Peer review of: Feeding-dependent tentacle development in the sea anemone Nematostella vectensis (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Ikmi et al. describe an intriguing feature of postembryonic development in Nematostella vectensis, 
the addition of tentacles during the growth phase of the animals. They describe the pattern in 
which pairs of tentacles are added after the first four have formed in the transition to the primary 
polyp. This pattern shows little variation and does not follow a simple sequence of symmetrical 
additions. The authors then focus on the first pair of new tentacles and show that these require 
feeding for their formation and that locally elevated levels of phosphorylated RPS6 and an increase 
in cell proliferation precede their outgrowth. Confirming that pRPS6 reflects a role for TOR 
signalling, incubation with rapamycin reduces both pRPS6 and EdU labelling at the site of tentacle 
formation. Lastly, animals mutant for FGFRb fail to form tentacles after the four tentacle stage, 
despite being able to feed, suggesting that the elevated levels of FGFRb transcript at the sites of 
tentacle addition are related to a function of FGF signalling in directing tentacle formation in 
primary polyps. 
Overall, this is a very well executed study with beautiful illustrations of interesting and novel 
observations. Some aspects are probably not surprising, e.g. that growth requires feeding and that 
this requires TOR signalling. Still, it has to be shown and this has been done convincingly. 
Technically, there are only some points that I would like the authors to consider for improvements, 
all concerning the role of FGFRb. 
 
1. The expression pattern in the colorimetric in situs in Fig S10 A and B (and in the Matus paper) 
resembles very much a potential in situ artefact of these late planula/early polyp stages. The 
stainings in S10C and D and in Fig 6 look more convincing, but they seem to be at a later stage. 
To support the proposed pre-metamorphic origin, would it be possible to test if these spots remain 
after shRNA injection? It is also not clear to me how the number of these “clusters” relate to the 
addition of the tentacles. The tentacles appear in pairs, but there seem to be more clusters of cells 
(also in the movie). This is a point that is not essential for the manuscript, but maybe the “pre-
metamorphic” origin of the clusters is not so clear. The authors probably also see a different way 
to show that this particular staining in the in situs is specific. 
 
2. Using FGFRb mutants for the analysis is great, but maybe the temporal control allowed by 
SU5402 treatment could be exploited to obtain more information about the temporal requirement 
of FGF signalling in this context. Obviously, the gene-specificity is lost, but maybe the comparison 
of the mutants with transient SU5402 treatments would be informative. 
 
3. A cross-section of the FGFRb mutants showing the organization of the gastric cavity and 
mesenteries would be informative to understand the phenotype better. Also, it would be good to 
add more information about the development of the phenotype: when does the animals first differ 
from the sibs? 
 
While these technical questions (and the minor points listed below) do not diminish my excitement 
for the data, I am more uncertain about the conceptual background and the implications of the 
manuscript. The authors embed their study in a context of “continuous developmental capacities” 
(introduction) and axial patterning (abstract). In the discussion, they mention developmental 
responses to changes in the environment (nutrition). There is a reference to regeneration, but the 
phenomenon under study is a regular developmental event that comes to an end at the 16 
tentacle stage. To me the broader context remained very unclear. To justify publication at this 
level, the authors would need to make a more convincing point (evolutionary or developmentally) 
that the data are important beyond the regulation of one step of tentacle addition in this species. 
 
Minor points: 
1. On page 3 the authors mention that they here study adult cnidarians, but this is not the case. 
2. The short folds are mentioned quite a while before there is a reference to them being shown in 

Fig S4. It might be helpful to have this reference earlier. How far do these short folds extend along 
the primary body axis? 
3. Is it unambiguous that Bodipy stains lipid droplets? 
4. What is the staining for the capsules? 
5. The description of the movie should be clearer. What are the two “episodes” of the movie 
(before and after rendering?). 
 
 
 
Reviewer #2: 
Remarks to the Author: 
Review on manuscript NCOMMS-19-17704 
 
The authors in this manuscript describe the post-embryonic tentacle patterning in sea anemone 
Nematostella, showing that patterning is dependent on nutrient availability, induction of the mTOR 
pathway and activation of FGFR developmental signaling pathways. 
 
The first part of the manuscript is a spatial and temporal description of the tentacles formation in 
Nematostella polyps (from 4 primary tentacles to up to 16-tentacles in the adult) and conclude on 
a stereotyped and reproducible spatial patterning system. The authors then show that 
development of a full set of tentacles is feeding-dependent, as unfed polyps arrests at the 4-
tentacles stage, and also mTOR dependent, by using molecular markers for cell proliferation and 
TOR activity. Finally they show that the FGFR expression is essential for post-embryonic tentacle 
formation, by generating CRISPR/Cas9 mutants. 
 
The authors suggest that mTOR-dependent growth activates the FGFR signaling pathway, which in 
turn generate a feed back loop to locally enhance mTOR pathway. It is not clear from the 
manuscript that there is a direct activation by mTOR of the FGFR expansion upon feeding. How is 
the FGFR pattern in presence of rapamycin? 
 
Overall, the data is well presented and convincing, the manuscript is well written. The work is 
important for the field of patterning in response to environmental cues (here nutrients) as it sets 
the ground to further explore the molecular mechanisms regulated by mTOR and FGFR. 
 
Minor remarks: 
p3: "bauplan" should be in italics 
Figure 5F: rapamycin concentration is 3uM and not 1uM as indicated in text? 
Figure S2: title sould implicitly indicates that these are unfed polyps 
 
 
 
Reviewer #3: 
Remarks to the Author: 
NCOMMS-19-17704 
 
Summary 
The authors examine the relationship between feeding, growth, and tentacle morphogenesis in the 
sea anemone Nematostella vectensis. They characterize the timing and sequence of tentacle 
formation in the juvenile polyp (after development of the four primary tentacles) and show that 
the onset of juvenile tentacle morphogenesis occurs only after the onset of feeding. Interestingly, 
they show that the initial process of primary tentacle formation is uncoupled from the process 
resulting in tentacles 5-16. Further, they suggest a role for the TOR pathway in both general 
growth and in juvenile tentacle morphogenesis and show that Rapamycin treatment is sufficient to 
block the formation of juvenile tentacle buds. They propose a role for FGF signaling in the 
development of specialized fields of cell proliferation and TOR signaling that demarcate the juvenile 

tentacle buds and further suggest that FGF is necessary to couple proliferation and TOR signaling 
during the general process of growth in these animals. 
 
General Concerns 
The description of the morphogenesis of tentacles 5-16 is thorough and while these data are novel, 
the phenomenon described is specific to Nematostella vectensis and as such, these results will not 
likely be of great importance to anyone outside of the Nematostella community. Furthermore, the 
claim that this study characterizes “lifelong axial patterning” is unfounded. In reality, the number 
of adult tentacles is largely invariant in this species (as noted by the authors and others before 
them) so the phenomenon they describe amounts to little more than a description of determinate 
post-embryonic growth. Although they have provided preliminary data to support a role for TOR 
and FGF signaling in the tentacle budding process, the data they provide are not of sufficient 
quality (or magnification) to assess the validity of these claims. Finally, throughout the 
manuscript, the authors use language that is imprecise and misleading and several key references 
have been excluded. Despite all of this, the amount of work that went into characterizing the 
spatial and temporal progression of tentacle morphogenesis is not trivial and these observations 
do constitute a valuable contribution to the continued development of this emerging model 
system. I hope the authors can provide additional data to support the very interesting potential 
role of TOR and FgfRb in regulating juvenile tentacle morphogenesis. The data provided with this 
manuscript are, unfortunately, not of sufficient quality to substantiate their major claims. I have 
provided specific comments below intended to help improve the clarity and rigor of this study. 
 
Specific Comments 
1. “Lifelong axial patterning” is unfounded 
It is not clear that tentacle morphogenesis is really feeding-dependent (abstract) or plastic 
(discussion). Feeding is clearly required to induce the onset of tentacle morphogenesis but the 
authors have not demonstrated that animals will arrest at a number of tentacles below 16 if they 
are starved. Indeed, this study shows that starvation reduces the number of proliferating cells 
throughout the body, but not in the tentacle bud primordia (Figure 4), which explicitly argues 
against the claim that this process is feeding-dependent. Also, the term plasticity does not apply 
when the total number of adult tentacles is determined genetically; unless the authors can 
demonstrate that tentacles are lost when nutrients are limited or that nutrient limitation in the 
juvenile stage translates into a permanent change in adult tentacle number, then the use of the 
term plasticity is inappropriate. Similarly, the authors suggest (page 7) that tentacle budding 
“scales with body size” despite showing that budding is a binary response to feeding, independent 
of body size. As presented, there is no validity to this claim either. 
 
2. Imprecise language 
On page 7, the authors state that “localized proliferation transformed the thin epithelium to a 
thickened outgrowth by generating an initial cell mass”. It is not clear what is meant by “initial cell 
mass” as these are simple epithelia - there is only a single layer of cells in the endoderm and there 
is a single layer of cells in the ectoderm. These cells are undoubtedly changing shape to facilitate 
the buckling required to form a bud but the implication that cells are “amassing” is incorrect. 
Likewise, their use of the term “placode” (page 4) to describe the tentacle tip is contentious and 
does not add anything to this paper. The authors have not demonstrated an effect on the 
thickness of the tentacle tip epithelium in any of their treatments and thus there is no reason to 
invoke this term or defend this idea in the present study. 
 
3. Lack of appropriate references 
The “zig-zag” distribution of tentacles, the number of tentacles, and the organization of the 
tentacles along the directive axis was described by Stephenson in 1935 and the “short 
gastrodermal folds” (which are actually called “microcnemes”) were described by both Stephenson 
1935 and Crowell 1945. Indeed, nothing presented in Figure 1 (or the first paragraph of the 
results) is novel. Figures 2 and 3 are novel as they are the first attempt to characterize the 
temporal and spatial progression of tentacle addition in this animal and the authors have done a 

very nice job with this description. The authors later discuss “hox dependent segmentation during 
embryonic development” (page 12) but fail to mention these observations were made previously 
by Finnerty et al 2004 and Matus et al 2006. Lastly, it should be noted that differentiating 
cnidocytes in the tentacle tips (page 8) were first described by Zenkert et al 2011 (not Babonis 
and Martindale, 2017); this should be cited appropriately. 
 
4. Insufficient data quality 
General - For all fluorescent images, the authors should provide the data in separate channels (in 
addition to composites) and provide high magnification images to demonstrate the specificity of 
each signal. For example, many images ostensibly show EdU-labeling in proliferating cells but 
some of the labels appear to be of the wrong shape/size to be nuclei (e.g., Figures 4F,5C). 
Furthermore, the authors suggest that cell proliferation increases in “both layers” in the 
presumptive tentacle bud (page 7), but their images appear to be max projections that show only 
ectoderm (Figure 4F-H). The number of EdU-labeled cells is used to assess effects on proliferation 
throughout the manuscript, but the authors report the number of EdU+ cells per “area” (Figure 
4I,5D) and it is not clear that these areas are comparable. EdU values should be assayed relative 
to the total number of nuclei in the selected area not the area of the selection itself. The authors 
should also provide the raw nuclear counts to show that the number of DAPI-labeled nuclei is 
approximately the same, independent of the number of EdU+ nuclei, when comparing various 
regions. 
 
Re: The role of TOR - It is not clear how the authors define some cells/tissues as “pRPS6-negative” 
when there seems to be a fair amount of red signal even in the rapamycin-treated animals (Figure 
5C,F). This should be addressed when the authors provide higher quality image data (above). 
There is no indication of why different doses of rapamycin were used for the experiments 
presented in Figures 5C,F and while the authors report that rapamycin-treated polyps were 
“smaller” this is not demonstrated or quantified in Figure 5. They also claim that the general 
growth that precedes tentacle bud specification is TOR-dependent, but do not provide sagittal 
views showing pRPS6+ or EdU+ cells throughout the body column. Additionally, the use of an anti-
ribosomal protein S6 kinase antibody without any validation is inappropriate. It is customary to 
perform a western blot the first time an antibody is used in a new system. At a minimum, the 
authors should confirm that Nematostella actually has an ortholog of this gene and provide 
evidence that it is expressed in the same tissue that is labeled by the antibody. 
 
Re: The role of FGF - The authors say they induce premature stop codons in FgfRb using 
CRISPR/Cas9 but they do not show that this manipulation results in a loss of WT FrfRb mRNA (by 
in situ or by PCR/qPCR). Additionally, they suggest FGF signaling modulates the expression of the 
TOR pathway and localizes cell proliferation to the tentacle buds but they did not actually quantify 
the effects of FgfRb knockout on the number of EdU+ or pRPS6+ cells in their mutants. Thus, they 
cannot really support a relationship between proliferation, TOR signaling, and FGF, as presented. 
The in situ patterns in Figures 6, S10 and the supplemental movie do not match. Figure 6 shows 
endodermal expression of FgfRb and Figure S10 shows what may be ectodermal expression in the 
polyps but the quality of these images is too poor to really evaluate. This supplemental movie is 
largely uninterpretable as structures of very different size and shape all seem to be labeled with 
the FgfRb probe and yet the magnification is not high enough to determine if any of these 
structures are co-labeled with a nuclear marker. 
 
In an effort to promote transparency in the peer review process, I choose to waive my anonymity.