Expression of an evolutionary conserved subset of Iroquois (Irx) genes during nephron development

The genes involved in regionalization and proximodistal patterning of the nephron are still poorly understood. Irx genes encode homeodomain-containing transcription factors, which spatially prepattern the neural system in vertebrates and invertebrates (Gomez-Skarmeta and Modolell 2002). Moreover, Irx genes have been implicated in the specification of heart chambers (Bao et al. 1999). Interestingly, some Irx genes are also expressed during vertebrate kidney development (Bellefroid et al. 1998; Houweling et al. 2001). Given this data, we hypothesized that Irx genes play a role in patterning the vertebrate nephron. To support this idea, we first performed a detailed analysis of the spatio-temporal expression during nephrogenesis in Xenopus and mouse. The genomes of both species contain six members of the Irx gene family organized into two clusters: IrxA (containing Irx1, Irx2, and Irx4) and IrxB (containing Irx3, Irx5, and Irx6) (Peters et al. 2000; de la Calle-Mustienes et al. 2005).

In Xenopus, pronephric kidney organogenesis is initiated during late gastrulation with the specification of the pronephric anlagen, and is completed by stage 37/38 (Brändli 1999). Expression of irx6 occurs late in Xenopus embryogenesis and is not associated with the developing pronephric kidney (de la Calle-Mustienes et al. 2005). Similarly, we failed to find any evidence for pronephric expression of irx4 and irx5 (L. Reggiani and A.W. Brändli, unpubl.; data not shown). In contrast, irx1, irx2, and irx3 were expressed during pronephric kidney development in highly characteristic patterns (Fig. 2; Supplementary Fig. 1). irx3 expression was initiated at stage 25 followed 8 h later at stage 29/30 by irx1 and irx2 (Fig. 2D; Supplementary Fig. 1). Interestingly, irx3 expression also preceded expression of segment-specific terminal differentiation markers such as slc5a11 (formerly SGLT-1L) and clnck (ClC-K) at stages 29/30 and 31, respectively (Eid et al. 2002; Vize 2003). While irx3 expression gradually ceased from stage 35/36 onward, irx1 and irx2 expression persisted in the pronephros (L. Reggiani and A.W. Brändli, unpubl.; data not shown). Most intriguingly, pronephric irx gene expression was highly regionalized and confined to a central region of the developing nephron (Fig. 2A–C). Mapping of the expression domains to distinct nephron segments of the stage 35/36 embryo revealed that irx1 and irx2 expression was confined to the intermediate tubule segment IT1, whereas irx3 was found in PT3, IT1, and IT2. Notably, sharp borders characterized the irx expression domains along the nephron. Taken together, we have identified a subset of irx genes whose expression patterns reveal subdivisions of the developing pronephric nephron. Most importantly, irx3 is expressed well before the segment-restricted expression of terminal differentiation markers.

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Figure 2.

Expression of irx genes is highly regionalized in the developing pronephric kidney. (A–C) Expression patterns of irx1 (A), irx2 (B), and irx3 (C) in the pronephric kidneys of stage 35/36 Xenopus embryos as determined by whole-mount in situ hybridization. Lateral views of whole embryos (left panels; arrowheads indicate pronephric expression), enlargements of the pronephric region (middle panels), and color-coded schematic representations of the segment-restricted expression domains (right panels) are shown. Note the sharp boundaries that limit the expression domains of irx genes in the developing nephron. (D) Summary of the temporal expression profiles of irx genes during pronephric kidney development. The embryonic stages of X. laevis development are indicated. High and low levels of irx gene expression are illustrated with thick and thin lines, respectively. The embryonic expression patterns in early embryos are shown in Supplementary Figure 1.

In the mouse, we examined expression of all six Irx genes in developing metanephric kidneys at embryonic day 18.5 (E18.5) and in adult kidneys. As in Xenopus, we detected renal expression of Irx1, Irx2, and Irx3 but not for Irx4, Irx5, and Irx6 (Figs. 3, ​,4;4; data not shown). During metanephric kidney development, signals from the ureteric bud initiate nephrogenesis by promoting condensation and epithelialization of the metanephric mesenchyme to form renal vesicles. Through a series of invaginations and elongations, renal vesicles are subsequently transformed first into comma-shaped and then into S-shaped bodies, which exhibit the first morphologic signs of nephron segmentation (Saxén 1987). Analysis of nephrogenesis in the E18.5 kidney revealed that Irx gene expression became first apparent in early comma-shaped bodies, where Irx3 transcripts were detected at low levels (Fig. 3G). In S-shaped bodies, transcripts of all three Irx genes were confined to intermediate domains of the developing nephron with high level of expression for Irx1, intermediate for Irx2, and low for Irx3 (Fig. 3E–G,I–K). In contrast, expression of Brn1 was broader covering both intermediate and distal domains of the S-shaped body at this stage (Fig. 3H,L), which is consistent with its role in the development of intermediate and distal nephron derivatives (Nakai et al. 2003).

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Figure 3.

Irx gene expression marks an intermediate region of the S-shaped body. In situ hybridizations were performed on paraffin sections of E18.5 kidneys. Whole sagittal sections (A–D) and magnifications (E–H) of the renal cortex are shown to illustrate gene expression in developing nephrons. (I–L) The corresponding gene expression domains in the S-shaped body are indicated in the schematic drawings. (A–C) Irx1 and Irx2 are expressed in newly forming nephrons in the cortex (arrows) and the developing intermediate tubule epithelia (arrowheads). Irx3 expression is similar to Irx1 and Irx2, but fainter and more restricted. (D) Brn1 expression in the newly forming nephrons (arrowhead) and developing distal tubule epithelia (arrow). Note that in contrast to Irx genes, Brn1 is also highly expressed in epithelia of the renal papilla (asterisk). (I–K) Irx transcripts are confined to intermediate regions of S-shaped bodies. (L) Brn1 transcripts are detected in intermediate and distal domains of the S-shaped body. (CB) Comma-shaped body; (SB) S-shaped body; (P) proximal pole of the nephron; (D) distal pole of the nephron.

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Figure 4.

Irx gene expression is confined to distinct segments of Henle’s loop in the adult metanephric kidney. (A–F) Expression of Irx genes in the adult kidney. In situ hybridizations were performed on paraffin sections. Whole sagittal sections (A–C) and magnifications (D–F) are shown to illustrate Irx gene expression in detail. (Co) Cortex; (OS) outer stripe of outer medulla; (IS) inner stripe of the outer medulla; (IM) inner medulla. (A,B,D,E) Irx1 and Irx2 transcripts are detected in S3 and TAL. (C,F) Irx3 transcripts are confined to S3 only. (G) Summary of Irx gene expression in the adult metanephric kidney. The segmental organization of the adult metanephric nephron is shown schematically. The expression domains of each Irx gene are indicated below the scheme. (ATL) Ascending thin limb; (CD) collecting duct; (CDS) collecting duct system; (CNT) connecting tubule; (DCT) distal convoluted tubule; (DTL) descending thin limb; (S1, S2, S3) segments of the proximal tubule; (TAL) thick ascending limb.

Regionalized Irx gene expression persisted in adult kidneys with expression domains being confined to specific nephron segments of the loop of Henle (Fig. 4). The loop of Henle is comprised of the S3 proximal tubule, the descending thin limb (DTL), the ascending thin limb (ATL), and the thick ascending limb (TAL) (Kriz and Bankir 1988). Irx1 and Irx2 transcripts were both present in S3 proximal tubule segments and in the TALs, whereas Irx3 expression was confined solely to the S3 segment (Fig. 4). Expression was not detected in the thin limbs of Henle of the adult kidney. In summary, a subgroup of Irx genes—namely, Irx1, Irx2, and Irx3—is expressed in specific and overlapping patterns in the developing and adult mouse kidney. Most importantly, the developing nephron segments expressing Irx genes comprise the prospective loop of Henle (Fig. 3). Moreover, the comparison between Xenopus and mouse has revealed striking similarities with regard to the scope of Irx gene family member expression, developmental timing of Irx gene expression, and spatial expression domains during kidney development. This indicates that the mechanisms responsible for patterning the intermediate compartment of the vertebrate nephron have remained remarkably conserved during tetrapod evolution.

Irx3 gene function is required for patterning of the nephron

The expression studies in Xenopus and mouse suggest a function for Irx genes in patterning the nephron by specifying intermediate tubule and Henle’s loop fates, respectively. Given the potential redundancy of Irx gene activities, we opted for a loss-of-function approach in Xenopus embryos to test this hypothesis. Morpholino (MO) antisense oligonucleotides were used to block irx gene functions by inhibiting mRNA translation either one by one or in combinations. Two independent MOs were designed for each Xenopus irx gene under investigation. Special care was taken to ensure that the selected MOs inhibit transcripts from both pseudoallelic irx genes typically present in the pseudotetraploid Xenopus laevis genome. All Irx MOs (Irx-MO) were able to inhibit in a concentration-dependent manner translation in cell-free coupled transcription–translation assays (Fig. 5). Importantly, Irx3-MO was unable to block translation of irx1 and irx2. Different doses of Irx-MOs, typically 5 ng, were injected into single V2 blastomeres of eight-cell-stage embryos to target the prospective pronephric anlage (Saulnier et al. 2002b). The resulting embryos were analyzed by visual inspection for externally visible defects and by in situ hybridization for changes in pronephric marker gene expression.

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Figure 5.

Inhibition of irx1, irx2, and irx3 translation in vitro by antisense MOs. Plasmids (500 ng) encoding the ORF of irx1, irx2, or irx3 were used as templates in cell-free coupled transcription–translation reactions. MOs were tested for inhibition of translation at the doses indicated. Cell-free transcription–translation reactions were performed in the presence of [³⁵S]methionine and analyzed by SDS-PAGE/autoradiography. (A,B) Dose-response analysis of inhibition of irx1 translation by Irx1-MO (A) and Irx1(2)-MO (B). (C,D) Dose-response analysis of inhibition of irx2 translation by Irx2-MO (C) and Irx2(2)-MO (D). (E,F) Dose-response analysis of inhibition of irx3 translation by Irx3-MO (E) and Irx3(2)-MO (F).

In a first set of experiments, we tested the effect of blocking irx1, irx2, and irx3 gene functions simultaneously in the Xenopus embryo by unilateral injection of 5 ng of each Irx-MO into V2 blastomeres. The resulting embryos exhibited severe developmental defects, such as unilateral shortening of the body axes, which precluded the analysis of possible pronephric phenotypes (Supplementary Fig. 2). Thus, loss of irx1, irx2, and irx3 gene functions appears to be incompatible with normal embryonic development in Xenopus. Next, we performed knockdowns of irx1 and irx2 gene functions either alone (5 ng and 10 ng MO) or in combination (5 ng MO each). In each case, we failed to observe any externally visible defects. Furthermore, the expression of pronephric marker genes (pax2, clcnk, slc5a11, and slc12a1) was overall normal (Supplementary Fig. 3A; data not shown). Similar results were obtained using alternate MOs directed against irx1 and irx2 (Supplementary Fig. 3B; data not shown). Taken together, our findings strongly suggest that both irx1 and irx2 are dispensable for morphogenesis and patterning of the Xenopus pronephric kidney. Interestingly, Irx2 is neither required for kidney organogenesis in mice, since homozygous Irx2 mutants are phenotypically normal (Lebel et al. 2003).

In contrast, injection of Irx3-MO led in the majority of the embryos to highly specific defects in pronephric kidney development, which are shown in Figure 5 and described in detail below. Irx3(2)-MO, a MO directed against the 5′ untranslated region (UTR) of irx3, caused similar pronephric phenotypes. With each Irx3-MO we observed in a smaller fraction, typically 25%, of the injected embryos malformations and absence of pronephric kidneys, indicating that irx3 may have an early essential function during Xenopus development, which will be explored elsewhere. Embryos injected with Irx3(mp)-MO, a control nonblocking Irx3-MO-containing mutation in four positions, were phenotypically normal (Figs. 5, ​,6H;6H; data not shown). Irx3 knockdown embryos with normal overall appearance were selected and probed for evidence of defects in nephron organization. The transcription factor pax2 is an early marker of the pronephric anlage, and its expression is subsequently found in all developing pronephric epithelia (Heller and Brändli 1997). Staining of Irx3-MO-injected embryos for pax2 expression revealed the presence of pronephric kidneys, indicating that the specification of the pronephric fate had occurred during gastrulation. The pronephric kidneys were, however, frequently (72%, n = 25) characterized by abnormal morphology of the looped central domain, whereas the flanking proximal and distal domains appeared unaffected (Fig. 6A). As shown in Figure 6B, this phenotype was also observed in 65% (n = 43) of the Irx3-MO injected embryos stained for fxyd2 (Na, K-ATPase γ subunit), which is expressed throughout the nephron (Eid and Brändli 2001). These findings indicate that loss of irx3 gene function did not cause a general block in terminal differentiation of pronephric epithelia.

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Figure 6.

Irx3 is required for intermediate tubule formation in the Xenopus pronephric kidney. (A–I) Irx3-MO (5 ng; A–G, I) or Irx3(mp)-MO (5 ng; H) and mRNA (0.25 ng) for the lineage tracer nuclear β-galactosidase were coinjected into single V2 blastomeres of eight-cell-stage embryos. Injected embryos were raised to the embryonic stage indicated, fixed, and processed for β-gal activity. Expression of marker genes was subsequently visualized by in situ hybridization. Control and injected sides are shown as lateral views with accompanying enlargements of the pronephric kidney region. (A,B) Irx3 knockdown affects pronephric morphogenesis. Arrowheads indicate the central looped region that is abnormal. Schematic representations show the outline of the nephron in normal and Irx3-MO-injected embryos. (C) Irx3 knockdown does not affect PT1 and PT2. (D–F) Irx3 knockdown causes a loss of the proximal tubule segment PT3. The arrowhead indicates the position of the PT3 segment. Examples of strong reduction (E) and complete loss (F) of slc7a13 expression are shown. (G–I) Irx3 knockdown causes loss of IT1 and IT2 but not of DT1. Arrowheads indicate the location of DT1. Note that slc12a1 expression remains unaffected in the presence of the control Irx3(mp)-MO (H). (J) Summary illustrating the nephron segmentation defects seen in irx3 knockdown embryos. See Figure 1C for the nomenclature of pronephric nephron segments and their abbreviations.

Next, we probed Irx3-MO-injected embryos for patterning defects along the proximodistal axis of the nephron using marker genes shown in Figure 1 and Table 1. Expression of slc5a2, a marker of PT1 and PT2, was retained, and there was no evidence indicating expansion of slc5a2 expression into more distal territories of the nephron (Fig. 6C). In the case of slc5a11 (Fig. 6D), the expression domains corresponding to PT1 and PT2 were again present, but expression in the distal PT3 domain was lost in the majority of the embryos (86%, n = 37). Analysis of irx3 knockdown embryos with the PT3 marker slc7a13 indicated that the loss of PT3 was less pronounced, but could still be detected in 36% (n = 55) of the injected embryos (Fig. 6E,F). Effects of irx3 knockdown on the formation of the intermediate tubule were analyzed by staining for slc12a1 and clcnk expression (Fig. 6G–I). The marker gene slc12a1 is expressed distal to slc5a11 and slc7a13 in the intermediate tubule and DT1. Interestingly, slc12a1 expression in the intermediate tubule segments IT1 and IT2 was no longer detectable, but residual staining remained associated with DT1 in the majority of Irx3-MO-injected embryos (72%, n = 138). Similarly, expression of clcnk was lost in IT1 and IT2 but retained in DT1 and the more distal nephron segments (82%, n = 33). In summary, our findings indicate that, consistent with its expression pattern (Fig. 2C), irx3 gene function is required for the formation of PT3 and intermediate tubules during nephron development (Fig. 6J).

Next we asked whether the expression of pronephric irx genes is dependent on irx3 gene function. For this purpose, irx3 knockdown embryos were raised until they reached stage 29/30, when expression of all three irx genes could be detected (Fig. 2D; Supplementary Fig. 1). The analysis revealed that irx3 knockdown embryos failed to initiate expression of irx1 as well as of irx2 in 71% of the injected embryos (n = 14 for each gene), indicating that expression of these genes requires irx3 gene function (Fig. 7A,B). In contrast, regionalized pronephric irx3 transcripts were readily detected in the majority of the injected embryos (63%, n = 16) (Fig. 7C). Hence, the block of irx3 translation does not impair irx3 transcription, indicating that the irx3 gene is not subjected to autoregulation during early pronephric kidney development. Importantly, the regionalized pronephric expression pattern of irx3 is retained in the irx3 knockdown embryos. This suggests that nephron patterning is initiated normally but subsequent steps of intermediate tubule development are disrupted, resulting in the loss of the PT3, IT1, and IT2 segments. Notably, since the expression of irx1 and irx2 requires irx3 gene function, they are not able to compensate for the loss of irx3.

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Figure 7.

Pronephric expression of irx1 and irx2 but not irx3 requires irx3 gene function. Irx3-MO (5 ng) and mRNA (0.25 ng) for the lineage tracer nuclear β-galactosidase were coinjected into single V2 blastomeres of eight-cell-stage embryos. Injected embryos were raised to the embryonic stage indicated, fixed, and processed for β-gal activity. Expression of marker genes was subsequently visualized by in situ hybridization. Control and injected sides are shown as lateral views with accompanying enlargements of the pronephric kidney region. (A,B) Irx3 knockdown disrupts irx1 and irx2 expression in the developing pronephric kidney. Arrowheads indicate the pronephric area devoid of irx1 (A) and irx2 (B) expression. (C) Irx3 knockdown does not affect irx3 expression.

Cloning of cDNAs, sequencing, and sequence analysis

Expressed sequence tag (EST) databases were screened to identify the cDNAs encoding slc transporters from X. laevis (D. Raciti, unpubl.). Among them, slc5a2 (GenBank accession no. CF520680), slc7a13 (BC060020), slc12a1 (BU904428), and slc12a3 (CA790325) were employed in this study as pronephric marker genes. Double-stranded DNA sequencing was performed in-house. Assembly of nucleotide sequence traces and analysis of nucleotides and protein sequences was performed using the DNAStar Lasergene software package (version 6.0).

Plasmid constructs

The following plasmids containing the complete ORF of Xenopus irx1 (Gomez-Skarmeta et al. 1998), irx2 (Gomez-Skarmeta et al. 1998), and irx3 (Bellefroid et al. 1998) were constructed for in vitro coupled transcription–translation reaction and in vitro RNA synthesis: pCS2-Irx1, pCS2-Irx2, and pCS2-Irx3. The cDNAs were amplified by PCR (Expand High-Fidelity PCR System, Roche Diagnostics) and subcloned into the pCS2⁺ vector (Turner and Weintraub 1994) using T4 DNA Ligase (Fermentas). All constructs were confirmed by DNA sequencing.

Xenopus embryo manipulation and in situ hybridization

In vitro fertilization, culture, and staging of Xenopus embryos were performed as previously described (Brändli and Kirschner 1995; Helbling et al. 1998). Probe synthesis, whole-mount in situ hybridization, β-galactosidase staining, and bleaching of embryos were carried out as described (Helbling et al. 1998, 1999; Saulnier et al. 2002b). Digoxigenin probes were generated from linearized plasmids encoding irx1 (Xiro) and irx2 (Xiro2) (Gomez-Skarmeta et al. 1998), irx3 (Xiro3) (Bellefroid et al. 1998), irx4 and irx5 (Garriock et al. 2001), pax2 (Heller and Brändli 1997), clcnk (ClC-K) (Maulet et al. 1999), fxyd2 (Na, K-ATPase γ-subunit) (Eid and Brändli 2001), slc5a11 (SGLT1-L) (Eid et al. 2002), slc7a13 (GenBank accession no. BC060020), slc12a1 (BU904428), slc12a3 (CA790325), slc5a2 (CF520680), and wnt4 (Saulnier et al. 2002a). Sense strand controls were prepared from all plasmids and then tested negative by in situ hybridization.

Contour model of the pronephric nephron and marker gene mapping

The pronephric expression patterns of 91 slc genes, along with a full account of how the segmental organization of the Xenopus pronephric kidney was determined and how the accompanying nomenclature was established, will be presented elsewhere (D. Raciti, L. Raggiani, and A.W. Brändli, in prep.). In brief, a first schematic representation of the contour of the stage 35/36 nephron was developed from Xenopus embryos stained by whole-mount in situ hybridization with a combination of probes for the pronephric marker genes fxyd2 (Eid and Brändli 2001), pax2 (Heller and Brändli 1997), and wnt4 (Saulnier et al. 2002a). Two dozen stained embryos were inspected to generate a two-dimensional contour drawing of the nephron on paper. Refinements to the initial contour model were made after inspection of hundreds of embryos stained with other pronephric marker genes. The final contour model of the nephron shown in Figure 1C was made with Illustrator CS2 (Adobe).

The pronephric expression patterns of 91 slc genes were projected onto the contour model to define the segments of the nephron. Unambiguous morphological features, such as the nephrostomes, a characteristically broad proximal tubule domain known as common tubule (Fox 1963) (subsequently named PT3), and the looped part of the pronephric nephron (IT1, IT2, and DT1), were used as landmarks to identify the relative location of the boundaries of the expression domains. The final borders between the nephron segments are defined by the boundaries of multiple marker genes.

Microinjection of Xenopus embryos

The following antisense MO oligonucleotides were ordered from GeneTools to inhibit translation of Xenopus mRNAs (sequence complementary to the predicted start codon is underlined; mispaired nucleotides are indicated with small letters): Irx1-MO, 5′-CATGTCTCTCCGGCAGGGAAATCGC-3′; Irx1(2)MO, 5′-CCCAGCTGCGGGAAGGACATGTCTC-3′; Irx2-MO, 5′-GGTAACCCTGAGGATAGGACATGGT-3′; Irx2(2)-MO, 5′-G CAGAAGCACAGAATCGCCGGGGCT-3′; Irx3-MO, 5′-AGCT GTGGGAAGGACATGGTGCAGC-3′; Irx3(mp)-MO, 5′-AGgTGT GGGtAGGACAgGGTGaAGC-3′; Irx3(2)-MO, 5′-GAATCCCCTT TTATGACCTGACTTT-3′.

In vitro coupled transcription translation assays were carried out as described previously (Saulnier et al. 2002b). When not indicated differently, 5 ng of individual MOs were injected in V2 blastomere of eight-cell-stage embryos (Moody and Kline 1990). RNA synthesis and microinjection were performed as described (Helbling et al. 1998) except that RNA purification was done by phenol-chloroform extraction. RNA encoding the lineage tracer nuclear β-galactosidase (nucβgal) was usually coinjected at 0.25 ng per blastomere.

In situ hybridization of mouse kidneys and kidney sections

Mouse embryos were obtained from matings of NMRI wild-type animals. For timed pregnancies, plugs were checked in the morning after mating, noon was taken as 0.5 d post-coitum (dpc). Microdissected metanephric kidneys were fixed in 4% PFA and stored in 100% methanol at −20°C prior to in situ hybridization analysis. In situ hybridization analysis of 10-μm paraffin sections of embryonic kidneys at E18.5 and 6-wk-old adult kidneys was performed according to an established protocol (Soufan et al. 2004). Digoxigenin-labeled probes were transcribed from linearized plasmids encoding the following mouse cDNAs: Irx1 (GenBank accession no. BU703212), Irx2 (BI111057), Irx3 (BI525434), Irx4 (BE367799), Irx5 (Bosse et al. 2000), and Irx6 (Peters et al. 2000). For probe synthesis, the mouse Brn1 cDNA (Hara et al. 1992) was subcloned by PCR into pGEM-TEasy (Promega).

We thank E. Bellefroid, J.L. Gómez-Skarmeta, P. Krieg, Y. Maulet, M. Nirenberg, and U. Rüther for providing plasmids; M. Petry for excellent technical assistance with mouse in situ hybridization analysis; F. Rechfeld for subcloning of Brn1; B. Kaissling for annotation of mouse Irx gene expression patterns; and M. Reggiani for assistance with computer graphics. This work was supported by grants from the German Research Council (DFG) to A.K. and the Swiss National Science Foundation (3100A0-101964) and European Community (EuReGene LSHG-CT-2004-005085) to A.W.B.