Megakaryocytes assemble a three-dimensional cage of extracellular matrix that controls their maturation and anchoring to the vascular niche

Reviewer #1 (Public review):

The authors report on a thorough investigation of the interaction of megakaryocytes (MK) with their associated ECM during maturation. They report convincing evidence to support the existence of a dense cage-like pericellular structure containing laminin γ1 and α4 and collagen IV, which interacts with integrins β1 and β3 on MK and serves to fix the perisinusoidal localization of MK and prevent their premature intravasation. As with everything in nature, the authors support a Goldilocks range of MK-ECM interactions - inability to digest the ECM via inhibition of MMPs leads to insufficient MK maturation and development of smaller MK. This important work sheds light on the role of cell-matrix interactions in MK maturation, and suggests that higher-dimensional analyses are necessary to capture the full scope of cellular biology in the context of their microenvironment.

There are several outstanding questions that this work does not address.

Major:

The authors postulate a synergistic role for Itgb1 and Itgb3 in the intravasation phenotype, because the single KOs did not replicate the phenotype of the DKO. However, this is not a correct interpretation in the opinion of this reviewer. The roles appear rather to be redundant. Synergistic roles would rather demonstrate a modest effect in the single KO with potentiation in the DKO.

Furthermore, the experiment does not explain how these integrins influence the interaction of the MK with their microenvironment. It is not surprising that attachment will be impacted by the presence or absence of integrins. However, it is unclear how activation of integrins allows the MK to become "architects for their ECM microenvironment" as the authors posit. A transcriptomic analysis of control and DKO MKs may help elucidate these effects.

Integrin DKO have a 50% reduction in platelets counts as reported previously, however laminin α4 deficiency only leads to 20% reduction in counts. This suggests a more nuanced and subtle role of the ECM in platelet growth. To this end, functional assays of the platelets in the KO and wildtype mice may provide more information.

There is insufficient information in the Methods Section to understand the BM isolation approach. Did the authors flush the bone marrow and then image residual bone, or the extruded bone marrow itself as described in PMID: 29104956?

The references in the Methods section were very frustrating. The authors reference Eckly et al 2020 (PMID: 32702204) which provides no more detail but references a previous publication (PMID: 24152908), which also offers no information and references a further paper (PMID: 22008103), which, as far as this reviewer can tell, did not describe the methodology of in situ bone marrow imaging.

Therefore, this reviewer cannot tell how the preparation was performed and, importantly, how can we be sure that the microarchitecture of the tissue did not get distorted in the process?

Reviewer #2 (Public review):

Summary:

This study makes a significant contribution to understanding the microenvironment of megakaryocytes (MKs) in the bone marrow, identifying an extracellular matrix (ECM) cage structure that influences MK localization and maturation. The authors provide compelling evidence for the presence of this ECM cage and its role in MK homeostasis, employing an array of sophisticated imaging techniques and molecular analyses. While the work is innovative and impactful, there are several points that require clarification or further data to fully support the conclusions.

Major Strengths:

Novelty: The identification of an ECM cage as a regulator of MK localization and maturation in the bone marrow is a novel and exciting finding.

Imaging Techniques: The use of advanced microscopy to visualize the 3D structure of the ECM cage and its role in MK homeostasis provides a strong visual foundation for the study's claims.

Comprehensive Analysis: The integration of in vivo and ex vivo approaches enhances the significance of the findings, offering valuable insights into the molecular mechanisms involved in ECM cage formation.

Areas for Improvement and Clarifications:

(1) ECM cage imaging:
a) The value or additional information provided by the staining on nano-sections (A) is not clear, especially considering that the thick vibratome sections already display the entirety of the laminin γ1 cage structure effectively. Further clarification on the unique insights gained from each approach would help justify its inclusion.
b) The sMK shown in Supplementary Figure 1C appears to be linked to two sinusoids, releasing proplatelets to the more distant vessels. Is this observation representative, and if so, can further discussion be provided?
c) Freshly isolated BM-derived MKs are reported to maintain their laminin γ1 cage. Are the proportions of MKs with/without cages consistent with those observed in microscopy?

(2) ECM cage formation:
a) The statement "the full assembly of the 3D ECM cage required megakaryocyte interaction with the sinusoidal basement membrane" on page 7 is too strong given the data presented at this stage of the study. Supplemental Figure 1C shows that approximately 10% of pMKs form cages without direct vessel contact, indicating that other factors may also play a role in cage formation.
b) The data supporting the statement that "pMK represent a small fraction of the total MK population" (cell number or density) could be shown to help contextualize the 10% of them with a cage.
c) How "the full assembly of the 3D ECM cage" is defined at this stage of the study should be clarified, specifically regarding the ECM components and structural features that characterize its completion.

(3) Data on MK Circulation and Cage Integrity: Does the cage require full component integrity to prevent MK release in circulation? Are circulating MKs found in Lama4-/- mice? Is the intravasation affected in these mice? Are the ~50% sinusoid associated MK functional?

(4) Methodology:
a) Details on fixation time are not provided, which is critical as it can impact antibody binding and staining. Including this information would improve reproducibility and feasibility for other researchers.
b) The description of 'random length measuring' is unclear, and the rationale behind choosing random quantification should be explained. Additionally, in the shown image, it appears that only the branching ends were measured, which makes it difficult to discern the randomness in the measurements.

(5) Figures:
a) Overall, the figures and their corresponding legends would benefit from greater clarity if some panels were split, such as separating images from graph quantifications.

Reviewer #3 (Public review):

In this manuscript, Masson, Scandola, et al investigate how interactions between megakaryocytes and the extracellular matrix contribute to the regulation of thrombopoiesis using primary murine bone marrow MK cultures, integrin B1/B3 knock-out mice, and high-resolution 2D and 3D imaging. They find that laminin and collagen iv create a 3D "cage" of ECM surrounding MKs and anchor them at the sinusoidal basement membrane, which contributes to MK maturation and proplatelet intravasation into circulation. Deletion of laminin a4 disrupts the localization of MKs and the endothelial basement membrane, reducing the number of MKs associated with the sinusoid while having no effect on MK-associated collagen IV. Deletion of B1/B3 integrin reduces the quantity, localization, and structural organization of multiple ECM components surrounding MKs, and reduces MK adhesion when subject to conditions of sinusoidal flow.

Further, using intravital microscopy of calvarial bone marrow and the pulmonary vasculature, they provide data suggesting that the stabilization of ECM around MKs (either in the BM or lung) prevents MKs from entering circulation as intact cells. Interestingly, deletion of B1 integrin reduces MK coverage in laminin y1, but deletion of both B1 and B3 independently results in increased MK intravasation into the sinusoidal space. Comparison of integrin KO MKs with GPVI KO MKs suggests that ECM cage formation, vessel adhesion, and intravasation are likely dependent on integrin activation/signaling rather than GPVI signals.

Further, they provide data that the balance of ECM synthesis and degradation is essential for MK maturation and also provide data showing that inhibition of ECM turnover (in vivo inhibition of MMPs) results in increased ECM cage components that correspond with reduced MK maturation, and reduced demarcation membrane development.

The conclusions of the paper are supported by the data, but there are some areas that would benefit from clarification or expansion.

(1) The data linking ECM cage formation to MK maturation raises several interesting questions. As the authors mention, MKs have been suggested to mature rapidly at the sinusoids, and both integrin KO and laminin KO MKs appear mislocalized away from the sinusoids. Additionally, average MK distances from the sinusoid may also help separate whether the maturation defects could be in part due to impaired migration towards CXCL12 at the sinusoid. Presumably, MKs could appear mislocalized away from the sinusoid given the data presented suggesting they leaving the BM and entering circulation. Additional data or commentary on intrinsic (ex-vivo) MK maturation phenotypes may help strengthen the author's conclusions and shed light on whether an essential function of the ECM cage is integrin activation at the sinusoid.

(2) The data demonstrating intact MKs inter circulation is intriguing - can the authors comment or provide evidence as to whether MKs are detectable in blood? A quantitative metric may strengthen these observations.

(3) Supplementary Figure 6 - shows no effect on in vitro MK maturation and proplt, or MK area - But Figures 6B/6C demonstrate an increase in total MK number in MMP-inhibitor treated mice compared to control. Some additional clarification in the text may substantiate the author's conclusions as to either the source of the MMPs or the in vitro environment not fully reflecting the complex and dynamic niche of the BM ECM in vivo.

(4) Similarly, one function of the ECM discussed relates to MK maturation but in the B1/3 integrin KO mice, the presence of the ECM cage is reduced but there appears to be no significant impact upon maturation (Supplementary Figure 4). By contrast, MMP inhibition in vivo (but not in vitro) reduces MK maturation. These data could be better clarified in the text, or by the addition of experiments addressing whether the composition and quantity of ECM cage components directly inhibit maturation versus whether effects of MMP-inhibitors perhaps lead to over-activation of the integrins (as with the B4galt KO in the discussion) are responsible for the differences in maturation.

Author response:

Reviewer #1 (Public review):

Point 1. The authors postulate a synergistic role for Itgb1 and Itgb3 in the intravasation phenotype, because the single KOs did not replicate the phenotype of the DKO. However, this is not a correct interpretation in the opinion of this reviewer. The roles appear rather to be redundant. Synergistic roles would rather demonstrate a modest effect in the single KO with potentiation in the DKO.

We agree that the interaction between Itgb1 and Itgb3 appears redundant and we will correct this point in the revised manuscript.

Point 2. The experiment does not explain how these integrins influence the interaction of the MK with their microenvironment. It is not surprising that attachment will be impacted by the presence or absence of integrins. However, it is unclear how activation of integrins allows the MK to become "architects for their ECM microenvironment" as the authors posit. A transcriptomic analysis of control and DKO MKs may help elucidate these effects.

We do not currently understand how α5β1 or αvβ3 integrins activation would contribute to ECM remodeling by megakaryocytes. Integrins are well known key regulators of ECM remodelling (https://doi.org/10.1016/j.ceb.2006.08.009). They can transmit traction force that provoques ECM remodelling (https://doi.org/10.1016/j.bpj.2008.10.009). We will discuss our previous study on the observed reduction in RhoA activation in double knockout (DKO) mice (Guinard et al., 2023, PMID: 37171626), which likely impact the organization of the ECM microenvironment. Alternatively, integrin signalling contribute to gene expression regulation involved in ECM remodelling (ECM proteins, proteases….). We do agree with the reviewer that the transcriptomic analysis could provide strong evidence; however, it is challenging to perform this analysis in vivo. Isolation of native megakaryocytes (MKs) from DKO mice is challenging due to their reduced numbers, requiring too many mice for sufficient RNA and risk of cell contamination. An alternative approach will be to analyze platelets, which are more abundant and easier to isolate, while still mimicking the characteristics of bone marrow MKs. We will use PCR array technology for selected ECM panels and adhesion molecules (from all players currently known to contribute to ECM remodelling), providing a practical way to address the reviewer's suggestions and provide valuable insights.

Point 3. Integrin DKO have a 50% reduction in platelets counts as reported previously, however laminin α4 deficiency only leads to 20% reduction in counts. This suggests a more nuanced and subtle role of the ECM in platelet growth. To this end, functional assays of the platelets in the KO and wildtype mice may provide more information.

The difference in platelet counts between integrin DKO and laminin α4 KO mice is not fully understood. Although our study specifically focuses on MK-ECM interactions in the bone marrow, we recognize the importance of providing additional information on platelet functionality. To address this, we will use flow cytometry to examine the levels of P-selectin surface expression and fibrinogen binding under basal conditions and after stimulation with collagen-related peptide and TRAP.

Point 4. There is insufficient information in the Methods Section to understand the BM isolation approach. Did the authors flush the bone marrow and then image residual bone, or the extruded bone marrow itself as described in PMID: 29104956?

Additional information on the methodology will be provided to clarify the BM isolation.

Point 5. The references in the Methods section were very frustrating. The authors reference Eckly et al 2020 (PMID: 32702204) which provides no more detail but references a previous publication (PMID: 24152908), which also offers no information and references a further paper (PMID: 22008103), which, as far as this reviewer can tell, did not describe the methodology of in situ bone marrow imaging.

To address this confusion, we will add the reference "In Situ Exploration of the Major Steps of Megakaryopoiesis Using Transmission Electron Microscopy" by C. Scandola et al. (PMID: 34570102), which provides a standardized protocol for bone marrow isolation.

Therefore, this reviewer cannot tell how the preparation was performed and, importantly, how can we be sure that the microarchitecture of the tissue did not get distorted in the process?

Thank you for pointing this out. While we cannot completely rule out the possibility of distortion, we will clarify the precautions taken to minimize it. We utilized a double fixation process immediately after extruding the bone marrow, followed by embedding it in agarose to preserve its integrity as much as possible. We will address this point in greater detail in Methods section of the revised version.

Reviewer #2 (Public review):

Point 1. ECM cage imaging

a) The value or additional information provided by the staining on nano-sections (A) is not clear, especially considering that the thick vibratome sections already display the entirety of the laminin γ1 cage structure effectively. Further clarification on the unique insights gained from each approach would help justify its inclusion.

Ultrathin cryosection allow high-resolution imaging (10x fold increased in Z), facilitating the analysis of signal superposition. This study explores the interactions between MKs and their immediate ECM microenvironment, located at a distance of less than one micrometer, making nano-sections optimal for precise analysis of ECM distribution both within and surrounding MKs. This high-resolution approach has revealed the presence of collagen IV, laminin, fibronectin, and fibrinogen near MKs, More importantly, ultrathin cryosection allow us to clearly show with high resolution the presence of activated integrin in contact with laminin an coll IV fibers (see Fig. 3)

We employed large-volume whole-mount imaging to clarify the overall three-dimensional architecture of the ECM interface, allowing us to identify the cages. Our findings emphasize the role of specific ECM components in facilitating proplatelet passage through the sinusoid barrier, an essential step for platelet production. Further details will be addressed in the revised manuscript.

b) The sMK shown in Supplementary Figure 1C appears to be linked to two sinusoids, releasing proplatelets to the more distant vessels. Is this observation representative, and if so, can further discussion be provided?

This observation is not representative; MKs can also be associated with just one sinusoid.

c) Freshly isolated BM-derived MKs are reported to maintain their laminin γ1 cage. Are the proportions of MKs with/without cages consistent with those observed in microscopy?

In the revised manuscript, we will include the quantification of the proportion of BM-derived MKs with/without cages.

Point 2. ECM cage formation

a) The statement "the full assembly of the 3D ECM cage required megakaryocyte interaction with the sinusoidal basement membrane" on page 7 is too strong given the data presented at this stage of the study. Supplemental Figure 1C shows that approximately 10% of pMKs form cages without direct vessel contact, indicating that other factors may also play a role in cage formation.

The reviewer is correct. We will modify the text to reflect a more cautious interpretation of our results.

b) The data supporting the statement that "pMK represent a small fraction of the total MK population" (cell number or density) could be shown to help contextualize the 10% of them with a cage.

New bar graphs will be provided to represent the density of MK in the parenchyma against the total MK in the bone marrow.

c) How "the full assembly of the 3D ECM cage" is defined at this stage of the study should be clarified, specifically regarding the ECM components and structural features that characterize its completion.

We recognize that the term ' full assembly' of the 3D ECM cage can be misleading, as it might suggest different stages of cage formation, such as a completed cage, one that is in the process of formation, or an incomplete cage. Since we have not yet studied this concept, we will eliminate the term "full assembly" from the manuscript to avoid any confusion. Instead, we will simply mention the presence of a cage.

Point 3. Data on MK Circulation and Cage Integrity: Does the cage require full component integrity to prevent MK release in circulation? Are circulating MKs found in Lama4-/- mice? Is the intravasation affected in these mice? Are the ~50% sinusoid associated MK functional?

These are very valid points. We will answer all these questions by performing a detailed analysis of MK localization, vessel association and intravascular MK detection using IF and high-resolution EM imaging of Lamα4^-/- mice. Additionally, we will analyze data from Lamα4-/- bone marrow explants to assess the capacity of MKs to extend proplatelets.

Point 4. Methodology

a) Details on fixation time are not provided, which is critical as it can impact antibody binding and staining. Including this information would improve reproducibility and feasibility for other researchers.

We will added this information in the methods section.

b) The description of 'random length measuring' is unclear, and the rationale behind choosing random quantification should be explained. Additionally, in the shown image, it appears that only the branching ends were measured, which makes it difficult to discern the randomness in the measurements.

The random length measurement method uses random sampling to provide unbiased data on laminin/collagen fibers in a 3D cage. Contrary to what the initial image might have suggested, measurements go beyond just the branching ends; they include intervals between various branching points throughout the cage.

To clarify this process, we will outline these steps: 1) acquire 3D images, 2) project onto 2D planar sections, 3) select random intersection points for measurement, 4) measure intervals using ImageJ software, and 5) repeat the process for a representative dataset. This will better illustrate the randomness of our measurements.

Point 5. Figures

a) Overall, the figures and their corresponding legends would benefit from greater clarity if some panels were split, such as separating images from graph quantifications.

Following the reviewer’s suggestion, we will fully update all the Figures and separate images from graph quantifications.

Reviewer #3 (Public review):

Point 1. The data linking ECM cage formation to MK maturation raises several interesting questions. As the authors mention, MKs have been suggested to mature rapidly at the sinusoids, and both integrin KO and laminin KO MKs appear mislocalized away from the sinusoids. Additionally, average MK distances from the sinusoid may also help separate whether the maturation defects could be in part due to impaired migration towards CXCL12 at the sinusoid. Presumably, MKs could appear mislocalized away from the sinusoid given the data presented suggesting they leaving the BM and entering circulation. Additional data or commentary on intrinsic (ex-vivo) MK maturation phenotypes may help strengthen the author's conclusions and shed light on whether an essential function of the ECM cage is integrin activation at the sinusoid.

The hypothesis of MK migration towards CXCL12 is interesting, although it has recently been challenged by Stegner et al. (2017), who found that MKs are primarily sessile. However, we cannot exclude this possibility. To address the reviewer's concerns, we will quantify the distance of MKs from the sinusoids. This could help to determine whether the maturation defects are due to impaired migration towards CXCL12 at the sinusoids or other factors, such as the ECM cage.

We would appreciate some clarification regarding the second point raised by the reviewer. Is the question specifically addressing whether the ECM cage has an effect on the activation of integrins in the sinusoids? If so, we will use immunofluorescence (IF) to investigate the relationship between the presence of an ECM cage and the activation of integrins on the surface of endothelial cells within the sinusoids. Thank you for your guidance on this matter.

Point 2. The data demonstrating intact MKs inter circulation is intriguing - can the authors comment or provide evidence as to whether MKs are detectable in blood? A quantitative metric may strengthen these observations.

We will conduct flow cytometry experiments and prepare blood smears to determine whether intact MKs are detectable in blood.

Point 3. Supplementary Figure 6 - shows no effect on in vitro MK maturation and proplt, or MK area - But Figures 6B/6C demonstrate an increase in total MK number in MMP-inhibitor treated mice compared to control. Some additional clarification in the text may substantiate the author's conclusions as to either the source of the MMPs or the in vitro environment not fully reflecting the complex and dynamic niche of the BM ECM in vivo.

This is a valid point. We will revise the text to include further clarification.

Point 4. Similarly, one function of the ECM discussed relates to MK maturation but in the B1/3 integrin KO mice, the presence of the ECM cage is reduced but there appears to be no significant impact upon maturation (Supplementary Figure 4). By contrast, MMP inhibition in vivo (but not in vitro) reduces MK maturation. These data could be better clarified in the text, or by the addition of experiments addressing whether the composition and quantity of ECM cage components directly inhibit maturation versus whether effects of MMP-inhibitors perhaps lead to over-activation of the integrins (as with the B4galt KO in the discussion) are responsible for the differences in maturation.

These are very good questions, but they are difficult to assess in situ. To approach this, we will perform in vitro experiments :

(1) We will vary collagenIV and laminin411 concentrations in the culture conditions to determine how this affects MK maturation ; and

(2) We will assess the integrin activation states on cultured MKs treated with MMP inhibitors to determine if MMP inhibitors could influence MK maturation through over-activation of integrins.