ECM-integrin signalling instructs cellular position sensing to pattern the early mouse embryo
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Evaluation Summary:
The importance of extracellular matrix (ECM) in multicellular organization has been studied in many different contexts. This study investigates the role of the ECM in the formation of the very first cell types to form in a mammalian embryo. The authors find that it may help stabilize the cell fate of early inside cells and play a role in promoting the epithelial organization of the next cell type to form within the inner cell mass. With appropriate extended experimental analysis, this study could be of interest to the specialist mammalian development community .
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM), which eventually forms the embryo itself. Yet, the molecular basis of how these cells recognise their ‘inside’ position to instruct their fate is unknown. Here, we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters the subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin β1 activity and involves apical-to-basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for ‘inside’ positional signalling and is required for correct EPI/PrE patterning. Thus, our findings highlight the significance of ECM-integrin adhesion in enabling position sensing by cells to achieve tissue patterning.
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Evaluation Summary:
The importance of extracellular matrix (ECM) in multicellular organization has been studied in many different contexts. This study investigates the role of the ECM in the formation of the very first cell types to form in a mammalian embryo. The authors find that it may help stabilize the cell fate of early inside cells and play a role in promoting the epithelial organization of the next cell type to form within the inner cell mass. With appropriate extended experimental analysis, this study could be of interest to the specialist mammalian development community .
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this paper, the authors address the underlying processes that lead to cell fate specification in the early mouse embryo. They explore the role of laminin-integrin signaling in specifying the fate of the inside cells to become the Inner Cell Mass and the subsequent separation of the epiblast and the primitive endoderm. They provide a nice description of the expression patterns of components of the integrin signaling pathway in early development, which adds to previously published work. Then using a combination of gain of function and loss of function experiments, they provide some evidence for integrin signaling playing a role in both lineage segregation events. Gain of function experiments involved culturing inside cells from morulae in the presence of Matrigel as a source of laminins, while loss of …
Reviewer #1 (Public Review):
In this paper, the authors address the underlying processes that lead to cell fate specification in the early mouse embryo. They explore the role of laminin-integrin signaling in specifying the fate of the inside cells to become the Inner Cell Mass and the subsequent separation of the epiblast and the primitive endoderm. They provide a nice description of the expression patterns of components of the integrin signaling pathway in early development, which adds to previously published work. Then using a combination of gain of function and loss of function experiments, they provide some evidence for integrin signaling playing a role in both lineage segregation events. Gain of function experiments involved culturing inside cells from morulae in the presence of Matrigel as a source of laminins, while loss of function experiments involved the use of blocking antibodies to integrinb1 and genetic studies with Itgb1-/- and Lamc1-/- embryos. They show that culture in Matrigel blocked the ability of inside cells to repolarize and form trophectoderm (TE) and that this was mediated by the integrin receptor. However, loss of function of both integrin and laminin had no effect on the formation of ICM and TE in the intact embryo, diminishing the impact of these results. The clearer results came from examining the formation and organization of the primitive endoderm where loss of function of both integrin and laminin led to a failure of the primitive endoderm to organize into a single epithelial layer.
Overall the claim that integrin signaling plays an active role in inducing ICM specification is not strongly supported. Rather, the role of Matrigel/integrin activation can be interpreted as blocking repolarization and thus blocking TE specification. They do not present any evidence of active induction of inside fate. Second, as they admit in the discussion, the culture of inside cells in Matrigel is an artificial situation, which may provide a non-physiological level of ECM/integrin activation. They might make a stronger case by testing directly the role of exogenous Laminin, rather than the complex Matrigel. Exogenous Laminin has been shown to rescue some phenotypic defects in laminin-null EBs (Li et al. 2002 J Cell Biol.). Given that embryos mutant for integrin or laminins (not shown) do not show any defects in ICM/TE specification at the blastocyst stage, the importance of integrin signaling for ICM/TE cell fate specification cannot be considered as a predominant influence on early patterning.
However both integrin and laminin mutant embryos do show defects during the next lineage differentiation event separating Epiblast (EPI) and primitive endoderm (PrE). The primitive endoderm and epiblast are formed and specified but PrE fails to form an organized epithelial layer overlying the EPI, suggestive of a role for the ECM in this process. This observation is interesting but not novel- similar defects in laminin mutant embryos and embryoid bodies have been previously reported. There is no new experimental insight into the actual mechanism of action of this ECM/integrin interaction.
Although this study provides some new information, the underlying mechanisms of action are not sufficiently explored and the study will be primarily of interest to specialists in mouse development.
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Reviewer #2 (Public Review):
This manuscript from the Hiragii Lab identifies two new roles for Integrins and Laminins in the preimplantation mouse embryo.
One of these roles is to influence trophectoderm-regenerating ability following removal of native trophectoderm by immunosurgery. In this context, the authors show that integrin/laminin can repress Hippo signaling in isolated inner cell masses.
The other role reported for Integrin/Laminin is to ensure that primitive endoderm cells form a monolayer after sorting out from epiblast cells within the inner cell mass. Unfortunately, however, there does not appear to be a role for integrin/laminin in regulating Hippo signaling or trophectoderm fate specification during normal development.
The evidence for the two roles for integrin/laminin comes from immunofluorescence (evaluation of …
Reviewer #2 (Public Review):
This manuscript from the Hiragii Lab identifies two new roles for Integrins and Laminins in the preimplantation mouse embryo.
One of these roles is to influence trophectoderm-regenerating ability following removal of native trophectoderm by immunosurgery. In this context, the authors show that integrin/laminin can repress Hippo signaling in isolated inner cell masses.
The other role reported for Integrin/Laminin is to ensure that primitive endoderm cells form a monolayer after sorting out from epiblast cells within the inner cell mass. Unfortunately, however, there does not appear to be a role for integrin/laminin in regulating Hippo signaling or trophectoderm fate specification during normal development.
The evidence for the two roles for integrin/laminin comes from immunofluorescence (evaluation of integrin/laminin and cell fate markers), loss of function studies (blocking antibodies and null alleles), and gain of function studies (where Matrigel contains laminins, among other things...).
Ultimately, this paper might interest preimplantation devotees, but will be less interesting to the broader scientific community in its current form. This is because the most compelling observations revolve around immunosurgery/regeneration, where the biological relevance is uncertain, while the embryo development phenotypes are minor and their analysis relatively superficial. If the study had revealed the mechanism by which integrin/laminin "talk to" the Hippo signaling pathway, it might have been more impactful.
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Reviewer #3 (Public Review):
Kim at al., proposed a model in which the extracellular matrix, a fibrous environment, serves as a niche for inner cell mass specification in the preimplantation mouse embryo. The presence of extracellular matrix components within the preimplantation embryo was demonstrated a few decades ago. However, our current knowledge on their role during early mammalian develpoment still remains limited to processes during peri- and postimplantation embryogenesis.
To simulate the in vivo environment, the authors removed the surrounding outer cells of the embryo by enzymatic digestion, called immunosurgery, and embedded the uncovered inner cell mass into Matrigel. Matrigel-embedded inner cell mass from embryos undergoing the first cell fate decision maintained their inner fate, shown by Sox2-positive immunostaining. In …
Reviewer #3 (Public Review):
Kim at al., proposed a model in which the extracellular matrix, a fibrous environment, serves as a niche for inner cell mass specification in the preimplantation mouse embryo. The presence of extracellular matrix components within the preimplantation embryo was demonstrated a few decades ago. However, our current knowledge on their role during early mammalian develpoment still remains limited to processes during peri- and postimplantation embryogenesis.
To simulate the in vivo environment, the authors removed the surrounding outer cells of the embryo by enzymatic digestion, called immunosurgery, and embedded the uncovered inner cell mass into Matrigel. Matrigel-embedded inner cell mass from embryos undergoing the first cell fate decision maintained their inner fate, shown by Sox2-positive immunostaining. In contrast, inner cell mass soaked in culture media (KSOM) developed into a proper blastocyst embryo with Sox2-positive inner and oblong CDX2-positive outer cells, and a blastocoel. The authors then underpinned their hypothesis by adding blocking antibodies against integrin beta1 and alpha6 into Matrigel or using embryos from integrin beta1-knockout mice. The manuscript takes then a sudden and contradictory twist. The authors proceed to perform similar experiments with inner cell mass isolated from blastocyst embryos to investigate the extracellular matrix-dependent processes on the second cell fate decision. At this developmental stage, the cells showed no altered cell fates but instead spatial mis-positioning. The authors finished with an interesting signalling mechanism but supported solely by co-expression analysis of Talin, Laminin gamma1 and integrin beta1 using immunostainings.
Overall, the strength of the paper is diminished by describing too many different aspects, but none of them in sufficient detail.
Sox2 has been extensively described to be involved in ICM specification. While the authors claim it as "the earliest marker of ICM specification", they then use it to visualise final internalised cells, several hours after the onset of internalisation. Therefore, the authors used a previously published Sox2-GFP mouse line. When checking the original paper on the generation of this Sox2-GFP line, it becomes clear that this is a reporter line in which GFP is under the regulatory elements of the Sox2 gene but not a Sox2-GFP fusion gene. Thus, measuring Sox2 expression levels as stated by the authors is not correct and it is questionable if the cytoplasmic GFP levels exactly reflect the physiological nuclear Sox2 expression levels.
The presence of extracellular matrix components, including integrins and laminins, have been shown in previous studies as cited by the authors in the introduction. Thus, the novelty of result chapter 2 is unclear.
It is an interesting approach to remove the outer cells from the morula embryos, unknown however if 16- or 32-cell stage embryos, by enzymatic digestion, allowing to study and manipulate the "naked" inner cell mass. The authors show one inner cell mass in which all cells maintained their inner cell fate. However, due to the lack of control experiments, the author's statement that "During the first lineage segregation, Matrigel is sufficient to drive ICM specification in an integrin-dependent manner,..." is not adequately supported. It is stated several times throughout the manuscript that the "extracellular matrix induces/drives ICM specification", contradictory to the subsequent chapter headed "integrin b1 is not required for initial ICM specification...".
The strength of the paper is a more comprehensible effect on the spatial organisation of epiblast and primitive endoderm cells in the more advanced blastocyst. A more in-depth analysis on this phenomenon, including the signalling mechanism, would have helped to substantiate the author's claims. The sorting of epiblast and primitive endoderm cells is position-independent as stated by the authors. Thus, the title of the manuscript is not in accordance with this finding.
A major drawback is the presentation of all imaging results in 2D. The preimplantation mouse embryo is a 3D object. If presented in 2D there is no reliable presentation of cell number of the embryos shown, no reliable quantification of expression intensities as it depends if cells are cut in the centre or at the edges and cropped outer cells can be mistaken by inner cells.
The study needs to be put into context with more recent embryo-related publications. Currently over 80% of all cited references in introduction and discussion are >10 years old. For instance, previous studies demonstrating the necessity of Sox2 in epiblast formation (Avilion et al., 2003) and Sox2-DNA dynamics rather than expression levels (Goolam et al., 2016; White et al., 2016) were not taken into consideration. Major advances in the molecular mechanisms of inner cell allocation have been achieved, including Yap signalling, E-cadherin, cytoskeleton-dependent tension, but neglected by the authors.
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