Response to Reviewers from Helman et al.
Below are our point-by-point responses to the reviewers’ comments on our manuscript,
“Extracellular matrix production and calcium carbonate precipitation by coral cells in
vitro.”
We have listed the reviewers’ concerns in bold followed by our specific response.
Responses to Reviewer 1 (Leo Buss):
“So how does this study stand up? The methods do not differ all that much from
prior methods or if they do the authors give no indication what materially differs
from their technique and others and why those differences are germane.”
The medium we used for cultivating coral cells is unique and although there are
similarities to other culture media that have been tried in the past, ours is supplemented
with moleucles that are commonly used for culturing human osteoblasts but have not
been used to culture coral cells. We agree with the reviewer that this essential point was
not properly called out in the paper and we added it to the discussion – Page 9, first
paragraph.
“They assume that the cells in the cultures are of cnidarian origin, but do not
confirm this (by simple 16S genotyping for example). This would be nice to confirm,
not so much because I doubt that the cells are cnidarian, but because the whole
history of this topic is so mixed.”
The cnidarian origin of the cells in culture was verified using universal eukaryotic 18S
primers followed by sequencing. Results were added to the text— page 4, first paragraph
of Results and Discussion and the sequences are provided as supplemental information
(Fig. 2SI).
“Given that the major result here is methodological, I am curious why we don't get
a more detailed account (say in a supplemental discussion) characterizing these
cultures. How many isolates are necessary to get one established? These are said to
be continuous cultures (p. 4), what does that mean precisely? What is the
survivorship of the cultures - how long do they last (mean, median, SD), how do they
fail, do they lose cell types, etc? Are the cells starved (various physiological
measures available)? Do the cells ever divide?”
A figure showing different cell types was added to supporting information Fig. *
A more detailed description of the method used to produce culture was added to material
and methods, including a description of the custom-made cell strainer – page 10.
We added a sentence emphasizing the fact that the coral cell cultures examined in this
study are primary cell cultures of non-dividing cells – page 4, first sentence of results and
discussion.
The viability of cell in culture was written as the mean ± SD at days 22 and 27.
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Responses to Reviewer #2 (Denis Allemand):
“Title: The authors did not really observe CaCO3 deposition by Xenia cell culture,
therefore I would suggest to specify in the title “by scleractinian coral cells” and just
show results concerning Xenia as a part of their study.”
The reviewer states that the title “Extracellular matrix production and calcium carbonate
precipitation by corals cells in vitro” might be misleading because CaCO3 deposition was
only observed in the M. digitata cell cultures. He suggests to specify in the title “by
scleractinian coral cells” and to show results concerning X. elongata as a part of the
study. However, we feel that the results described for the X. elongata cell cultures are as
important as those described for M. digitata and since the title does not specify coral
types, we feel it is not misleading. To prevent any possible confusion we have changed
the abstract to emphasize the fact that calcification was observed only in cultures of M.
digitata cells. We have further clarified this point on page 7.
“Cell culture characterization: Cell identification is based on "typical morphology",
but I know by experience that cells in culture completely loose their initial shape,
excepted if there is a morphological characteristics (such as the presence of
zooxanthellae inside - you have to show these endodermic cells in culture, or
Cnidoblasts).”
A figure showing different cell types was added to supporting information Fig. *
“Extracellular matrix (ECM) production: In order to examine the composition of
these ECM, authors used Sirius red and FITC-lectin conjugates of concanavalin A
and wheat germ agglutinin. However, they have to verify that these dyes effectively
label coral mesoglea since coral collagen is a short collagen.”
The reviewer expressed his concern regarding the use of Sirius red to stain coral collagen
since corals posses only short chain collagens. However, of Sirius red does stain intimal
collagen, which is short chain collagen (see references below). Due to the extensive use
of Sirius red for collagen (short and long chain) detection in various fields, we do not
think it is necessary to show that it is also appropriate for coral collagen identification.
We further added an SEM image to the supporting information (Fig. 4SI a),
demonstrating the existence of collagen-like fibers within the cell aggregates.
References:
 The discoidin domain receptor tyrosine kinase DDR1 in arterial
wound repair
Guangpei Hou, Wolfgang Vogel, and Michelle P. Bendeck
J Clin Invest. 2001 March 15; 107(6): 727–735.

Further Characterization of the Three Polypeptide Chains of Bovine
and Human Short-Chain Collagen (Intima Collagen)
Renate Jander, Jiirgen Rauterberg, and Robert W. Glanville
Eur. J. Biochem. (1983) 133, 39-46.
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“If it is possible to add more Supporting information, I would also suggest to show
pictures for both corals.”
We have changed figure 3 in supporting information to show lectin binding to ECM of
both corals. We have also added a reference to the result section (extracellular matrix
production - page 6, top paragraph) demonstrating the occurrence of mannose, and
glucosamine in collagens of another anthozoan - the sea anemone Metridium dianthus.
“Page 6, the authors should precise that collagen production is expressed as percent
of total ECM production (which is not indicated in the results section).”
In page 6 we give the amount of collagen as percent of total protein.
“CaCO3 production: The authors said that cultured cells produced amorphous
calcium carbonate (page 7, line 2), however, later in the same paragraph they said
that these granules are composed of aragonite, which is a crystal. Since there is
presently a very interesting debate concerning the initial state of CaCO3, amorphous
or crystalline, it is important to make clear this point.”
We agree with the reviewer that describing the shape of the calcium carbonate particles
as amorphous may be misleading. We meant to indicate that the particles did not appear
to exhibit a specific shape (circle, square…) and we were not referring to the crystal
morphology. However, due to the ongoing debate concerning the initial state of CaCO3,
amorphous or crystalline this adjective is not appropriate and was therefore removed
from the text.
“It is not clear on your picture (Fig 3) if the deposited CaCO3 is totally surrounded
by cells or not. It would be interesting to provide a TEM pictures across the CaCO 3
particles and a SEM details on the skeletal particle itself.”
In order to further describe the CaCO3 particles, an SEM image of particles and cells was
added to supporting information (Fig. 4SI). Since the number of cells surrounding the
particles varied a lot between the different aggregates a TEM would not be
representative. A sentence describing the variability of cell distribution around the
different CaCO3 particles was added to the results (page 8, first paragraph in section of
Calcium carbonate particles)
“The authors compared the rate of 45Ca incorporation obtained in their in vitro
culture with published data obtained with coral nubbins. However, I don’t think
that this comparison is relevant since present data are expressed by mg of cell
protein while data obtained in coral nubbins were standardized by mg of total
protein (zooxanthellae + coral including the whole mesoglea…) which is far more
important than just some cell protein.”
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Comparisons between in vitro rates of
from the text.
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Ca incorporation and in situ rates were removed.
“Skeletal organic matrix (SOM): As for ECM, the authors have to confirm that
SOM obtained is similar to that normally found in coral skeleton. To my knowledge,
Alcian blue was not used to label SOM in situ. I suggest to also using acridine
orange that was successfully used by Gautret et al. (2000) Organic components of
the skeleton of scleractinian corals - Evidence from in situ acridine orange staining.
Acta Palaeontol. Pol. 45:107-118).”
Alcian blue is widely used for staining of mucopolysaccharides and glycoproteins and
was used several times to stain SOM extracted from corals (relevant references are
presented in the text - page 8, end of first paragraph in section skeletal organic matrix). In
order to demonstrate the existence of mucopolysaccharides within particle's SOM, we
stained SOM with alcian blue after dissolution with acid and do not see any reason to
stain the SOM with an additional stain such as acridine orange, which is less specific and
thus less informative.
“In their conclusions, the authors said that “SOM of .. corals is composed of acidic
amino acids … (46, 47, present study)”. I am not sure that staining with alcian blue
will prove the presence of acidic amino acid and polysaccharides. I therefore suggest
to remove this sentence, as well as the two other following sentences which are
unnecessary generalization.”
We removed the words "present study" from the references but left the sentence since the
composition of coral skeleton was described in the referenced papers. Although we agree
with the reviewer that the following sentences are generalized, we disagree that they are
unnecessary since they are used to point out the similar aspects of coral and vertebrate
mineralization, a point we think is important when studying coral biomineralization.
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