A subset of megakaryocytes regulates development of hematopoietic stem cell precursors

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

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Referee #2:
The manuscript by Lan et al focus on the role of megakaryocytes (Mk) in the onset of HSC embryonic development.They first characterize the presence of Mk by surface markers and show the specific expression of Pf4 to be used as a reporter and a cell deleter in the embryo.Next, they will use genetic DTA expression in these cells to assess the function of Mk in the early hematopoietic development.They find that hemogenic, preHSC and HSCs are affected after deletion of Mk and functional HSCs are also underrepresented.They characterize the cells by single cell RNAseq, identify CD226 as a marker for the functional subpopulation of Mk and show that Tnfsf14 can rescue the Mk depletion effect on hematopoietic production.While Mk are important in the maintenance of adult HSCs, the role of Mk during embryonic hematopoietic development is unknown.Thus, it is a novel and important observation, however some of the experiments are not convincing or at least they should address some issues to rule out some possible misinterpretation of the data.Major issues: 1-There have been previous reports showing that PF4 is also expressed in fetal liver and adult HSCs (PMID: 23300543).This possibility or the fact that it could apply to early HSC development seriously affects most of the conclusions of this work and has to be thoroughly investigated and ruled out.Although authors check the presence of the reporter in AGM (Fig S1D ) and lin-sca+CD201+E 12.5 FL cells, they should further demonstrate that it is not expressed in any of the pre-HSC and HSC populations.If this is done by flow cytometry, populations should be sufficiently represented.In addition, the reporter may not cover the same cells as the PF4-cre, thus cre expression in the PF4-Cre;Rosa-DTA HSC or preHSC populations in these embryos should be tested at different times of development from E10.5 HSC-like to E14.5 HSC and HPC development.2-Related to the previous point, the authors do not see any effect in the PF4-Cre;Rosa-DTA E10.5 embryos.Is that also due to expression of PF4 in HSPCs?They should explain why EHT at E10.5 is not affected but it is at E11.5.3-What is the effect of PF4+ cell deletion in the adult?Can the authors explain if this is not possible due to PF4 expression on adult HSCs? 4-All flow cytometry gates should be shown for all the analysis.Eg. in figure 1A, the Hoechst dots were previously gated for negative cells, but not sure why.Moreover, negative controls for PF4, but specially for CD42d should be included in supplementary.Other issues: 1-In many figures, the percentage of cells is shown in graphs, however this is a relative number and for examples in case of AGM, the percentage of Mk in total cells depends on the dissection area.They should show number per embryo equivalent shown in other figures.2-In the yolk sac determinations, are the vitellin/umbilical are included?Please specify.3-In text corresponding to figure1, the authors mention the analysis of CD31, but they do not show anywhere.Are Mk CD31?Please show.4-In figure 1I-1J, most Mk cells are circulating.They should distinguish between circulating and endothelial Mk.In addition, can they test whether Cd226 is different between both populations?An Immunostaining of CD226 would help.5-Transplantation assays: the engraftment in Fig 3H is very low.Although the reviewer recognizes the challenge of AGM transplants, if this is E11.5 could be improved.The authors use 200000 cells as support, but they may be outcompeting the few HSCs.It is hard to see a clear effect when transplantation is so low.In addition, the transplantation experiments are only meaningful if the expression of PF4 is totally rule out from the HSC population.6-The authors refer to HEC as CD31+CD44+, to my knowledge these are arterial cells enriched in HECs.7-English language should be revised 8-Discussion is quite repetitive of results, it should be shortened and focused.

Referee #3:
In this manuscript by Lan and colleagues, the authors use mouse genetic tools to examine a novel role for early megakaryocytes in regulating HSC emergence from hemogenic endothelium.The Pf4-Cre model was used to both label megakaryocytes in the developing embryos (YS, AGM, and FL) as well as delete megakaryocytes by crossing to stop-lox-DTA model.The authors nicely demonstrate that Pf4-labeled MKs are present near hemogenic regions during hematopoietic development, and that deletion of Mks results in a decrease in the frequency of both Mks as well as hematopoietic progenitors in the AGM, particularly at slightly later stages (E11.5).Leveraging scseq analysis, the authors identify two clusters of Mks, CD226+ and CD226-, and determine that the CD226+ Mks that express gene programs related to more mature Mk expression programs are the primary regulators of HSC emergence.Furthermore, they propose that CD226+ Mks regulate HSC emergence via Tnfsf14 secretion.Overall, there are many interesting observations in this manuscript.There are also some aspects that could be clarified further.
In general, there is a lot of variation in the N used for different experiments, and the number of independent experiments is not indicated.For example, in Fig. 2 N is quite high whereas in Fig 3A-E and some of supplemental Figures (S2, S4), N is quite low (N = 4), and it's unclear across experiments (particularly for experiments with low N) if data represent a single experiment/litter.This should be clarified and the use of multiple independent experiments/litters would strengthen the data in cases where N is low.Statistical tests being used should also be indicated for all figures.
In Figure 3, it is unclear why cKIT is not being used to define HSCs and HSPCs.That is standard of the field.3H, it is very difficult to draw conclusions from such a low number of mice transplanted (2/5 vs 1/3, where 5% is the metric for engraftment -this metric is very high).Furthermore, in the secondary transplants, it is unclear which primary recipient(s) were selected for secondary transplant.If only one primary recipient was selected, then, it is possible that the single recipient in the control group with the highest chimerism in primary accounts for differences in secondary (which are also very close, generally).These experiments need to be clarified and/or performed with better controls.

Also in Figure
In Figure 4, clusters are defined as CD34 Runx1/CKit.Are they both?Either?It is unclear from the text.
In the text referring to Fig. 6, when discussing CD226 expression, data are discussed in the results section that are not referred to (e.g."Flow cytometric analysis displayed that 29.8{plus minus}2.1% of Mks were positive and more than half of Mks were negative for CD226 in the E11.5 AGM region, which is different from E10.5 Mks and similar to E11.5 fetal liver."It would be helpful if data were referenced as they were mentioned in the text, as well as labeled above data/FACS panels for YS/AGM/FL (Fig 6E , for example, and throughout Fig. 6).
I think Fig S6S is an important piece of data (given that overall Mks don't decrease at E10.5) and the authors may want to consider including it in the main figure!Perhaps the weakest link of the paper is data in Figure 7.While the authors convincingly demonstrate that CD226+ MKs express Tnfsf14, and their data in Fig 6M -O also strongly suggest that Mks are releasing a secreted factor, the data presented stop short of demonstrating definitively that Tnfsf14 is the factor released by MKs that regulates HSC emergence.Enhancement of HSC emergence or rescue of the DTA phenotype by the addition of Tnfsf14 is not the same experiment as MK-specific genetic deletion of Tnfs14 in vivo.While that experiment is not absolutely required, some of the language in the manuscript and in the discussion should be toned down to reflect that that experiment has not been directly performed.
In the discussion: the AGM explant experiment in Fig. 4 suggested that depletion of Mks regulates B-cell output upon transplant.Early B-cell output may arise from distinct progenitors during development.Perhaps the authors want to speculate on that.
Dear Reviewer and Editor, Thanks a lot for your kind consideration and critical comments.In this version, we have addressed carefully all the points one by one.Please check these details in the following pages.In this manuscript, the authors investigated the potential role of megakaryocytes (Mks) on hematopoietic stem and progenitor cell development in the embryonic AGM region.They first applied platelet factor 4 (PF4)-Cre;Rosa-tdTmaoto+ mouse model to show that Mks were enriched in tdTomato+ cells.The authors then conducted a series of experiments to investigate embryonic hematopoiesis after MKs deletion by using PF4-Cre;Rosa-DTA(DTA) embryos, with endpoints of phenotypic HEC, pre-HSC, and HSC quantification, as well as repopulating HSCs.The authors also applied the ex-vivo co-culture system to investigate the role of Mks on endothelial to hematopoietic transition process.scRNA-seq was then performed to characterize Mks, where authors identified Mk progenitors and mature Mks based on the expression of cell surface marker CD226.Co-culturing with HECs with CD226+ Mks, rather than CD226-Mks, promoted the endothelial to hematopoietic transition, with endpoints of total CD45+ cells and CFU-C number.Cell Chat analysis predicted the lineagereceptor interactions between Mks and HECs.Finally, the authors chose TNFSF14 for functional validation experiment, showing that adding TNFSF14 promoted % CD45+ cells, increasing CFU-C number.Overall, the authors identified Mks as potential positive niche cells for embryonic hematopoiesis, in agreement with previous findings of Mks on HSCs in adult BM.However, there are some concerns about this study.
1.The main concern is that no significant difference was found between control and DTA groups in all in vivo transplantation assays, including characterize the cells by single cell RNAseq, identify CD226 as a marker for the functional subpopulation of Mk and show that Tnfsf14 can rescue the Mk depletion effect on hematopoietic production.
While Mk are important in the maintenance of adult HSCs, the role of Mk during embryonic hematopoietic development is unknown.Thus, it is a novel and important observation, however some of the experiments are not convincing or at least they should address some issues to rule out some possible misinterpretation of the data.

Major issues:
1-There have been previous reports showing that PF4 is also expressed in fetal liver and adult HSCs (PMID: 23300543).This possibility or the fact that it could apply to early HSC development seriously affects most of the conclusions of this work and has to be thoroughly

7-English language should be revised
Re: Thanks a lot for your comments.We have modified the English language by asking for the help of English native speakers.
8-Discussion is quite repetitive of results, it should be shortened and focused.
Re: Thanks a lot for your comments.We have modified and shortened the discussion.

Referee #3:
In this manuscript by Lan and colleagues, the authors use mouse genetic tools to examine a novel role for early megakaryocytes in regulating HSC emergence from hemogenic endothelium.
The Pf4-Cre model was used to both label megakaryocytes in the developing embryos (YS, AGM, and FL) as well as delete megakaryocytes by crossing to stop-lox-DTA model.The authors nicely demonstrate that Pf4-labeled MKs are present near hemogenic regions during hematopoietic development, and that deletion of Mks results in a decrease in the frequency of both Mks as well as hematopoietic progenitors in the AGM, particularly at slightly later stages (E11.5).Leveraging scseq analysis, the authors identify two clusters of Mks, CD226+ and CD226-, and determine that the CD226+ Mks that express gene programs related to more mature Mk expression programs are the primary regulators of HSC emergence.Furthermore, they propose that CD226+ Mks regulate HSC emergence via Tnfsf14 secretion.Overall, there are many interesting observations in this manuscript.There are also some aspects that could be clarified further.
In general, there is a lot of variation in the N used for different experiments, and the number of independent experiments is not indicated.For example, in Fig. 2  In Figure 3, it is unclear why cKIT is not being used to define HSCs and HSPCs.That is standard of the field.
Re: Thanks a lot for your comments.This comment is similar to the comment #6 from Reviewer Also in Figure 3H, it is very difficult to draw conclusions from such a low number of mice transplanted (2/5 vs 1/3, where 5% is the metric for engraftment -this metric is very high).Furthermore, in the secondary transplants, it is unclear which primary recipient(s) were selected for secondary transplant.If only one primary recipient was selected, then, it is possible that the single recipient in the control group with the highest chimerism in primary accounts for differences in secondary (which are also very close, generally).
These experiments need to be clarified and/or performed with better controls.
Re: Thanks a lot for your comments.Th is a similar comment to that from Reviewers 1 and 2. In the text referring to Fig. 6, when discussing CD226 expression, data are discussed in the results section that are not referred to (e.g."Flow cytometric analysis displayed that 29.8{plus minus}2.1% of Mks were positive and more than half of Mks were negative for CD226 in the E11.5 AGM region, which is different from E10.Perhaps the weakest link of the paper is data in Figure 7.While the authors convincingly

21st Feb 2024 1st Revision -Editorial Decision
Dear Dr Zhuan Li, Thank you for submitting your revised manuscript (EMBOJ-2023-115554R) to The EMBO Journal.Your amended study was sent back to the three referees for their scientific re-evaluation, and we have received detailed comments from all of them, which I enclose below.
As you will see, the experts state that the work has been substantially improved by the revisions and they are now in favour of publication, pending minor revision.Thus, we are pleased to inform you that your manuscript has been accepted in principle for publication in The EMBO Journal.
Please consider the remaining minor comment of referees #2 and #3 regarding data annotation carefully and amend the manuscript figures and text accordingly.Also, we now need you to take care of a number of issues related to formatting and data presentation as detailed below, which should be addressed at re-submission.
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Referee #2:
I acknowledge the work that the authors have performed for the revisions of the manuscript and the transplantation data is slighly improved, however the related text should be revised in page 6: Whereas only 3 out of 6 mice receiving control AGM cells were engrafted and 1 out of 5 recipients were engrafted in the DTA group at 4 weeks post-transplantation.If the authors have the data of engraftment of Bone Marrow, I would recommend them to include it.BM can show better engraftment.
I also agree that possible expression of PF4 in the HSCs seems not too important.
There is one thing that worries me with the revisions, this is the inclusion of ckit marker in the HEC population.The idea of using ckit is just to restric hematopoietic cells to intraortic cluster cells which all express ckit, but, to my knowledge, HECs do not express ckit yet.In any case, it is hard to understand which populations have they measured in FIG3 I-N since the gating strategy has not been included.
Their definition for each subpopulation is: HEC (CD41-CD45-CD31+CD44+c-Kit+), pre-HSC I (CD41lowCD45-CD31+CD201+c-Kit+), and pre-HSC II (CD45+CD31+CD201+c-Kit+) If they use ckit+ for their definition of HECs, they are referring to a similar pre-HSC I population, likely different than the one that includes CD201, but they are excluding HECs.They need to count CD41-CD45-CD31+CD44+ckit-for HECs!In any case, they should include one example of the gating for all these populations.Referee #3: The authors have made a strong effort to address all of the reviewers' previous concerns and have improved the manuscript.Most notably, the authors have increased the number of mice used in transplantation assays and have also clarified number of mice and independent experiments performed throughout the manuscript.
For the transplant data in Figure 3, it's still not entirely clear why a cutoff of 5% engraftment was chosen to indicate "engrafters" -this is a very high cut-off and seems somewhat random.Can the authors instead report total engraftment for both groups?Are these differences statistically significant (or was a test of significance applied?)I think that although these experiments are technically challenging, the data are best presented as transparently as possible.
Data for AMG explant transplants are not correctly referenced in the text (reference is missing).
While the additional data with explant culture transplants in Figure 7 provide some additional support for the author's claim that tnfsf14 regulates HSC emergence, the experimental numbers for transplantation experiments are still very low -it is very difficult to make a determination from one engrafted experiments vs zero in the DTA only group at 20 weeks.I do feel that the authors still need to directly indicate the limitation of this experiment and the conclusions of these data in their discussion.The final paragraph in their discussion still suggests that they have shown directly that MKs regulate HSC emergence through the production of tnfsf14.I think it would be more judicious to state what was discovered and what was not shown directly.revised manuscript.Thirdly, no significant statistics were found between both groups based on all chimerism data, that is why we didn't add any statistic asterisk.
Data for AMG explant transplants are not correctly referenced in the text (reference is missing).
Re: Thanks a lot for your suggestion.We have modified them.
While the additional data with explant culture transplants in Figure 7 provide some additional support for the author's claim that tnfsf14 regulates HSC emergence, the experimental numbers for transplantation experiments are still very low -it is very difficult to make a determination from one engrafted experiments vs zero in the DTA only group at 20 weeks.I do feel that the authors still need to directly indicate the limitation of this experiment and the conclusions of these data in their discussion.
The final paragraph in their discussion still suggests that they have shown directly that MKs regulate HSC emergence through the production of tnfsf14.I think it would be more judicious to state what was discovered and what was not shown directly.
Re: Thanks a lot for your critical point.We have modified the related text.We emphasized HPC, but not HSCs.

28th Feb 2024 2nd Revision -Editorial Decision
Dear Dr Zhuan Li, Thank you for submitting the revised version of your manuscript.I have now evaluated your amended manuscript and concluded that the remaining minor concerns have been sufficiently addressed.
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Fig 3H and Fig 4C&D.In addition, there are only 5 control mice and 3 mice in DTA group in Fig 3H, making it difficult to make a solid conclusion.The authors observed the declined phenotypic HECs and pre-HSCs after Mk deletion.However, cell surface markers cannot completely purify functional HSCs and pre-HSCs.Therefore, as transplantation is the gold-standard assay to quantify functional HSCs, whether Mk deletion affects HSC development is still unclear.Instead of doing more experiments to investigate the role of Mks on functional HSC development, the authors could weaken their statement like 'Mk deletion influences EHT, as evidenced by declined cd45+ cells and CFU number.'Re: Thanks a lot for your comments and kind consideration.Approximately one adultrepopulated HSCs were observed in the AGM region (Medvinsky et al., Development, 2011), and in direct transplantation experiments, normally around 60% of recipients were engrafted even if two embryo equivalent AGM cells were transplanted.Meanwhile, we have added more recipients for testing HSC and the maturation of pre-HSCs into HSCs.In direct transplantation, 3/6 recipients were engrafted (>5%) after 4 weeks transplantation in the control group, but only one out of 5 recipients in the DTA group.After 20 weeks transplantation, no recipients (0/5) were engrafted in the DTA group, but 5/6 recipients in the control group with the average chimerism 19.04±12.60%(the following Figure A), suggesting the possibility of reduced HSC activity.Please check all the details in Figure 3H and the modified text in the revised manuscript.Explant cultures are used to test the HSC precursors (including pre-HSCs).7/8 recipients had positive engraftment in the control group, however, 5/9 recipients were engrafted in the DTA group at 4 weeks post-transplantation.After 16 weeks transplantation, the chimerism was deceased in the DTA group compared to control group (the following Figure B).These data indicate the reduction of pre-HSC to HSC maturation in the DTA group.Please check all the details in the Figure 4C-4D and the modified text in the revised manuscript.

2 .Referee # 2 :
Figure A), similar to the treatment of Tnfsf14 and/or Tnfa.Methylcellulose culture data showed an increase in total CFU-C number from cocultures in the Mks and/or Macs group (the following Figure B) compared to HEC group.However, the existence of Mac and Mk in Triple group failed to enhance the production of CFU-C as well as CD45 + cells compared to the group at the presence of each of them.These data suggest the promotion of Mks or Macs in EHT process.Since we didn't see the obvious enhancement by combining Macs and Mks, no transplantation was performed after coculture.In addition, we have modified the texts in the revised manuscript.Please see the details in Figure EV5I-5J in the revised manuscript.
investigated and ruled out.Although authors check the presence of the reporter in AGM (Fig S1D) and lin-sca+CD201+E 12.5 FL cells, they should further demonstrate that it is not expressed in any of the pre-HSC and HSC populations.If this is done by flow cytometry, populations should be sufficiently represented.In addition, the reporter may not cover the same cells as the PF4-cre, thus cre expression in the PF4-Cre;Rosa-DTA HSC or preHSC populations in these embryos should be tested at different times of development from E10.5 HSC-like to E14.5 HSC and HPC development.Re: Thanks a lot for your comments.Firstly, we have checked the expression of tdTomato (tdT+) in the earlier stage of hematopoietic development E10.5 PF4-Cre; Rosa-Tdt AGM region.The percentage and cell number of Tdt + cells in the EC and pre-HSCs are hardly detected (the following Figure A-C), in line with E11.5 AGM cells(Figure 1G-1H).Secondly, according to your suggestion, we have checked the expression of HSCs in the fetal liver from E12.5 to E14.5 by using the cocktails of HSC (E12.5 HSCs, Lin − Sca-1 + Mac-1 low CD201 + ; E13.5-14.5 HSCs, Lin − Sca-1 + c-Kit + CD150 + CD48 − ).TdT + cells were found in the E12.5-14.5 fetal liver (the following Figure A-B).A few tdT + HSCs were found in E12.5-14.5 fetal liver, although the percentage and cell number of tdT + HSCs was very low (the following Figure C-E), lower than that in the previous report (Calaminus et al., PLOS one, 2012).In the fetal liver and further late stage, we can't exclude the effects of PF4-Cre labeling HSCs non-specifically.are right.PF4-Cre labels distinct cells between AGM and fetal liver.In the AGM region, PF4-Cre is specifically expressed in the MKs according to our data.However, in the fetal liver, PF4-Cre;tdT labels not only Megakaryocytes but also some parts of HSCs from E12.5-14.5 fetal liver.2-Related to the previous point, the authors do not see any effect in the PF4-Cre;Rosa-DTA E10.5 embryos.Is that also due to expression of PF4 in HSPCs?They should explain why EHT at E10.5 is not affected but it is at E11.5.Re: Thanks a lot for your critical comments.As we showed in the last comment above, no tdT + signals were found in the E10.5-E11.5 ECs and pre-HSCs of AGM region (in the following Figure and Figure 1G-1H).All these data are in Figure 1G-1H ang Figure EV 1H-1J in the revised manuscript.From our data, the percentage and cell number of Mks were very low and decreased from E10.5 to E11.5 in the AGM of PF4-Cre;RosaDTA compared to the control group in Figure 2A-2G.The reduction started in the E10.5 AGM region, but not in the E9.5 AGM.And the effects of Mks on the hematopoiesis developed gradually and need some time.Especially, our data showed that the more mature Mks (CD226 + Mks) played roles in the EHT.Mks require a mature pattern and then affect the EHT process depending on the time course.That is the reason we didn't observe the change in the E10.5 or even earlier stage of AGM region.More details were modified in the revised manuscript.3-What is the effect of PF4+ cell deletion in the adult?Can the authors explain if this is not possible due to PF4 expression on adult HSCs?Re: Thanks a lot for your critical comments.As we mentioned above (comments 1-2), although some HSCs in the fetal liver (E12.5-14.5)were positive for tdT + ;PF4-Cre, which is lower than that in the previous publication (Calaminus et al., PLOS one, 2012), but we can't exclude the effects by PF4-non specific labeling HSCs.Calaminus et al. reported that a higher percentage of HSCs from bone marrow expressed tdT + , along with our data, we didn't continue to check the expression in bone marrow.The effects of PF4 deletion will be not specific to Mks in bone marrow.Cre is not specific to HSCs in the bone marrow, it is specific to Mks in the embryos, which is not expressed in the pre-HSC/HSCs in the AGM region.Taken together, PF4-Cre is useful for checking the Mks in the earlier stage of embryos, not suitable for adults.4-All flow cytometry gates should be shown for all the analysis.Eg. in figure 1A, the Hoechst dots were previously gated for negative cells, but not sure why.Moreover, negative controls for PF4, but specially for CD42d should be included in supplementary.Re: Thanks a lot for your kind comments.We gated live cells by Hoechst negative and showed the expression of Hoechst and other antibodies.To be more clear, we have labeled the live cell fractions by Hoechst -in Figure 1A.According to your suggestion, flow analysis data was added as the negative control and PF4-Cre;tdT AGM regions(in the following Figures A and B, respectively).These data are in Figure EV1D, please check all the details in the revised manuscript.Other issues: 1-In many figures, the percentage of cells is shown in graphs, however this is a relative number and for examples in case of AGM, the percentage of Mk in total cells depends on the dissection area.They should show number per embryo equivalent shown in other figures.Re: Thanks a lot for your comments.The total cell number of PF4-Cre;Rosa-tdT AGM and yolk sac was analyzed (in the following Figure A-B), which was added to Figure EV1A.Additionally, we have shown the cell number of AGM, YS, FL, which is comparable between control and DTA group (AGM in Figure 2A, YS in Figure EV2A and E10.5-E12.5 FL in Figure EV2B).

2 - 3 - 4 -)RFP+ 6 -
Figure B-D).So most Mks are circulating cells and CD226 fails to distinguish them due to few Mks interacting with endothelial cells.Please see all the details in Figure EV 4L-4N in the revised manuscript.
N is quite high whereas in Fig 3A-E and some of supplemental Figures (S2, S4), N is quite low (N = 4), and it's unclear across experiments (particularly for experiments with low N) if data represent a single experiment/litter.This should be clarified and the use of multiple independent experiments/litters would strengthen the data in cases where N is low.Statistical tests being used should also be indicated for all figures.Re: Thanks a lot for your comments.In Fig2, we have shown the embryos from more than 8 times experiments.Because the percentage is very low in the pre-HSC I and II fromAGM region, we repeated more times than that in fetal liver.Meanwhile, we also repeated these experiments for fetal liver analysis (the following Figure A-F).Please see the details in Figure 3A-3F.Additionally, we added all the details in methods.The experimental times, and statistics were shown in the figure legends.Please see all the details in the revised manuscript.

In Figure 4 ,
HSCs were observed in the AGM region (Medvinsky et al., Development, 2011), and in direct transplantation experiments, normally around 60% of recipients were engrafted even if two embryo equivalent AGM cells were transplanted.Meanwhile, we have added more recipients for testing HSC and the maturation of pre-HSCs into HSCs.In direct transplantation, 3/6 recipients were engrafted (>5%) after 4 weeks transplantation in the control group, but only one out of 5 recipients in the DTA group.After 20 weeks transplantation, no recipients (0/5) were engrafted in the DTA group, but 5/6 recipients in the control group with the average chimerism 19.04±12.60%(the following Figure A), suggesting the possibility of reduced HSC activity.Because of no engrafted recipients in the DTA group, we didn't perform the secondary transplantation.Please check all the details in Figure 3H and the modified text in the revised manuscript.clusters are defined as CD34 Runx1/CKit.Are they both?Either?It is unclear from the text.Re: Thanks a lot for your comments.In Figure 4B, clusters were calculated for the number of both CD34 + Runx1 + and CD34 + c-Kit + clusters.In supplementary Figure 4A, the cell number of CD34 + Runx1 + or CD34 + c-Kit + cluster was displayed, respectively.The text was modified clearly, please see the modified manuscript.

5
Fig. 6).Re: Thanks a lot for your comments.We have added Figure 6E-6F and Figure EV4O-4Q in the revised manuscript.
MKs express Tnfsf14, and their data in Fig 6M-O also strongly suggest that Mks are releasing a secreted factor, the data presented stop short of demonstrating definitively that Tnfsf14 is the factor released by MKs that regulates HSC emergence.Enhancement of HSC emergence or rescue of the DTA phenotype by the addition of Tnfsf14 is not the same experiment as MK-specific genetic deletion of Tnfs14 in vivo.While that experiment is not absolutely required, some of the language in the manuscript and in the discussion should be toned down to reflect that that experiment has not been directly performed.Re: Thanks a lot for your comments.This is similar to the comment 2 from reviewer 1. Yes, we only foucus one of secreted factors(Tnfsf14) to test the rescue function in vitro.It's better to establish MK specifically deleted Tnfs14 mouse model for checking the roles of Tnfsf14 derived from Mks.It is a pity no mouse model is available until now and This will be in plan in future.We have added Tnfsf14 into explant culture for checking the pre-HSC maturation.At 4 weeks transplantation, 2 out of 4 recipients were engrafted with chimerism (22.9% and 35.3%) in the DTA group by Tnfsf14 treatment, similar to control group (3/4 engrafted recipients with chimerism 23.7%, 10%, 7%) and one recipient with 16.4% chimerism in the DTA group (the following Figure A).Furthermore, three out of four recipients were engrafted with high chimerism in the control group, and in the DTA+Tnfsf14 group, one out of three recipients (unfortunately, one recipient died before 16 weeks transplantation) was engrafted with high chimerism (87.9%) after 16 weeks transplantation, however, the chimerism of one positive recipient (1/4) was 7.5% ( the following Figure B).Although the rescued function was not obvious, the possible trend existed with the Tnfsf14 treatment in the DTA group compared to the control group, likely due to the limited recipients.Meanwhile, we have modified the language and shortened the discussion according to your suggestion.In the discussion: the AGM explant experiment in Fig. 4 suggested that depletion of Mks regulates B-cell output upon transplant.Early B-cell output may arise from distinct progenitors during development.Perhaps the authors want to speculate on that.Re: Thanks a lot for your comments.You are correct that some parts of B cells derive from other B cell progenitors in the yolk sac during development (Yoshimoto et al., PNAS, 2011).We have added more recipients and modified the lineage output in the Figure 4E.We have corrected the text and please see all the details in the revised manuscript.
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