 expression in gastruloids following WNT/NOTCH inhibition (Figure 4B, 4D, 4E) would be more compelling if the authors include supplementary videos of the time-lapse imaging.
In lines 322-324, the authors conclude that Tbxt-cells are the driving cells. Please elaborate on the interpretation that this is a cell-autonomous effect driven by TBXT levels. The observation that Sox2 levels increase ~10-15 hours after WNT/NOTCH inhibition is interesting (Figure 4D, 4E). Could the authors discuss this upregulation?
Significance
General Assessment
In the development of a novel dual Sox2/Tbxt reporter cell line, which provides a powerful tool for quantitatively understanding the dynamics of cell fate specification during gastrulation and potentially in other developmental contexts. However, a key limitation is the study's primary focus on in vitro models. The findings will require further validation in an in vivo context.
Advance
This study provides a technical advance that provides a new resource available to the field for stem cell and developmental biology.
Audience
This paper will primarily interest a specialized audience, particularly developmental and stem cell biologists who study the fundamental mechanisms of embryogenesis, cell fate specification, and axis elongation.
Field of expertise
Stem cell biology and developmental biology. I do not have the expertise to evaluate their mathematical modeling.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors generate a novel reporter mouse ESC line to track SOX2 and TBXT dynamics in neuromesodermal progenitors. The authors leverage multiple systems (in vitro differentiation, chimeric embryos and gastruloids) to address how SOX2/TBXT levels impact the contribution of neuromesodermal progenitors towards neural versus mesodermal fates. They show that the levels of TBXT can predict differentiation outcomes, whereby certain thresholds of TBXT influence the differentiation towards a mesodermal identity. In gastruloids, perturbing either WNT or NOTCH signalling coincides with diminished axial …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors generate a novel reporter mouse ESC line to track SOX2 and TBXT dynamics in neuromesodermal progenitors. The authors leverage multiple systems (in vitro differentiation, chimeric embryos and gastruloids) to address how SOX2/TBXT levels impact the contribution of neuromesodermal progenitors towards neural versus mesodermal fates. They show that the levels of TBXT can predict differentiation outcomes, whereby certain thresholds of TBXT influence the differentiation towards a mesodermal identity. In gastruloids, perturbing either WNT or NOTCH signalling coincides with diminished axial elongation. In vitro, the bias mediated by TBXT is also somewhat influenced by the substrate.
Major comments:
There are some key items to clarify that are important to resolve the interpretation of the results and clarify the main advances for a broader readership.
With respect to the mESC differentiation into NMPs (using an established protocol from Gouti et al 2014), after Day 3, cells are cultured in conditions where they are exposed to both CHIR and FGF for a further two days. As these extended CHIR/FGF conditions don't appear to be characterised by Gouti et al., 2014, what proportions and progenitors are generated in the dish under these conditions? The loss of SOX2 by day 5 suggests mesodermal progenitors are the main derivatives but further characterization (eg Meox1/Msgn1) would be needed to verify this claim.
Further validation of the generated derivatives would also be useful in the re-plating experiments (Figure 3) to test whether the double negative cells are transitioning to a mesodermal (eg Meox1/Msgn1) or neural derivative (eg Sox1/Pax6). Similarly, at day 5 and 6 of the differentiation, there appears to be a loss of Sox2 expression in some of the replated cells from the Sox2-positive population (see Figure 3D). Could the authors please clarify whether the double negative cells represent neural progenitors, and/or alternative cell types? Do the replated cells transiently adopt Tbxt? This would be possible by staining with neural (SOX1), or (pre)somitic mesoderm genes (MSGN1,MEOX1) or adjusting the text to reflect the uncertainty.
At line 172 "The cells posterior to the node expressed only TBXT." Do these cells have low SOX2 expression that is hard to detect? Are these TBXT-positive cells derived from the primitive streak? Would staining with the primitive streak marker TBX6 enable visualization of these distinct cell types, and/or could the authors please label the figure in more detail.
Could the authors please comment on the design of the reporter system in a bit more detail? For example, please clarify the necessity to generate a TBXT reporter that includes a H2B-GFP, unlike Sox2, which does not include a H2B. Can the authors distinguish between an increase in the threshold of TBXT, versus an increase detected due to the stability of the H2B-GFP? The low versus high TBXT cells may reflect early versus late TBXT expressing cells. Are the changes in TBXT expression (eg Supplementary Figure 2) significantly changed between the low versus high GFP populations? Additionally, why is the level of GFP similar across low and high GFP populations in Sup Fig 2? Could the in vivo data be used to quantify differences in TBXT (similar to what has been shown previously in Ivanovitch et al., 2021 PMID: 33999917)?
Optional
- Altering extrinsic cues (such as RA/CHIR) could clarify how reversible the high TBXT state is as cells progress towards mesoderm. Can you redirect the TBXT-high cells to a neural fate or are they already irreversibly committed?
- Line 283: Flk1 is also expressed in TBXT-low cells. It would be interesting to test whether the TBXT-low cells and the SOX2neg/TBXTpos can generate lateral plate mesoderm or whether this competence to generate lateral plate mesoderm is limited to TBXT-high cells.
- Line 324: The lack of elongation in gastruloids following the inhibition of WNT/NOTCH is clear. Do the authors expect that the reintroduction of TBXT would rescue axis elongation in the WNT/NOTCH inhibited gastruloids?
Minor comments:
Figure 3B - The SOX2+/TBXT- population only shows a moderate level of SOX1 by RT-qPCR. Using pre-neural markers (e.g. NKX1-2) might be helpful here to show progression towards a neural progenitor identity.
Line 310 - Can you comment on the general efficiency in generating elongating gastruloids compared to WT cells and/or previous literature?
Line 236 - Add "at constant SOX2 levels" (when comparing orange and yellow populations)
Figure 5
Fig5C and E take time to understand. Potentially expanding the figure legend slightly could be helpful to the reader.
Supplementary Figure 2
Line 864 - refer to Fig3A
Line 350 - The link with testing the different substrates is a bit abrupt. Please can you make it clearer by modifying the text to explain the hypothesis being tested here.
Line 363 - Could you comment in relation to what happens in the embryo to future mesoderm progenitors that seem to have a more motile phenotype compared to neural progenitors (eg Romanos et al 2021 PMID: 34607629)?
Significance
In this work, the authors have explored how the dynamics of SOX2/TBXT impact the decision process of neuromesodermal progenitors (NMPs). They have engineered and validated a novel dual fluorescent reporter ESC line to track SOX2 and TBXT. The work combines in vitro, in vivo and modelling approaches to understand how NMPs make decisions - a highly relevant and important question for multiple fields spanning developmental and quantitative biology, and the engineering of cell types in vitro from pluripotent cells. The authors propose a critically important finding: that discrete thresholds of TBXT influence the outcome of NMPs. However, further clarification is required to solidify these claims (discussed above).
The data generated from this study also suggests that in contrast to Tbxt, the level of Sox2 does not appear to impact the NMP fate decision. While these are interesting and important findings, it is not entirely clear in this version of the manuscript how these advances relate with previous studies that have highlighted critical roles mediated by Sox2 and its level of expression (including the work of Koch et al 2017 - PMID: 28826820 and Blassberg et al PMID: 35550614). We expect that a broader discussion will in turn broaden the general interest and value of the work to a wider readership.
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Referee #1
Evidence, reproducibility and clarity
The work of Binagui-Casas, Granes and colleagues, investigates in a rigorous way the origin and potential of murine NMPs. The authors first validated a dual reporter system, which monitors Sox2 and Tbxt expression. Next, they identified the location in the embryo and the sequence of gene activation (Sox2 first, then Tbxt) leading to NMPs specification. Importantly, the in vitro model faithfully mimics the in vivo ontogeny of NMPs. Among Sox2+ NMCs, the authors observed different levels of Tbxt, which they proposed mark different stages of mesoderm maturation, with high Tbxt corresponding to more mature mesodermal state. Through …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
The work of Binagui-Casas, Granes and colleagues, investigates in a rigorous way the origin and potential of murine NMPs. The authors first validated a dual reporter system, which monitors Sox2 and Tbxt expression. Next, they identified the location in the embryo and the sequence of gene activation (Sox2 first, then Tbxt) leading to NMPs specification. Importantly, the in vitro model faithfully mimics the in vivo ontogeny of NMPs. Among Sox2+ NMCs, the authors observed different levels of Tbxt, which they proposed mark different stages of mesoderm maturation, with high Tbxt corresponding to more mature mesodermal state. Through sorting and replating of different populations, the authors convincingly showed that Sox2+/Tbxt-low cells are still bipotent, while Sox2+/Tbxt-high are committed towards the mesoderm lineage. Using a gastruloid model, the authors then showed that Tbxt expression correlates with axis elongation, as both are reduced upon inhibition of either WNT or NOTCH Finally, single-cell sorting followed by differentiation in FGF/CHIR and high throughput microscopy confirmed that double Sox2/Tbxt positive cells behave as NMPs and that high levels of Tbxt predispose cells towards mesoderm differentiation. These conclusions were further supported by mathematical modelling. The manuscript is easy to read, and figures are very clear. I have only minor suggestions, as I find the manuscript quite solid and complete.
Minor points:
- The reporter system is based on stable fluorescent proteins, whose half-life is generally much longer than the endogenous proteins. This could generate a discrepancy between expression of reporters and the endogenous proteins. I find this relevant for Tbxt, as it is very clear that Tbxt reporter levels dictates the differentiation propensity, but I wonder whether, Tbxt low cells actually express TBXT protein or not. It might be the case that only a fraction of Tbxt low cells actually express TBXT protein, or none. It would enough to sort the populations showed in Figure 3A and perform immunostaining for endogenous SOX2 and TBXT. This could reveal even better correlations between their levels and cell behaviour.
- In the experiments in which IWP2 and LY411575 are used, I would suggest to asses cell viability, as the two inhibitors could induce toxicity. Staining for cleaved caspase-3 or a TUNEL assay would be enough. It would also be important to confirm that IWP2 blocks WNT signalling (by looking at WNT target genes or staining for active beta-cateinin) and that LY411575 blocks NOTCH signalling.
- I would define in the figure legends what the black line in figure 5E represents.
Referees cross-commenting
I agree with Reviewer #2 comment about "Further validation of the generated derivatives" by staining for additional markers.
I also made a comment related to GFP stability, as Reviewer #2 did (i.e. The low versus high TBXT cells may reflect early versus late TBXT expressing cells ).
Significance
Overall, the manuscript uses an elegant approach and address an important question about NMPs behaviour. The results presented are an important advance in knowledge of NMP biology. I am confident that both stem cell and developmental biologist would be interested in this manuscript. I am an expert of pluripotency, signaling and models of early mammalian development.
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