Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta

Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta–gonad–mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5–E11.5 aorta–gonad–mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta–gonad–mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

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In particular, it would be essential to: A) Acknowledge and discussion related literature: Reviewer 2 "Previous studies by Ding https://doi.org/10.1002/dvdy.23923 with knockout animals revealed that loss of PDGFRa had no effect on fetal liver HSCs. However, present study has found a profound effect of Pdgfra knockout or inhibition with APA5 antibody on HSC and blood production. How this could be explained?"

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Reviewers' Comments:
Reviewer #1: Remarks to the Author: The authors investigated whether cells with mesenchymal stem cell-like activity, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta6 gonad-mesonephros (AGM) and contribute to the structural development of the dorsal aorta and endothelial to hematopoietic transition (EHT). Using transgenic mice, they identify a lineage hierarchy for AGM stromal cells and traced E10.5/E11.5 aortic endothelium and HSCs to mesoderm derived (Mesp1) PDGFRA+ stromal cells (Mesp1der PSCs). They determined that these cells dominate the sub-endothelial and ventral stroma in the E10.5-E11.5 AGM but by E13.5 were replaced by neural crest (Wnt1) derived PDGFRA+ stromal cells (Wnt1der PSCs). Co-aggregating non-hemogenic embryonic and adult endothelial cells with Mesp1der PSCs but not with Wnt1der 13 PSCs resulted in activation of a hematopoietic transcriptional program in embryonic and adult endothelial cells accompanied by EHT and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA signaling or BMP, WNT, NOTCH signaling interrupted this reprogramming event. The authors postulate that such endothelial and PSC interactions may prove beneficial in translating generation of HSC in vitro. This is novel insights into an area that has been of interest to developmental and stem cell scientists for many years. They directly contributed to new knowledge about the origins of stromal cells that regulate the endothelial cell behavior in becoming hemogenic endothelium with HSC emergence. The methods used are state of the art and appropriate controls are used throughout. The flow of studies is easy to follow and the salient points summarized at key steps. The conclusions are supported by the data presented.
A key deficiency in the presentation of the results are the sporadic use of statistical tests. To bolster confidence, the authors should statistically analyze all quantitative data for level of significance.
There are some questions and points to consider: 1) The authors should quantitate the total number of CFU-F present in the AGM region over the stages E9.5-13.5. It is important to know the total number present in a tissue (at each embryonic stage) and then compare this number to the total number recovered in the various sorted cell populations. Only then can the authors claim (page 6 line 20/21) "Taken together, these data show that PDGRFA marks all CFU-Fs in the E11.5 AGM....
2) The authors show numerous growth curves (example Fig. 1E) but average results of 3 studies. All of the individual growth curves for n=3 should be shown to assess the variance among the individual groups.
3) There is no indication how many times the clonal analyses (Fig. 1F) were performed. 4) Fig. 1J and Movie 2 do not convince this reviewer that the cells formed lumenized vessel structures in vivo. One would need to see intraluminal red blood cells or evidence of systemically infused fluorescent dyes to be sure these are vessels.

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Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Fig. 2C(ii) does not indicate which are primary or secondary recipient mice. 6) The doses used in combination in Ext. data Fig. 6C (i-ii) were near dose limiting when used as single agents. One would question whether the inhibition of CFU-C and LT-HSC activity is because all of the cells died. Some evidence of cell viability at those combined doses (or lowered combined doses that assure cell viability) are required.

5)
Reviewer #2: Remarks to the Author: In this manuscript, Chandrakanthan et al. investigated the origin and properties of stromal cells in AGM region. They show that periaortic stromal cells in this location are derived from Mesp1+ progenitors (Mesp1der) but later replaced by stromal cells derived from neural crest (Wnt1der). Coaggregation of Mesp1der or Wnt1der stromal cells with endothelial cells revealed that aggregates of Mesp1der stromal cells with AGM endothelium including nonhemogenic E13.5 endothelium and endothelium from hearts and aorta of 8-12 weeks old animals produced HSCs with robust engraftment potential. This effect was observed only with E10.5 or E11.5 Mesp1der stromal cells but not with E13.5 Wnt1der stromal cells. In addition, authors demonstrated that treatment of endothelial/stromal aggregates or ex vivo cultured E10.5 embryos with PDGFRa inhibitor reduced blood and HSC formation. The differences in Mesp1der stromal cells from ventral and dorsal fraction of were also revealed. Overall, these studies are well executed and make a significant novel contribution to our understanding of HSC development.
1. Previous studies by Ding https://doi.org/10.1002/dvdy.23923 with knockout animals revealed that loss of PDGFRa had no effect on fetal liver HSCs. However, present study has found a profound effect of Pdgfra knockout or inhibition with APA5 antibody on HSC and blood production. How this could be explained?
2. Another important difference between findings presented in this manuscript and the Ding paper is the developmental window at which contribution of PAGFRa+ cells to blood cells was observed. Ding found no contribution of PDGFRa+ cells to HSCs when tamoxifen was injected at E9.5. In contrast, this study revealed a substantial labeling blood cells following tamoxifen treatment at E9.5. This should be discussed.
3. PDGFRa-positive cells in the AGM region of E9.5 embryos could still contain multipotential precursors, including Flk1+ mesodermal precursors which are known to contribute to HSC in the embryo proper. What is the proportion of FLK1+ cells within PDGFRa+ cells at E9.5 in the mouse model used in this study? 4. Tracing experiments demonstrated that blood and hemogenic endothelium originate from PDGFRa+ cells found in periaortic mesenchyme. Any evidence that these cells possess hematopoietic potential in vitro, for example in coculture with OP9? 5. How embryos were exposed to APA5 and APB5 antibodies. Were they injected with antibodies or 7 Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
antibodies simply added to ex vivo cultures? In the later scenario, any evidence that antibodies can reach AGM by diffusion?
6. Page 11 lines 20-23. Pdgfra-nGFP+/Mesp1-DsRed mice are introduced here for the first time. However, it is unclear whether Mesp1-DsRed is knock-in reporter or it traces Mesp1-Cre recombination. If Mesp1-DsRed is knock-in reporter, why DsRed signal is seen in blood and endothelial cells. Expression of Mesp1 is downregulated in endothelial and blood cells.
7. Transcriptional analysis of adult heart and aortic endothelium before and after coaggregation would be very interesting.
Reviewer #3: Remarks to the Author: Chandrakanthan, V. et al. investigated the origin and contribution of Pdgfra+ cells to hematopoietic stem cell (HSC) development in the dorsal aorta. The study shows Pdgfra+ cells from mesodermal origin contribute to endothelial cells and HSC in the dorsal aorta, and to sub-endothelial mesenchyme at the time HSC emerge. This mesoderm-derived mesenchyme supports endothelial-to-hematopoietic transition (EHT) from hemogenic endothelium and is later replaced by neural crest-derived Pdgra+ cells that lack this ability, coinciding with the cessation of hematopoiesis in the DA. Mesoderm-derived Pdgfra+ cells not only enable EHT from hemogenic endothelium but also from non-hemogenic embryonic and adult endothelial cells, in a process dependent on Pdgfra signaling and BMP, WNT and Notch pathways.
The manuscript includes interesting, novel findings and builds upon previous reports, such as the contribution of Mesp1-mesoderm and Pdgfra+ cells to the dorsal aorta and to adult hematopoiesis, with transplantation assays that bring more robustness to the previous reports (references below) that were based on lineage tracing. The findings that Mesp1-derived PSC can instruct adult EC to undergo EHT and generate LT-HSC in vitro are interesting and significant, though this reviewer suggests the inclusion of more experimental details and the performance of experiments that might confer more robustness to the findings. Several results show no statistical analysis, or they lack the reference that no statistical significance was observed. Overall, the manuscript is well written and presents highquality and significant data, but the clarification of some points and the performance of additional experiments, suggested below, may confer more robustness and validity to the findings and conclusions of the manuscript.  Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 7. The authors' findings that co-aggregation of DA endothelium and, particularly adult endothelium with embryo-derived Mesp1der PSC instructs EHT and HSC emergence are very interesting. There are experimental details and inclusion of more data that can clarify/strengthen the findings: 7.1. There is inconsistency in the cell numbers presented to form the co-aggregates in the figure legends and the methods section. 7.2. The authors should include the flow plots showing how the sorting strategy for isolation of EC and DSRed+ PSC was performed (including the fluorophores used), as well as transplants with freshly isolated EC populations to discard any possibility of contamination with UBC-GFP+ hematopoietic cells in the input population that could have expanded in co-culture with Mesp1-PSC. 7.3. In addition to the confocal imaging showing CD45 expression within the co-aggregate, the authors should include flow cytometry data after culture to enable a more precise characterization of the cell populations and quantification. 7.4. The authors should include the efficiency of EC conversion to HSC. Are significant differences being observed between hemogenic EC and non-hemogenic EC? 7.5. Although EC derived from the IVC were able to originate HSC, the levels of chimerism after transplantation were low, suggesting some EC might not be able to become hemogenic. Is EHT similarly efficient between arterial and venous EC or in adult EC from a distinct organ, such the brain? 7.6. The authors should include the results from the transplants of co-aggregates formed with DsRedneg PSC in Figure4. 7.7. Richard, C et al., (doi: 10.1016/j.devcel.2013.02.011.) have shown that the subaortic mesenchyme controls endothelial Runx1 in the dorsal aorta, enabling EHT. The authors show an upregulation of Runx1 during culture suggesting that a similar mechanism might be occurring. The use of EC isolated from a Runx1 reporter mouse would unequivocally demonstrate that EC turn on endogenous Runx1 expression and undergo EHT and it would allow a quantification of how many non-Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. hemogenic EC undergo reprogramming to hemogenic fate. 7.8. The scRNA-Seq suggests that different hematopoietic lineages are derived during the reprograming process. Do the authors think that HSC are derived within the 4 days of coculture or an HSC precursor that is able to complete the process of maturation in vivo, or both? Flow cytometric characterization of the co-aggregates and transplantation of the different cell subsets within the coaggregate could bring more clarity of what cells are endowed with long-term reconstitution ability, or if transplantation of the entire co-aggregate is required to support engraftment after transplantation.

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13
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---------Please don't hesitate to contact NCB@nature.com should you have queries about any of the above requirements ---------Author Rebuttal to Initial comments Editorial comments: A) Acknowledge and discussion related literature: Reviewer 2 "Previous studies by Ding https://doi.org/10.1002/dvdy.23923 with knockout animals revealed that loss of PDGFRa had no effect on fetal liver HSCs. However, present study has found a profound effect of Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Pdgfra knockout or inhibition with APA5 antibody on HSC and blood production. How this could be explained?" "Another important difference between findings presented in this manuscript and the Ding paper is the developmental window at which contribution of PAGFRa+ cells to blood cells was observed. Ding found no contribution of PDGFRa+ cells to HSCs when tamoxifen was injected at E9.5. In contrast, this study revealed a substantial labeling blood cells following tamoxifen treatment at E9.5. This should be discussed."

B) Improve quantification and statistical analysis:
Reviewer 1 "The authors should quantitate the total number of CFU-F present in the AGM region over the stages E9.5-13.5. It is important to know the total number present in a tissue (at each embryonic stage) and then compare this number to the total number recovered in the various sorted cell populations. Only then can the authors claim (page 6-line 20/21) "Taken together, these data show that PDGRFA marks all CFU-Fs in the E11.5 AGM...." "The authors show numerous growth curves (example Fig. 1E) but average results of 3 studies. All of the individual growth curves for n=3 should be shown to assess the variance among the individual groups." "There is no indication how many times the clonal analyses (Fig. 1F) were performed." Reviewer 3 "...Several results show no statistical analysis, or they lack the reference that no statistical significance was observed..." Response: We have addressed these requests in the revised manuscript. Importantly, all our data have now been reviewed and analysed by a statistician-Dr Jake Olivier, Professor of Mathematics and Statistics at UNSW Sydney, who is now an author on this manuscript. The raw data corresponding to each figure panel and statistical method and codes applied to generate significance have now been included as supplemental worksheets. The figure legends and methods sections have been updated. Please see the point-by-point rebuttal to reviewer comments.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

C) Provide sufficient experimental details as requested by Reviewer 3:
"The authors should include information of how many embryo equivalents per recipient are used for direct transplantation of AGM cells (Figure 2)." "The authors' findings that co-aggregation of DA endothelium and, particularly adult endothelium with embryo-derived Mesp1der PSC instructs EHT and HSC emergence are very interesting. There are experimental details and inclusion of more data that can clarify/strengthen the findings..." Response: We have addressed all these points by including new experimental data. Please see the point-by-point rebuttal to reviewer comments. D) All other referee concerns pertaining to strengthening existing data, providing controls, methodological details, clarifications, and textual changes as applicable should also be addressed.

Reviewer comments (point-by-point response)
Reviewer #1: Remarks to the Author: The authors investigated whether cells with mesenchymal stem cell-like activity, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta6 gonad-mesonephros (AGM) and contribute to the structural development of the dorsal aorta and endothelial to hematopoietic transition (EHT). Using transgenic mice, they identify a lineage hierarchy Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. for AGM stromal cells and traced E10.5/E11.5 aortic endothelium and HSCs to mesoderm derived (Mesp1) PDGFRA+ stromal cells (Mesp1der PSCs). They determined that these cells dominate the sub-endothelial and ventral stroma in the E10.5-E11.5 AGM but by E13.5 were replaced by neural crest (Wnt1) derived PDGFRA+ stromal cells (Wnt1der PSCs). Co-aggregating non-hemogenic embryonic and adult endothelial cells with Mesp1der PSCs but not with Wnt1der 13 PSCs resulted in activation of a hematopoietic transcriptional program in embryonic and adult endothelial cells accompanied by EHT and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA signaling or BMP, WNT, NOTCH signaling interrupted this reprogramming event. The authors postulate that such endothelial and PSC interactions may prove beneficial in translating generation of HSC in vitro. This is novel insights into an area that has been of interest to developmental and stem cell scientists for many years. They directly contributed to new knowledge about the origins of stromal cells that regulate the endothelial cell behaviour in becoming hemogenic endothelium with HSC emergence. The methods used are state of the art and appropriate controls are used throughout. The flow of studies is easy to follow, and the salient points summarized at key steps. The conclusions are supported by the data presented.
A key deficiency in the presentation of the results are the sporadic use of statistical tests. To bolster confidence, the authors should statistically analyze all quantitative data for level of significance. Response: We apologise for any omissions in this regard. To properly address this concern, we have collaborated with Dr Jake Olivier, Professor of Mathematics and Statistics at UNSW Sydney to perform a root and branch analysis of all the quantitative data.
The raw data corresponding to each relevant figure panel is now included as a separate sheet in the data file (Supplementary Table with numerical source data) along with the relevant statistical tests and codes that was used to assess the level of significance. The figure legends and methods section have also been revised accordingly. Professor Olivier is now a co-author of this manuscript.
There are some questions and points to consider: 1) The authors should quantitate the total number of CFU-F present in the AGM region over the stages E9.5-13.5. It is important to know the total number present in a tissue (at each embryonic stage) and then compare this number to the total number recovered in the various sorted cell populations. Only then can the authors claim (page 6-line 20/21) "Taken together, these data show that PDGRFA marks all CFU-Fs in the E11.5 AGM....". Response: Thank you for this important suggestion. We have now assessed bulk CFU-Fs at these embryonic time points (Extended data figure 1B(ii); page 6; lines 1-3). As shown in Figure 1D, bearing in Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. mind that sorted cells are subjected to additional stress, in contrast to PDGFRA+ cells, PDGFRA-cells in the AGM produced no large or small colonies ( Figure 1D).
We have rephrased the sentence to "Taken together, these data show that CFU-F potential in the E11.5 AGM resides largely within PDGFRA+ cells and that Nestin expression marks a sub-population of PDGFRA + cells with more restricted CFU-F and differentiation potential" (page 6; lines 20-23).
2) The authors show numerous growth curves (example Fig. 1E) but average results of 3 studies. All of the individual growth curves for n=3 should be shown to assess the variance among the individual groups. Response: Thank you for this suggestion. We have revised all the relevant figures ( Figure 1E, 4C (iii), Extended data figures 1H and 2C(iii)) accordingly.
3) There is no indication how many times the clonal analyses (Fig. 1F) were performed. Response: We apologise for this omission. The primary colonies were from triplicate experiments and 144-385 single cells were plated from 54 micro or 30 small or 27 large colonies, to assess secondary to quaternary CFU-Fs.
This information is now summarised in the figure legend and detailed in the methods and supplemental numerical source data table. Fig. 1J and Movie 2 do not convince this reviewer that the cells formed lumenized vessel structures in vivo. One would need to see intraluminal red blood cells or evidence of systemically infused fluorescent dyes to be sure these are vessels. Response: We performed this experiment by mixing 250,000 PDGFRA + Nestin-GFP -CD31 -PDGFRB -FACsorted cells from E11.5 Nestin-GFP + AGMs with matrigel (100uL), followed by sub-cutaneous implantation in the nuchal and inguinal regions of mice. Vascularised plugs were removed at 4-weeks post-implantation, fixed with 4% PFA, washed and embedded in OCT prior to cryosection, immunofluorescence staining and confocal microscopy. Unfortunately, most intraluminal red blood cells would have been lost during processing and perfusion of fluorescent dyes in conjunction with live imaging had not been planned. However, we have now included longitudinal and cross sections of lumenized vessel-like structures (Extended data Figure 1I) to support the data in Figure 1J. We have also amended the text from vessels to vessel-like structures (page 7; line 15). Fig. 2C(ii) does not indicate which are primary or secondary recipient mice. Response: We apologise for this omission. This Figure panel (now Fig. 2D (ii)) has been appropriately labelled.

5)
6) The doses used in combination in Ext. data Fig. 6C (i-ii) were near dose limiting when used as single Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. agents. One would question whether the inhibition of CFU-C and LT-HSC activity is because all of the cells died. Some evidence of cell viability at those combined doses (or lowered combined doses that assure cell viability) are required. Response: Thank you for this observation and comment. Cell viability was assessed by flow cytometrythere was no loss of viability with inhibitors even at the highest concentration (n=3).
This finding is presented in Extended data Figure 6C (i) of the revised manuscript (page 19, lines 8-10).

Reviewer #2:
Remarks to the Author: In this manuscript, Chandrakanthan et al. investigated the origin and properties of stromal cells in AGM region. They show that periaortic stromal cells in this location are derived from Mesp1+ progenitors (Mesp1der) but later replaced by stromal cells derived from neural crest (Wnt1der). Coaggregation of Mesp1der or Wnt1der stromal cells with endothelial cells revealed that aggregates of Mesp1der stromal cells with AGM endothelium including nonhemogenic E13.5 endothelium and endothelium from hearts and aorta of 8-12 weeks old animals produced HSCs with robust engraftment potential. This effect was observed only with E10.5 or E11.5 Mesp1der stromal cells but not with E13.5 Wnt1der stromal cells. In addition, authors demonstrated that treatment of endothelial/stromal aggregates or ex vivo cultured E10.5 embryos with PDGFRa inhibitor reduced blood and HSC formation. The differences in Mesp1der stromal cells from ventral and dorsal fraction of were also revealed. Overall, these studies are well executed and make a significant novel contribution to our understanding of HSC development.
1. Previous studies by Ding https://doi.org/10.1002/dvdy.23923 with knockout animals revealed that loss of PDGFRa had no effect on fetal liver HSCs. However, present study has found a profound effect of Pdgfra knockout or inhibition with APA5 antibody on HSC and blood production. How this could be explained? Response: Thank you for this comment. Using Pdgfra-nGFP KI embryos, we showed that E10.5 and E11.5 AGM lacked CFU-Cs and transplantable HSCs in homozygotes (Extended data figure 2L (i)-(iii)). Ding et al (ref #36 in the submission) did not investigate AGM hematopoiesis or HSCs per se. Ding et al showed that FL LSK numbers in heterozygous and homozygous E12.5 Pdgfra-mer-Cre-mer KI were comparable by flow cytometry. It is important to note that the cellular composition of the FL LSK population is functionally heterogeneous (containing a mix of progenitor cells and HSCs), only a small fraction of these cells are HSCs (50-60; ref 18#). Because the functional status of Pdgfra-null HSCs was not investigated by Ding et al, it is not possible to conclude if HSCs were formed (or functionally healthy). For example, yolk sac-derived progenitors are though to contribute to the FL LSK compartment, it is conceivable that in the absence HSCs the E12.5 FL LSKs could appear immunophenotypically normal.

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Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
2. Another important difference between findings presented in this manuscript and the Ding paper is the developmental window at which contribution of PDGFRa+ cells to blood cells was observed. Ding found no contribution of PDGFRa+ cells to HSCs when tamoxifen was injected at E9.5. In contrast, this study revealed a substantial labelling blood cells following tamoxifen treatment at E9.5. This should be discussed. Response: Thank you for this comment. Ding et al showed that PDGFRA mesodermal contributions to FL and adult hematopoiesis was limited to cre-activation at E7.5-E8.5, whereas our data clearly showed PDGFRA contributions to AGM endothelium, and transplantable HSCs, when Pdgfra Cre-recombinase was activated at E9.5. These variations could be due to differences in cre-recombinase efficiency or Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 4. Tracing experiments demonstrated that blood and hemogenic endothelium originate from PDGFRa+ cells found in periaortic mesenchyme. Any evidence that these cells possess hematopoietic potential in vitro, for example in coculture with OP9? Response: Thank you for this suggestion. We have now performed OP9 co-culture assays with E9.5 whole AGM and FACS-purified PSCs for orthogonal validation of our in vivo lineage tracing and transplant experiments and show robust CD31 + /45 + generation. These data are presented in Figure 2B (i)-(iii) of the revised manuscript. 5. How embryos were exposed to APA5 and APB5 antibodies. Were they injected with antibodies or antibodies simply added to ex vivo cultures? In the later scenario, any evidence that antibodies can reach AGM by diffusion? Response: E10 ex vivo embryo cultures were performed only with APA5 ( Figure 6C (i)-(iii)). The antibody was added to cultures and AGM staining was confirmed in E11.5 cryosections. This is in keeping with a previous study (Takakura et al, PDGFR alpha expression during mouse embryogenesis: immunolocalization analyzed by whole-mount immunohistostaining using the monoclonal anti-mouse PDGFR alpha antibody APA5) which demonstrated that APA5 could deeply penetrate E6.5-16.5 embryos incubated with the antibody.
We have included this reference ( Supplementary Information ref #6) in the revised manuscript.
6. Page 11 lines 20-23. Pdgfra-nGFP+/Mesp1-DsRed mice are introduced here for the first time. However, it is unclear whether Mesp1-DsRed is knock-in reporter, or it traces Mesp1-Cre recombination. If Mesp1-DsRed is knock-in reporter, why DsRed signal is seen in blood and endothelial cells. Expression of Mesp1 is downregulated in endothelial and blood cells. Response: Thank you for pointing this out and we apologise for the confusion. These images were from an E11.5 AGM dissected from a compound transgenic mouse-Mesp1-cre x ZRed; Pdgfra-nGFP and show cells that were derived from Mesp1 expressing cells (rather than those currently expressing Mesp1) in Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. conjunction with those currently/recently expressing Pdgfra (Pdgfra-nGFP (KI); GFP half-life ~ 24-36 hours).
In the revised manuscript, we have included a schematic showing the mouse cross along with the image (Extended data figure 3B (i)) for clarity.
7. Transcriptional analysis of adult heart and aortic endothelium before and after coaggregation would be very interesting.
8. Please spell out PSC abbreviation. Response: Thank you. We have ensured that PSC is defined at first use both in the abstract and main text (page 4; lines 6/7).

Reviewer #3:
Remarks to the Author: Chandrakanthan, V. et al. investigated the origin and contribution of Pdgfra+ cells to hematopoietic stem cell (HSC) development in the dorsal aorta. The study shows Pdgfra+ cells from mesodermal origin contribute to endothelial cells and HSC in the dorsal aorta, and to sub-endothelial mesenchyme at the time HSC emerge. This mesoderm-derived mesenchyme supports endothelial-to-hematopoietic transition (EHT) from hemogenic endothelium and is later replaced by neural crest-derived Pdgra+ cells that lack this ability, coinciding with the cessation of hematopoiesis in the DA. Mesoderm-derived Pdgfra+ cells not only enable EHT from hemogenic endothelium but also from non-hemogenic embryonic and adult endothelial cells, in a process dependent on Pdgfra signaling and BMP, WNT and Notch pathways.
The manuscript includes interesting, novel findings and builds upon previous reports, such as the contribution of Mesp1-mesoderm and Pdgfra+ cells to the dorsal aorta and to adult hematopoiesis, with transplantation assays that bring more robustness to the previous reports (references below) that were based on lineage tracing. The findings that Mesp1-derived PSC can instruct adult EC to undergo EHT and generate LT-HSC in vitro are interesting and significant, though this reviewer suggests the inclusion of more experimental details and the performance of experiments that might confer more robustness to the findings. Several results show no statistical analysis, or they lack the reference that no statistical significance was observed. Overall, the manuscript is well written and presents high-quality and significant data, but the clarification of some points and the performance of additional experiments, Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. suggested below, may confer more robustness and validity to the findings and conclusions of the manuscript. We show that PDGFRA+ cells labelled at E9.5 also contribute to YFP+ AGM endothelial and blood clusters at E11.5 and that these AGM cells include transplantable HSCs (primary/secondary transplants).
We have now discussed our findings in relation to those by Ding et al (page 8, lines 4-6 and page 23, lines 11-16).
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. DsRed+Pdgfra+ isolated from dorsal and ventral aorta suggest PSC cells are located both dorsally and ventrally. Do the authors see differences in numbers and a preferential localization in the ventral aorta? Response: The reviewer is correct. PDGFRA + ; Mesp1-DsRed cells were seven-fold more abundant in the ventral half of the dorsal aorta (AoV) compared with the dorsal half (AoD). We have now included confocal and flow cytometry data to clarify and enumerate this difference (Extended data figure 5E (i)-(ii); page 16, lines 18-20).
6. Most Pdgfra+ cells present in the aorta at E11.5 are not derived from Mesp1-mesoderm or Wnt1 neural crest. Are Mesp1-derived PSC more limited in differentiation potential compared to Mesp1neg PSC? Assessment of Nestin expression in Mesp1der PSCs should be shown. Response: The reviewer is correct. Although the origins of these non-Mesp1 and non-Wnt1 PSCs are not clear, Mesp1 and Wnt1 derived PSCs collectively accounted for most CFU-Fs in the AGM ( Figure 3A (iv), 3B (iv) and Extended data figure 3F(ii), page 12, lines 21-24). The in vitro differentiation potential of Mesp1 der PSCs was broader than that of Wnt1 der PSCs (Extended data figure 3G). Furthermore, Mesp1 der PSCs and Wnt1 der PSCs each mirrored the differentiation potential of non-Wnt1 der PSCs and non-Mesp1 der PSCs respectively. These data are now included in the revised manuscript (Extended data figure  3G).
We have also now included E11.5 AGM confocal microscopy and flow cytometry data from Mesp1-DsRed; Pdgfra-nGFP and from Mesp1-DsRed; Nestin-GFP compound transgenic mice side by side for the readers to appreciate the distribution of Pdgfra-nGFP and Nestin-GFP in Mesp1-DsRed embryos (Extended data figure 3B). Flow cytometry panels from Nestin-GFP; Mesp1-DsRed compound transgenic embryos are included to show Nestin-GFP expression in Mesp1 der PCS (Extended data figure 3B, page 12, lines 1-5).
7. The authors' findings that co-aggregation of DA endothelium and, particularly adult endothelium with embryo derived Mesp1der PSC instructs EHT and HSC emergence are very interesting. There are experimental details and inclusion of more data that can clarify/strengthen the findings: Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Response: Thank you for this suggestion. The sorting strategy (Extended data figure 4C(i)-(ii)) is now included.
We agree with the reviewer that transplants with freshly isolated EC populations is an important control. We had included this control with our co-aggregate transplants but had inadvertently left this out of the original figure. This has been corrected in the revised manuscript ( Figure 4A (iv)). 7.3. In addition to the confocal imaging showing CD45 expression within the co-aggregate, the authors should include flow cytometry data after culture to enable a more precise characterization of the cell populations and quantification. Response: Thank you for this suggestion. These data are now included (Extended data figure 4D).
7.4. The authors should include the efficiency of EC conversion to HSC. Are significant differences being observed between hemogenic EC and non-hemogenic EC? Response: The efficiencies of various input EC to HSC conversion (calculated from transplantation assays using serial dilutions of co-aggregates and re-aggregates) are now shown along with the statistical significance of any observed difference between input EC type (Extended data figure 5B(i)-(ii)). 7.5. Although EC derived from the IVC were able to originate HSC, the levels of chimerism after transplantation were low, suggesting some EC might not be able to become hemogenic. Is EHT similarly efficient between arterial and venous EC or in adult EC from a distinct organ, such the brain? Response: The number of starting endothelial cells in each co-aggregate was 25,000 and recipients in Figure 4A (iv) were all injected with one co-aggregate/recipient. E10.5 and E11.5 whole AGM reaggregates showed the highest donor chimerism (51-69% and 54-71%) respectively. From purified EC/PSC co-aggregates, donor chimerism varied from 42-58% (E10.5 AGM ECs/10.5 PSCs) to 2-8% (adult IVC EC/ 10.5 or 11.5 PSCs) with those from the E13.5 AGM, adult Heart, adult Aorta ECs ranging between these extremes. We have now performed co-aggregate cultures with ECs purified from adult lung (as a distinct organ) with E10.5 and E11.5 PSC followed by CFU-C and transplantation assays as suggested. Donor chimerism was comparable with that seen with E11.5AGM ECs and PSCs ( Figure 4A (iv)).
Taken together, although it is possible that some endothelial cells may not be amenable to EHT by coaggregation with PSCs, those tested by us were all amenable to transformation, albeit at varying efficiency. These data were suggestive of inherent differences in endothelial cell types that made them more or less receptive to Mesp1 der PSC induced transformation and worthy of further investigation although beyond the scope of this manuscript. We have included this interpretation in the revised manuscript (page 16, lines 1-3).
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work. The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 7.6. The authors should include the results from the transplants of co-aggregates formed with DsRedneg PSC in Figure4. Response: These data were inadvertently left out and have been included in the revised manuscript ( Figure 4A (iv)). 7.7. Richard, C et al., (doi: 10.1016/j.devcel.2013.02.011.) have shown that the subaortic mesenchyme controls endothelial Runx1 in the dorsal aorta, enabling EHT. The authors show an upregulation of Runx1 during culture suggesting that a similar mechanism might be occurring. The use of EC isolated from a Runx1 reporter mouse would unequivocally demonstrate that EC turn on endogenous Runx1 expression and undergo EHT and it would allow a quantification of how many non-hemogenic EC undergo reprogramming to hemogenic fate. Response: Although we agree that such a reporter mouse could serve as an excellent tool to demonstrate Runx1 expression in EC following co-aggregation with PSCs, we do not have this at our disposal. However, our single cell gene expression data already serves this purpose. We tracked endogenous Runx1 transcripts in FACS purified CD31+/VE-CAD+ ECs harvested from PSC co-aggregates over 96 hours ( Figure 5D-H). As Runx1 expression is switched on in ECs, there is a concomitant decrease in Pecam 1 (CD31)/Cdh5 (VE-CAD) gene expression (although the corresponding proteins linger on the cell surface allowing FACS based isolation) and increase in Itga2b (CD41), and Ptprc (CD45) expression in Runx1 expressing cells.
We have now included a new heat-map showing these changes in single cells ( Figure 5H) and used these data to quantify the number of non-hemogenic cardiac endothelial cells that underwent reprogramming to a hemogenic fate when co-aggregated with E11.5 Mesp1 der PSCs. 12% and 21% of ECs expressed Runx1 at >1 count per ten thousand (cptt) or >0 cptt respectively (page 20, lines 16-18). 7.8. The scRNA-Seq suggests that different hematopoietic lineages are derived during the reprograming process. Do the authors think that HSC are derived within the 4 days of coculture or an HSC precursor that is able to complete the process of maturation in vivo, or both? Flow cytometric characterization of the co-aggregates and transplantation of the different cell subsets within the co-aggregate could bring more clarity of what cells are endowed with long-term reconstitution ability, or if transplantation of the entire co-aggregate is required to support engraftment after transplantation. Response: Thank you for this excellent suggestion. We have included flow cytometry data of the coaggregates (Extended data figure 4D) and performed transplantation assays to investigate the contributions of EC (UBC-GFP) derived CD45 + and CD45cells with/without Mesp1-DsRed PSCs to donor chimerism ( Figure 4B (i)-(ii)). We were able to conclude the following (i) endothelial cell derived (GFP+) CD45+ cells in the co-aggregates could engraft and contribute to long-term reconstitution without requiring on-going support from Mesp1-DsRed PSCs. (ii) co-transplantation of Mesp1-DsRed PSCs could not facilitate the engraftment/in vivo maturation of CD45cells. (iii) There was a modest increase in donor chimerism when CD45 + cells, were co-transplanted with Mesp1-DsRed PSCs (page 16, lines 4-16).
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