,EricJ.Bellefroid MassimoNichane ,ClaudeVanCampenhout ,He´le`nePendeville ,MarianneL.Voz Xenopus andzebrafishembryos TheNa /PO cotransporter SLC20A1 genelabelsdistinctrestrictedsubdomainsofthedevelopingpronephrosin

The Na ⁺ /PO ₄ cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros

in Xenopus and zebrafish embryos

Massimo Nichane

, Claude Van Campenhout

, He´le`ne Pendeville

, Marianne L. Voz

, Eric J. Bellefroid

aLaboratoire d’Embryologie Mole´culaire, Universite´ Libre de Bruxelles, Institut de Biologie et de Me´decine Mole´culaires (IBMM), rue des Profs. Jeener et Brachet 12, B-6041 Gosselies, Belgium

bLaboratoire de Biologie Mole´culaire et Ge´nie Ge´ne´tique, Institut de Chimie, Universite´ de Lie`ge, Baˆtiment B6, B-4000 Lie`ge (Sart-Tilman), Belgium

Received 29 July 2005; received in revised form 21 December 2005; accepted 17 January 2006 Available online 10 March 2006

Abstract

The embryonic pronephric kidneys of

and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the

pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and pos- terior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the

and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na

/PO

cotransporter

(PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in

and zebrafish. In

mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25,

is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish,

is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of

expression is also observed in the posterior cardinal and caudal veins. Based on the

expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and

pronephros have non-identical proximo-distal organizations.

Ó2006 Elsevier B.V. All rights reserved.

Keywords: SLC20A1;PiT1;Xenopus; Zebrafish; Pronephros; Kidney; Phosphate; Cotransporter

1. Results and discussion

Studies of pronephros development are currently mainly conducted in the frog, Xenopus laevis, and the zebrafish, Danio rerio, due to the unique advantages those experimen- tal systems offer for studies of organogenesis (Drummond, 2003, 2005; Ryffel, 2003; Jones, 2005). In the frog, distinct domains and subdomains within the tubules and duct

compartments have been defined based on the localized expression of various membrane transporter genes (Vize, 2003; Zhou and Vize, 2004, 2005a,b). However, the segmen- tation of the zebrafish nephron has been much less studied.

Three types of Na

/PO

cotransporters have been char- acterized in higher vertebrate kidneys. The first two types of Na

/PO

cotransporters include SLC17A1 (also known as Npt1 and NaPi-1) and SLC34A1–3 (also known as Npt2a-c and NaPi-2a-c) (Werner et al., 1991; Magagnin et al., 1993). In mammalians, those type I and II genes are highly expressed in the kidney. Only low level of expres- sion is detected in a few other organs such as liver and

1567-133X/$ - see front matter Ó2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.modgep.2006.01.005

* Corresponding author. Tel.: +32 2 650 97 32; fax: +32 2 650 97 33.

E-mail address:ebellefr@ulb.ac.be(E.J. Bellefroid).

1 These authors contributed equally to this work.

www.elsevier.com/locate/modgep

intestine. Within the kidney they are almost exclusively expressed in the brush-border of proximal tubular cells (Biber et al., 1993; Custer et al., 1994; Tenenhouse et al., 1998). Moreover, using laser-assisted microdissection and quantitative real-time PCR, it was recently shown that the expression of SLC34A1 and SLC34A3 is heterogenous with respect to proximal tubular segmentation and neph- ron generation (Madjdpour et al., 2004). Sodium phos- phate transporters from the third type consist of the SLC20A1 (also called PiT-1 and GLVR1) and SLC20A2 (also called PiT-2 and Ram-1) proteins originally described as a cell surface receptor for gibbon ape leukemia virus (Kavanaugh et al., 1994; Olah et al., 1994). In contrast to type I and II Na

/PO

cotransporters, type III cotrans- porters appear more ubiquitously expressed. The SLC20A1 and SLC20A2 genes are expressed in mammalians in a wide variety of tissues including kidney, brain, heart, liver, muscle, and bone marrow (Johann et al., 1992; Kavanaugh and Kabat, 1996). Within the kidney, SLC20A1 transcripts are found throughout the kidney by in situ hybridization (Tenenhouse et al., 1998). SLC20A2 appears also expressed stably throughout development in all regions of the kidney as detected by RT-PCR analysis (Leung et al., 2005).

We have recently shown by comparing in Xenopus and zebrafish the expression of several proximal and distal tubule/duct specific pronephric markers that in zebrafish, the anterior and posterior segments of the pronephros have characteristics of proximal tubule and distal tubule/duct compartments, respectively (Van Campenhout et al., unpublished data). To further analyze the compartmental- ization of the Xenopus and zebrafish pronephros, we search for novel segment-specific pronephric markers and found in Xenopus one cDNA clone whose predicted full-length protein is 80% (673) identical to human SLC20A1 (O’Hara et al., 1990). In zebrafish, consistent with a genome dupli- cation at the root of teleosts, we identified two distinct zeb- rafish cDNA clones whose predicted full-length proteins are 63–69% (673) identical to human SLC20A1. As in both cases human SLC20A1 represents the closest match identi- fied by blast search, they are both likely to correspond to orthologs of the mammalian SLC20A1 gene. The two SLC20A1 genes identified in the zebrafish genome (SLC20A1a and SLC20A1b) which share 68% amino acid identity are found on distinct locus (SLC20A1a, chromo- some 8, Zv_5 scaffold 425; SLC20A1b, unidentified chro- mosome, Zv_5 scaffold NA8672), which show no conserved syntenies to the segment of the human chromo- some 2 harboring the SLC20A1 locus. Analysis of the expression profile of the two zebrafish genes using NCBI Unigene’s EST profile viewer revealed that the two genes may have distinct spatio-temporal expression patterns, SLC20A1a being the only one strongly and specifically expressed in the kidney in the embryo. We used whole- mount in situ hybridization to analyze the expression pat- tern of the identified Xenopus xSLC20A1 and zebrafish SLC20A1a clones in the developing Xenopus and zebrafish embryos. In Xenopus, expression of xSLC20A1 was first

detected in neurula embryos (st 13) in the neural plate (Fig. 1A). At tailbud and early tadpole stages, expression of xSLC20A1 was prominent in the somites with lower level of expression detected in the neural tube, eye, and neural crest cells (Figs. 1B and C). As shown in Figs.

1C–F, while expression from stage 25 in the somites decreases, staining was also observed in the anterior part of the pronephros. To identify where within the proneph- ros xSLC20A1 is expressed, we compared in stage 38 embryos its expression to that of the chloride conductance channel xClC-K gene that labels the duct and distal tubule (Vize, 2003), to the sodium bicarbonate cotransporter XNBC1 (SLC4A4) gene which is predominantly expressed in the late distal tubule segment (Zhou and Vize, 2004) and to the xPDZK1 gene encoding a multi–PDZ domain con- taining adaptator protein that is expressed in the proximal tubules (Van Campenhout et al., unpublished data).

xSLC20A1 staining is similar to that of XNBC1 in the late distal portion of the pronephric tubules (Figs. 1F–I).

In zebrafish, SLC20A1 expression was detected from the 18-somite stage in a short region of the anterior part of the pronephric duct. Weaker staining was also observed more caudally that does not appear to correspond to the pro- nephric duct (Figs. 2A and C). Transverse sections of stage 24 h post-fertilization embryos revealed that this weaker expression occurs outside the pronephric duct in the pos- terior cardinal and caudal veins (Fig. 2D). At 48 h post-fer- tilization, which is the latest stage analyzed, SLC20A1 expression has decreased in the posterior and cardinal veins but is maintained in the anterior part of pronephric duct (Figs. 2E and F). To determine more precisely where in the pronephros SLC20A1 is expressed, as in Xenopus we compared in 24 hpf embryos its expression to that of the ClC-K, PDZK1 (Van Campenhout et al., unpublished data), and NBC1 genes. The zebrafish NBC1 clone we iso- lated encodes a protein showing 79% (263) to the human NBC1 protein. Figs. 2G and H show that NBC1 staining, like PDZK1 staining, is only observed in the anterior part of the pronephros. Weaker NBC1 staining was also observed in the somites. In contrast, as previously described, ClC-K labels only the posterior part of the pro- nephric duct (Fig. 2I). SLC20A1 staining corresponds to the rostral part of PDZK1 and NBC1 expression domains (Fig. 2 compare A and B and G, H, and J). PDZK1 staining appears to extend more posteriorly than the NBC1 staining (Figs. 2H and J). To confirm this observation, we performed double staining with NBC1 or PDZK1 and ret1 that label the most caudal part of the pronephros as a reference (Mar- cos-Gutie´rrez et al., 1997). The data show that while PDZK1 and ret1 label the entire pronephric duct, a gap of staining appears in between the NBC1 and ret1 expression domains (Figs. 2K and L). Two-color double in situ hybrid- ization with ClC-K and PDZK1 was also performed. Figs.

2L and N show that the expression of PDZK1 extends fur- ther than the anterior limit of ClC-K expression.

Thus, SLC20A1 in both Xenopus and zebrafish embryos

appears to be expressed in a restricted region of the

pronephros, which contrasts with the ubiquitous expression observed in mouse kidney by in situ hybridization (Tenen- house et al., 1998). It is, however, possible that using our in situ conditions, we detect only the sites of particular high expression and that SLC20A1 has low-level ubiquitous expression. In Xenopus, our data indicate that xSLC20A1 constitutes a novel useful marker of the late distal segment of the pronephros together with XNBC1 and the carbonic anhydrase type 2 CA2 gene (Zhou and Vize, 2004, 2005a).

Its expression in Xenopus in the late distal segment of the pronephros suggests that this portion not only plays a role in the recovery of filtered bicarbonate but may also function in P

homeostasis. In zebrafish, our data indicate that NBC1 is another tubule-specific marker whose expression is restricted to the anterior part of the pronephros, thus fur- ther demonstrating that the anterior segment of the zebra- fish pronephros has proximal tubule characteristics. In accordance with those results, mAb 3G8 that is used in Xenopus to visualize the proximal tubules also marks specif- ically in zebrafish the anterior pronephros (Majumdar et al., 2000). We found that SLC20A1 is also expressed only in the anterior part of the pronephros and that it is found in a short region corresponding to the rostral end of the expres- sion of the PDZK1 and NBC1 proximal tubule markers. We also observed that PDZK1 staining extends further posteri-

orly than that of NBC1 and further than the anterior limit of ClC-K expression. These observations indicate that the zebrafish pronephric epithelium can be subdivided into four segments based on the expression of the cotransporters used in this study as summarized in Fig. 3. This segmentation of the zebrafish pronephros is in accordance with previous studies that revealed different segments within the meso- nephric nephron of different fish species (Miyazaki et al., 2002; Hentschel et al., 2003; Hyodo et al., 2004). In the kid- ney of winter flounder, for example, only the late proximal tubule segment PII expresses high level of a protein corre- sponding to the SLC34A1–3 mammalian proteins (Elger et al., 1998). Our observations suggest that Xenopus and zebrafish pronephros have non-identical proximo-distal organization. Analysis of the expression of additional seg- ment-specific pronephric markers is necessary to get a better understanding of the compartmentalization of the Xenopus and zebrafish nephrons.

2. Experimental procedures

ESTCF924882, encoding the zebrafish SLC20A1a full-length proteins, was identified through a search of the ZFIN expression database and cor- responds to the uncharacterizedsb:cb1011clone (Thisse et al., 2001). The full-length sequence of the protein was obtained through the identification of additional overlapping EST clones (UnigeneDr.34731andDr.23911).

St.28 St.38

XNBC1

St.38 xClC-k

St.38

xSLC20A1 xSLC20A1

xSLC20A1

St.16

xSLC20A1

St. 22

xSLC20A1

St.25 np

e

nc s

nc

dl de

dl p

s

e nc

s

xSLC20A1

St.34

XPDZK1

St.38 p

A B C

D E F

G H I

Fig. 1. SLC20A1 expression in Xenopus laevisembryos. Whole-mount in situ hybridization with the indicated DIG-labeled antisense probes was performed on embryos at the indicated stages (Nieuwkoop and Faber, 1997). (A–C) Lateral views of embryos with anterior towards the left. (D–I) High magnification views of the pronephric region. Arrows indicateSLC20A1expression in the late distal tubule compartment of the developing pronephros.

Abbreviations: dl, distal late pronephric compartment; de, distal early pronephric compartment; e, eye; nc, neural crest; np, neural plate; p, proximal pronephric compartment; s, somites.

The zebrafish clone encoding the SLC20A1b full-length protein (EST BC57497; UnigeneDr.5307), theXenopusclone encoding the full-length xSLC20A1 protein (ESTBC059957), and the zebrafish clone encoding the NBC1 and ret1 protein (ESTCK236285andAI793987) were identified by BLAST mining of the EST database.

Whole-mount in situ hybridization using digoxygenin antisense ribo- probes inXenopusand zebrafish embryos was performed as described pre- viously (Harland, 1991; Broadbent and Read, 1999). Xenopus and zebrafish SLC20A1 plasmids were linearized withSalI and transcribed with T7. ZebrafishNBC1plasmid was linearized withEcoRV and tran-

scribed with T7. Zebrafish ret1 plasmid was linearized withSalI and tran- scribed with SP6. Plasmids used to generate the other in situ probes have been described previously:xClC-K(Vize, 2003),XNBC1(Zhou and Vize, 2004). Zebrafish ClC-K (EST BC053277) and zebrafish PDZK1 (EST BC066762) plasmids were linearized with EcoRV and transcribed with T7.XenopusxPDZK1 (EST BQ731430) plasmid was linearized withSalI and transcribed with T7. FISH was performed as previously described (Mavropoulos et al., 2005). For sections, embryos after completion of the whole-mount procedure were gelatin-embedded and vibratome- sectioned at 30lm.

Fig. 2. SLC20A1 expression during zebrafish embryogenesis. Whole-mount in situ hybridization with the indicated DIG-labeled antisense probes at the indicated stages. (A,B,E,G–J) Lateral views with anterior towards the right. (C,D) Transversal sections of the embryo shown in B at the levels indicated. (F) Dorsal view. (K,L) Costaining forNBC1orClC-Kand ret1.ClC-Kposterior limit of expression extends further than that of NBC1 and appears to correspond to the anterior limit of ret1. (M–O). Double in situ hybridization withClC-K(green) andPDZK1(red) indicates that a cell population in the middle of the pronephros expresses both ClC-K and PDZK1 (yellow). Arrowheads show the distinct posterior limit of the expression of the indicated markers. Abbreviations: cv, caudal vein; pcv, posterior caudal vein; pnd, pronephric duct segment; s, somites.

Acknowledgements

We thank Bernard and Christine Thisse and Bernard Peers for critical reading of the manuscript and the review- ers for helpful comments on the manuscript. We are also grateful to Peter Vize for providing us with the xClC-K probe and to Sadia Kricha for technical assistance. This work was supported by the Belgian program Inter-univers- itary attraction poles from the Prime Minister’s Office, Sci- ence Policy Programming (Grant P5/35 to E.B.), by the Communaute´ Franc¸aise de Belgique (Grant No. ARC 00/05-250 to E.B.), by the Fund for Scientific Medical Re- search (FRSM 3.4555.01 and 3.4556.01), by the Interna- tional Brachet Stiftung (Grant No. 01-4/l), and by the Fondation Reine Elisabeth (Grant 11256). M.N. and C.V. are fellow of the Fonds pour la Formation a` la Recherche dans l’Industrie et l’Agriculture.

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