Many marine organisms such as mussels, sea urchins or corals, have skeletons and shells, which – due to their beautiful colors and shapes – are often desirable collector pieces. These structures are made from calcium and carbonate ions that react to form calcium carbonate crystals in a process known as biomineralization.

In sea urchin larvae, for example, the skeleton is built by so-called primary mesenchyme cells, which work similar to the bone forming cells in mammals. These mesenchyme cells use calcium from the sea water, which travels to the site where the shell starts to form. About half of the carbonate comes from carbon dioxide that the animals make as they breathe, but it is not known how the other half gets to the site of biomineralization.

Producing a skeleton generates acid, and marine animals need to be able to regulate their pH levels, as too acidic environments can dissolve the calcium carbonate and threatening to destroy the developing shell. How cells accumulate enough carbonate to make their shells, and how they cope with acidity is still poorly understood.

Here, Hu et al. address this problem by studying purple sea urchin larvae, revealing that they use ion transporters to gather bicarbonate from seawater. These structures are part of a group of bicarbonate transporters known as the ‘SLC4 transporter family’, which sit across the membrane of the mesenchyme cells and move the bicarbonate ions along. As the sea urchin larvae develop, the levels of the transporter protein start to rise in mesenchyme cells, peaking around the time they are producing the skeleton.

Stopping the production of the transporter hindered the larvae from building normal skeletons and also made their cells more acidic. It turns out that bicarbonate is more than a skeleton ingredient – it also helps to buffer the acid made in the process. Bicarbonate ions can combine with acidic molecules to form water and carbon dioxide. Bicarbonate pumped in from the sea neutralises the acidic molecules made during calcium carbonate formation, which helps to stabilize pH levels.

When the acidity of the water was experimentally increased, it prompted the sea urchins to produce more of the SLC4 transporters, revealing that they may have another role to play. Their acid-neutralizing capability helped the animals to cope with changes in their environment. Taking on more bicarbonate could therefore help to compensate for rising acidity, allowing skeleton production to carry on as normal.

This last finding is important in the context of ocean acidification. As the amount of carbon dioxide in the atmosphere increases, more of the gas dissolves in the sea. The chemical reactions that follow make the water more acidic and decreases the pH levels of the sea. Understanding how animals make their skeletons and shells, and manage acid, could reveal how they will cope as the environment changes in the future.

Sea urchin larvae with their elaborate calcareous endoskeleton have been studied by embryologists for over a century to promote our understanding of calcification in biological systems (Boveri, 1901; Decker and Lennarz, 1988; Wilt, 2002). Similar to mammalian osteoblasts that arise from mesenchymal stem cells (MSC), the sea urchin larval skeleton is also produced by a specific cell line – the primary mesenchyme cells (PMCs) (Wilt, 2002). During the blastula stage, PMCs ingress into the blastocoel and migrate in a stereotypical pattern, forming a posterior ring around the blastopore (Ettensohn, 1990; Gustafson and Kinnander, 1956). Amorphous calcium carbonate (ACC) is precipitated within intracellular vesicles that exocytose their content into the lumen of a syncytial cable formed by the PMCs. This process involves at least 40 distinct skeletal matrix proteins supporting the formation of the mature calcite spicules within this extracellular space (Beniash et al., 1997; Benson et al., 1986). Some of these matrix proteins are also present in intracellular compartments where they may play a role in the stabilization of ACC (Urry et al., 2000; Wilt, 2002). Furthermore, recent findings suggest that Ca²⁺ also enters calcification vesicles by endocytosis of seawater into a vesicular network within the PMCs (Vidavsky et al., 2016).

During de novo formation of the larval skeleton in the mid-gastrula stage, calcium uptake increases ten-fold, compared to the blastula stage. 70% of this calcium is incorporated into the newly formed spicules (Nakano et al., 1963), while the remaining 30% reflects the uptake of calcium into cell organelles, including mitochondria and the smooth endoplasmatic reticulum (Decker et al., 1987). Besides the availability of Ca²⁺, dissolved inorganic carbon (DIC, i.e. CO₂, HCO₃^- and CO₃^2-) is required at equimolar levels for the precipitation of CaCO₃. Radioisotopic measurements demonstrate that up to 63% of carbon for spicule formation derives from respiratory CO₂, while the remaining DIC that is incorporated into the spicules derives from seawater (Sikes et al., 1981). Based on the assumption that HCO₃^- rather than CO₂ or CO₃^2- supplies the remaining 37% of DIC for calcification, 1.6 protons are generated per molecule CO₃^2- precipitated. An even stronger local acidification at the site of ACC formation can be expected considering CO₂ as the major source of DIC. Based on these calculations, it has been suggested that PMCs are capable of efficiently handling this proton load using pH regulatory mechanisms (Sikes et al., 1981). However, such potential acid-base regulatory mechanisms in PMCs remain poorly understood despite their importance in the formation of the larval skeleton.

A previous study demonstrated that pH_i regulation of PMCs is dependent on external Na⁺ as well as HCO₃^- to compensate for an intracellular acidosis indicating Na⁺-dependent HCO₃^- buffering mechanisms (Stumpp et al., 2012). The importance of HCO₃^- transport in the calcifying PMCs is underlined by the observation that DIDS (4,4´-diisothiocyano-2,2´´-disulfonic acid stilbene), an inhibitor for most SLC4 family transporters (Romero et al., 2004) inhibits uptake and deposition of ⁴⁵Ca into spicules (Yasumasu et al., 1985). Accordingly, it has been concluded that these compounds block the supply of HCO₃^- for ACC formation rather than the ability of PMC cells to form their syncytium as shown for other blockers (Basse et al., 2015; Mitsunaga et al., 1986). Furthermore, H⁺-export pathways involving Na⁺/H⁺ exchange were suggested to remove protons from the cytosol of PMCs although amiloride, an inhibitor for Na⁺-dependent H⁺ exchange had no effect on spicule formation and pH_i regulation of PMCs (Mitsunaga et al., 1987; Stumpp et al., 2012). Here, we hypothesize that HCO₃^- transport pathways, via a so far unidentified HCO₃^- transport mechanism affect the calcification process in different ways: (i) HCO₃^- transport supplies the cell with substrate for the precipitation of CaCO₃ and (ii) the regulation of intracellular HCO₃^- homeostasis is critical to buffer excess protons generated by the intravesicular precipitation of CaCO₃.

Apart from an endogenous generation of protons through biomineralization, pH regulatory mechanisms of sea urchin larvae have received considerable attention in the context of CO₂ driven sea water acidification (Byrne et al., 2013; Stumpp et al., 2012). The sea urchin larva has been extensively studied with respect to their potential for physiological acclimation and evolutionary adaptation to predicted near-future ocean acidification. These studies suggested that energy allocations and ion regulatory efforts are key processes that determine the resilience to reductions in seawater pH (Pan et al., 2015; Pespeni et al., 2013; Stumpp et al., 2012). These studies also indicated that despite moderate impacts at the organismal level, the compensatory reactions at the cellular level are substantial. Thus, a better mechanistic understanding for cellular processes affected by changes in seawater pH is essential to explain energy allocations in the sea urchin larva exposed to experimental ocean acidification.

Four Slc4 transporters were identified in the genome of the purple sea urchin, Strongylocentrotus purpuratus (Tu et al., 2012). To date, little is known regarding the function and tissue-specific localization of Slc4 transporters in the sea urchin larva. During the period of early skeleton formation in the sea urchin embryo (30–48 hr post fertilization), highest transcript abundance was detected for the SpSlc4a10 gene compared to all other Slc4 transporters (Tu et al., 2014). This gene shares highest sequence identity with the mammalian Slc4a10 (NBCn2) gene that encodes an electroneutral sodium-dependnet Cl^-/HCO₃^- exchanger with Cl^- self-exchange activity (Parker et al., 2008). This prompted us to hypothesize that SpSlc4a10 may be critically involved in HCO₃^- transport during formation of the larval skeleton. Here, we test the role of SpSlc4a10 in the maintenance of intracellular pH homeostasis and as a DIC concentration mechanism in the calcifying PMCs of the sea urchin embryo. Since pH_i regulation is intrinsically linked to precipitation of ACC, these mechanisms will fill an important knowledge gap regarding the fundamental principles of biomineralization in the sea urchin larva.

This study identified a SLC4-type bicarbonate transporter specifically expressed by primary mesenchyme cells (PMCs) of the sea urchin larva and demonstrated its role in the formation of the larval skeleton. In mammals, the SLC4 familiy can be categorized into three major clades of HCO₃^- and/or CO₃^2- transporters including Na⁺-driven Cl^-/HCO₃^- exchangers, electrogenic Na⁺ coupled HCO₃^- transporters and electroneutral Na⁺ coupled HCO₃^- transporters (Romero et al., 2013; Romero et al., 2004). Our phylogenetic analysis based on deduced amino acid sequences suggest that SpSlc4a10 has highest identity with the mammalian clade of electroneutral Na⁺ coupled HCO₃^-transporters, including Na⁺/HCO₃^- cotransporters (NBCn) and Na⁺-driven Cl^-/HCO₃^- exchangers (NCBE) that were demonstrated to control pH_i in neurons (Wang et al., 2000) and enable the transcellular passage of HCO₃^- across epithelial cells of the pancreatic duct and the renal proximal tubule (Guo et al., 2017; Ko et al., 2002). The human NBCn2 encoded by Slc4a10 has absolute requirements for Na⁺ and HCO₃^- and appears to be a Na⁺-dependent Cl^-/HCO₃^- exchanger that is blocked in the presence of 200 µM DIDS (Parker et al., 2008). Earlier studies demonstrated that pH_i regulation in PMCs is also highly Na⁺ and HCO₃^--dependent thereby supporting the involvement of an Na⁺-dependent HCO₃^- transport mechanism in the regulation of PMC pH_i (Stumpp et al., 2012). Together with findings of the present work demonstrating that pH_i regulation in PMCs is DIDS sensitive (IC₅₀ = 120 µM) and that knock-down of SpSlc4a10 reduces the ability to compensate an intracellular acidosis, there is strong evidence that SpSlc4a10 plays a central role in controlling pH_i of PMCs.

ACC is precipitated in intracellular vesicles which are subsequently exocytosed into the luminal space of the syncycial cable to form and maintain the larval skeleton (Vidavsky et al., 2014). Accordingly, protons generated during the formation of ACC must be removed from the vesicles into the cytosol to avoid an increasing intravesicular acidification leading to inhibition of CaCO₃ precipitation. This uptake of protons into the cytosol may cause disturbances in pH_i homeostasis if not actively compensated by buffering and secretion of protons. Here, the PMCs of the sea urchin larva share many common features with mammalian osteoblasts where precipitation of the calcium phosphate mineral, hydroxyapatite, is initiated in vesicles (Boonrungsiman et al., 2012). These vesicles are exocytosed from osteoblasts to deliver mineral to the protein matrix of the bone (Anderson, 2003). During mineral precipitation large amounts of acid are produced that need to be removed in order to maintain the slightly alkaline conditions at the site of bone formation (Brandao-Burch et al., 2005; Liu et al., 2011). Thus, osteoblasts also possess a high pH_i regulatory capacity during biomineralization that is achieved by a basolateral Na⁺/H⁺ exchanger 1 (NHE1) and NHE6 as well as HCO₃^-/Cl^- exchange mechanisms by the anion exchanger 2 (AE2) (Liu et al., 2011; Mobasheri et al., 1998). The present work demonstrates that similar to mammalian osteoblasts, PMCs of the sea urchin larva also possess pH_i regulatory mechanisms to promote biomineralization. Highest expression of the PMC-specific bicarbonate transporter SpSlc4a10 is accompanied by the onset of skeleton formation in the sea urchin embryo and expression levels decrease in later stages that have lower calcification rates. The progressive down regulation of SpSlc4a10 is accompanied by a migration of SpSlc4a10 expressing cells toward the tips of the spicules during further development.

Immunocytochemical analyses using a custom made antibody designed against the sea urchin SpSlc4a10 demonstrated high concentrations of this protein in PMCs. This suggests substantial HCO₃^- transport capacities by PMCs that may be associated with pH_i regulation and accumulation of HCO₃^- to promote precipitation of CaCO₃. Besides a localization of SpSlc4a10 in the plasma membrane, large intracellular compartments demonstrated positive immunoreactivity as well. In mammalian skeletal tissues AE2 has been associated with the Golgi apparatus, where it is hypothesized to control pH within the Golgi complex (Bronckers et al., 2009; Holappa et al., 2001). By controlling pH inside the Golgi, AE2 is believed to mediate posttranslational modification of matrix proteins, their packaging, transfer to the outer plasma membrane and exocytosis (Bronckers et al., 2009; Jansen et al., 2009). A lack of this protein in skeletogenic cells is often associated with osteoporosis-like skeletal defects (Jansen et al., 2009). Accordingly, future studies will address the function of the SpSlc4a10 transporter in subcellular compartments and organelles of PMCs.

The disturbance of skeletogenesis in the SpSlc4a10 morphants may be due to multiple factors, including (i) disturbance of PMC migration (ii) reductions in HCO₃^- supply (iii) decreased control of pH_i. The phenotype of the SpSlc4a10 morphants was characterized by decreased calcification abilities and abnormally formed spicules, lacking or having irregular branchings from the primary rods. Localization of SpSlc4a10 positive PMCs in control and morphants indicated an abnormal migration of PMCs. Disturbance of migratory patterns and syncytium formation has been documented in several earlier studies using pharmacological approaches. For example loop diuretics, blocking the Na⁺/K⁺/2Cl^- cotransporter (NKCC) inhibited the fusion and formation of cytoplasmic cords (Basse et al., 2015). Also the H⁺/K⁺-ATPase (HKA) inhibitor SCH28080 decreased calcification through impaired PMC fusion. However, while inhibition of HKA decreased pH_i of PMCs, no changes in the migratory pattern were observed (Schatzberg et al., 2015). In the present study, the disruption of cell migration in the SpSlc4a10 morphants may be explained by changes in pH_i gradients that can be critical for cell polarization (Martin et al., 2011). It has been demonstrated that inhibition of NHE1, an important regulator of pH_i, caused pH_i gradients to flatten or disappear. These gradients serve as an axis of movement in migrating cells and so their absence would likely affect migration (Martin et al., 2011). This hypothesis would be underlined by the down regulation of the Na⁺/H⁺-exchanger (SpSlc9a2) in the SpSlc4a10 morphants. Although these are likely explanations for the observed migratory defects, intracellular pH disturbances were demonstrated to be associated with a wide range of cellular dysfunctions (e.g. Kurkdjian and Guern, 1989; Madshus, 1988; Roos and Boron, 1981) that may also systemically disturb PMC migration.

The present work provides further evidence for the requirement of bicarbonate transport pathways to regulate pH_i that are likely the primary cause for reduced and abnormal skeleton formation (Figure 7). We propose that reductions in calcification observed for the SpSlc4a10 morphants resulted from the decreased pH_i regulatory ability of PMCs. The uncompensated proton load generated by the formation of ACC leads to an intracellular acidosis impairing futher precipitation of CaCO₃. Furthermore, since 40% of the dissolved inorganic carbon (DIC) that is incorporated into spicules derives from the seawater (Sikes et al., 1981), it can be suggested that SpSlc4a10 may be also involved in a HCO₃^- concentrating mechanism to promote intracellular calcification. Corroborating ealier studies that demonstrated reductions in Ca⁴⁵ uptake into spicules in the presence of the anion exchange inhibitor, DIDS (Yasumasu et al., 1985), the present work could show reduced incorporation of calcein in larvae treated with this compound. These reductions in calcification rates are accompanied by decreased pH_i regulatory abilities under DIDS treatment. However, DIDS is a broad-band inhibitor for most SLC4 transporters, and has been demonstrated to potentially also inhibit the gastric-type H⁺/K⁺-ATPase in mammals (Sugita et al., 1999). Accordingly, it remains unresolved whether decreased pH_i regulatory abilities under DIDS treatment can be attributed to an inhibition of HCO₃^- or H⁺ transport. Nonethless, the knock-down of the HCO₃^- transporter SpSlc4a10 caused similar reductions in pH_i regulatory capacities and calcein incorporation into spicules providing strong evidence for SpSlc4a10 to be a key player controlling intracellular ACC formation in the sea urchin larva.

Figure 7

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CO₂-perturbation experiments relevant for near-future ocean acidification scenarios demonstrated that sea urchin larvae are capable of maintaining calcification rates despite reductions in seawater pH. Reductions in seawater pH were demonstrated to negatively impact calcification in many marine organisms due to a reduced availability of calcification substrates (e.g. HCO₃^- and CO₃^2-) accompanied by enhanced dissolution of CaCO₃ (Kroeker et al., 2013). To maintain calcification rates under acidified conditions PMCs must allocate energy to increase HCO₃^- / CO₃^2- transport to the site of calcification and to export protons against steeper H⁺ gradients (Stumpp et al., 2012). While the concept of energy allocation toward compensatory processes (i.e. pH regulation) is widely accepted, the identification of key acid-base transporters remains largely unknown. This work has identified SpSlc4a10 as a key candidate gene that is critical for calcification under acidified conditions in the sea urchin larva.

All data generated or analysed during this study are included in the manuscript and supporting files.

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[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "A SLC4 family bicarbonate transporter regulates intracellular pH critical for biomineralization in the sea urchin embryo" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Martin Tresguerres (Reviewer #1).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. However, should the authors address the concerns raised by the reviewers with respect to experimental controls and specificity, we would entertain a revised paper in the future. This would take the form of a new submission, linked to the previous one within the system and would be assessed by the same experts.

Reviewer #1:

This study attempted to characterize the involvement of the bicarbonate transporter SpSlc4a10 in sea urchin larvae calcification. The hypothesis was that SpSlc4a10 is preferentially expressed in primary mesenchyme cells (PMCs), which are responsible for calcification, where it alkalanizes intracellular pH (pHi) to promote calcification. The study also addressed the potential involvement of SpSlc4a10 under ocean acidification (OA)- like conditions.

The topic is important and timely because information about molecular and cellular calcification mechanisms in marine organisms is scarce, and because this is essential for understanding and predicting potential effects of OA. The choice of experimental organism is excellent because sea urchins have very well characterized developmental biology and genome, and genetic manipulations are possible.

Unfortunately, this study has one major flaw in that the specificity and efficacy of SpSlc4a10 morpholino knockdown was not validated.

Figure 2D shows a 4-fold upregulation of SpSlc4a10 mRNA, however this is not a reliable validation. In addition, those larvae had significant NHE downregulation (which would also explain the results). It is likely that mRNA levels for other ion transporting proteins that were not examined were also up or downregulated. This makes it impossible to attribute the effects to SpSlc4a10.

Morpholinos should be validated in some of the following ways:

The gold standard would be to determine protein abundance using validated antibodies; SpSlc4a10 protein level should be reduced. Antibodies against SpSlc4a10 would have the additional advantage of determining subcellular localization -i.e. basolateral or apical membrane, or intracellular calcifying vesicles.

If making antibodies is not possible, a second morpholino against another region of the SpSlc4a10 gene should be used.

Alternatively, CRISPR-Cas9 or siRNA could be used.

In any case, control "scrambled morpholino" should be used and those larvae must be used for at least some of the key experiments. Unfortunately, skeleton defects is a very common phenotype seen in stressed sea urchin larvae, so determining a specific effect on calcification requires very carefully controlled experiments.

Another issue is the use of DIDS. While it is true this drug inhibits bicarbonate transporters, it is very broad so it will inhibit all bicarbonate transporters (not just SpSlc4a10). Furthermore and even more worrying is that DIDS has multiple and significant off-target effects, which explains why cutting-edge biomedical research has stopped using DIDS at least a decade ago. For example, DIDS inhibits the H⁺/K⁺-ATPase at concentrations as low as 1μM (Sugita, et al. 1999).

One way to circumvent this problem would be to assess the effect of DIDS on cells from sea urchin larvae morphants (which should not be sensitive to DIDS).

Moreover, the effects of DMSO (the vehicle for DIDS) on pHi should be determined.

Unless these issues are resolved, I will not provide further feedback about the experiments or conclusions. I repeat this is a very interesting and important topic, and that the choice of experimental organism is excellent. However, the issues listed above must be addressed.

Reviewer #2:

This manuscript suggests that a member of the SLC4 HCO3 transporter family, SpSlc4a10, has an important role in pH regulatory and HCO3 concentrating mechanism is sea urchin.

This is a very interesting and important information relating to the biomineralization mechanism in animals. This is a very elegant and well design experiment, the authors studied each relevant aspect that HCO3 transporter can affect.

Reviewer #3:

This manuscript addressed the role of bicarbonate transport in developing sea urchins, in particular on biomineralization. The manuscript is well written and easy to follow. The manuscript supports a key role of the bicarbonate transporter slc4a10 in pH regulation and biomineralization processes in developing sea urchin. The only real problem with the manuscript is a logical gap as to why the research has chosen to focus on slc4a10 as opposed to any other bicarbonate transporter of sea urchins? The data do support a central, but not unique, role of slc4a10 in the processes mentioned above.

1) Fig, 1B is too small to be of value. It should be greatly increased in size and included as a figure supplement.

2) Figure 1C needs an X-axis label (presumably time).

3) Subsection “Expression pattern of the sea urchin SpSlc4a10 bicarbonate transporter”, first sentence. The term "ubiquitous" may be too strong. Perhaps "widespread" would be better supported by the data.

4) The manuscript does not provide sufficient text rationalizing the focus on slc4a10, to the exclusion of other bicarbonate transporters in sea urchin. Please explain why the analysis was confined to this transporter.

5) Figure 2 legend and elsewhere: Ideally legends should explain the experiment, but not interpret the results. The phrase "demonstrate developmental defects in the formation of the larval skeleton in the pluteus larva (4 dpf)" clearly belongs in the Results section, not legend. Same issue arises in panel D and E legend. Please revise this and other legends accordingly.

6) The established convention in the SLC4 field is that NBC refers to "sodium bicarbonate co-transporter". Family member SLC4A10 is an electroneutral transporter called NBCn2, as the second recognized electroneutral sodium bicarbonate cotransporter. If the sea urchin gene product studied here is orthologous to mammalian SLC4A10, it may very well share the same transport function. If so, that function is electroneutral sodium dependent Cl^-/HCO₃^- exchange. This point should form part of the introduction. Moreover, in the manuscript where the term "NBC" is used to described slc4a10, NBCn2 might be preferable.

The manuscript needs to provide the evidence in the form of amino acid sequence alignments or literature citations that identifies the gene under investigation as orthologous to SLC4A10.

7) The third paragraph of the Discussion would be enhanced by citing literature discussing the myriad effects of pH on cellular function. So powerful is the effect of pH that it is difficult to assess the mechanism by which it acts.

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

This study attempted to characterize the involvement of the bicarbonate transporter SpSlc4a10 in sea urchin larvae calcification. The hypothesis was that SpSlc4a10 is preferentially expressed in primary mesenchyme cells (PMCs), which are responsible for calcification, where it alkalanizes intracellular pH (pHi) to promote calcification. The study also addressed the potential involvement of SpSlc4a10 under ocean acidification (OA)- like conditions.

The topic is important and timely because information about molecular and cellular calcification mechanisms in marine organisms is scarce, and because this is essential for understanding and predicting potential effects of OA. The choice of experimental organism is excellent because sea urchins have very well characterized developmental biology and genome, and genetic manipulations are possible.

We would like to thank reviewer #1 for a generally positive but also critical review on our manuscript. We spent a lot of time and efforts to carefully address and resolve all issues raised by reviewer #1, which has significantly increased the quality and scientific value of this work.

We were able to generate and validate an antibody raised against the sea urchin SpSlc4a10 protein. Using this antibody we validated our MO-knock-down by western blot analysis and could confirm our in situ hybridization experiments demonstrating that also the protein of SpSlc4a10 is exclusively located in PMCs. We performed additional experiments using scrambled morpholinos to demonstrate that skeletal defects can be specifically attributed to the SpSlc4a10 MO. In addition, in the context of another project, we are currently working with several other MOs targeting proteins expressed in the endoderm of the sea urchin larva. Using these MOs we found no skeletal defects making us very confident that skeletal deformations and reductions in calcification seen in this study can be attributed to the specificity of our SpSlc4a10 MO. We also explained the rationale for choosing DIDS in our pharmacological experiments and the implications of these observations for the research community.

Unfortunately, this study has one major flaw in that the specificity and efficacy of SpSlc4a10 morpholino knockdown was not validated.

Figure 2D shows a 4-fold upregulation of SpSlc4a10 mRNA, however this is not a reliable validation. In addition, those larvae had significant NHE downregulation (which would also explain the results). It is likely that mRNA levels for other ion transporting proteins that were not examined were also up or downregulated. This makes it impossible to attribute the effects to SpSlc4a10.

Morpholinos should be validated in some of the following ways:

The gold standard would be to determine protein abundance using validated antibodies; SpSlc4a10 protein level should be reduced. Antibodies against SpSlc4a10 would have the additional advantage of determining subcellular localization -i.e. basolateral or apical membrane, or intracellular calcifying vesicles.

If making antibodies is not possible, a second morpholino against another region of the SpSlc4a10 gene should be used.

Alternatively, CRISPR-Cas9 or siRNA could be used.

We agree with reviewer #1 that a compensatory expression, by increasing SpSlc4a10 mRNA levels in morphants, merely represents a weak validation for the specificity of our knock-down. According to the reviewer´s suggestion we now included additional experiments to validate the specificity of our SpSlc4a10 knock-down by demonstrating that protein levels are decreased in SpSlc4a10 morphants. We generated a polyclonal antibody designed against AA205-231 (LTRHRHHKQKKKKEPENKAYNKGRRKS) of the SpSlc4a10 protein of the purple sea urchin (Strongylocentrotus purpurtus). Western blot analyses demonstrate specific immunoreactivity of our SpScl4a10 antibody with a 130 KDa protein which is in the predicted size range, and which is similar to the molecular weight of the mammalian Slc4a10 orthologue. SpSlc4a10 protein levels are significantly reduced by 70% (normalized to ß-actin) in morphants compared to control larvae. Immunocytological analyses demonstrate high concentration of this protein in primary mesenchyme cells and associated filopodia. The protein is localized in cellular membranes as well as in the cytosol (vesicles). We added this data as a new figure (Figure 3) to the revised manuscript and added the respective paragraphs to the manuscript describing and discussing these new findings.

In any case, control "scrambled morpholino" should be used and those larvae must be used for at least some of the key experiments. Unfortunately, skeleton defects is a very common phenotype seen in stressed sea urchin larvae, so determining a specific effect on calcification requires very carefully controlled experiments.

We used “scramble morpholino” at the same concentration as we applied our SpSlc4a10 MO and could not detect any morphological defects during larval development. We added a new supplemental figure to this work comparing primary rod-length in KCL and “scramble-MO” injected larvae (Figure 2—figure supplement 1). Furthermore, using other MOs (at the same concentration) targeting endodermal genes we did not find skeletal defects underlining the specificity of skeletal deformations in our SpSlc4a10 morphants.

Another issue is the use of DIDS. While it is true this drug inhibits bicarbonate transporters, it is very broad so it will inhibit all bicarbonate transporters (not just SpSlc4a10). Furthermore and even more worrying is that DIDS has multiple and significant off-target effects, which explains why cutting-edge biomedical research has stopped using DIDS at least a decade ago. For example, DIDS inhibits the H⁺/K⁺-ATPase at concentrations as low as 1μM

Sugita, et al. (1999).

One way to circumvent this problem would be to assess the effect of DIDS on cells from sea urchin larvae morphants (which should not be sensitive to DIDS).

Moreover, the effects of DMSO (the vehicle for DIDS) on pHi should be determined.

We agree that DIDS is a rather broad-band compound inhibiting most Slc4 transporters and potentially also other transporters relevant for pH homeostasis. There are two main purposes why we used DIDS in addition to our specific knock-down of Slc4a10. First, this inhibitor has been used in studies addressing the formation of the larval sea urchin skeleton (Yasumasu et al. 1986 Exp Cell Res 159:80-90). These studies demonstrated skeletal deformations and the authors concluded the involvement of HCO₃^- transport in the biomineralization process with the underlying mechanisms remaining unresolved. In this way the present work helps demonstrating that DIDS affects pHi regulatory abilities that are likely responsible for skeletal defects in the sea urchin larva. Second, based on our previous studies demonstrating that pH regulation in PMCs is HCO₃^- and Na⁺ dependent, we used DIDS to provide a complete chain of evidences (Na+, HCO₃^- manipulations, pharmacology and morpholino knock-down of SpSlc4a10) demonstrating the importance of HCO₃^- transport in pHi regulation.

In addition we added a paragraph discussing potential off-target effects of DIDS mentioned by reviewer #1: “These reductions in calcification rates are accompanied by decreased pH_i regulatory abilities under DIDS treatment. […] Accordingly, it remains unresolved whether decreased pH_i regulatory abilities under DIDS treatment can be attributed to an inhibition of HCO₃^- or H⁺ transport.”

In our controls we always added the vehicle (DMSO) at the same concentration as in the ones with DIDS. We have this information in the Materials and methods section as well as in the respective figure legends.

Unless these issues are resolved, I will not provide further feedback about the experiments or conclusions. I repeat this is a very interesting and important topic, and that the choice of experimental organism is excellent. However, the issues listed above must be addressed.

We appreciate that reviewer #1 sees the importance and timeliness of this work and hope he is satisfied with the revised version now including the requested experiments.

Reviewer #3:

This manuscript addressed the role of bicarbonate transport in developing sea urchins, in particular on biomineralization. The manuscript is well written and easy to follow. The manuscript supports a key role of the bicarbonate transporter slc4a10 in pH regulation and biomineralization processes in developing sea urchin. The only real problem with the manuscript is a logical gap as to why the research has chosen to focus on slc4a10 as opposed to any other bicarbonate transporter of sea urchins? The data do support a central, but not unique, role of slc4a10 in the processes mentioned above.

We are grateful for the positive and constructive comments made by reviewer #3 that significantly improved the quality of our manuscript. We addressed and accepted all concerns raised and corrected them in the revised version of our manuscript. In particular, we explained our rationale for choosing SpSlc4a10 out of the six Slc4 transporters annotated in the sea urchin genome (see comments below). Furthermore we corrected all legends in that we now focus on explaining the experiments rather than describing the data. We also separated the Introduction and Discussion into shorter focused units and added a paragraph introducing the function of the mammalian NBCn2. We hope reviewer #3 is happy with the revised manuscript and we would like to once again thank him/her for constructive criticism.

1) Fig, 1B is too small to be of value. It should be greatly increased in size and included as a figure supplement.

We enlarged Figure A2 and moved it to the supplemental material part.

2) Figure 1C needs an X-axis label (presumably time).

We added the axis label (days post fertilization).

3) Subsection “Expression pattern of the sea urchin SpSlc4a10 bicarbonate transporter”, first sentence. The term "ubiquitous" may be too strong. Perhaps "widespread" would be better supported by the data.

We agree and replaced ubiquitous with widespread: “The SpSlc4a10 has widespread expression in blastula embryos.”

4) The manuscript does not provide sufficient text rationalizing the focus on slc4a10, to the exclusion of other bicarbonate transporters in sea urchin. Please explain why the analysis was confined to this transporter.

We agree with the reviewer’s comment and added information as to why we specifically addressed the role of SpSlc4a10 in this study. A developmental transcriptome (Tu, Cameron and Davidson, 2014) indicates that the SpSlc4a10 gene is highly expressed in the early sea urchin embryo during skeleton formation. We added the following paragraph to the Introduction: “Six Slc4 transporters including four putative anion exchangers and two sodium-bicarbonate co-transporters were identified in the genome of the purple sea urchin (Tu et al., 2012). […] This prompted us to hypothesize that SpSlc4a10 may be critically involved in HCO₃^- transport during formation of the larval skeleton.”

5) Figure 2 legend and elsewhere: Ideally legends should explain the experiment, but not interpret the results. The phrase "demonstrate developmental defects in the formation of the larval skeleton in the pluteus larva (4 dpf)" clearly belongs in the Results section, not legend. Same issue arises in panel D and E legend. Please revise this and other legends accordingly.

We agree with the concern raised by reviewer #3 and carefully corrected and rephrased all respective figure legends.

6) The established convention in the SLC4 field is that NBC refers to "sodium bicarbonate co-transporter". Family member SLC4A10 is an electroneutral transporter called NBCn2, as the second recognized electroneutral sodium bicarbonate cotransporter. If the sea urchin gene product studied here is orthologous to mammalian SLC4A10, it may very well share the same transport function. If so, that function is electroneutral sodium dependent Cl^-/HCO₃^- exchange. This point should form part of the introduction. Moreover, in the manuscript where the term "NBC" is used to described slc4a10, NBCn2 might be preferable.

The manuscript needs to provide the evidence in the form of amino acid sequence alignments or literature citations that identifies the gene under investigation as orthologous to SLC4A10.

We added information regarding the function of the mammalian NBCn2 to the introduction. We carefully checked our text and replaced Slc4a10 by NBCn2 where necessary. We also added an amino acid sequence alignment of the human NBCn2 and the sea urchin SpSlc4a10 to the supplemental part (Figure 1—figure supplement 3).

7) The third paragraph of the Discussion would be enhanced by citing literature discussing the myriad effects of pH on cellular function. So powerful is the effect of pH that it is difficult to assess the mechanism by which it acts.

We added a sentence to this paragraph referring to many cellular defects associated with intracellular pH disturbances: “However, intracellular pH disturbances were demonstrated to be associated with a wide range of cellular dysfunctions (e.g. Kurdjian and Guern, 1989; Madshus, 1988; Roos and Boron, 1981) that may contribute to the disturbed PMC migration observed in this study.”

Author details

1. Marian Y Hu

   Institute of Physiology, Christian-Albrechts University of Kiel, Kiel, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   m.hu@physiologie.uni-kiel.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8914-139X

2. Jia-Jiun Yan

   1. Institute of Physiology, Christian-Albrechts University of Kiel, Kiel, Germany
   2. Institute of Cellular and Organismic Biology, Taipei, Taiwan

   Contribution

   Conceptualization, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

3. Inga Petersen

   Institute of Physiology, Christian-Albrechts University of Kiel, Kiel, Germany

   Contribution

   Data curation, Formal analysis, Validation, Methodology

   Competing interests

   No competing interests declared

4. Nina Himmerkus

   Institute of Physiology, Christian-Albrechts University of Kiel, Kiel, Germany

   Contribution

   Conceptualization, Data curation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

5. Markus Bleich

   Institute of Physiology, Christian-Albrechts University of Kiel, Kiel, Germany

   Contribution

   Visualization, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

6. Meike Stumpp

   Comparative Immunobiology, Institute of Zoology, Christian-Albrechts University of Kiel, Kiel, Germany

   Contribution

   Conceptualization, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

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