Tissue-specific expression of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) 1, 2, 3 during Xenopus laevis development

Gene Expression Patterns 11 (2011) 122–128

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Tissue-specific expression of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) 1, 2, 3 during Xenopus laevis development Caterina Pegoraro a,b, Nicolas Pollet c, Anne H. Monsoro-Burq a,b,⇑ a

Institut Curie, Research division, CNRS UMR3347, INSERM U1021, Centre Universitaire, Orsay F-91 405, France Université Paris Sud-11, Orsay F-91 405, France c CNRS, Genopole, Université d’Evry Val d’Essonne, F91 058 Evry, France b

a r t i c l e

i n f o

Article history: Received 26 April 2010 Received in revised form 12 October 2010 Accepted 22 October 2010 Available online 11 November 2010 Keywords: Calcium SERCA ATP2A Muscle Neural plate Neural crest Ectoderm Embryo Xenopus MyoD CaP60A

a b s t r a c t Calcium-ATPase pumps are critical in most cells, to sequester calcium into intracytoplasmic stores and regulate general calcium signalling. In addition, cell-specific needs for calcium signals have been described and employ a diversity of calcium ATPases in adult tissues and oocytes. A major family of such calcium pumps is ATP2A/SERCA family, for Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases. Although largely studied in adults, the developmental expression of the atp2a/serca genes remains unknown. Here, we provide genome organisation in Xenopus laevis and tropicalis and phylogeny of atp2a/serca genes in craniates. We detail embryonic expression for the three X. laevis atp2a/serca genes. We found that the three atp2a/serca genes are strongly conserved among vertebrates and display complementary and tissue-specific expression in embryos. These expression patterns present variations when compared to the data reported in adults. Atp2a1/serca1 is expressed as soon as the end of gastrulation in a subset of the myod-positive cells, and later labels prospective slow muscle cells in the superficial part of the somite. In contrast atp2a2/serca2 is found in a larger subset of cells, but is not ubiquitous as reported in adults. Notably, atp2a2/serca2 is prominently expressed in the neural-related tissues, i.e. the neural plate, cement gland, but is excluded from premigratory neural crest. Finally, atp2a3/serca3 expression is restricted to the ectoderm throughout development. Ó 2010 Elsevier B.V. All rights reserved.

Calcium is an essential second messenger in cell homeostasis, cell growth and differentiation as well as for specialized functions in specific cells such as muscles and oocytes (Lechleiter et al., 1998; Lipskaia et al., 2009). Cytoplasmic calcium levels are maintained low in the absence of signal, by the activity of calcium-ATPase pumps at the plasma membrane or on intracellular organelles, which either eject calcium out of the cell or sequester and concentrate it into sub cellular compartments. P-ATPases transport different compounds across a membrane using the hydrolysis of ATP (Gunaratne and Vacquier, 2006). Sarcoplasmic/ Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) are evolutionary conserved transmembrane calcium transporters, found from yeast to mammals in the sarcoplasmic and endoplasmic reticulum, that belong to the family of the P-ATPases; (Cho et al., 2000; Gunaratne and Vacquier, 2006). In this family there are also other proteins involved in Ca2+ transport across a membrane such as PMCA, Plasma Membrane Calcium ATPases, in charge of removing Ca2+ from the cells (Brini, 2009). ATP2A/SERCA proteins are Abbreviation: SERCA, sarcoplasmic and endoplasmic reticulum calcium ATPases.

⇑ Corresponding author. Tel.: +33 1 69 86 71 52.

E-mail address: [email protected] (A.H. Monsoro-Burq). 1567-133X/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2010.10.006

involved in maintaining calcium homeostasis, using the hydrolysis of 1 ATP to reuptake 2 Ca2+ into the sarco/endoplasmic reticulum (Periasamy and Kalyanasundaram, 2007). Crystal structure and biochemical analysis show that these proteins have 10 transmembrane alpha helices, 5 luminal loops and 3 cytoplasmic domains: A, actuator; P, phosphorylation; N, nucleotide binding (Brini, 2009; Gunaratne and Vacquier, 2006; Periasamy and Kalyanasundaram, 2007). While a single gene is found in invertebrates (Periasamy and Kalyanasundaram, 2007), the three ATP2A/SERCA genes found in mammals encode a dozen of isoforms, which mainly differ by their affinity to calcium and their calcium turnover rate (Lipskaia et al., 2009). The different genes/isoforms also differ in their tissue expression in adults. In humans, atp2a1/serca1 is expressed in the fast-twitch skeletal muscle; atp2a2/serca2 is expressed in cardiomyocytes, vascular myocytes and slow-twitch skeletal muscle and its isoform 2b is globally ubiquitous; and atp2a3/serca3 is found in multiple tissues and cell types (endothelial cells, lymphocytes, fibroblasts, platelets, epithelial cells) (Lipskaia et al., 2009; Periasamy and Kalyanasundaram, 2007). In mammals, abnormalities in the structure or in the amount of these proteins have been mainly related to cardiac malfunction (Brini, 2009; Sanyal et al.,

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2006; Schwinger et al., 1999). In addition, human mutations in serca1 are linked to the autosomal recessive form of Brody’s disease, a rare inherited muscle function disorder characterized by exercise-induced impairment of skeletal muscle relaxation, stiffness and cramps (MacLennan et al., 1997; Odermatt et al., 1996). Moreover, splicing defects in serca1 are found in type 1 myotonic dystrophy (DM1), one of the most common form of adult muscular dystrophy (Hino et al., 2007). Embryonic and fetal expression of these genes was not previously recorded, except for atp2a2/serca2 expression in rat fetus heart (Anger et al., 1994). Here, we have studied the three ATP2A/SERCA genes during embryogenesis in the amphibian Xenopus laevis, a non-mammalian vertebrate species. We have identified ATP2A/SERCA orthologs in Xenopus laevis and Xenopus tropicalis, compared their evolutive organisation in vertebrates (including mammals and novel chick and zebrafish orthologs identified in this work), and provide their genome organisation. We here detail their expression in Xenopus laevis, from blastula to tadpole stages. 1. Results 1.1. ATP2A/SERCA gene family in craniates 1.1.1. Isolation of ATP2A/SERCA sequences from genomic databases We surveyed the ATP2A gene family using sequence similarity searches and datamining on public repositories of chordate genomic

data using previously known members as initial seeds. After some iteration of the searches, we eventually identified a set of full-length ATP2A sequences in cephalochordate, urochordate and craniate species (listed in Table 1). A single ATP2A gene sequence was found in two urochordates (sea squirts Halocynthia roretzi and Ciona savignyi), but several co-orthologs are present in the Ciona intestinalis genome (not shown, see Metazome). We found a single ATP2A gene in a cephalochordate, the amphioxus Branchiostoma floridae. In all craniates species with substantial genomic sequences, we found more than one gene encoding an ATP2A-type protein. In mammals, alternative splicing gives rise to two isoforms of ATP2A1: the adult ATP2A1A of 994 aa encoded by a longer transcript and the neonatal one ATP2A1B of 1001 aa encoded by a shorter transcript. We found evidence that the Xenopus ATP2A1 gene is similarly transcribed as either a short (Gurdon 1004794465, JGI model estExt_fgenesh1_kg.C_8290001) or a long transcript (MGC107776, NP_001027498.1). Like ATP2A1, ATP2A2 transcription unit gives rise to alternative transcripts expressed in a tissue-specific manner. Evidences for two forms are described in mammals and chick, ATP2A2a being muscle-specific and ATP2A2b being expressed ubiquitously. In X. tropicalis, the genomic locus containing the ATP2A2 transcription unit encomprise at least 22 exons and therefore is able to generate the two ATP2A2 isoforms. Similarly, ATP2A3 transcription unit gives rise to several transcripts and evidence for six isoforms have been described in man. Variations are exclusively located at the carboxyl terminus of the protein, and expression is

Table 1 Craniate ATP2A/SERCA family. Accession numbers and details for the ATP2A/SERCA family members analyzed in this work. Species

Gene symbol

Sequence name

Size

Accession number

Source

Anolis carolensis Anolis carolensis Bos taurus Canis familiaris Canis familiaris Ciona savignyi Danio rerio Danio rerio Drosophila melanogaster Felis cattus Gallus gallus Gallus gallus Gallus gallus Gasterosteus aculeatus Gasterosteus aculeatus Halocynthia roretzi Homo sapiens Homo sapiens Homo sapiens Monodelphis domestica Mus musculus Mus musculus Oryctolagus cuniculus Oryctolagus cuniculus Rana clamitans Rana esculenta Rana sylvatica Rattus norvegicus Rattus norvegicus Sus scrofa Taeniopygia guttata Taeniopygia guttata Tetraodon nigroviridis Tetraodon nigroviridis Tetraodon nigroviridis Xenopus laevis Xenopus laevis Xenopus laevis Xenopus tropicalis Xenopus tropicalis Xenopus tropicalis

ENSACAG00000005552 ENSACAG00000016106 ATP2A1 ATP2A1 ATP2A2 Cs-ATP2A1/2/3 atp2a2a atp2a1 Ca-P60A ATP2A2 ATP2A1 ATP2A2 ATP2A3 ENSGACG00000017543 ENSGACG00000020226 HrSERCA ATP2A1 ATP2A2 ATP2A3 ENSMODG00000011679 Atp2A2 Atp2a3 ATP2A1 ATP2A2 atp2A1 ATP2A1 atp2A1 atp2A1 Atp2a3 SERCA2 LOC100227680 ENSTGUG00000004042 ENSTNIG00000011586 ENSTNIG00000011446 ENSTNIG00000008543 ca-p60a LOC495046 check LOC495440/atp2a3 mgc107776 atp2a2 atp2a3

AT2A1_ANOCAR AT2A2_ANOCAR AT2A1_BOVIN AT2A1_CANFA AT2A2_CANFA Q75UU1_CIOSA A9C3Q4_DANRE Q5U3A4_DANRE ATC1_DROME AT2A2_FELCA AT2A1_CHICK AT2A2_CHICK AT2A3_CHICK Gac_ENSGACP00000023207 Gac_ENSGACP00000026723 Q8IAC0_HALRO AT2A1_HUMAN AT2A2_HUMAN AT2A3_HUMAN AT2A2_MONDO AT2A2_MOUSE AT2A3_MOUSE AT2A1_RABIT AT2A2_RABIT Q9DDB9_RANCL AT2A1_RANES Q9DDB8_RANSY AT2A1_RAT AT2A3_RAT AT2A2_PIG AT2A2_TGUT AT2A3_TGUT AT2A1_TETNI AT2A2_TETNI AT2A3_TETNI AT2A1_XENLA AT2A2_XENLA AT2A3_XENLA AT2A1_XENTR AT2A2_XENTR AT2A3_XENTR

990 995 993 1069 997 1000 1042 1005 1020 997 994 1041 1042 1042 990 1003 1001 1042 1043 1056 1044 1038 1001 1042 994 994 994 994 999 1042 1043 957 1005 1038 1004 996 1042 1033 994 1047 1033

ENSACAP00000005618 ENSACAP00000015925 Q0VCY0 XP_536925.2 O46674 Q75UU1 A9C3Q4 Q5U3A4 P22700 Q00779 P13585 Q03669 Q9YGL9 ENSGACP00000023207 ENSGACP00000026723 Q8IAC0 O14983 P16615 Q93084 ENSMODP00000014631 O55143 Q64518 P04191 P20647 Q9DDB9 Q92105 Q9DDB8 Q64578 P18596 P11607 XP_002192568.1 ENSTGUP00000004279 ENSTNIP00000014536 ENSTNIP00000014393 ENSTNIP00000001466 NP_001080404.1 NP_001088218.1 NP_001088563.1 NP_001027498.1 1012072482 NP_001072333.1

ENSEMBL ENSEMBL UniprotKB Refseq UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB ENSEMBL ENSEMBL UniprotKB UniprotKB UniprotKB UniprotKB ENSEMBL UniprotKB UniprotKB UniprotKB/PDB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB UniprotKB Refseq ENSEMBL ENSEMBL ENSEMBL ENSEMBL Refseq/Xenbase Refseq/Xenbase Refseq/Xenbase Refseq/Xenbase Gurdon/Xenbase Refseq/Xenbase

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found in a variety of tissues. Evidences are lacking in the Xenopus genomic data to identify these isoforms. 1.1.2. Phylogeny We found 3 groups of genes that together form the ATP2A gene family in craniates: ATP2A1 (syn. SERCA1), ATP2A2 (syn. SERCA2) and ATP2A3 (syn. SERCA3) (Fig. 1). In actinopterygians, the ancestral genome duplication (Brunet et al., 2006) led to the existence of additional paralogs for each ATP2A subfamily (not shown, see metazome). The evolutionary history of ATP2A genes in craniates and in terms of duplication events could not be reconstructed with high confidence (Fig. 1). We inspected the 3 chromosomal loci in metazome (http://www.metazome.net) and found evidences for conservation of synteny between teleosts and mammals for these 3 loci. 1.2. Developmental expression of SERCA 1 We have compared atp2a1/serca1 mRNA expression to one of the earliest myotome markers, the basic helix-loop-helix encoding gene myod, using in situ hybridization (Frank and Harland, 1991; Hopwood et al., 1989). At late blastula stage (stage 9, Fig. 2C a–c), none of the two transcripts is found. Myod appears first around the blastopore at gastrula stage 10.5 (not shown) and is robustly expressed by stage 11, except in the dorsal midline area while serca1 remains undetected (Fig. 2C d–f). Serca1 is first detected at early neurula stage 12, in the posterior part of the paraxial mesoderm, while myod also labels the more anterior paraxial structures (Fig. 2C g–i). As neurulation proceeds, through stages 14–18, serca1 and myod essentially overlap in dorsal whole mount views (Fig. 2C–D j–n). Transverse sections show that in fact, myod labels most of the somite, while serca1 is restricted to the most superficial cells (Fig. 2D m0 –n0 and not shown at stage 14). In the course of organogenesis, myod remains strongly expressed in the

newly formed posterior somites, but fades out from the most anterior ones (Fig. 2D o, o0 , q and q0 ), while serca1 remains strongly expressed along the whole length of the paraxial mesoderm, in the most superficial cells as shown in transverse sections (Fig. 2D p, p0 , r and r0 ). Both myod and serca1 are expressed in the migrating hypaxial muscle cells and head muscle, and neither of them labels heart muscle (Fig. 2D q and r). In conclusion, while there are a few spatial differences between myod and serca1, serca1 stands as a novel, very early and robust muscle marker in Xenopus embryos. 1.3. Developmental expression of SERCA 2 and SERCA 3 We have next analyzed the developmental expression of atp2a2/serca2 and atp2a3/serca3 in X. laevis embryos, by whole mount in situ hybridization followed by analysis on transverse sections. We observe a strikingly tissue-specific expression for these two genes, as noted for serca1. During cleavage and gastrulation, neither serca2 nor serca3 were expressed significantly above background (Fig. 3A a–f). At neurulation, both genes exhibit a complementary expression pattern: at stage 14, serca2 is expressed in the neural plate (Fig. 3A g and g0 ) while serca3 is restricted to the ectoderm, outside the neural plate (Fig. 3A o and o0 ). As the neural tube closes, the ectoderm covers it. During this process, the premigratory cranial neural crest lies on both sides of the neural plate. Serca2 is excluded from the neural crest, being robustly expressed in the neural plate/tube and faintly in the lateral non-neural ectoderm (Fig. 3A h and h0 ). Serca2 is also detected in the prospective cement gland, anterior to the neural plate (not shown). Serca3 is expressed in the lateral non-neural ectoderm as well as in the ectoderm overlying the neural tube (Fig. 3A p and p0 ). None of the two transcripts is found in the endoderm. During organogenesis, a dynamic serca2 pattern is observed, while serca3 remains exclusively expressed in the ectoderm. Serca2 is found in the neural tube (Fig. 3A i, i0 , i00 , j, j0 and l), in the eye (lens

Fig. 1. Phylogenetic analysis. Phylogenetic tree built using the maximum likelihood method implemented in the PhyML program. The LG substitution model was selected for the analysis, and aLRT statistics was used to compute branch supports. See details in Section 3. Gene names are given in Table 1.

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Fig. 2. Atp2a1/serca1 embryonic expression compared to myod pattern in Xenopus laevis embryos. (A). Schematic of predicted Xenopus laevis SERCA1 protein structure. The different domains of the protein are indicated: Actuator (A) domain: from aa 1 to aa 34 and from aa 122 to aa 239; Nucleotide (N) domain: from aa 360 to aa 599; Phosphorylation (P) domain: from aa 318 to aa 353 and from aa 604 to aa 753 and transmembrane (TM) domains: M1 aa 57–76; M2 aa 87–103; M3 aa 260–276; M4 aa 292– 310; M5 aa760–777; M6 aa 789–806; M7 aa 832–849; M8 aa 898–914; M9 aa 931–948; M10 aa 967–984. (B). Conservation of ATP2A1–3/SERCA1–3 proteins between species. Percentage of identities and positives between Xenopus laevis SERCA1, SERCA2, SERCA3 and Homo sapiens, Mus musculus, Xenopus tropicalis SERCA1, SERCA2, SERCA3. (C–D). Atp2a1/serca1 mRNA expression from cleavage to tadpole stage in Xenopus laevis. Myod and serca1 mRNA expression were compared on stagematched sibling embryos by in situ hybridization. Whole mount (a–r) and transverse sections (m0 ,n0 ,o0 ,p0 ,q0 ,r0 ) are shown for myod (b,e,h,k,m,o,q) and serca1 (c,f,i,l,n,p,r). Blastula stage embryo (stage 9) are shown in animal pole view (a, control unstained embryo, b, c). Gastrula stage embryos are shown in vegetal view with blastopore facing top (d, control unstained embryo, e, f). Neurula stage embryos are shown in dorsal view, blastopore (yellow arrowhead) to the left (j, control unstained embryo, k–n). Tadpoles are shown in side views, head to the right. Yellow arrows in m–r indicate the level of the transverse section shown in m0 –r0 . n00 -r00 are schematic drawings of the transverse sections. Fin, np, neural plate, nt, neural tube, so, somite, no, notochord, ec, ectoderm. Green arrows indicate hypaxial muscle cells, red arrows indicate head muscles. Scale bar: a–r 0.5 mm; m0 –r0 25 lm.

and inner retina, Fig. 3A j and k), in the branchial arch mesenchyme, in the heart (Fig. 3A i, j and m) and in the pronephros (Fig. 3A i, i0 , j and n). In order to detail which part of the pronephros expresses serca2 (Fig 3B a and b), we have compared serca2 with two other pronephric markers: nkcc2 which labels the distal segment of the tubule (Fig. 3B c) and Evi1 which stains the distal segment of the tubule and the duct (Fig. 3B d). Serca2 is mainly

expressed in the proximal segment of the tubule. While serca2 is expressed at low level in the ectoderm (Fig. 3A j), serca3 is robustly expressed, first in the entire ectoderm (mid tadpole stage 26, Fig. 3A q, q0 and q00 ), then restricts to evenly spaced ectoderm cells, likely sensory skin cells (Fig. 3A r, r0 , u and v). Serca3 is also expressed in the cement gland (Fig. 3A q–s). Serca1–3 patterns are summarized in Table 2.

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Fig. 3. Atp2a2/serca2 and atpa3/serca3 embryonic expression. (A). Wholemount in situ hybridization (a–j, j0 , o–r, r0 ) or transverse sections (g0 –n and o0 –v) are shown for atp2a2/serca2 (a–c, g–n) and atpa3/serca3 (d–f, o–v). Neither genes are significantly expressed at cleavage and gastrulation stages (a–f). Neurula (g–h0 ; o–p0 ) show serca2 expression in the neural tissue (arrow1) but not in the neural crest (arrow 2); while serca3 is found in the ectoderm. Tadpoles (stage 26, i–i00 and q–q00 ; stage 37, j–n and r–v) show dynamic serca2 expression in the neural tube (arrow 1, section in i0 , i00 , l), the cement gland (arrow 3), the eye (section in k), the pronephros (arrow 4, section in i0 , n), the heart and branchial arches mesenchyme (arrow 6, section in m) and the proctodeum (arrow 5). Serca3 is restricted to the ectoderm (q–q00 ), the cement gland (arrow3, q, r, s) then to the sensory ectodermal cells (r–v). See text for details. Scale bar: a–j, j0 , o–r, r0 0.5 mm; g0 –n, o0 –v 0.1 mm. (B). Whole mount in situ hybridization (b,c,d) on embryos at stage 37–38 for atp2a2/serca2, nkcc2 and evi1 and scheme of the structure of the pronephros at tadpole stage (a, modified from (Raciti et al., 2008)). Atp2a2/serca2 is expressed in the proximal part of the tubule (PT); nkcc2 is expressed in the distal part of the tubule (DT); evi1 is expressed in the distal part of the tubule and in the duct (D). Scale bar: b– d 0.5 mm.

2. Discussion Calcium is an essential second messenger from the earliest developmental stages and throughout adult life, regulating multiple aspects of cell biology. High calcium affinity ATP2A/SERCA pumps are essential to terminate the signalling and sequester calcium into intracytoplasmic stores. We describe here the family of three ATPA2/SERCA genes in amphibian (X. laevis and X. tropicalis) and provide their evolutionary relationships with the other craniate orthologs. Moreover, we describe the embryonic expression of one isoform in each family. The most striking observation is that, as early as the end of gastrulation, these isoforms are expressed and predominantly localised in distinct and complementary tissues: myotome, neural plate and ectoderm and that this distribution does not fully match the expression patterns described in adults. First, in humans, serca1 isoforms are expressed in fast-twitch muscles: serca1a in adult cells and serca1b in neonatal ones (Brini,

2009). In Xenopus embryos, trunk somites develop similarly as somites in amniotes (mammals, birds), by generation of superficial slow muscle fibers from the dermomyotome (Grimaldi et al., 2004). Here we find serca1 appearing shortly after myogenesis regulator myod, but in contrast to myod, serca1 is restricted to the superficial myotome cells (Fig. 2). At later stage, serca1 remains at high expression levels in those superficial cells that differentiate into slow muscle fibers with high oxidative metabolism (Grimaldi et al., 2004). Moreover, it was believed so far that the first wave of myogenesis in Xenopus was fast fibers, since the available markers for slow fibers do not stain the somite before stage 22 (using two monoclonal antibodies against specific myosin heavy chain isoforms, Grimaldi et al., 2004). In contrast, our results suggest that serca1 specifically labels a population of prospective slow-type myoblasts as early as midneurula stage. In addition, our observations suggest that the assignment of serca pumps to specific muscle fibers types (i.e. fast versus slow) varies between embryos and adults. It would be interesting to compare serca1 expression to that of another early muscle marker,

C. Pegoraro et al. / Gene Expression Patterns 11 (2011) 122–128 Table 2 Summary of SERCA genes expression. Gene name/embryonic structure

serca1

serca2

serca3

 + 

+ + 

+  

    ++  

++  + ++  (+/) ++ +

  ++ ++   

General expression (by embryonic layer) Ectoderm Mesoderm Endoderm Main expression sites Neural plate/tube Neural crest Superficial ectoderm Cement gland Paraxial mesoderm Pronephros Heart

cardiac alpha actin, with the observation that serca1 does not label heart muscle (Mohun et al., 1986). Further analysis comparing non-amniote and amniote embryos will be useful to better understand the variability of serca1 mutants phenotypes, resulting in Brody’s disease and myotonic dystrophy in human, respiratory failure in mice and behavior defects in zebrafish (Drogemuller et al., 2008; Hino et al., 2007; MacLennan et al., 1997; Odermatt et al., 1996; Olson et al., 2010). Secondly, in humans, serca2a is found in the heart and slowtwitch muscles while isoform 2b, being ubiquitous, is considered as a housekeeping isoform. Serca2c is found in monocytes and fetal heart. Serca2 defects are linked to several cardiac malfunctions and heart failure (Sanyal et al., 2006). In agreement with these data, we find serca2 expression in heart region (Fig. 3A, at tadpole stages 26–37). However, serca2 expression is not ubiquitous (except possibly at very low levels in blastula embryos). Rather, serca2 is clearly upregulated in specific tissues during development: serca2 is strongly expressed in the neural plate during neurulation then in the neural tube, excluded from premigratory neural crest (compare to Fig. 1 in (Monsoro-Burq, 2005). Serca2 is also upregulated in heart and in the proximal tubule of the pronephros and expressed at low level in the ectoderm until tadpole stage (Fig. 3A– B). The role and importance of serca2 downregulation in the neural crest awaits further investigation. Thirdly, serca3 is less well described in human. Here, we find that serca3 is strongly expressed in and restricted to the ectoderm as early as neurula stage then is restricted to ciliated epidermal cells in tadpoles. Thus low-level expression of serca2 and high-level expression of serca3 coexist in neurula and early tadpole ectoderm. This differs from the situation in humans: in Darier’s disease, a rare skin disorder due to mutations in serca2, the phenotype restriction to the skin is thought to come from lack of overlap with serca3 expression in this tissue, contrarily to other cell types (Brini, 2009; Dhitavat et al., 2003; Tavadia et al., 2004). The embryo situation described here is opposite to this adult situation. It is only in later tadpoles that serca3 expression gets restricted in a few epidermal cells, maybe distantly related to another restricted serca3 expression in human skin eccrine glands (Tavadia et al., 2004). Our study suggests the importance of fine tissue- and stagespecific analysis for each isoform expression: similar conclusions emerge in recent studies in mammals where finer analysis shows a more complex and dynamic expression of serca2 and serca3 in heart and other tissues than previously described (Dally et al., 2010; Garside et al., 2010). On a functional point of view, previous work has shown that the different SERCA proteins display distinct affinity for calcium and different transport kinetics. SERCA2b has the highest Ca2+ affinity (Vandecaetsbeek et al., 2009), due to a longer C-terminal tail that we find conserved in the Xenopus isoform. This high affinity allows regulation of free intracytoplasmic calcium contents to very low

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levels. In contrast, SERCA3 has the lowest calcium affinity (Chandrasekera et al., 2009). Our observations raise the interesting possibility that such differences in how calcium is controlled in the cell may be of importance for the patterning and development of various cell types in the embryo. Indeed, calcium waves during gastrulation are critical for neural induction and induction of the early neural gene zic3 (Leclerc et al., 2003; Leclerc et al., 2000; Moreau et al., 2008). Our observation of high serca2 expression in the neural plate shortly after neural induction may indicate that the high affinity SERCA2 pumps may participate in tight control of calcium once neural induction has finished. Similarly, pronephros development involves increased calcium signals in tailbud stage embryos (Leclerc et al., 2008) whereas our results show increased serca2 expression at shortly later tadpole stages. Collectively, our data suggest that the requirements for calcium signalling in different embryonic structures may vary during development and vary between embryos and adults. In this context, free calcium levels may be differentially controlled by distinct ATPA2/SERCA isoforms. Our observations that serca2 displays a dynamic expression during development may relate to calcium homeostasis developmental changes recently described in mouse heart cells and explants (Kawamura et al., 2010; Korhonen et al., 2010). Future functional studies, including tissue-targeted gain- or loss-of-function experiments will allow validating these hypotheses. 3. Materials and methods 3.1. Genomics analysis: search for ATP2A1, ATP2A2 and ATP2A3 gene family members and sequence analyses ATP2A1, ATP2A2 and ATP2A3 protein sequences were extracted from Refseq, metazome, Hovergen (Penel et al., 2009), Gurdon Institute X. tropicalis Full-Length Database, Xenbase, and UniprotKB databases by virtue of similarity or pre-computed orthology relationships. X. laevis SERCA (NP_001080404.1) was used as a query in BLASTP (BLAST, basic local alignment search tool) searches of completed genome sequences (in Ensembl database) and Genbank/European Molecular Biology Laboratory/DNA Data Bank of Japan repository. Global multiple alignments of protein sequences were obtained using TCoffee. Phylogenetic analyses were performed using PhyML (Guindon and Gascuel, 2003) (Dereeper et al., 2008), TNT or the neighbor joining method implemented in the BioNJ program. The LG substitution model was used in PhyML. Distances were calculated using ProtDist and the JTT substitution model was selected for the analysis using BioNJ. Synteny was searched using the metazome database (http://www.metazome. net) by virtue of name search and sequence similarities (BLAST). 3.2. Embryos, in situ hybridization and sectioning The full length sequence-verified cDNAs for serca1 (Accession #BC044063, isoform serca1a), serca2 (#BC133185, isoform serca2b) and serca3 (#BC084962, isoform serca3b) were purchased from Open Biosystems and partially resequenced. Since the whole cDNA was used to generate in situ hybridization probes, each clone is likely to recognise various splice isoforms in each family (Brini, 2009): we will thus use either serca1a, serca2b and serca3b isoforms, but our results will be discussed as serca1, serca2 and serca3 expression. Digoxigenin-labeled cRNA was synthesised in vitro to generate RNA probes. Evi1 and nkcc2 cDNAs were kindly provided by M. Umbhauer. Evi1 RNA probe was synthesized according to (Van Campenhout et al., 2006). Nkcc2 RNA probe was synthesized according to (Zhou and Vize, 2004). X. laevis embryos were collected and fixed according to standard procedures (Sive et al.,

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2000). They were then processed following a modified in situ hybridization procedure (Monsoro-Burq, 2007). Sections (50– 60 lm thick) were cut using a Leica VT1000 vibratome after gelatin-albumin embedding. Pictures were captured using a Scion camera on Leica MZFLIII stereoscope and Olympus microscope, and imported using ImageJ or Photoshop CS software. Scans levels were homogenized using levels and brightness options in PhotoshopCS.

Acknowledgements The authors are grateful to the Monsoro-Burq lab members for their support. This work was funded by Ligue contre le Cancer, Association pour la Recherche contre le Cancer, Centre National de la Recherche Scientifique (ATIP program) awarded to AMB, Genopole ATIGE program awarded to NP. C. P. is an Institut Curie Ph. D. fellow. A. M.-B. is Professor at Université Paris Sud-11 and N. P is a CNRS researcher.

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