Genome-wide slc gene expression analysis defines a large panel of pronephric marker genes
A genome-scale, whole-mount in situ hybridization screen was performed to evaluate the expression of solute carrier (slc) genes during Xenopus pronephric kidney development. We mined public databases to identify cDNAs encoding Xenopus laevis slc genes. In total, 225 unique slc Xenopus cDNAs were identified that encoded genuine orthologs of human SLC genes, based on phylogenetic analyses and synteny mapping (DR and AWB, unpublished data). The retrieved Xenopus slc orthologs represent 64% of all human SLC genes (total 352).
Gene expression patterns were analyzed by whole-mount in situ
hybridization using Xenopus
embryos at selected developmental stages, in accordance with the terminology established by Nieuwkoop and Faber (1956) [28
]: 20 (22 hours postfertilization [hpf]), 25 (28 hpf), 29/30 (35 hpf), 35/36 (50 hpf), and 40 (66 hpf). The stages were chosen to cover the key steps of pronephric kidney organogenesis: initiation of nephrogenesis (stage 20), onset of cellular differentiation (stage 25), maturation and terminal differentiation (stages 29/30 and 35/36), and acquisition of full excretory organ functions (stage 40; Figure ) [14
Figure 1 Pronephric kidney development and the global expression of slc and cldn genes. (a) Hallmarks of pronephric kidney development in Xenopus laevis. Schematic representations of Xenopus embryos are shown with the embryonic stages and hours postfertilization (more ...)
Of the 225 slc
genes identified, we detected expression of 210 genes during the embryonic stages tested, and thereof 101 genes (48%) were expressed specifically during pronephric kidney development (Figure ). The first evidence for pronephric expression of slc
genes was identified at stage 25, at which ten genes could be detected (Figure ). These included the Na-K-Cl transporter slc12a1
), the facilitated glucose transporter slc2a2
) and the amino acid transporters slc6a14
, and slc7a7
(Additional data file 1). By stage 29/30, expression of 65 genes - representing the majority (64%) of the slc
genes tested - could be detected. This correlates well with the onset of epithelial differentiation and lumen formation [14
]. The number of expressed slc
genes increases to 91 and 89 at stages 35/36 and 40, respectively (Figure ), as the pronephric nephron undergoes terminal differentiation and acquires characteristics of a functional excretory organ. Complete lists of slc
genes expressed for each stage of pronephric development tested are provided in Additional data file 1.
A comprehensive model for pronephric segmentation revealed by slc gene expression mapping
Our gene expression studies indicated that all 101 slc genes exhibited spatially restricted expression patterns in the developing pronephric kidney. Because slc genes encode terminal differentiation markers, we reasoned that a systematic analysis of the slc gene expression domains could reveal the underlying segmental organization of the differentiated pronephric nephron.
The nephron of the stage 35/36 pronephric kidney was selected for slc gene expression mapping along the proximodistal axis. Robust expression of most slc genes was evident by this stage, which preceded the onset of pronephric functions by about 3 hours. Furthermore, the stage 35/36 nephron retains a simple structure, lacking areas of extensive tubular convolution. It is largely a linear epithelial tube stretched out along the anteroposterior body axis. Characteristic morphological landmarks (somites, thickenings, and looped areas of the nephron) facilitate the mapping of the gene expression domains that can be performed on whole embryos without need for sectioning. A contour map of the stage 35/36 nephron was developed from embryos subjected to whole-mount in situ hybridization with fxyd2, pax2, and wnt4 probes (see Materials and methods, below, for details). The obtained model covered the three nephrostomes, which mark the most proximal end of the nephron, followed by three tubules, which merge to form a long-stretched duct that connects at its distal end to the cloaca. Subsequently, the expression domains of each slc gene were carefully mapped onto the stage 35/36 model nephron.
The segmental organization that emerged from slc
gene expression mapping is shown in Figure . It revealed a previously unappreciated complexity and extends an older model reported by Zhou and Vize [19
]. In addition to the nephrostomes, which connect the pronephric nephron to the coelomic cavity and the glomerular filtration apparatus, eight functionally distinct segments were defined. Cross-species gene expression comparisons were performed to delineate similarities between the Xenopus
pronephric and mammalian metanephric nephron (see below). These studies revealed striking analogies, allowing us to adopt a nomenclature for the pronephric segments that largely follows the widely accepted one used for the mammalian metanephros [2
], which is shown in Figure . The pronephric nephron of Xenopus
is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. The proximal tubule (PT) is divided into three segments (PT1, PT2, and PT3), whereas the intermediate tubule (IT) and the distal tubule (DT) are both composed of two segments IT1 and IT2, and DT1 and DT2, respectively. In contrast, the connecting tubule (formerly known as pronephric duct) does not appear to be further subdivided. The molecular evidence supporting the proposed segmentation model and nomenclature are discussed in detail below.
Figure 2 Models of the segmental organization of the Xenopus pronephric and mammalian metanephric nephrons. The color coding of analogous nephron segments is based on the comparison of marker gene expression as shown in Figure 7. (a) Schematic representation of (more ...)
Distribution of slc gene expression in the pronephric kidney
The complete annotation of the pronephric expression domains for each slc gene can be found in Additional data file 2. The slc gene expression domains were characterized by sharp, conserved expression boundaries, which define the limits of the segments and tubules. A given expression domain could either be confined to a single segment, comprise an entire tubule, or spread over more than one tubule. Of the 91 slc genes analyzed for expression in the stage 35/36 pronephric kidney, we detected expression of 75 genes in the proximal tubule, 27 genes in the intermediate tubule, 24 genes in the distal tubule, and 13 genes in the connecting tubule (Additional data files 3 to 6).
Expression domains of slc genes define three segments in the proximal tubule
With 75 genes, the proximal tubules exhibited the greatest complexity of slc
gene expression. This underscores their importance in reabsorbing diverse classes of solutes from the glomerular ultrafiltrate. We identified 26 genes with exclusive expression throughout the proximal tubule compartment. Among these, 18 were strongly expressed and included slc2a2
, and slc26a1
(Figure and Additional data file 2). The expression domains of other slc
genes revealed a further subdivision of the proximal tubule into three distinct segments (PT1, PT2, and PT3). This tripartite organization is reminiscent of mammalian proximal tubules, which are commonly subdivided into S1, S2, and S3 segments [2
Figure 3 The expression domains of slc genes identify three distinct segments in the proximal tubule. Stage 35/36 Xenopus embryos were stained for marker gene expression by whole-mount in situ hybridization. For each distinct class of expression pattern obtained, (more ...)
Two genes were predominantly expressed in PT1 (the most proximal segment of the proximal tubule), namely slc7a7
. Low levels of expression could also be detected in PT2 (Figure and Additional data file 2). Interestingly, all three PT1 segments appear to be equivalent, because we do not have evidence for differential expression of marker genes. Two genes, namely slc25a10
, were exclusively expressed in PT2 (Figure ), and slc1a1
were confined to PT3 (Figure ). Furthermore, we found several examples of slc
gene expression encompassing two segments. Twelve genes including slc5a2
, and slc15a2
were expressed in PT1 as well as PT2 (Figure and Additional data file 2). In contrast, 13 slc
genes, including slc2a11
, were detected in both PT2 and PT3 (Figure and Additional data file 2). The molecular subdivision of the proximal tubule revealed by segment-specific markers is also evident morphologically. Three PT1 segments connect the nephrostomes to a single PT2 segment. The adjacent distal region corresponds to PT3 and can be identified as a bulging of the proximal tubule, which is also known as the broad or common tubule [29
Expression of slc genes delineate the intermediate tubule as a bipartite structure
The intermediate tubule, an S-shaped structure, follows distal to the proximal tubule in the stage 35/36 pronephric nephron (Figure ). It is characterized molecularly by the expression of the thiamine transporter slc19a2 (Figure ). In addition, slc genes with nonexclusive expression in the intermediate tubule include slc4a11, slc12a1, slc16a7, and slc25a11 (Figure and Additional data file 2). For example, slc12a1 expression extends into the distal tubule to include DT1 (Figure ). The boundaries of the intermediate tubule are also defined by slc4a4, which was not detected in the intermediate tubule but was prominently expressed in the flanking proximal and distal tubule domains (Figure ).
Figure 4 Slc gene expression defines segmentation of the intermediate, distal, and connecting tubules. Stage 35/36 Xenopus embryos were stained for marker gene expression by whole-mount in situ hybridization. Lateral views of stained embryos are shown accompanied (more ...)
The intermediate tubule is comprised of two segments, namely IT1 and IT2. The molecular evidence for this subdivision was provided by the expression of slc20a1
in the proximal part (IT1) and slc5a8
in the distal part (IT2; Figure ). Although slc5a8
expression occurs also in the proximal tubules (PT2 and PT3) and in the distal tubule (DT1), the expression domain in the intermediate tubules defines unequivocally the boundary between IT1 and IT2 (Figure ). The bipartite nature of the intermediate tubule is further supported by the expression of irx
transcription factor family members irx1
, and irx3
Organization of distal and connecting tubules revealed by slc gene expression
The distal tubule occupies roughly the proximal half of the stretch-out part of the pronephric nephron (Figure ). To date we have failed to identify an slc gene with expression in the entire distal tubule only. However, the distal expression domain of slc16a6 comprises the entire distal tubule (Additional data file 2). The distal tubule is composed of two distinct segments: DT1 and DT2. Molecularly, DT1 was defined by the expression of the sodium bicarbonate transporter slc4a4; however, this transporter also has a second expression domain in the proximal tubule (Figure ). In addition, several slc genes were identified that have DT1 as their most distal expression domain. These included slc4a11, slc5a8, and slc12a1 (Figure and Additional data file 2). DT2 was demarcated by expression of the ammonia transporter rhcg/slc42a3 (Figure ). Furthermore, slc12a3 shared DT2 as its most proximal expression domain (Figure ).
The connecting tubule links the pronephric kidney to the rectal diverticulum and the cloaca. Two slc genes exhibited exclusive expression in this compartment, namely the sodium/calcium exchanger slc8a1 and the zinc transporter slc30a8 (Figure and Additional data file 2). To date, we have not obtained any evidence supporting further subdivision of the connecting tubule.
Validation of the pronephric segmentation model
We extended our gene expression analysis to the claudin (cldn
) gene family and selected other genes to validate the proposed model of pronephric segmentation. Claudins are key components of epithelial tight junctions, where they are responsible for the selectivity and regulation of paracellular permeability [30
]. In the mammalian kidney, several claudin genes are expressed in segment-specific patterns along the nephron [30
]. We profiled the claudin gene family for evidence of nephron segment-specific gene expression in Xenopus
. We retrieved 14 distinct Xenopus
claudin cDNAs from database searches, which covers 64% of the complement of 22 claudin genes typically found in vertebrate genomes. We analyzed the expression of 13 claudin genes by whole-mount in situ
hybridization and found that eight genes were expressed in the developing pronephric kidney (Figure and Additional data file 2). No pronephric expression of claudin genes was detected at stage 20. Induction of cldn6
expression occurred at stage 25, and by stage 35/36 all eight cldn
genes were expressed (Figure and Additional data file 1). The temporal profile of claudin gene expression during pronephric kidney development therefore mirrors the situation reported for the slc
genes (Figure ). Four cldn
, and cldn12
) were expressed throughout the entire stage 35/36 nephron. In contrast, expression of the other cldn
genes was highly regionalized. Interestingly, all shared expression in the intermediate tubule. The cldn8
gene had the most restricted expression, being present only in the IT2 segment (Figure ). Apart from the intermediate tubule, the expression domains of cldn14
extended distally to include DT1 (Figure ). Finally, transcripts for cldn19
were present not only in the intermediate tubule but also in the nephrostomes (Figure ).
Figure 5 Expression domains of selected molecular marker genes validates the pronephric segmentation model. Whole-mount in situ hybridizations of stage 35/36 Xenopus embryos were performed. Lateral views of whole embryos (left panels), enlargements of the pronephric (more ...)
We also studied the expression of the kidney-specific chloride channel clcnk
, the potassium channel kcnj1
(also known as romk
), and the calcium-binding protein calbindin 1 (calbindin 28 kDa; calb1
). Previously, we reported clcnk
to be a marker of the pronephric duct [17
], and more recently mapped its expression to cover the intermediate, distal, and connecting tubules [22
] (Figure ). Expression of kcnj1
was similar to that of clcnk
, with the exception that kcnj1
was not present in IT2 (Figure ). Finally, calb1
expression was restricted to the connecting tubule with highest expression at the distal tip (Figure ). Expression throughout the connecting tubule segment became more apparent by stage 40 (data not shown). In summary, the analysis of additional pronephric marker genes fully supports our proposed model of pronephric nephron segmentation. For example, cldn8
expression provides further evidence for the bipartite nature of the intermediate tubule compartment. Furthermore, we failed to detect any evidence for additional subdivisions of the nephron other than the ones reported here.
Gene expression comparisons reveal striking analogies of nephron segmentation between pronephric and metanephric kidneys
We performed cross-species gene expression comparisons to identify similarities between the nephron organization of the Xenopus pronephros and the mammalian metanephros. We selected 23 marker genes with highly regionalized expression in the Xenopus pronephric kidney to compare their renal expression domains with the corresponding mammalian orthologs. As shown in Table , the list included 18 slc genes, calb1, cldn8, cldn16, clcnk, and kcnj1. Information on the expression of the mammalian counterparts in either the adult mouse or rat kidney was obtained in part from the published literature (Table ). In addition, we determined independently the expression patterns for many of the selected genes by in situ hybridization analysis. Selected examples of stained adult mouse kidney sections are shown in Figure . We determined the previously unknown renal expression domains of Slc5a9, Slc6a13, Slc13a3, and Slc16a7 (Figure and data not shown). Furthermore, we confirmed the expression domains of many others, including Slc5a2, Slc7a13, Slc8a1, Slc12a1, Slc12a3, Cldn8, and Calb1 (Table , Figure , and data not shown).
Selected marker genes of the stage 35/36 Xenopus pronephric nephron
Selected marker genes of the adult rodent metanephric nephron
Figure 6 Expression of selected renal marker genes in the adult mouse kidney. In situ hybridizations were performed on paraffin sections of adult kidneys taken from 12-week old mice. Whole transverse sections (upper panels) and magnifications (lower panels) are (more ...)
A comparison of the expression domains of the selected marker genes between the Xenopus
pronephros and the rodent metanephros is shown schematically in Figure . Overall, a remarkable conservation of segmental gene expression was observed. This was most striking for the proximal tubule. All 13 mammalian genes with expression in the proximal tubule were also expressed in the Xenopus
proximal tubule. Generally, only minor differences between Xenopus
and mammalian marker genes were observed. In many cases, however, we found complete conservation of segmental expression domains. This is best illustrated by the low-affinity and high-affinity Na-glucose transporters Slc5a2
, which are sequentially expressed along the proximodistal axis of the proximal tubule [33
localizes to S1 and S2 in mouse and to PT1 and PT2 in Xenopus
, whereas Slc5a1
was detected in S2 and S3, and PT2 and PT3, respectively (Figure , Figure , and data not shown).
Figure 7 Expression domains of selected marker genes in the Xenopus pronephros and the rodent metanephros. The expression domains of selected marker genes in the nephrons of (a) Xenopus stage 35/36 pronephric kidneys and (b) adult rodent metanephric kidneys are (more ...)
The comparison of gene expression in the intermediate tubule revealed a more complex picture. Importantly, there was clear evidence for expression of Cldn8 and Clcnk in the intermediate tubules of Xenopus and mouse. The Cldn8 gene, which in mouse is expressed in the descending thin limb, was confined to IT2 in Xenopus (Figure and Figure ). With regard to Clcnk, the broad expression domain (IT1 → connecting tubule) of the single Xenopus clcnk gene was comparable to the combined expression domains of Clcnka (ascending thin limb) and Clcnkb (thick ascending limb [TAL] to collecting duct) in mouse kidney (Figure and data not shown). Moreover, we observed that the Xenopus intermediate tubule shares some transport properties with the mammalian TAL. In Xenopus, slc12a1, slc16a7, cldn16, and kcnj1 - whose murine counterparts are markers of the TAL (Table ) - exhibited striking proximal expansions of their expression domains to include segments of the intermediate tubule (Figure ).
The distal tubule in mammals can be divided structurally into two compartments: the TAL and the distal convoluted tubule (DCT). Molecularly, it is defined by the differential expression of the Na-K-Cl transporter Slc12a1 in the TAL and the Na-Cl cotransporter Slc12a3 in the DCT (Figure ). We found that this was also the case for the Xenopus distal tubule. Note that the junctions between the slc12a1 and slc12a3 expression domains define the boundary between DT1 and DT2 (Figure ). We also noticed that the Xenopus orthologs of mouse TAL markers were expressed in the Xenopus DT1. When comparing the mouse DCT with the Xenopus DT2, striking similarities became apparent. As mentioned above, we could demonstrate expressions of the key marker slc12a3 and of clcnk, kcnj1, and the ammonia transporter Rhcg/Slc42a3 in DT2. However, we found one exception relating to the expression of the calcium-binding protein encoded by Calb1, which is a marker of the mouse DCT and connecting tubule (Figure ). In Xenopus, calb1 was not expressed in DT2 but in the connecting tubule only (Figure ). Taken together, the Xenopus DT1 and DT2 are analogous to the mouse TAL and DCT, respectively.
In mouse kidney, the connecting tubule can be identified on the basis of Slc8a1 expression (Figure ). Interestingly, expression of Xenopus slc8a1 also defines a distinct compartment adjacent to the distal tubule, which we termed connecting tubule. As in the mouse, the Xenopus connecting tubule expresses slc16a7, calb1, clcnk, and kcnj1, which further emphasize the similarities between the connecting tubules of the pronephros and metanephros (Figure , Figure , and data not shown).
The Xenopus pronephric kidney lacks a nephron segment analogous to the mammalian collecting duct
We assessed Xenopus
embryos for the expression of marker genes of the mammalian collecting duct. First, we analyzed three aquaporin genes, namely aqp2
, and aqp4
, which are markers of principal cells in the mammalian collecting duct [34
]. None of the tested genes were expressed in the pronephric kidney (data not shown). Subsequently, we analyzed slc4a1
(AE1), a marker of type A intercalated cells of the cortical and medullary collecting ducts [38
], and slc26a4
(pendrin), which is expressed in type B intercalated cells of the connecting tubule and cortical collecting duct in the adult mammalian kidney [39
]. No expression could be detected in the stage 35/36 pronephric kidney (data not shown). We therefore conclude that there is presently no molecular evidence indicating that the stage 35/36 Xenopus
pronephric kidney harbors a nephron segment that shares molecular characteristics with the mammalian collecting duct.
A public resource: XGEbase
The present study has generated a large, unique dataset of temporal and spatial gene expression patterns. We organized these data online as a public resource in EuReGene in the form of an interactive database. XGEbase [27
] currently contains whole-mount in situ
hybridization data on 210 slc
genes. The embryonic expression patterns are documented by more than 1,200 representative microscopic in situ
hybridization images. The pronephric expression patterns are fully annotated in accordance with our model. Moreover, we also identified more than 100 genes expressed in spatially restricted patterns within other non-renal tissues such as brain, liver, and heart (DR and AWB, unpublished data). OK! Although not the explicit focus of the present study, the obtained expression patterns were fully annotated in accordance with the Xenopus
Anatomy Ontology [40
] and deposited in XGEbase. Hence, XGEbase provides not only a unique resource for future studies on pronephric kidney development and function, but also enhances our general understanding of organogenesis in the Xenopus