Hu MY et al. (2018), A SLC4 family bicarbonate transporter is critic...

ECB-ART-46294

Elife 2018 May 01;7. doi: 10.7554/eLife.36600.

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A SLC4 family bicarbonate transporter is critical for intracellular pH regulation and biomineralization in sea urchin embryos.

Hu MY , Yan JJ , Petersen I , Himmerkus N , Bleich M , Stumpp M .

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Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown. Using the sea urchin larva, this work identified the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations. Reductions in seawater pH, resembling ocean acidification scenarios, led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates. We propose a first pH regulatory and HCO3- concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO3. This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment.

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Species referenced: Echinodermata
Genes referenced: irak1bp1 LOC100887844 LOC115919910 LOC580912 LOC581395 LOC590371 mos NBC SpZ12-1
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	Figure 1â€”figure supplement 1. Expression of SpSlc4a10 in the syncytial cables of the PMCs.(A) Whole-mount in situ hybridization using the SpSlc4a10 antisense probe demonstrated high expression levels of this gene in the cell bodies of PMCs and staining is also found in the syncytial cables. (B) High-magnification image of four PMCs and the filopodia of the PMC syncytium that are positively stained.
	Figure 1â€”figure supplement 2. Phylogeny of vertebrate and sea urchin Slc4 transporters.Phylogenetic analysis performed on deduced amino acid sequences of the four S. purpuratus SLC4 family transporters and those from vertebrates and other marine invertebrates (see Supplementary file 1-Table S1 for sequence information).
	Figure 1â€”figure supplement 3. Sequence alignment of the human NBCn2 (Slc4a10) and the sea urchin SpSlc4a10.Alignment of deduced amino acid sequences demonstrates 44% identity between the human Slc4a10 and the sea urchin SpSlc4a10.
	Figure 1. Expression pattern of the SpSlc4a10 gene from blastula through pluteus larva in Strongylocentrotus purpuratus.(A) Localization of SpSlc4a10 expression in the sea urchin larva along the larval development until 3 days post fertilization (dpf). Expression was detected in primary mesenchyme cells (PMCs) of the late blastula stage forming a ring around the blastopore. In the early pluteus larva SpSlc4a10 expression is exclusively found in PMCs located at ends of the spicules. (B) SpSlc4a10 expression levels and total calcium content along the early development of sea urchin larvae raised under control conditions. Bars represent meanÂ Â±Â SD; nÂ =Â 3. dpf: days post fertilization; LV, lateral view; VV, vegetal view; OV, oral view.
	Figure 2â€”figure supplement 1. Comparison of KCL and scramble morpholino (MO) injected larvae.The primary rod length was used as an indicator for skeletal deformations. Fertilized eggs were injected with 0.5 M KCL, the vehicle of the MOs used in the present work. The concentration of the scramble morpholino was adjusted to 150 ÂµM which corresponds to the concentration of SpSlc4a10 MO used for all experiments.


	Figure 4â€”figure supplement 1. Mortality and growth of sea urchin larvae raised under different pH treatments.(A) Larval densities were constant at 140 to 160 larvae per ml over the experimental duration of 5 days. No effects on the larval densities were observed between the three pH treatments. Larval growth expressed as body length (BL) followed a logarithmic curve along the period of 5 days. Regression analyses demonstrated significant differences in BL between the three pH treatments. Values are presented as meanÂ Â±Â SD.
	Figure 4. Development, calcium accumulation and expression of SpSlc4a10 in sea urchin larvae raised under experimental ocean acidification.(A) Total Ca2+ content of larvae raised under three different pH conditions. (B) Larval Ca2+ content plotted as a function of body length to normalize for the developmental delay caused by acidified conditions (for morphometric analyses see Supplemental information Figure 4â€”figure supplement 1). (C) SpSlc4a10 mRNA levels normalized to the housekeeping gene SpZ12 during development under different pH conditions. (D) Expression pattterns for SpSlc4a10 along the early development plotted as a function of body length. Different letters denote significant differences between treatments. Bars represent meanÂ Â±Â SD; nÂ =Â 3.
	Figure 5. Intracellular pH regulatory abilities of primary mesenchyme cells.(A) Ratiometric fluorimetry in primary mesencyme cells (PMCs) using the pH sensitive dye BCECF-AM. False colour images superimposed on transmission images at time points 1, 2, 3, 4 as indicated in (B). (B) Summarized data from the control period (control (1)), after addition and removal of NH3/NH4+ (alkalosis (2) and acidification (3); ammonium pulse), and during pHi recovery (4). (C) The recovery rate of the SpSlc4a10 morphants was significantly reduced (see Supplementary file 1-table S2 for summary of prameters measured). (D) Calibration curve of BCECF-AM in PMCs obtained at different pH levels in the presence of the ionophore nigericin and 150 mM K+ allowing the translation of ratios to pH values. (E) Acidâ€“base regulatory abilities of PMCs in the presence of 500 ÂµM DIDS or only the vehicle (DMSO) as control. (F) The recovery rate from an intracellular acidosis is inhibited by DIDS in a dose-dependent manner with an IC50 value of 120 ÂµM. Bars represent meanÂ Â±SD; *p<0.05 (nÂ =Â 4â€“5 larvae with 3â€“5 cells measured per larvae).

	Figure 7. Hypothetical model for pH regulation and bicarbonate transport in PMCs of the sea urchin larva.PMCs form a syncytium within the extraxellular matrix (ECM) that has a pH the same as sea water (SW) in which the larva develops. Amorphous calcium carbonate (ACC) is precipitated in intracellular vesicles and exocytosed to the growing calcite spicule. Dissolved inorganic carbon (DIC) is provided through endogenous (i.e. respiratory CO2) as well as exogenous (from the sea water) sources. SpSlc4a10 (NBC) is proposed to mediate the import of bicarbonate from the seawater and to buffer protons generated during the precipitation of CaCO3. Protons are exported from the vesicles through so far unknown pathways. Protons accumulating in the cytoplasm are potentially exported by the Na+/H+-exchanger (NHE) SpSlc9a2. Both secondary active transporters, NBC and NHE are driven by the Na+/K+-ATPase (NKA; SpAtp1a3) that is highly expressed by PMCs. In addition to its localization in the plasma membrane, NBC is associated with intracellular compartments including vesicles (1), vesicular networks (2) and vesicles fusing with the plasma membrane (3). n; nucleus.

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Anderson, Matrix vesicles and calcification. 2003, Pubmed