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There are no data looking at co-expression of various HOX genes. Summary scheme in Figure 6. However, the characterization of those sympathetic neurons is very limited and lacks basic co-expression data e. Such assays are commonly presented by other studies reporting on the derivation of sympathetic neurons from hPSCs e. Friauf, In addition, the authors define culturing conditions that allow efficient production of peripheral sympathetic neurons without the need for additional purification schemes, such as FACS.

Overall, the authors present their work very well, with excellent command of the literature, and have advanced our knowledge for generating trunk NC cells and their derivatives that I expect will be used by researchers in the field. However, in its current form, the conclusions are over-stated and the impact of the study is lessened by the fact that similar protocols for generating trunk NC and peripheral neurons and other NC-cell types have recently been published as noted by the authors.

In addition, the experimental approaches are not overly innovative cell culture, immunohistochemistry and RNA-Seq and largely focus on sufficiency outcomes with no experiments addressing necessity. As one of the main conclusions from the study concerns the origin of trunk NC cells from axial progenitors, there should be some spatial data to confirm the molecular signatures.

For example, transplantation of anterior versus posterior NC cells into avian embryos to show the observed transcriptional changes impact the fate of cells at different axial positions. This technique is commonly used in the field to test axial identity and NC competence e. Indeed, many of the genes used by the authors to define axial NC identity are expressed in other cell types, particularly other neural, neuroectodermal and mesoderm derivatives that develop adjacent to NC cell in vivo.

Such data would significant enhance the impact of the study and strengthen the conclusion that human axial progenitors generate trunk NC. The authors should perform in situ hybridization or immunohistochemistry on vertebrate animals mouse, chick or fish to localize ASLX3 or its conserved equivalent to trunk NC cells in vivo. This would strengthen their argument that their approach used to classify axial identity in vitro which is reliant on HOX gene expression can also produce new knowledge on the genetic regulation of trunk NC in vivo.

The authors show solid work that clearly indicate that this progenitor can be guided into producing NC cells with trunk identity. More sophisticated experiments, such as following cells over time using reporter lines to show that the NMP truly is giving rise to the trunk-NC population as well as in vivo work showing the same thing would definitively prove this interesting hypothesis. Therefore, some of the conclusions should be worded a bit more carefully. Nevertheless, this is the first report to my knowledge of NC cells derived from NMPs with trunk NC character and it seems a highly efficient one as well.

Thus, I think this is an important report that should be published. This could be strengthened by showing that these PXM cells can further be differentiated into definitive mesodermal cell types. This notion should be strengthened by staining to show the same at the protein level. What are these cells?

RIC | Bannister Gallery | “Trunks and Tails”: Stuart Diamond

Maybe they are younger cells that have not yet turned on Phox2B, which would mean that at day 18 there is a mixture of cells in terms of developmental stage. Could that be showed more? Without confirming in vivo data, one cannot definitively prove that axial progenitors are converted into posterior NC in embryogenesis. Thus, to investigate the persistence of pluripotent cells, we examined the presence of cells expressing more definitive pluripotency markers such as NANOG Osorno, Tsakiridis et al. We have included these data in new Figure 3—figure supplement 3.

We have now verified the functionality of the axial progenitor-derived trunk NC cells using the following assays:. These data are shown in new Figure 5G. These data are shown in new Figure 5F and Figure 5—figure supplement 1D. We have now included a number of additional functional experiments to address this issue, see our response above to point B. These results are now part of figure 2C, Figure 2—figure supplement 1B, Figure 3—figure supplement 3A, B; see also our responses above. The reviewer is raising some very interesting points. Furthermore, our new data show that BMP-inhibited axial progenitors can still generate trunk NC at a similar efficiency to untreated controls See new Figure 3—figure supplement 2D.

These data indicate that endogenous BMP signalling alone is not the mediator of NC potential in axial progenitor cultures and we have therefore amended our statements in the new manuscript version regarding the role of this pathway. The question of NMP subpopulations is very interesting and requires further investigation but it is outside the scope of this manuscript. Recent lineage tracing and single cell RNA-seq studies have revealed that mouse embryonic NMP subpopulations exhibit expression of more definitive lineage-specific markers such as the paraxial mesoderm gene Tbx6 Javali et al.

We are confident about the specificity of the monoclonal anti-HOXC9 antibody we employ. We did not obtain any positive signal when we used the antibody on undifferentiated hES cells suggesting that it is specific. We have included these data in new Figure 3—figure supplement 1A, B.

The reviewer is raising a valid point. Development and Arenkiel et al. This may be due to the action of endogenous RA signalling since our microarray data revealed the upregulation of RA signalling components in axial progenitor-derived trunk NC even though no exogenous retinoic acid was added to the differentiation medium see Supplementary file 2 in submitted manuscript.

Unfortunately, we have not found reliable antibodies against HOX group members that we can use together with HOXC9 in order to distinguish between these two possibilities. See our response to point B. We have now also included data showing co-expression of TH and DBH in axial progenitor-derived sympathetic neurons see new Figure 5—figure supplement 1B. We have tried to be careful about our conclusions and we are happy to consider rephrasing them on a case-to-case basis if required. We disagree that the impact of our study is lessened by the publication of similar protocols we presume the reviewer refers to the manuscripts by Denham et al.

Apart from establishing a highly efficient method for generating trunk NC cells we believe that our manuscript also provides an insight into the basis of anterior-posterior regionalisation in the human neural crest and therefore, as reviewer 1 correctly noted, goes beyond the Denham et al.

We do not understand this point.

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We believe that the additional data we have included e. In our opinion, the use of chick embryo transplantation as a comparative assay for quantifying differences in the axial identity status of heterogeneous hPSC-derived donor cell populations is very challenging to set up and possibly hard to interpret. In fact, we are not aware of any study where chick embryo grafting was employed to compare in a quantitative manner distinct PSC-derived cell types in terms of axial identity.

However, as stated earlier we have performed grafting of axial progenitor-derived trunk NC cells into chick embryos to confirm in qualitative terms that these behave similarly to their in vivo counterparts, i. However, the characterisation of its expression domains in vivo will be time-consuming since we are not aware of any published antibodies or in situ hybridisation probes that could be used for ASXL3 detection and we would have to generate these reagents from scratch.

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Consequently, while of clear interest, we think that carrying out these in vivo experiments are beyond the scope of the current study which is focused on the generation of trunk neural crest cells by differentiation of hPSC in vitro. We would like to thank the reviewer for recognising the importance of our manuscript.

Dev Cell. Cheung M, Briscoe J. Neural crest development is regulated by the transcription factor Sox9.

Human axial progenitors generate trunk neural crest cells in vitro

PLoS Genet. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Neural crest regionalisation for enteric nervous system formation: implications for Hirschsprung's disease and stem cell therapy. Dev Biol. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

Article citation count generated by polling the highest count across the following sources: Crossref , Scopus , PubMed Central. Cited 5 Views 2, Annotations Open annotations.

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