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.

1. 1. Anderson HC

   (2003) Matrix vesicles and calcification

   Current Rheumatology Reports 5:222–226.

   https://doi.org/10.1007/s11926-003-0071-z

   • PubMed
   • Google Scholar
2. 1. Basse WC
   2. Gutowska MA
   3. Findeisen U
   4. Stumpp M
   5. Dupont S
   6. Jackson DJ
   7. Himmerkus N
   8. Melzner F
   9. Bleich M

   (2015) A sea urchin Na ⁺ K ⁺ 2Cl − cotransporter is involved in the maintenance of calcification-relevant cytoplasmic cords in Strongylocentrotus droebachiensis larvae

   Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 187:184–192.

   https://doi.org/10.1016/j.cbpa.2015.05.005

   • Google Scholar
3. 1. Beniash E
   2. Aizenberg J
   3. Addadi L
   4. Weiner S

   (1997) Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth

   Proceedings of the Royal Society B: Biological Sciences 264:461–465.

   https://doi.org/10.1098/rspb.1997.0066

   • Google Scholar
4. 1. Benson SC
   2. Benson NC
   3. Wilt F

   (1986) The organic matrix of the skeletal spicule of sea urchin embryos

   The Journal of Cell Biology 102:1878–1886.

   https://doi.org/10.1083/jcb.102.5.1878

   • PubMed
   • Google Scholar
5. 1. Boonrungsiman S
   2. Gentleman E
   3. Carzaniga R
   4. Evans ND
   5. McComb DW
   6. Porter AE
   7. Stevens MM

   (2012) The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

   PNAS 109:14170–14175.

   https://doi.org/10.1073/pnas.1208916109

   • PubMed
   • Google Scholar
6. 1. Boveri T

   (1901)

   Über die polarität des Seeigel-Eies

   Ver Phys Med Ges 34:145–176.

   • Google Scholar
7. 1. Brandao-Burch A
   2. Utting JC
   3. Orriss IR
   4. Arnett TR

   (2005) Acidosis inhibits bone formation by osteoblasts in vitro by preventing mineralization

   Calcified Tissue International 77:167–174.

   https://doi.org/10.1007/s00223-004-0285-8

   • PubMed
   • Google Scholar
8. 1. Bronckers AL
   2. Lyaruu DM
   3. Jansen ID
   4. Medina JF
   5. Kellokumpu S
   6. Hoeben KA
   7. Gawenis LR
   8. Oude-Elferink RP
   9. Everts V

   (2009) Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents

   Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 312B:375–387.

   https://doi.org/10.1002/jez.b.21267

   • PubMed
   • Google Scholar
9. 1. Byrne M
   2. Lamare M
   3. Winter D
   4. Dworjanyn SA
   5. Uthicke S

   (2013) The stunting effect of a high CO2 ocean on calcification and development in sea urchin larvae, a synthesis from the tropics to the poles

   Philosophical Transactions of the Royal Society B: Biological Sciences 368:20120439.

   https://doi.org/10.1098/rstb.2012.0439

   • PubMed
   • Google Scholar
10. 1. Decker GL
    2. Lennarz WJ

    (1988)

    Skeletogenesis in the sea urchin embryo

    Development 103:231–247.

    • PubMed
    • Google Scholar
11. 1. Decker GL
    2. Morrill JB
    3. Lennarz WJ

    (1987)

    Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

    Development 101:297–312.

    • PubMed
    • Google Scholar
12. 1. Ettensohn CA

    (1990) The regulation of primary mesenchyme cell patterning

    Developmental Biology 140:261–271.

    https://doi.org/10.1016/0012-1606(90)90076-U

    • PubMed
    • Google Scholar
13. 1. Guo YM
    2. Liu Y
    3. Liu M
    4. Wang JL
    5. Xie ZD
    6. Chen KJ

    (2017) Na+/HCO3- cotransporter NBCn2 mediates HCO3- reclamation in the apical membrane of renal proximal tubules

    Journal of the American Society of Nephrology : JASN 28:2409–2419.

    https://doi.org/10.1681/ASN.2016080930

    • Google Scholar
14. 1. Gustafson T
    2. Kinnander H

    (1956) Gastrulation in the sea urchin larva studied by aid of time-lapse cinematography

    Experimental Cell Research 10:733–734.

    https://doi.org/10.1016/0014-4827(56)90050-7

    • Google Scholar
15. 1. Holappa K
    2. Suokas M
    3. Soininen P
    4. Kellokumpu S

    (2001) Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts

    Journal of Histochemistry & Cytochemistry 49:259–269.

    https://doi.org/10.1177/002215540104900213

    • PubMed
    • Google Scholar
16. 1. Jansen ID
    2. Mardones P
    3. Lecanda F
    4. de Vries TJ
    5. Recalde S
    6. Hoeben KA
    7. Schoenmaker T
    8. Ravesloot JH
    9. van Borren MM
    10. van Eijden TM
    11. Bronckers AL
    12. Kellokumpu S
    13. Medina JF
    14. Everts V
    15. Oude Elferink RP

    (2009) Ae2(a,b)-deficient mice exhibit osteopetrosis of long bones but not of calvaria

    The FASEB Journal 23:3470–3481.

    https://doi.org/10.1096/fj.08-122598

    • PubMed
    • Google Scholar
17. 1. Ko SB
    2. Shcheynikov N
    3. Choi JY
    4. Luo X
    5. Ishibashi K
    6. Thomas PJ
    7. Kim JY
    8. Kim KH
    9. Lee MG
    10. Naruse S
    11. Muallem S

    (2002) A molecular mechanism for aberrant CFTR-dependent HCO₍₃₎ transport in cystic fibrosis

    The EMBO journal 21:5662–5672.

    https://doi.org/10.1093/emboj/cdf580

    • PubMed
    • Google Scholar
18. 1. Kroeker KJ
    2. Kordas RL
    3. Crim R
    4. Hendriks IE
    5. Ramajo L
    6. Singh GS
    7. Duarte CM
    8. Gattuso JP

    (2013) Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming

    Global Change Biology 19:1884–1896.

    https://doi.org/10.1111/gcb.12179

    • PubMed
    • Google Scholar
19. 1. Kurkdjian A
    2. Guern J

    (1989) Intracellular pH: measurement and importance in cell activity

    Annual Review of Plant Physiology and Plant Molecular Biology 40:271–303.

    https://doi.org/10.1146/annurev.pp.40.060189.001415

    • Google Scholar
20. 1. Liu L
    2. Schlesinger PH
    3. Slack NM
    4. Friedman PA
    5. Blair HC

    (2011) High capacity Na⁺/H⁺ exchange activity in mineralizing osteoblasts

    Journal of Cellular Physiology 226:1702–1712.

    https://doi.org/10.1002/jcp.22501

    • PubMed
    • Google Scholar
21. 1. Madshus IH

    (1988) Regulation of intracellular pH in eukaryotic cells

    Biochemical Journal 250:1–8.

    https://doi.org/10.1042/bj2500001

    • PubMed
    • Google Scholar
22. 1. Martin C
    2. Pedersen SF
    3. Schwab A
    4. Stock C

    (2011) Intracellular pH gradients in migrating cells

    American Journal of Physiology-Cell Physiology 300:C490–C495.

    https://doi.org/10.1152/ajpcell.00280.2010

    • PubMed
    • Google Scholar
23. 1. Mitsunaga K
    2. Fujino Y
    3. Yasumasu I

    (1987) Distributions of H+,K+-ATPase and Cl-,HCO3(-)-ATPase in micromere-derived cells of sea urchin embryos

    Differentiation 35:190–196.

    https://doi.org/10.1111/j.1432-0436.1987.tb00168.x

    • PubMed
    • Google Scholar
24. 1. Mitsunaga K
    2. Makihara R
    3. Fujino Y
    4. Yasumasu I

    (1986) Inhibitory effects of ethacrynic acid, furosemide, and nifedipine on the calcification of spicules in cultures of micromeres isolated from sea-urchin eggs

    Differentiation 30:197–204.

    https://doi.org/10.1111/j.1432-0436.1986.tb00781.x

    • Google Scholar
25. 1. Mobasheri A
    2. Golding S
    3. Pagakis SN
    4. Corkey K
    5. Pocock AE
    6. Fermor B
    7. O'BRIEN MJ
    8. Wilkins RJ
    9. Ellory JC
    10. Francis MJ

    (1998) Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts

    Cell Biology International 22:551–562.

    https://doi.org/10.1006/cbir.1998.0299

    • PubMed
    • Google Scholar
26. 1. Nakano E
    2. Okazaki K
    3. Iwamatsu T

    (1963) Accumulation of eadioactive calcium tn larvae of the sea urchin pseudocentrotus depressus

    The Biological Bulletin 125:125–132.

    https://doi.org/10.2307/1539295

    • Google Scholar
27. 1. Pan TC
    2. Applebaum SL
    3. Manahan DT

    (2015) Experimental ocean acidification alters the allocation of metabolic energy

    PNAS 112:4696–4701.

    https://doi.org/10.1073/pnas.1416967112

    • PubMed
    • Google Scholar
28. 1. Parker MD
    2. Musa-Aziz R
    3. Rojas JD
    4. Choi I
    5. Daly CM
    6. Boron WF

    (2008) Characterization of human SLC4A10 as an electroneutral Na/HCO3 cotransporter (NBCn2) with Cl^- self-exchange activity

    Journal of Biological Chemistry 283:12777–12788.

    https://doi.org/10.1074/jbc.M707829200

    • PubMed
    • Google Scholar
29. 1. Pespeni MH
    2. Sanford E
    3. Gaylord B
    4. Hill TM
    5. Hosfelt JD
    6. Jaris HK
    7. LaVigne M
    8. Lenz EA
    9. Russell AD
    10. Young MK
    11. Palumbi SR

    (2013) Evolutionary change during experimental ocean acidification

    PNAS 110:6937–6942.

    https://doi.org/10.1073/pnas.1220673110

    • PubMed
    • Google Scholar
30. 1. Romero MF
    2. Chen A-P
    3. Parker MD
    4. Boron WF

    (2013) The SLC4 family of bicarbonate transporters

    Molecular Aspects of Medicine 34:159–182.

    https://doi.org/10.1016/j.mam.2012.10.008

    • Google Scholar
31. 1. Romero MF
    2. Fulton CM
    3. Boron WF

    (2004) The SLC4 family of HCO₃ transporters

    Pflugers Archiv European Journal of Physiology 447:495–509.

    https://doi.org/10.1007/s00424-003-1180-2

    • PubMed
    • Google Scholar
32. 1. Roos A
    2. Boron WF

    (1981) Intracellular pH

    Physiological Reviews 61:296–434.

    https://doi.org/10.1152/physrev.1981.61.2.296

    • Google Scholar
33. 1. Schatzberg D
    2. Lawton M
    3. Hadyniak SE
    4. Ross EJ
    5. Carney T
    6. Beane WS
    7. Levin M
    8. Bradham CA

    (2015) H⁽⁺)/K⁽⁺) ATPase activity is required for biomineralization in sea urchin embryos

    Developmental Biology 406:259–270.

    https://doi.org/10.1016/j.ydbio.2015.08.014

    • PubMed
    • Google Scholar
34. 1. Sikes CS
    2. Okazaki K
    3. Fink RD

    (1981) Respiratory CO₂ and the supply of inorganic carbon for calcification of sea urchin embryos

    Comparative Biochemistry and Physiology Part A: Physiology 70:285–291.

    https://doi.org/10.1016/0300-9629(81)90181-X

    • Google Scholar
35. 1. Stumpp M
    2. Dupont S
    3. Thorndyke MC
    4. Melzner F

    (2011) CO2 induced seawater acidification impacts sea urchin larval development II: gene expression patterns in pluteus larvae

    Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 160:320–330.

    https://doi.org/10.1016/j.cbpa.2011.06.023

    • PubMed
    • Google Scholar
36. 1. Stumpp M
    2. Hu M
    3. Casties I
    4. Saborowski R
    5. Bleich M
    6. Melzner F
    7. Dupont S

    (2013) Digestion in sea urchin larvae impaired under ocean acidification

    Nature Climate Change 3:1044–1049.

    https://doi.org/10.1038/nclimate2028

    • Google Scholar
37. 1. Stumpp M
    2. Hu MY
    3. Melzner F
    4. Gutowska MA
    5. Dorey N
    6. Himmerkus N
    7. Holtmann WC
    8. Dupont ST
    9. Thorndyke MC
    10. Bleich M

    (2012) Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

    PNAS 109:18192–18197.

    https://doi.org/10.1073/pnas.1209174109

    • PubMed
    • Google Scholar
38. 1. Stumpp M
    2. Hu MY
    3. Tseng YC
    4. Guh YJ
    5. Chen YC
    6. Yu JK
    7. Su YH
    8. Hwang PP
    9. My H
    10. Jk Y

    (2015) Evolution of extreme stomach pH in bilateria inferred from gastric alkalization mechanisms in basal deuterostomes

    Scientific Reports 5:1–9.

    https://doi.org/10.1038/srep10421

    • PubMed
    • Google Scholar
39. 1. Sugita Y
    2. Nagao T
    3. Urushidani T

    (1999) Nonspecific effects of the pharmacological probes commonly used to analyze signal transduction in rabbit parietal cells

    European Journal of Pharmacology 365:77–89.

    https://doi.org/10.1016/S0014-2999(98)00855-3

    • PubMed
    • Google Scholar
40. 1. Tu Q
    2. Cameron RA
    3. Davidson EH

    (2014) Quantitative developmental transcriptomes of the sea urchin Strongylocentrotus purpuratus

    Developmental Biology 385:160–167.

    https://doi.org/10.1016/j.ydbio.2013.11.019

    • PubMed
    • Google Scholar
41. 1. Tu Q
    2. Cameron RA
    3. Worley KC
    4. Gibbs RA
    5. Davidson EH

    (2012) Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis

    Genome Research 22:2079–2087.

    https://doi.org/10.1101/gr.139170.112

    • PubMed
    • Google Scholar
42. 1. Urry LA
    2. Hamilton PC
    3. Killian CE
    4. Wilt FH

    (2000) Expression of spicule matrix proteins in the sea urchin embryo during normal and experimentally altered spiculogenesis

    Developmental Biology 225:201–213.

    https://doi.org/10.1006/dbio.2000.9828

    • PubMed
    • Google Scholar
43. 1. Vidavsky N
    2. Addadi S
    3. Mahamid J
    4. Shimoni E
    5. Ben-Ezra D
    6. Shpigel M
    7. Weiner S
    8. Addadi L

    (2014) Initial stages of calcium uptake and mineral deposition in sea urchin embryos

    PNAS 111:39–44.

    https://doi.org/10.1073/pnas.1312833110

    • PubMed
    • Google Scholar
44. 1. Vidavsky N
    2. Addadi S
    3. Schertel A
    4. Ben-Ezra D
    5. Shpigel M
    6. Addadi L
    7. Weiner S

    (2016) Calcium transport into the cells of the sea urchin larva in relation to spicule formation

    PNAS 113:12637–12642.

    https://doi.org/10.1073/pnas.1612017113

    • PubMed
    • Google Scholar
45. 1. Wang CZ
    2. Yano H
    3. Nagashima K
    4. Seino S

    (2000) The na+^-driven ^cl-/HCO₃^- exchanger. cloning, tissue distribution, and functional characterization

    The Journal of Biological Chemistry 275:35486–35490.

    https://doi.org/10.1074/jbc.C000456200

    • PubMed
    • Google Scholar
46. Book

    1. Warner J
    2. McClay DR

    (2014) Perturbation of the Hedgehog pathway in sea urchin embryos

    In: Carroll D. J, Stricker S. A, editors. Developmental Biology of the Sea Urchin and Other Marine Invertebrates. New York: Humana Press. pp. 211–223.

    https://doi.org/10.1007/978-1-62703-974-1_14

    • Google Scholar
47. 1. Wilt FH

    (2002) Biomineralization of the spicules of sea urchin embryos

    Zoological Science 19:253–261.

    https://doi.org/10.2108/zsj.19.253

    • PubMed
    • Google Scholar
48. 1. Yasumasu I
    2. Mitsunaga K
    3. Fujino Y

    (1985) Mechanism for electrosilent ^Ca2+ transport to cause calcification of spicules in sea urchin embryos

    Experimental Cell Research 159:80–90.

    https://doi.org/10.1016/S0014-4827(85)80039-2

    • PubMed
    • Google Scholar
49. 1. Zoccola D
    2. Ganot P
    3. Bertucci A
    4. Caminiti-Segonds N
    5. Techer N
    6. Voolstra CR
    7. Aranda M
    8. Tambutté E
    9. Allemand D
    10. Casey JR
    11. Tambutté S

    (2015) Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification

    Scientific Reports 5:9983.

    https://doi.org/10.1038/srep09983

    • PubMed
    • Google Scholar

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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