Peer review of: Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Summary: 
While actin cytoskeletal remodeling is important in hematopoietic cell motility and migration, the 
precise mechanisms by which hematopoietic stem and progenitor cells (HSPCs) establish residency 
following homing to the bone marrow (BM) niche remain unclear. Two major protein families regulate 
actin-nucleation and cellular motility in mammalian cells, (1) formin-related proteins and (2) WASP 
protein complexes. mDia1, mDia2, and mDia3 belong to a highly conserved family of diaphanous-
related formins proteins that modulate a number of cell processes in addition to actin polymerization, 
including the modulation of microtubule networks, mRNA trafficking, serum response factor (SRF)-
mediated gene expression, and cytokinesis. In this manuscript, Mei et. al. explore mechanisms of 
mDia2 (Diaph3) and, to a lesser extent, mDia1 (Diaph1)-mediated HSPC transmigration and 
engraftment into the HSPC-supportive bone marrow microenvironment using in vivo knockout 
strategies paired with genetic complementation to rescue HSPC deficiencies in a bone marrow 
transplantation (BMT) model system. In competitively transplanted recipients, loss of mDia2 (using a 
hematopoietic-specific Vav1-cre and Mx1-cre) results in distinct hematopoietic failure in non-
competitive and competitive BMTs. Interestingly, impaired HSPC function is not due to a homing 
defect, but rather a failure of HSPCs to properly transmigrate from the peripheral blood to the BM 
parenchyma, and vice versa. This was confirmed using a combination of BM imaging following BMT, in 
vivo hematopoietic mobilization assays, and ex vivo migration assays of mDia2 knockout HSPCs. 
mDia1 knockout animals display a significant, but more mild engraftment phenotype (when compared 
to mDia2 knockouts). HSPC transmigration was not examined in mDia1 knockouts. Downstream of 
mDia2 activity, MAL-SRF signaling displayed functional deficiencies including a decrease in some 
canonical SRF targets and non-canonical beta2 integrins CD11a, CD11b, and CD18 targets. The 
authors confirmed enhancer binding activity in both CD11b and CD18 integrins using mutational 
enhancer analysis in a luciferase assay. Genetic complementation (overexpression) of SRF in mDia2 
knockout HSPCs restored the observed engraftment phenotype. Moreover, the authors confirmed the 
function of SRF-dependent integrins by deleting CD11b and CD18 using CRISPR-Cas9 phenocopying 
the mDia2 knockout BMT mice. Genetic complementation (overexpression) of CD11b in mDia2 
knockout HSPCs also partially restored the observed engraftment phenotype. The approach to 
examining the mechanism of mDia2 loss of function in HSPC engraftment in this manuscript is 
exhaustive and provides a significant springboard for future studies. The findings also provide 
significant insights into HSPCs migration and lodgement phenotypes, and should be of interest to the 
general readership of Nature Communications. The inclusion of data from mDia1 knockout mice is 
some cause for concern: (1) The phenotype in mDia1 mice are not examined in detail (compared to 
mDIa2 knockout mice), (2) Analysis of mDia1/mDia2 double knockouts is incomplete, and (3) the 
mDia1 knockout model is a global knockout that may have additional niche-mediated effects on HSPCs. 
Moreover, the discussion points out a number of obvious questions that can be explained by mDia1 
overlapping function. The inclusion of mDia1 in this story demands experimental answers to these 
questions (e.g. expansion of HSPCs phenotype in SRF knockouts, page 19 and partial SRF target gene 
dysregulation in mDia2 knockouts, page 20, and proper HSPC experimental phenotyping/MAL-SRF 
signaling investigation). As currently presented by the authors, the manuscript is incomplete. If the 
authors choose to leave mDia1 knockout model in the manuscript, they should address the questions 
raised in Major Points #5. Alternatively, this manuscript can focus on the mDia2 story and address 
mDia1 more completely in another manuscript. 

Major Points: 
1. Can the authors explain the rationale for examining non-competitive BMT (Fig 1) at eight weeks 

Editorial Note: Parts of this peer review file have been redacted as indicated to remove third-party material where no permission to publish could be obtained.post-transplantation (as opposed to standard 16-24 week time points in which homeostatic 
hematopoiesis reestablished)? Considering the dramatic engraftment phenotype in Vav1-cre; 
mDia2fl/fl competitive BMT (Fig 2), does engraftment of HSPCs in Mx1-cre; mDia2fl/fl in non-
competitive BMT represent non-recombined HSPCs? 

2. The initial description and experimental usage of mouse models in this manuscript is confusing. 
Model generation: The methods and text do not adequately describe the generation of these models. 
Please provide source (catalog/vendor) information for the Mx1-cre and Vav1-cre. Is the Vav1-cre 
constitutively active during development or an inducible model? Experimental usage: What is the 
reasoning for utilizing both Mx1-cre and Vav1-cre models interchangeably when making hematopoietic 
comparisons (e.g. steady state Sup 1C, E vs BMT stress Fig 1A). What is the significance of comparing 
results from the two models (i.e. what new information does it tell the reader)? Why was Mx1-cre and 
Vav1-cre chosen for this study? In some cases, data is left out entirely for comparisons between 
models (e.g. page 6, Mx1-cre steady state analysis). 

3. The observation that HSPCs fail to transmigrate into the in vivo bone marrow niche (Fig 2G-H) is 
critical piece of information for this manuscript. In addition to the in vitro migration assay, it directly 
implicates a lodgement phenotype. However, the images (Fig 2G) do not give the reader any 
magnitude of this observation. It is important to have a much more representative series of images to 
support the quantification (Fig 2H). This data can be amended as a supplementary Figure. Scale bars 
also needs to be added to all microscopic images. It would also be beneficial to test this hypothesis; 
does intrafemoral injection of mDia2 knockout HSPCs circumvent the observed engraftment phenotype? 

4. No cell cycle differences were observed in steady state mDia2 knockout. Are there any cell cycle 
differences noted following hematopoietic recovery in non-competitive mDia2 BMT mice (Fig 1A-C)? 

5. The interplay between mDia1 and mDia2 in this manuscript are very interesting, but raise a series 
of questions concerning their mutual (compensatory) and mutually exclusive functions that should be 
addressed experimentally. (1) mDia2 functions through the MAL-SRF signaling axis, but only alters a 
subset of canonical/integrin SRF targets. Are the remaining targets altered in mDia1 knockout animals? 
(2) Is HSPC frequency and absolute numbers altered in under steady state and BMT conditions? (3) 
What is the status of canonical/integrin SRF targets in the mDia1/mDia2 double knockout HSPCs? (4) 
As noted by the authors, SRF knockout mice have a significant expansion of phenotypic HSPCs at 
steady state that was not observed in mDia2 knockouts. Do mDia1/mDia2 double knockouts display 
HSPC expansion under homeostatic conditions? (5) Does SRF rescue mDia1 single knockout or 
mDia1/mDia2 double knockout HSPCs? (6) Do mDia1 knockout mice display an transmigration defect 
similar to mDia2 knockouts (imaging, mobilization, Transwell assays)? (7) Is the transmigration 
phenotype more pronounced in mDia1/mDia2 double knockout HSPCs? 

Minor Points: 
1. The intermittent inclusion of heterozygous deletion of mDia2 (e.g. Fig 2C) is distracting and does 
not add any value to the conclusions of the paper. 

2. Figures use a combination of listed p-values and asterisks in bar graphs to indicate significance. 
Please correct for consistency. 

3. Figure legends should have indications of which statistics were used to generate p-values. 

4. Include significance for engraftment in Fig 2C. 

5. Include significance for engraftment in Fig 7A. 

6. An ANOVA analysis (repeated measures) may provide statistical power to Fig 7C. 

7. The Methods sections lacks experimental detail in many sections (e.g. Clear explanations the 
mice/models used, how were the animals preconditioned for BMT, etc.). Please ensure transparency 
and clarity for the readers. 

Reviewer #2 (Remarks to the Author): 

Homing and establishment of HS/PCs in the bone marrow is critical for establishment of hematopoiesis, 
but the molecular mechanisms involved are poorly understood. 

In this study, Mei et al investigate the role of mDia1 and mDia2 formins in bone marrow engraftment 
of hematopoietic stem and progenitor cells (HSPCs). First they show that mDIa2 KO cells show 
defective reestablishment of LSK and LT/ST-HSC populations, with LSK KO cells showing loss of 
quiescence. Similar but weaker phenotypes were seen with mDia1 KO., which appears to synergise 
with mDia2. They show that mDia2KO did not affect homing of the HSPC to the bone marrow 
vasculature but led to impaired transendothelial migration and niche engraftment, and defective 
mobilization by GCSF. This was associated with inactivation of the MAL transcriptional coactivator, 
which became refractory to serum stimulation, and reduced expression of MAL-SRF target genes. The 
authors provide evidence that Itgb2 and ItgaM are direct targets for SRF. They go on to show that 
retroviral transduction of SRF restores classic SRF target gene expression as well as integrin 
expression, adhesion, and engraftment. 
Next they use CRISP/Cas of ItgaM or Itgb2 to show that these integrins are required for recruitment, 
their combined deletion being synergistic. Ectopic expression of ItgaM can partially rescue the 
mDia2KO phenotype. 

EVALUATION 
The paper nicely shows that MAL-SRF signalling is downstream of mDia2 in HSCP engraftment, and 
that integrin expression is an important aspect of this with ItgaM and ItgbB2 playing imporant roles. It 
also shows redundancy with mDia1. What it does not do is convincingly show that MAL-SRF signalling 
activates the integrins directly, as opposed to indirect effects arising thorugh MAL-SRF’s well known 
role in regulating expression of multiple cytoskeletal genes. The paper is marred by an unconvincing 
analysis of the integrin regulatory sequences and particularly poor presentation of this section. 

MAJOR ISSUES 
Ectopic expression of ItgaM can partially rescue the mDia2KO phenotype, but Itgb2 is not tested, 
alone or incombination with ItgaM. What happens with Itgb2 ± ItgaM? Moreover, they authors do not 
test ItgaL either. These experiments should be done and the results shown. 

A critical point of the authors’ model is that mDia2KO cells should be defective in recruitment of MAL 
to SRF binding sites (ANY SRF binding sites that recruitment MAL). This can be demonstrated by ChIP, 
but no data are provided. This is essential to vailidate the proposed integrin SRF binding sites, and for 
canonical target genes such as Acta2 or SRF itself. 

The SRF binding analysis is not adequately presented and does not cite the appropriate literature. 

OTHER POINTS (not in order of importance) 

Order of presentation. Two points: 
- The presentation of the Dia1 data occurs in Figure S3(p8), then in Figure 7 (p17), and as a result the 
Figure 7 data reads as an afterthought. Should present the mDia1 combination with mDia2 before 
dealing with mechanism. 
- The Integrin deletion and rescue data – strong data that show that it is integrin expression is 
limitiing upon mDiaKO – could be presented before the data showing that they are SRF targets. 

Absolute cell counts should be displayed in Figure 1D 
The transwell assay in Figure 2F could be performed on a monolayer of endothelial cells as well, which 
would better mimick the in vivo context and confirm whether the transendothelial migration of KO 
cells is defective in vitro 

Figure 3E/F. The MAL and SRF and gene expression data is not convincing. 
3E -The localisation figure needs quantitation, it is only shown as images of representative cells, but it 
should also be quantified over 50-100 cells in Figure 3E, with markers for the cytoplasm. 
3F - some these genes are MAL-controlled and others by the Elk1 transcription factor but it looks like 
there are Elk targets affected by mDia2KO (eg Egr1) and MAL targets that are not (eg Vcl, Tgln). What 
is going on? What happens if the serum-stimulated cells in 3E are analysed by RT-PCR? 
3F – relative expression data do not agree with Costello et al. which estimated relative WT/KO as 
Itgb2: 1.14; Itgal: 1.01; Itgam: 0.75. Please comment. 

Figure 3K – adhesion should also be performed on an ICAM substrate, which is one of the main 
physiological substrate of integrins alphaM/beta2 

Figure 4 is not acceptable, and the authors appear to be unaware of the classical SRF literature. 
- Figure 4A. The SRE sequence consensus is CC(A/T)6GG, which should be referenced properly. There 
is no good fit to this consensus in either of these two sequences. What do the arrows indicate? 
- Figure 4C/D. These are not consistent. Why does SRF cause a 3fold effect on ITGAM2+ in C, but an 
8fold effect in D? 
- Figure 4F Assuming that the CCTTGAGAGG is a consensus, there is NO published literature 
supporting the notion that the C at position +6 downstream would have any effect on SRF binding – 
why was this mutant chosen? Where are two sites, and what is the evidence that they increase DNA 
binding activity? 
- Figure 4G –Figure 4A bottom should be modified to match this. Where are the proposed SRE 
elements in this sequence, and why were the point mutations chosen? And what is a “G/C to T” 
mutation? 
- Figure 4H. This experiment is not explained. What is the significance of the +10% FBS? SRF is 
meant to bind constitutively to its targets. Surely the authors should see a signal in the “-“ lane too? A 
control with isotype-matched irrelevant antibody should be shown. What is the ChIP signal on a bona 
fide SRF target such as Acta2 or SRF itself? The authors argue that mDIa2 controls MAL to regulate 
these integrins, so should also show a MAL ChIP. Finally a critical point in the authors’ model is that 
mDia cells should show a defeect in MAL recruitment to SRF sites. This should be done. 
- Figure 4I. The CRISPR/Cas experiment is not adequately explained in the text or figure legends. It 
appears that there are multiple edits in the population (Figure 4D), yet a single population is shown 
for CD11B analysis - explain. Moreover, the effect of the deletion should be evaluated by qRT-PCR on 
ITGAM2, not by CD11 MFI. 

The competitive BMT experiment in Fig S3D made use of GFP labelled KO cells, tracking them 2 
months post-transplantation; are cells infected with GFP expressing viral particles, or is GFP 
transgenically expressed? if cells were transduced, to control for the fact that the cells could have lost 

the GFP during this time, the reverse labelling experiment should be performed, with unlabelled KO 
cells and GFP labelled WT cells; alternatively, a CD45.1/CD45.2 system could be used 

Figure S3E The second competitive BMT here has not been initiated with a 1:1 mix, but with only 20% 
of KO cells, which seems to be the ratio at which they are found 6 months after the first cBMT; please 
clarify in the legend 

Figures 2G&H It would have been good to have both WT & KO labelled with different dyes & injected 
into the same recipient. Limited information is presented - just two cells for the visual representation. 
The Figure needs to include details on numbers of cells counted per field, how many cells are there in 
5-6 fields? 

General Figure issues 
- Statistical data (p-values and n) are missing in some panels, they should be added to all figure 
panels, and the statistical test used should be stated in the legends 
- for the Kaplan-Meier survival curves, median survival for each condition (including control) should be 
clearly indicated in Figures 1F, 1G, 1H, 5J, 6G, 7B, 7H 
- the y-axis should not be discontinued in graphs, it artificially amplifies small differences between 
conditions: please correct accordingly Figures 1C, 4J, S6A, S6E 
- red and blue columns have been swapped in Figure 3H, please correct 

- the legends of the x-axis (0 to 7 months post-cBMT) in Figure 7A should be shifted towards the left, 
consistent with pIpC injections one month after transplantation 

- Figure S5, please specify in the legend what the blue shading indicates 

Results p10: 'mDia2 regulates HSPC engraftment through SRF signaling'. This statement is too strong 
– these results shows that loss of mDia2 in HSPC results in defective F-actin, cell polarity, adhesion, 
MAL-SRF pathway and integrin beta2 expression, but not that this is cause of the engraftment defect. 
Please rephrase. 

P10§3 should read ”… reported to affect cell functions through INactivation of the MAL-SRF pathway in 
cell based assays” 

reference 40 Is not about MAL. Replace. 

Numerous typos – correct please. 

Refs 27 and 66 are the same! 

The comment in the discussion that transcription factors binding sites are located 5' of the TSS on 
promoter regions, and that an intronic binding site is unusual, is simply not true. Remove. 

Reviewer #3 (Remarks to the Author): 

In their manuscript, Mei et al. demonstrate that mDia1 and 2 -proteins involved in the regulation of 
actin- are involved in HSPCs homing, engraftment, and repopulation potential of lethally-irradiated 
mice. Whereas the transendothelial migration of leukocytes has been studied in detail before, this is 
not the case for HSPCs. Therefore, the manuscript substantially adds to the field. However, how mDia 
deletion impacts transplanted HSPCs is not so clear. Below are some points aimed at sharing clarity in 
this regard and improve the manuscript. 

1) Endothelial cells lining BM sinusoids have been identiﬁed as critical for HSCs homing and 
engraftment [1]–[3] . However, it is well known that irradiation damages the niche, in particular 
sinusoidal endothelial cells [3]–[7]. Therefore it is unclear to what extent the findings reflect 
physiological migration rather than BM engraftment and reconstitution after myeloablation. Protocol in 
non-irradiated mice have been described [8]. Although the authors state that “the bone marrow 
vasculature remained intact”, Fig 2G-H shows disruption of BM vessels stained with CD31 which, 
together with increased vessel permeability, makes difficult to localize cells inside/outside of vessels. 
To address this, HSPC transmigration through vessels could be analyzed through intravital imaging in 
non-irradiated mice <15h after transplantation [8] for confirmation. 

2) While it is clear that mDia1 and mDia2 have a role in the maintenance of transplanted HSCs and 
their repopulation potential, the mechanism seems unclear. mDia deletion could lead to impairment in 
transendothelial migration, or in the maintenance of HSPCs inside of their niche by altering their 
adhesion to specific ECM protein (like fibronectin as you describe) or specific stromal cells, or both. For 
instance, the effects of mDia on HSC might be triggered by defective adhesion (rather than/besides 
transmigration impairment), which could disrupt HSC quiescence leading to exhaustion. The different 
possibilities and the most important mechanism should be better demonstrated and explained. It 
would be interesting to assess if Itg B2 re-expression can restore the proliferation pattern in 
transplanted HSCs mDia2-deficient HSCs. 

3) Fig 5: It would be better to show Itg surface expression by flow cytometry (rather than qpcr). 
Coculture assays of mDia2-deficient HSCs with endothelial cells (and not only fibronectin) with/without 
Itg B2 inhibition would help substantiate the contention that mDia2 regulates trans-endothelial 
migration through Itg B2. 

4) The introduction can be improved. The authors state that the bone marrow repopulation is a two-
step process, with homing occurring within 24h, followed by a long-term engraftment requiring 
lodgment to specific niches. However, the current bibliography indicates that immediately (1 hour) 
following a HSPC transplantation in lethally-irradiated mice the majority of donor cells locate in the 
central marrow region. This is followed by a rapid redistribution of the cells within 5h, resulting in a 
preferential seeding of LT-HSC-enriched cells closest to the bone surface, whereas more mature cells 
progressively engraft further away from bone [9], [10]. The bibliographic reference that is supposed 
to support the statement (ref 4 of the manuscript) is a review about the development of CXCR4 and 
VLA-4 inhibitors and how they may improve the utility and convenience of peripheral blood stem cell 
transplantation, which seems disconnected. Even if scarce, some studies regarding HSPC 
transmigration have been reported and should be presented in the introduction. 

5) Fig 3H: Does the frequency of cells expressing these integrins change upon mDia2 deletion? It has 
been reported that VE-cadherin regulates hHSPCs trans endothelial migration [11]. Does VE-cadherin 
cell surface expression change in mDia2fl/fl Vav-Cre mice? 

6) Fig 5A,F might be better placed in supplementary data for more streamlined presentation of the 
main data. 

7) Describing more clearly the relationships between CD11a/CD11b/CD18 and Itgb2 in the manuscript 
would help the reader. Few typos should be corrected throughout the manuscript. This sentence needs 
to be rephrased: “how transendothelial migration of the HSPCs in vivo are rarely investigated”. 

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