Endogenous FGFs drive ERK-dependent cell fate patterning in 2D human gastruloids

Reviewer #1 (Public review):

Summary:

This is an interesting study on the role of FGF signaling in the induction of primitive streak-like cells (PS-LC) in human 2D-gastruloids. The authors use a previously characterized standard culture that generates a ring of PS-LCs (TBXT+) and correlate this with pERK staining. A requirement for FGF signaling in TBXT induction is demonstrated via pharmacological inhibition of MEK and FGFR activity. A second set of culture conditions (with no exogenous FGFs) suggests that endogenous FGFs are required for pERK and TBXT induction. The authors then characterize, via scRNA-seq, various components of the FGF pathway (genes for ligands, receptors, ERK regulators, and HSPG regulation). They go on to characterize the pFGFR1, receptor isoforms, and polarized localization of this receptor. Finally, they perform FGF4 inhibition and use a cell line with a limited FGF17 inactivation (heterozygous null) and show that loss of these FGFs reduces PS-LC and derivative cell types.

Strengths:

(1) As the authors point out, the role of FGF signaling in gastrulation is less well understood than other signaling pathways. Hence this is a valuable contribution to that field.

(2) The FGF4 and FGF17 loss-of-function experiments in Figure 5 are very intriguing. This is especially so given the intriguing observation that these FGFs appear to be dominating in this model of human gastrulation, in contrast to what FGFs dominate in mice, chicks, and frogs.

(3) In general this paper is valuable as a further development of the Human gastruloid system and the role of FGF signaling in the induction of PS-CLs. The wide net that the authors cast in characterizing the FGF ligand gene, receptor isoforms, and downstream components provides a foundation for future work. As the authors write near the beginning of the Discussion "Many questions remain."

Weaknesses:

(1) FGFs are cell survival factors in various aspects of development. The authors fail to address cell death due to loss of FGF signaling in their experiments. For example, in Figure 1E (which requires statistical analysis) and 1G (the bottom FGFRi row), there appears to be a significant amount of cell loss. Is this due to cell death? The authors should address the question of whether the role of FGF/ERK signaling is to keep the cells alive.

(2) Regarding the sparse cells in 1G, is there a reduction in cell number only with FGFRi and not MEKi? Is this reproducible? Gattiglio et al (Development, 2023, PMID: 37530863) present data supporting a "community effect" in the FGF-induced mesoderm differentiation of mouse embryonic stem cells. Could a community effect be at play in this human system (especially given the images in the bottom row of 1G)? If the authors don't address this experimentally they should at least address the ideas in Gattoglio et al.

(3) Do the FGF4 and FGF17 LOF experiments in Figure 5 affect cell numbers like FGFRi in Figure 1? Why examine PS-LC induction only in FGF17 heterozygous cells and not homozygous FGF17 nulls?

(4) The idea that FGF8 plays a dominant role during gastrulation of other species but not humans is so intriguing it warrants deeper testing. The authors dismiss FGF8 because its mRNA "...levels always remained low." (line 363) as well as the data published in Zhai et al (PMID: 36517595) and Tyser et al (PMID: 34789876). But there are cases in mouse development where a gene was expressed at levels so low, that it might be dismissed, and yet LOF experiments revealed it played a role or even was required in a developmental process. The authors should consider FGF8 inhibition or inactivation to explore its potential role, despite its low levels of expression.

(5) Redundancy is a common feature in FGF genetics. What is the effect of inhibiting FGF4 in FGF17 LOF cells?

(6) I suggest stating that the authors take more caution in describing FGF gradients. For example, in one Results heading they write "Endogenous FGF4 and FGF17 gradients underly the ERK activity pattern.", implying an FGF protein gradient. However, they only present data for FGF mRNA , not protein. This issue would be clarified if they used proper nomenclature for gene, mRNA (italics), and protein (no italics) throughout the paper.

Reviewer #2 (Public review):

Summary:

The role of FGFs in embryonic development and stem cell differentiation has remained unclear due to its complexity. In this study, the authors utilized a 2D human stem cell-based gastrulation model to investigate the functions of FGFs. They discovered that FGF-dependent ERK activity is closely linked to the emergence of primitive streak cells. Importantly, this 2D model effectively illustrates the spatial distribution of key signaling effectors and receptors by correlating these markers with cell fate markers, such as T and ISL1. Through inhibition and loss-of-function studies, they further corroborated the needs of FGF ligands. Their data shows that FGFR1 is the primary receptor, and FGF2/4/17 are the key ligands for primitive streak development, which aligns with observations in primate embryos. Additional experiments revealed that the reduction of FGF4 and FGF17 decreases ERK activity.

Strengths:

This study provides comprehensive data and improves our understanding of the role of FGF signaling in primate primitive streak formation. The authors provide new insights related to the spatial localization of the key components of FGF signaling and attempt to reveal the temporal dynamics of the signal propagation and cell fate decision, which has been challenging.

Weaknesses:

Given the solid data, the work only partially clarifies the complex picture of FGF signaling, so details remain somewhat elusive. The findings lack a strong punchline, which may limit their broader impact.

Reviewer #3 (Public review):

Jo and colleagues set out to investigate the origins and functions of localized FGF/ERK signaling for the differentiation and spatial patterning of primitive streak fates of human embryonic stem cells in a well-established micropattern system. They demonstrate that endogenous FGF signaling is required for ERK activation in a ring-domain in the micropatterns, and that this localized signaling is directly required for differentiation and spatial patterning of specific cell types. Through high-resolution microscopy and transwell assays, they show that cells receive FGF signals through basally localized receptors. Finally, the authors find that there is a requirement for exogenous FGF2 to initiate primitive streak-like differentiation, but endogenous FGFs, especially FGF4 and FGF17, fully take over at later stages.

Even though some of the authors' findings - such as the localized expression of FGF ligands during gastrulation and the importance of FGF/ERK signaling for cell differentiation in the primitive streak - have been reported in model organisms before, this is one of the first studies to investigate the role of FGF signaling during primitive streak-like differentiation of human cells. In doing so, the paper reports a number of interesting and valuable observations, namely the basal localization of FGF receptors which mirrors that of BMP and Nodal receptors, as well as the existence of a positive feedback loop centered on FGF signaling that drives primitive-streak differentiation. The authors also perform a comparison of the role of different FGFs across species and try to assign specific functions to individual FGFs. In the absence of clean genetic loss-of-function cell lines, this part of the work remains less strong.

Author response:

Reviewer #1 (Public review):

Summary:

This is an interesting study on the role of FGF signaling in the induction of primitive streak-like cells (PS-LC) in human 2D-gastruloids. The authors use a previously characterized standard culture that generates a ring of PS-LCs (TBXT+) and correlate this with pERK staining. A requirement for FGF signaling in TBXT induction is demonstrated via pharmacological inhibition of MEK and FGFR activity. A second set of culture conditions (with no exogenous FGFs) suggests that endogenous FGFs are required for pERK and TBXT induction. The authors then characterize, via scRNA-seq, various components of the FGF pathway (genes for ligands, receptors, ERK regulators, and HSPG regulation). They go on to characterize the pFGFR1, receptor isoforms, and polarized localization of this receptor. Finally, they perform FGF4 inhibition and use a cell line with a limited FGF17 inactivation (heterozygous null) and show that loss of these FGFs reduces PS-LC and derivative cell types.

Strengths:

(1) As the authors point out, the role of FGF signaling in gastrulation is less well understood than other signaling pathways. Hence this is a valuable contribution to that field.

(2) The FGF4 and FGF17 loss-of-function experiments in Figure 5 are very intriguing. This is especially so given the intriguing observation that these FGFs appear to be dominating in this model of human gastrulation, in contrast to what FGFs dominate in mice, chicks, and frogs.

(3) In general this paper is valuable as a further development of the Human gastruloid system and the role of FGF signaling in the induction of PS-CLs. The wide net that the authors cast in characterizing the FGF ligand gene, receptor isoforms, and downstream components provides a foundation for future work. As the authors write near the beginning of the Discussion "Many questions remain."

We thank the reviewer for these positive comments.

Weaknesses:

(1) FGFs are cell survival factors in various aspects of development. The authors fail to address cell death due to loss of FGF signaling in their experiments. For example, in Figure 1E (which requires statistical analysis) and 1G (the bottom FGFRi row), there appears to be a significant amount of cell loss. Is this due to cell death? The authors should address the question of whether the role of FGF/ERK signaling is to keep the cells alive.

Indeed, FGF also strongly affects cell number and it is an interesting question to what extent this depends on ERK. Our manuscript focuses instead on the role of FGF/ERK signaling in cell fate patterning. However, as mentioned in our discussion, figure 1de show that doxycycline induced pERK leads to more TBXT+ cells than the control without restoring cell number, suggesting the role of FGF in controlling cell number is independent of the requirement for FGF/ERK in PS-LC differrentiation. Unpublished data below showing a MEK inhibitor dose response further supports this: low doses of MEKi are sufficient to inhibit differentiation without affecting cell number. To address the reviewer’s question we will include this data in the revised manuscript and perform several additional experiments to determine in more detail how cell death and proliferation depend on FGF.

Author response image 1.

MEK affects differentiation and cell number at different doses. a-c) control and MEKi (0.3uM) treated colonies with similar cell number but different TBXT expression. d-f) quantification of cell number per colonies (d), percentage of TBXT-positive cell per colony (e), and the distribution of pERK intensities for different doses of MEK inhibitor (f). N>6 colonies per condition. MEKi = PD0325901. Scalebar = 50 micron.

(2) Regarding the sparse cells in 1G, is there a reduction in cell number only with FGFRi and not MEKi? Is this reproducible? Gattiglio et al (Development, 2023, PMID: 37530863) present data supporting a "community effect" in the FGF-induced mesoderm differentiation of mouse embryonic stem cells. Could a community effect be at play in this human system (especially given the images in the bottom row of 1G)? If the authors don't address this experimentally they should at least address the ideas in Gattoglio et al.

Indeed, FGFRi reproducibly affects cell number more than MEKi, in line with the fact that pathways downstream of FGF other than MAPK/ERK (e.g. PI3K) play important roles in cell survival and growth. We think the lack of differentiation in MEKi and FGFRi in Fig.1g cannot be attributed to a loss of cells combined with a community effect. This is because without FGFRi or MEKi cells also differentiate to primitive streak at much lower densities than those shown, consistent with the data we show above in response to (1), which argue against a primarily indirect effect of FGF on PS-LC differentiation through cell density. In the context of directed differentiation (rather than 2D gastruloids), we will show this in a controlled manner by repeating the experiment in Fig.1g while adjusting cell seeding densities to obtain similar final cell densities in all three conditions. We will also include Gattoglio et al. in our revised discussion.

(3) Do the FGF4 and FGF17 LOF experiments in Figure 5 affect cell numbers like FGFRi in Figure 1?

It seems the effect on cell number is small but we will analyze this carefully and include it in the revised manuscript. A small effect would be consistent with our unpublished data below showing a near uniform proliferation rate. This in turn suggests that low levels of pERK in the center are sufficient to maintain proliferation there while the much higher pERK levels in the PS-LC ring (that we think depend on FGF4 and FGF17) do not signifcantly increase the proliferation rate (see Fig.1 in the manuscript for the pERK pattern). Thus, loss of high pERK in PS-LC ring while maintaining low pERK throughout would not be expected to have a major impact on cell number but would impact differentiation. In contrast, loss of all FGF signaling through FGFRi does dramatically affect cell number. This is again consistent with the data provided in response to (1) showing that ERK levels can be reduced to a point where PS-LC differentiation is lost without significantly affecting cell number. We will include the data below in the revised manuscript.

Author response image 2.

Why examine PS-LC induction only in FGF17 heterozygous cells and not homozygous FGF17 nulls?

We were unable to obtain homozygous FGF17 nulls, it is not clear if there is a reason for this. We will try again and otherwise attempt to corroborate our findings with further knockdown data.

(4) The idea that FGF8 plays a dominant role during gastrulation of other species but not humans is so intriguing it warrants deeper testing. The authors dismiss FGF8 because its mRNA "...levels always remained low." (line 363) as well as the data published in Zhai et al (PMID: 36517595) and Tyser et al (PMID: 34789876). But there are cases in mouse development where a gene was expressed at levels so low, that it might be dismissed, and yet LOF experiments revealed it played a role or even was required in a developmental process. The authors should consider FGF8 inhibition or inactivation to explore its potential role, despite its low levels of expression.

We agree with the reviewer that FGF8 is worth investigating further and we will now pursue this.

(5) Redundancy is a common feature in FGF genetics. What is the effect of inhibiting FGF4 in FGF17 LOF cells?

We will attempt to do the experiment the reviewer suggests.

(6) I suggest stating that the authors take more caution in describing FGF gradients. For example, in one Results heading they write "Endogenous FGF4 and FGF17 gradients underly the ERK activity pattern.", implying an FGF protein gradient. However, they only present data for FGF mRNA , not protein. This issue would be clarified if they used proper nomenclature for gene, mRNA (italics), and protein (no italics) throughout the paper.

We will edit the paper to more clearly distinguish protein and mRNA.

Reviewer #2 (Public review):

Summary:

The role of FGFs in embryonic development and stem cell differentiation has remained unclear due to its complexity. In this study, the authors utilized a 2D human stem cell-based gastrulation model to investigate the functions of FGFs. They discovered that FGF-dependent ERK activity is closely linked to the emergence of primitive streak cells. Importantly, this 2D model effectively illustrates the spatial distribution of key signaling effectors and receptors by correlating these markers with cell fate markers, such as T and ISL1. Through inhibition and loss-of-function studies, they further corroborated the needs of FGF ligands. Their data shows that FGFR1 is the primary receptor, and FGF2/4/17 are the key ligands for primitive streak development, which aligns with observations in primate embryos. Additional experiments revealed that the reduction of FGF4 and FGF17 decreases ERK activity.

Strengths:

This study provides comprehensive data and improves our understanding of the role of FGF signaling in primate primitive streak formation. The authors provide new insights related to the spatial localization of the key components of FGF signaling and attempt to reveal the temporal dynamics of the signal propagation and cell fate decision, which has been challenging.

Weaknesses:

Given the solid data, the work only partially clarifies the complex picture of FGF signaling, so details remain somewhat elusive. The findings lack a strong punchline, which may limit their broader impact.

We thank this reviewer for their valuable feedback and the compliment on the solidity of our data. The punchline of our work is that FGF4- and FGF17-dependent ERK signaling plays a key role in human PS-LC differentiation, and that these are different FGFs than those thought to drive mouse gastrulation. A second key point is that like BMP and TGFβ signaling, FGF signaling is restricted to the basolateral sides of pluripotent stem cell colonies due to polarized receptor expression, which is crucial for understanding the response to exogenous ligands added to the cell medium. Indeed, many facets of FGF signaling remain to investigated in the future, such as how FGF regulates and is regulated by other signals, which we will dedicate a different manuscript to.

Reviewer #3 (Public review):

Jo and colleagues set out to investigate the origins and functions of localized FGF/ERK signaling for the differentiation and spatial patterning of primitive streak fates of human embryonic stem cells in a well-established micropattern system. They demonstrate that endogenous FGF signaling is required for ERK activation in a ring-domain in the micropatterns, and that this localized signaling is directly required for differentiation and spatial patterning of specific cell types. Through high-resolution microscopy and transwell assays, they show that cells receive FGF signals through basally localized receptors. Finally, the authors find that there is a requirement for exogenous FGF2 to initiate primitive streak-like differentiation, but endogenous FGFs, especially FGF4 and FGF17, fully take over at later stages.

Even though some of the authors' findings - such as the localized expression of FGF ligands during gastrulation and the importance of FGF/ERK signaling for cell differentiation in the primitive streak - have been reported in model organisms before, this is one of the first studies to investigate the role of FGF signaling during primitive streak-like differentiation of human cells. In doing so, the paper reports a number of interesting and valuable observations, namely the basal localization of FGF receptors which mirrors that of BMP and Nodal receptors, as well as the existence of a positive feedback loop centered on FGF signaling that drives primitive-streak differentiation. The authors also perform a comparison of the role of different FGFs across species and try to assign specific functions to individual FGFs. In the absence of clean genetic loss-of-function cell lines, this part of the work remains less strong.

We thank the reviewer for emphasizing the value of our findings in a human model for gastrulation. We agree more loss-of-function experiments would provide further insight into the role of different FGFs, and we plan to provide additional data along these lines in the revised manuscript.