Segmental expression of Notch and Hairy genes in nephrogenesis
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《美国生理学杂志》
Departments of Medicine and Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, New York
ABSTRACT
Notch signaling pathway genes are required for nephrogenesis, raising the possibility that Notch effector Hairy-related genes should also control nephron formation. We performed in situ hybridization of Hairy transcription factors with segment-specific lectins and/or antibodies during early nephrogenesis to identify their possible roles in segment identity of the nephron. We found that among all of Notch downstream Hairy genes, only Hes1, Hes5, Hey1, and HeyL were expressed in a segment-specific manner in early nephrons and their expression pattern changed dynamically during metanephric development. Based on these patterns of expression, it was possible to propose a pairwise association of specific ligand and receptor and to suggest that the effector of this association is one of the Hairy transcription factors. We found that Hes5 is specifically expressed in the anlage of the loop of Henle, suggesting that it might be involved in the determination of its cell identity. We also examined the morphological appearance of kidneys from mice where the Hes1 or Hes5 genes were deleted and found that at least at the gross morphological level, there was little difference from wild-type kidneys. Because Hairy genes associate with other transcription factors to exert their effect, it is necessary to examine a more complete array of genetic deletions before a conclusion can be reached regarding their role in kidney development. These studies provide the basis for the future development of strategies to examine the role of individual effector molecules in the determination of the differentiation pattern of the nephron.
in situ hybridization; Hairy-related genes; renal expression pattern; Hes mutant
THE NOTCH SIGNALING PATHWAY was initially identified in Drosophila when a mutation in Notch was found to cause an increase in the number of neuroblasts at the expense of epidermal cells (20). However, it is now clear that this pathway is present in all metazoa where it functions as one of the major pathways that determine cell identity during development (3, 13). Notch is a transmembrane protein that interacts with a ligand of the Jagged and Delta family (13, 17). There are four Notch members in mammals (Notch 1–4), two Jagged (Jag), and four Delta-like genes (8). Each of these proteins shows a cell type- and tissue-specific expression during development. Interaction of these ligands with Notch activates the proteolytic cleavage of the Notch intracellular domain (NICD) (39). Transport of NICD to the nucleus allows it to bind to a transcription factor, RBP-Jk, which then mediates the program of cell identity. In the absence of NICD, RBP-Jk binds to a number of corepressor molecules that repress transcription from the DNA bound to RBP-Jk (26). The RBP-Jk NICD complex activates the transcription of a number of effector molecules, the most important of which are themselves transcription factors of the HES family, a group of basic helix loop helix (bHLH) genes that are orthologs of the Drosophila gene enhancer of split (4). Studies of localization of Notch genes and HES genes showed that some cells do not express HES genes but express Notch and its ligands, suggesting that other effectors might be present. This led to the identification of Hey genes that are bHLH genes closely related to the HES genes (23, 29, 35). Among the seven Hes members and three Hey members isolated in mammals, Hes1, -5, -7 and Hey1, -2, -L are potential target genes of Notch (22).
Deletion of Notch is embryonically lethal, which has limited the study of the role of these receptors in organogenesis (18). Notch 3 mutations have been implicated in the human disease CADASIL, a cerebral arteriopathy (25). However, a hypomorphic allele of Notch 2 shows a vascular anomaly in the kidney with an aneurysmal dilatation of the glomerular capillary (32). Jagged 1 mutations are associated with the Alagille syndrome, where there are a large number of skeletal and cardiovascular anomalies (37, 42).
The mammalian kidney develops in three waves, in only the last of which the metanephros continues as the adult kidney. The metanephric kidney begins when the ureteric bud (UB), an outgrowth of the Wolffian duct, invades the metanephric mesenchyme. Factors secreted by the UB induce this mesenchyme to aggregate and then convert to an epithelial structure (5, 34). The induced mesenchyme, in turn, sends signals back to the UB to cause it to divide and grow. This reciprocal induction proceeds in an ordered manner to produce several generations of branches and eventually all the nephrons of the kidney. The tip of each UB first induces the development of the renal vesicle, which is an epithelial cyst that undergoes morphogenetic transformation to eventually form the nephron from the glomerulus to the end of the distal tubule while the UB eventually forms the collecting tubule. During the morphogenesis of the S-shaped body, the proximal end becomes invaded by blood vessels and its epithelial cells differentiate into podocytes and parietal epithelial cells. Simultaneously, the middle segment of the nephron begins to express several proteins (or sugars) that are characeristic of the proximal tubule. The most distal segment of the primitive nephron becomes connected to the UB epithelium (1, 14). Similar to the UB, the primitive distal tubule also expresses E-cadherin at the lateral junctions of the epithelium. The molecular mechanism by which the S-shaped body undergoes segmentation is unknown at present. We embarked on a study that aims at examination of pathways that determine cell identity in kidneys. The Notch signaling pathway is one of these pathways, and we now describe the segment-specific expression of these genes in the hope that they will help in designing new studies that examined the mechanism of determination of cell identity during nephrogenesis.
MATERIALS AND METHODS
Animals. Embryos were obtained from timed matings of Swiss-Webster mice (Taconic). Day 0.5 of gestation (E0.5) was considered to be at noon of the day on which the vaginal plug was detected.
Hes1 and Hes5 mutant mice were a generous gift of Dr R. Kageyama (Institute for Virus Research, Kyoto University, Kyoto, Japan). Hes1 –/–, Hes5 –/–, Hes1 –/– Hes5 +/–, and Hes1 +/– Hes5 –/– embryos were acquired as previously described (10).
Culture of kidney cell line and embryonic kidneys. The UB cell line was derived from transgenic mice for SV40 T-antigen (6). UB cells were grown in minimal essential media (MEM; GIBCO) with 10% FCS (Hyclone). A stromal cell line was isolated from mice in which -galactosidase was knocked into the BF-2/Foxd1 locus (19). Stromal cell lines were grown in DMEM (low glucose) with 10% FCS.
Embryonic mice kidneys were microdissected at E11.5. Kidneys were applied to the top of polyester Transwell filters (Costa) and grown in MEM (GIBCO) supplemented with 10% FCS at 37°C in an atmosphere containing 5% CO2.
RT-PCR analysis. Total RNA was isolated from kidneys of different embryonic days by using RNeasy kit (Qiagen) and then digested with DNase I to get rid of genomic DNA. First-strand cDNA synthesis was primed by MMLV reverse transcriptase with the antisense primers. The primers used are described in Table 1. For negative controls, reverse transcriptase was omitted and PCR was performed directly from the RNA. Amplification of cDNAs was performed using 36 cycles with denaturation at 95°C for 30 s, annealing at 63°C for 30 s, and extension at 68°C for 60 s. After completion of the PCR cycles, reactions were subjected to 68°C for 10 min and the products were subjected to electrophoresis and visualized with ethidium bromide.
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Probe synthesis. Probes were synthesized with the appropriate RNA polymerase in the presence of digoxigenin-UTP (Boehringer-Mannheim Biochemicals). The probes were precipitated and resuspended in the hybridization buffer. We used full-length cDNA of Hey1 and Hey2, partial coding sequence of Dll1 (466 bp), Jagged1 (494 bp), and Notch2 (986 bp) as templates for the generation of digoxygenin-labeled antisense and sense riboprobes with either T7 or SP6 polymerase (Promega). Both of the above probes were synthesized from PGEMT-Easy vector (Promega). The Notch1 probe was produced by linearization of plasmid (a gift from Dr. G. Weinmaster) by EcoRI and priming with T3. Hes1 and Hes5 plasmids were made by linearization of plasmids (generous gifts from Dr. F. Guillemot) either by EcoRI and then priming with T7 or by HindIII and then priming with T3. The Hes7 probe was produced by linearization of plasmid (a kind gift from Dr. R. Kageyama) by SpeI and transcribed using T7. The HeyL probe (a kind gift from Dr. D. Srivastava) was produced by transcribing EcoRV-linearized CDNA with SP6.
In situ hybridization and whole mount in situ hybridization. Embryos were fixed in 4% paraformaldehyde (PFA), infiltrated with 20% sucrose, embedded in OCT, and 10-μm sections were cut, postfixed for 10 min, treated with proteinase K (1 μg/ml), and acetylated at room temperature. Prehybridization was carried out for 2 h at room temperature. Hybridization was performed overnight at 68°C. The prehybridization and hybridization solution was 50% formamide, 5x SSC, 5x Denhard’s, 250 μg/ml yeast tRNA (Sigma), and 500 μg/ml salmon sperm DNA (Sigma). Posthybridization washes were done at 68°C in 0.2x SSC for 1 h. Sections were then blocked with 10% sheep serum and incubated overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) at a 1:5,000 dilution. Alkaline phosphatase activity was detected by developing slides in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT; Roche). Control staining was performed with the sense riboprobes.
Whole mount in situ hybridization was performed as described before (33). Briefly, kidneys were fixed in 4% PFA in 0.1 M phosphate buffer and treated with proteinsase K (1 μg/ml). Prehybridization was carried out for 2 h at room temperature. Hybridization and posthybridization were performed at 68°C in the water bath. Kidneys were then incubated overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin antibody and developed in BCIP and NBT (Roche). Control staining was performed with the sense riboprobes.
Photographs were taken under a Spot imaging camera (RT slider) using a Nikon Microphot-SA microscope (Olympus Optical, Tokyo, Japan).
Immunofluorescence. The mouse monoclonal E-cadherin antibody and Pax-2 antibody were purchased from Zymed. Mice cryostat sections were fixed in 4% PFA, rinsed in PBS, and pretreated with 10% donkey normal serum. Primary antibodies were diluted 1:200 in PBS and incubated overnight at 4°C. Sections were then incubated with FITC-conjugated secondary antibodies at 1:200 dilution, rinsed in PBS, and mounted with Vectashield mounting medium. The fluorescent images were electronically captured with a Spot imaging camera (RT slider) using a Nikon Microphot-SA microscope (Olympus Optical).
Combined lectin staining with in situ hybridization. Dolichos biflorus agglutinin (DBA) and LTA lectin conjugated to FITC were used to detect collecting ducts and proximal tubules. Peanut lectin (PNA) conjugated to rhodamine was used to detect glomerular podocytes. To perform lectin staining, we performed in situ hybridization on sections as described above. Slides were then washed three times with PBS, treated with gelatin buffer (0.075% saponin detergent, 0.2% gelatin, 1 mmol/l MgCl2, and 0.1 mM CaCl2 in PBS) for 30 min. Slides were washed twice in neuraminidase buffer (50 mmol/l Na acetate, 150 mmol/l NaCl, 9 mmol/l CaCl2, pH 5.5) and incubated for 4 h at 37°C with 1 IU/ml of neuraminidase (Sigma). After being rinsed with gelatin buffer, sections were labeled for 1 h at 37°C with rhodamine-coupled PNA diluted in gelatin buffer (1:100). After several washes, slides were incubated with FITC-conjucated DBA (dilution 1:50) or lotus lectin (Sigma, 1:50) for 1 h at 37°C. Slides were mounted in Vectashield mounting medium and viewed under a Nikon Microphot-SA microscope (Olympus Optical).
Hematoxylin and periodic acid-Schiff staining. Kidney sections were fixed, incubated with 0.5% periodic acid for 5 min, and treated with Schiff’s reagent (PAS). Slides were then stained with hematoxylin for 2 min and blued in tap water. Slides were viewed under a Nikon Microphot-SA microscope (Olympus Optical). These studies were approved by the institutional animal care committee, AC-IACUC protocol AAAA2035.
RESULTS
Expression of Hairy and Notch-related genes using RT-PCR. To characterize the expression of the Hairy family of genes, we initially performed a screening assay using RT-PCR in embryonic kidneys at different stages of development and in two cell lines derived from mouse kidneys. We found that the Hairy-related genes Hes1, Hes5, Hes7, Hey1, Hey2, and HeyL were all expressed. However, Hes7 was only expressed at a very low level and then transiently, and no signal could be detected by in situ hybridization (data not shown). Because Hairy-related genes are a target of Notch signaling, we also studied the expression of Notch and its ligands by RT-PCR. These results indicated that mRNAs for Dll1, Jagged1, and all Notch receptors were present in different stage of metanephric development. Jagged2 and Dlk were only expressed at a low level and then only transiently. Dll3 was not expressed in metanephroi, although it was expressed in the brain of the same mice. Identity of these products was confirmed by sequencing of the PCR products. No RT-PCR products were detected in the H2O or non-RT controls of any sample. The results are summarized in Table 2.
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To test whether cultured embryonic kidney cells maintain similar expression as in vivo kidneys, we examined Notch and Hairy-related gene expression in UB cell and stromal cell lines by RT-PCR. As shown in Table 3, Hes1, Hes7, Hey1, and Hey2 transcripts were detected in the renal stromal cell lines, but only Hes1 transcripts were observed in the UB cell lines. The stromal cell line also expressed Dll4, Dlk, Jagged1, Notch1, Notch2, and Notch3 mRNA. Jagged1, Notch1, and Notch2 were expressed in UB cells. However, Jagged1 was not detected in the ureteric branches by in situ hybridization (data not shown).
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Hes1 is expressed in both mesenchymal and epithelial structure. In E13.5 kidneys, Hes1 transcripts were present in most cells during the early stages of kidney development. The signal was prominent in both the condensed mesenchyme, UB tips, ureteric branches, primary vesicles, comma-shaped bodies, and S-shaped bodies. A weak signal was also observed in the capsule and subcapsular interstitium (Fig. 1A).
To identify segment-specific expression in developing nephrons, we stained E15.5 kidneys with lectins known to bind to specific segments. By colabeling ureteric branches and tips with DBA (in green), a marker of the UB (27), we confirmed that while high levels of expression were present in UB tips in E15.5 and the nephrogenic zone of newborn kidneys (data not shown), only faint expression was detected in more developing collecting ducts (Fig. 1B). In the nephrogenic zone, all regions of the S-shaped bodies expressed Hes1 mRNA, but in the more developing segments, the pattern changed such that the expression disappeared from the developing proximal tubules (LTA-positive tubules in green) but remained in the other segments of the developing tubules (Fig. 1, C-E). Expression of Hes1 mRNA also appeared to decrease with glomerular maturation. A strong signal was detected throughout the early capillary loop stage of glomeruli including podocyte progenitors (stained with PNA in red), mesangial cell progenitors, and vascular wall (Fig. 1, F-H), while expression was hardly detectable in more mature capillary loops (Fig. 1, I-K, arrows). No signal was detected in the corresponding section hybridized with sense cRNA probe. We also analyzed by RT-PCR an embryonic kidney stromal cell line and found that Hes1 was also present, suggesting that it might be expressed also in developing renal stromal cells. (Table 2).
Hes5. Hes5 was observed in comma- and S-shaped bodies. To precisely identify the boundary of the different segments, in situ hybridization sections of E13.5 kidneys were triple labeled with the podocyte marker PNA and the proximal tubule marker LTA (27). As can be seen in Fig. 2, A-C, there was no colocalization of the in situ signal with these tubular markers. At this stage of development, that leaves the loop of Henle and the distal tubule. We performed serial sections and stained adjacent ones by in situ hybridization or E-cadherin, a marker for distal and collecting tubules. We found that Hes5 was only expressed in the most proximal end of these E-cadherin-expressing segments (Fig. 2, D and E). Because the proximal end of E-cadherin staining was colocalized with anti-uromucoid antibody, a marker for the ascending limb of the loop of Henle (data not shown), we think Hes5 was expressed in the prospective loop of Henle segment not the proximal tubules as stated by others (30, 38). This is best seen in Fig. 2F where the in situ signal did not colocalize with either the distal tubular elements (long arrow) or the proximal tubular elements (short arrow). The strongest staining was thus seen in the segment that will develop into the loop of Henle. There was also some faint staining in an adjacent region of the prospective proximal tubules, but whether this was simply background staining or low level of expression is difficult to tell (Fig. 2, A-C).
As defined by Neiss (36), three distinct stages of developing loop of Henle were present in the developing nephron. Hes5 mRNA was only detected in the first stage (the anlage) of the developing loop in the nephrogenic zone and absent in primitive loop and immature loops (Fig. 2, G-J).
Hey1. Whole mount in situ hybridization analysis of cultured embryonic kidneys demonstrated that Hey1 first appeared at E11.5. Initially, Hey1 was expressed in regions surrounding the UB tips and in pretubular aggregates. The strongest signals were often located below the UB tips, an area that will eventually become the renal vesicle. Early nephron development progresses in an ordered manner from loose mesenchyme, to condensed mesenchyme, and finally to renal vesicle, a pattern that recurs after each generation of branches (Fig. 3). It is likely that after each branch of the UB the mesenchyme next to each tip has reached a similar level of induction. With that in mind, we found a surprising result that the expression of Hey1 in each mesenchyme surrounding the UB often was not identical (Fig. 3, top, compare 1 and 3 and 2 and 4). The signal could be categorized into four different patterns (1–4 in the figure): 1, no signal in mesenchyme; 2, signal in the mesenchyme surrounding the tip; 3, transcripts in the mesemchyme below the UB and partially surrounding the tip; and 4, signal only in the mesenchyme below the UB. This suggested that the Hey1 expression pattern might oscillate in mesenchyme. Similar oscillations in presomitic mesoderm during somite segmentation have attracted much attention. However, due to a large variation between left and right kidneys when cultured in vitro, we were unable to determine the precise timing of each cycle using the similar method as in the presomitic mesoderm. However, this unique expression pattern only occurred in the very early stage. After 2 days of in vitro culture, no signal was detected in condensed mesenchyme and pretubular aggregates (Fig. 3).
In situ hybridization showed that Hey1 mRNA was localized in renal vesicles, comma-shaped bodies, and S-shaped bodies. In the S-shaped bodies, it localized into the more proximal regions of the developing nephron, as shown by the lack of colocalization with E-cadherin in adjacent sections (Fig. 4, A and B). In early S-shaped bodies, the expression colocalized with the podocyte marker PNA (Fig. 4, C-E, arrows). As the nephrons matured and acquired a strong lectin-staining pattern, the expression of Hey1 was downregulated (Fig. 4, F-H). Similar to Hes5, Hey1 was only observed in the nephrogenic region (Fig. 4, F-H). Hey1 was also detected in vascular structures (not shown).
HeyL. HeyL mRNA was present in the developing glomerular cleft but not in the PNA-positive podocytes. Cells that expressed HeyL, presumably the vascular elements of primitive glomeruli, were located within the glomerulus (Fig. 5, A-C, long arrows), but we occasionally found cells that surrounded the pododcytes (Fig. 5, A-C, short arrows). It was only expressed in early capillary loop stage of glomeruli including both endothelial cells and mesangial cell progenitors but not in the maturing glomeruli (Fig. 5D, short arrows). HeyL mRNA was also present in primary vesicles (Fig. 5, A-C), comma-shaped body, and S-shaped body. Figure 5E indicated the structure of an S-shaped body located below the right side of F in E. It clearly showed that only the segment of the developing proximal tubule in the S-shaped body had a signal. However, its expression in proximal tubules was transient, disappearing in more mature stages (Fig. 5, H-J).
There was no HeyL expression in collecting ducts (cd) or distal tubules (db) as determined by the lack of colocalization with E-cadherin (performed in adjacent sections; Fig. 5, E and G). This is different from the findings of the Gessler group (30), who stated that they found HeyL expressed in the collecting duct; however, they did not use colocalization with markers of the collecting duct. We also found a strong signal in various vascular elements (Fig. 5, A and C, ). No specific signal was found for the sense probes of HeyL.
Hey2 and Hes7. In situ hybridization studies showed that Hey2 was localized in large blood vessels, probably smooth muscle cells. Hey2 expression initially appeared at late E12.0 kidneys (not shown). In vitro culture of E11 kidneys for 48 h showed no staining of Hey2 (not shown). No specific signal was found for the sense probe of Hey2. No Hes7 signal was detected by in situ hybridization (not shown).
Comparison of Notch receptor expression pattern. The renal expression pattern for Notch was examined in several recently published reports; unfortunately, they came to differing conclusions regarding the site of localization (30, 38). We reexamined the expression pattern with other markers including E-cadherin, PNA, or lotus lectin to clarify the precise segment of these transcripts.
We found that Notch1 was expressed inside PNA-positive regions, suggesting that it is expressed in the prospective mesangium but not in the podocytes (Fig. 6, A-C). There was good colocalization with E-cadherin in adjacent sections, suggesting that it was expressed by the prospective distal tubule and collecting ducts (Fig. 6, D and E, arrows). However, prospective proximal tubules had no expression (Fig. 6F).
Notch2 was present in the primitive proximal tubule (Fig. 6, G-L, arrows) with very faint colocalization with podocyte progenitors. It was expressed in the early capillary loop stage of glomerular podocytes with faint staining in the mesangial region probably in both endothelial and mesangial cells (Fig. 6J, arrow). More mature podocytes had no expression of Notch2 (Fig. 6J, star). There was good colocalization with E-cadherin as judged by E-cadherin staining in adjacent sections (Fig. 6, K and L, arrows).
Notch3 was expressed inside PNA-positive regions (Fig. 6, M and N, long arrows), suggesting that it is expressed in the capillary loop vascular wall including the prospective mesangium and endothelial precursors. Notch3 was also detected in UB tips and large blood vessels (Fig. 6, N and O). However, the prospective podocytes did not express Notch3.
Expression of the Notch ligands Delta and Jagged. Both Dll1 and Hes5 first appeared at E12.75 kidneys. As shown in Fig. 7, A-C, the tissue distribution patterns were quite similar for Hes5 and Dll1 transcripts, with expression in the prospective loop of Henle and proximal tubules in the nephrogenic zone. In these adjacent sections, Hes5 was expressed at a very faint level in proximal tubules, only appearing after prolonged color development, raising the question of whether it reflected background staining rather than actual mRNA expression. In contrast, a Dll1 signal was strong in both the prospective proximal tubules and loop of Henle. We also noted that the Hes5 signal was ubiquitously distributed in the prospective loop of Henle, whereas a Dll1 signal seems not.
Jagged1 was expressed at a faint level in primary vesicles, comma-shaped bodies, and S-shaped bodies. In more advanced nephrons, it was observed in developing distal tubules and prospective loop of Henle but absent in collecting ducts (Fig. 7, G and H). Its signal was also detected in proximal tubules; however, no expression was observed in podocytes (Fig. 7, H and I). Jagged1 signals were located in the apical region of the tubular cells.
Hes1 and Hes5 null mice have grossly normal developing kidney. We examined embryonic mouse kidneys using hematoxylin/PAS staining and found that both Hes1 and Hes5 knockout mice have normal development of nephrogenesis (Fig. 8). We also stained sections with LTA for the development of proximal tubules and with PNA staining for podocytes formation in both Hes1 and Hes5 mutant mice. We did not detect any significant difference between wild-type and Hes1 or Hes5 knockout mice (data not shown). To test whether Hes5 mutants had defects in the loop of Henle, we stained Hes5 knockout and wild-type mouse kidneys with anti-Tamm-Horsfall protein antibody to examine them for a differentiated marker of the loop of Henle. Positive staining was first detected in wild-type E16.5 kidneys. However, no difference was detected in both embryonic kidneys (E15.5, E16.5, and E17.5) and postnatal kidneys (newborn, 2 wk, and 1 mo). Hes1 mutant and wild-type kidneys were also stained with E-cadherin for distal tubules and collecting ducts and with Pax-2 for early nephron formation. Again, no difference was found (data not shown).
We then crossbred Hes1 and Hes5 mice. We were unable to get Hes1 –/– Hes5 –/– mice because the double mutants die around E11.5 before nephron formation. We examined Hes1 +/– Hes5 –/– and Hes1 –/– Hes5 +/– and wild-type mice using HE or hematoxylin/PAS sections. No obvious defect was detected in these compound mutant embryonic kidneys (Fig. 8). However, we noticed that some of the mutant kidneys (Hes5 –/–, Hes5 +/– Hes1 +/–, Hes1 +/– Hes5 –/–) were smaller.
DISCUSSION
The Notch signaling pathway controls cell identity in a variety of systems. Initially described in the fruit fly, mutations in Notch resulted in an excess of neuroblasts at the expense of epidermal cells (20). Since then, Notch signaling in vertebrates has been implicated in the control of cell identity in a variety of organs derived from all three germ layers (15). Mutations in Notch pathway genes lead to anomalies in blood vessel development, hematopoiesis, thymus, central nervous system, heart, somites, ribs, craniofacial development, and the kidney (7, 10–12, 21, 24, 32, 42, 43). Because the Notch pathway is involved in specifying cell identity in different organs, it was possible that it might also mediate such an effect in the kidney.
Detailed functional and morphological analysis shows that the kidney and the epithelium of the nephron in particular are composed of a variety (as many as 15) specific cell types (40). The determination of cell identity during this process is unknown. It is likely that these cell types are specified early in development (2). Examination of the morphology of the developing nephron shows that after conversion of the mesenchyme to an epithelium, all the cells of the S-shaped body resemble each other. However, lectin staining and immunocytochemistry showed that at that stage the primitive nephron had begun to differentiate as LTA lectins bound to the future proximal tubule (27), PNA lectins bound to the future podocyte (27), and E-cadherin began to be expressed in the future distal tubule. Hence if a cell identity gene were going to be expressed, it would have to appear at that stage or earlier in the specific segment.
It is well known that Hairy-related genes are Notch downstream effectors. Previous studies examined their renal localization (30–32, 38). However, in both studies (32, 38), the segmental boundaries for those genes were not definitively identified. To more precisely define the segmental expression of these genes, we performed a survey of the expression pattern of members of the Hairy-related genes and used triple staining or serial sections with segment-specific markers. Dynamic and segmental expression patterns were observed at critical stages of nephron development. Figure 9 provides a summary cartoon of the expression of Notch effectors HES and HEY genes, whereas Fig. 10 shows the same for Notch receptors and ligands expressed in the kidney. Our results also show that HeyL was not expressed in collecting ducts as reported (30) and Hes5 was expressed in the prospective loop of Henle instead of proximal tubules as reported before (38) by staining serial sections with E-cadherin, a distal tubule and collecting duct marker.
As one can see, Hairy-related genes show segment-specific expression in nephrogenesis. Hes5 is only expressed in the region of the S-shaped body destined to become the loop of Henle with very low expression in the prospective proximal tubules. Hence, we suggest that Hes5 might contribute to the identity of the loop of Henle differentiation. Hes1 was present throughout the metanephros, including the ureteric epithelium and S-shaped bodies and the developing podocytes, indicating that it might have a broader role in kidney development.
HeyL was expressed in the glomerular cleft, renal vesicle, and endothelial cells surrounding the early developing glomerulus and also in the segment of prospective proximal tubules, suggesting that HeyL might function in regulating glomerular vascular wall formation. Hey1 was expressed in pretubular aggregates and renal vesicles, suggesting the possible role of Hey1 in early nephron formation. The repeated Hey1 expression pattern in each cycle of nephron formation in the cultured kidneys resembled its oscillating expression pattern found during somite formation. However, we were unable to determine the timing of one cycle due to the large variation between left and right kidneys in the culture system. The mechanism and role of this specific expression pattern in early nephron segmentation are unknown. Considering that each cycle of gene expression in the presomitic mesoderm is associated with an intrinsic segmentation clock that coordinates somite segmentation, Hey1 might play a similar role in determining the precise timing of the transformation of the renal vesicle to the comma-shaped bodies. Hey1 was also abundantly expressed in the S-shaped body from the region that will form the loop of Henle to the developing podocytes, suggesting it might also contribute to glomerular formation.
Recent studies showed that presenilins and Notch1 were required for the progression from renal vesicles to comma- and S-shaped bodies (9, 41). A hypomorphic allele of Notch2 was found to express defects in vascular development of the glomerulus (32). These data suggest that Notch pathway genes likely have a critical role in various stages of nephron development. The study of deletion mutants in the hairy-related genes suffers from the fact that many such mutations are embryonic lethal. We examined Hes5 knockout mice and found that they survived through nephrogenesis but we were unable to detect any defects in the loop of Henle or in other segments. One expectation was that, similar to studies in neurogenesis, the expression of differentiation markers of the loop of Henle would be accelerated in these mutant mice. However, using expression of Tamm-Horsfall protein, we did not find an earlier expression in mutant mice. Similarly, we did not find any gross anomalies in the Hes1 mutant mice and Hey1 mice had already been reported to have no nephron defect (16).
Hairy-related genes function by forming homo- or heteromultimers with other bHLH transcription factors (23, 28). Notch signaling thus may rely on both homodimer and heterodimer formation between different hairy genes to suppress the cell identity genes. Because the compound homozygotes for both Hes1and Hes5 died before kidney development, we are unable to study the double knockout mice to test this hypothesis. Furthermore, Hey1 was also detected in the prospective loop of Henle; hence, we could not exclude the possibility that Hey1 might compensate for Hes5 function in the Hes5 null kidneys. Hence, the lack of defect in early nephrogenesis is most likely due to overlapping expression of these genes.
In summary, our survey provides a comprehensive expression map of Notch signaling molecules during early nephron development. Future evaluation of targeted disruption of these genes using conditional knockout mice and misexpression of various components of Hairy signaling will help determine the functional role of these genes in renal segmentation.
GRANTS
This work was supported by National Institutes of Health Grant DK-55388.
ACKNOWLEDGMENTS
We are grateful to Dr. R. Kageyama from the Institute for Virus Research, Kyoto University, Japan, for a generous gift of Hes5 and Hes1 mutant mice.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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ABSTRACT
Notch signaling pathway genes are required for nephrogenesis, raising the possibility that Notch effector Hairy-related genes should also control nephron formation. We performed in situ hybridization of Hairy transcription factors with segment-specific lectins and/or antibodies during early nephrogenesis to identify their possible roles in segment identity of the nephron. We found that among all of Notch downstream Hairy genes, only Hes1, Hes5, Hey1, and HeyL were expressed in a segment-specific manner in early nephrons and their expression pattern changed dynamically during metanephric development. Based on these patterns of expression, it was possible to propose a pairwise association of specific ligand and receptor and to suggest that the effector of this association is one of the Hairy transcription factors. We found that Hes5 is specifically expressed in the anlage of the loop of Henle, suggesting that it might be involved in the determination of its cell identity. We also examined the morphological appearance of kidneys from mice where the Hes1 or Hes5 genes were deleted and found that at least at the gross morphological level, there was little difference from wild-type kidneys. Because Hairy genes associate with other transcription factors to exert their effect, it is necessary to examine a more complete array of genetic deletions before a conclusion can be reached regarding their role in kidney development. These studies provide the basis for the future development of strategies to examine the role of individual effector molecules in the determination of the differentiation pattern of the nephron.
in situ hybridization; Hairy-related genes; renal expression pattern; Hes mutant
THE NOTCH SIGNALING PATHWAY was initially identified in Drosophila when a mutation in Notch was found to cause an increase in the number of neuroblasts at the expense of epidermal cells (20). However, it is now clear that this pathway is present in all metazoa where it functions as one of the major pathways that determine cell identity during development (3, 13). Notch is a transmembrane protein that interacts with a ligand of the Jagged and Delta family (13, 17). There are four Notch members in mammals (Notch 1–4), two Jagged (Jag), and four Delta-like genes (8). Each of these proteins shows a cell type- and tissue-specific expression during development. Interaction of these ligands with Notch activates the proteolytic cleavage of the Notch intracellular domain (NICD) (39). Transport of NICD to the nucleus allows it to bind to a transcription factor, RBP-Jk, which then mediates the program of cell identity. In the absence of NICD, RBP-Jk binds to a number of corepressor molecules that repress transcription from the DNA bound to RBP-Jk (26). The RBP-Jk NICD complex activates the transcription of a number of effector molecules, the most important of which are themselves transcription factors of the HES family, a group of basic helix loop helix (bHLH) genes that are orthologs of the Drosophila gene enhancer of split (4). Studies of localization of Notch genes and HES genes showed that some cells do not express HES genes but express Notch and its ligands, suggesting that other effectors might be present. This led to the identification of Hey genes that are bHLH genes closely related to the HES genes (23, 29, 35). Among the seven Hes members and three Hey members isolated in mammals, Hes1, -5, -7 and Hey1, -2, -L are potential target genes of Notch (22).
Deletion of Notch is embryonically lethal, which has limited the study of the role of these receptors in organogenesis (18). Notch 3 mutations have been implicated in the human disease CADASIL, a cerebral arteriopathy (25). However, a hypomorphic allele of Notch 2 shows a vascular anomaly in the kidney with an aneurysmal dilatation of the glomerular capillary (32). Jagged 1 mutations are associated with the Alagille syndrome, where there are a large number of skeletal and cardiovascular anomalies (37, 42).
The mammalian kidney develops in three waves, in only the last of which the metanephros continues as the adult kidney. The metanephric kidney begins when the ureteric bud (UB), an outgrowth of the Wolffian duct, invades the metanephric mesenchyme. Factors secreted by the UB induce this mesenchyme to aggregate and then convert to an epithelial structure (5, 34). The induced mesenchyme, in turn, sends signals back to the UB to cause it to divide and grow. This reciprocal induction proceeds in an ordered manner to produce several generations of branches and eventually all the nephrons of the kidney. The tip of each UB first induces the development of the renal vesicle, which is an epithelial cyst that undergoes morphogenetic transformation to eventually form the nephron from the glomerulus to the end of the distal tubule while the UB eventually forms the collecting tubule. During the morphogenesis of the S-shaped body, the proximal end becomes invaded by blood vessels and its epithelial cells differentiate into podocytes and parietal epithelial cells. Simultaneously, the middle segment of the nephron begins to express several proteins (or sugars) that are characeristic of the proximal tubule. The most distal segment of the primitive nephron becomes connected to the UB epithelium (1, 14). Similar to the UB, the primitive distal tubule also expresses E-cadherin at the lateral junctions of the epithelium. The molecular mechanism by which the S-shaped body undergoes segmentation is unknown at present. We embarked on a study that aims at examination of pathways that determine cell identity in kidneys. The Notch signaling pathway is one of these pathways, and we now describe the segment-specific expression of these genes in the hope that they will help in designing new studies that examined the mechanism of determination of cell identity during nephrogenesis.
MATERIALS AND METHODS
Animals. Embryos were obtained from timed matings of Swiss-Webster mice (Taconic). Day 0.5 of gestation (E0.5) was considered to be at noon of the day on which the vaginal plug was detected.
Hes1 and Hes5 mutant mice were a generous gift of Dr R. Kageyama (Institute for Virus Research, Kyoto University, Kyoto, Japan). Hes1 –/–, Hes5 –/–, Hes1 –/– Hes5 +/–, and Hes1 +/– Hes5 –/– embryos were acquired as previously described (10).
Culture of kidney cell line and embryonic kidneys. The UB cell line was derived from transgenic mice for SV40 T-antigen (6). UB cells were grown in minimal essential media (MEM; GIBCO) with 10% FCS (Hyclone). A stromal cell line was isolated from mice in which -galactosidase was knocked into the BF-2/Foxd1 locus (19). Stromal cell lines were grown in DMEM (low glucose) with 10% FCS.
Embryonic mice kidneys were microdissected at E11.5. Kidneys were applied to the top of polyester Transwell filters (Costa) and grown in MEM (GIBCO) supplemented with 10% FCS at 37°C in an atmosphere containing 5% CO2.
RT-PCR analysis. Total RNA was isolated from kidneys of different embryonic days by using RNeasy kit (Qiagen) and then digested with DNase I to get rid of genomic DNA. First-strand cDNA synthesis was primed by MMLV reverse transcriptase with the antisense primers. The primers used are described in Table 1. For negative controls, reverse transcriptase was omitted and PCR was performed directly from the RNA. Amplification of cDNAs was performed using 36 cycles with denaturation at 95°C for 30 s, annealing at 63°C for 30 s, and extension at 68°C for 60 s. After completion of the PCR cycles, reactions were subjected to 68°C for 10 min and the products were subjected to electrophoresis and visualized with ethidium bromide.
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Probe synthesis. Probes were synthesized with the appropriate RNA polymerase in the presence of digoxigenin-UTP (Boehringer-Mannheim Biochemicals). The probes were precipitated and resuspended in the hybridization buffer. We used full-length cDNA of Hey1 and Hey2, partial coding sequence of Dll1 (466 bp), Jagged1 (494 bp), and Notch2 (986 bp) as templates for the generation of digoxygenin-labeled antisense and sense riboprobes with either T7 or SP6 polymerase (Promega). Both of the above probes were synthesized from PGEMT-Easy vector (Promega). The Notch1 probe was produced by linearization of plasmid (a gift from Dr. G. Weinmaster) by EcoRI and priming with T3. Hes1 and Hes5 plasmids were made by linearization of plasmids (generous gifts from Dr. F. Guillemot) either by EcoRI and then priming with T7 or by HindIII and then priming with T3. The Hes7 probe was produced by linearization of plasmid (a kind gift from Dr. R. Kageyama) by SpeI and transcribed using T7. The HeyL probe (a kind gift from Dr. D. Srivastava) was produced by transcribing EcoRV-linearized CDNA with SP6.
In situ hybridization and whole mount in situ hybridization. Embryos were fixed in 4% paraformaldehyde (PFA), infiltrated with 20% sucrose, embedded in OCT, and 10-μm sections were cut, postfixed for 10 min, treated with proteinase K (1 μg/ml), and acetylated at room temperature. Prehybridization was carried out for 2 h at room temperature. Hybridization was performed overnight at 68°C. The prehybridization and hybridization solution was 50% formamide, 5x SSC, 5x Denhard’s, 250 μg/ml yeast tRNA (Sigma), and 500 μg/ml salmon sperm DNA (Sigma). Posthybridization washes were done at 68°C in 0.2x SSC for 1 h. Sections were then blocked with 10% sheep serum and incubated overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) at a 1:5,000 dilution. Alkaline phosphatase activity was detected by developing slides in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT; Roche). Control staining was performed with the sense riboprobes.
Whole mount in situ hybridization was performed as described before (33). Briefly, kidneys were fixed in 4% PFA in 0.1 M phosphate buffer and treated with proteinsase K (1 μg/ml). Prehybridization was carried out for 2 h at room temperature. Hybridization and posthybridization were performed at 68°C in the water bath. Kidneys were then incubated overnight at 4°C with alkaline phosphatase-conjugated anti-digoxigenin antibody and developed in BCIP and NBT (Roche). Control staining was performed with the sense riboprobes.
Photographs were taken under a Spot imaging camera (RT slider) using a Nikon Microphot-SA microscope (Olympus Optical, Tokyo, Japan).
Immunofluorescence. The mouse monoclonal E-cadherin antibody and Pax-2 antibody were purchased from Zymed. Mice cryostat sections were fixed in 4% PFA, rinsed in PBS, and pretreated with 10% donkey normal serum. Primary antibodies were diluted 1:200 in PBS and incubated overnight at 4°C. Sections were then incubated with FITC-conjugated secondary antibodies at 1:200 dilution, rinsed in PBS, and mounted with Vectashield mounting medium. The fluorescent images were electronically captured with a Spot imaging camera (RT slider) using a Nikon Microphot-SA microscope (Olympus Optical).
Combined lectin staining with in situ hybridization. Dolichos biflorus agglutinin (DBA) and LTA lectin conjugated to FITC were used to detect collecting ducts and proximal tubules. Peanut lectin (PNA) conjugated to rhodamine was used to detect glomerular podocytes. To perform lectin staining, we performed in situ hybridization on sections as described above. Slides were then washed three times with PBS, treated with gelatin buffer (0.075% saponin detergent, 0.2% gelatin, 1 mmol/l MgCl2, and 0.1 mM CaCl2 in PBS) for 30 min. Slides were washed twice in neuraminidase buffer (50 mmol/l Na acetate, 150 mmol/l NaCl, 9 mmol/l CaCl2, pH 5.5) and incubated for 4 h at 37°C with 1 IU/ml of neuraminidase (Sigma). After being rinsed with gelatin buffer, sections were labeled for 1 h at 37°C with rhodamine-coupled PNA diluted in gelatin buffer (1:100). After several washes, slides were incubated with FITC-conjucated DBA (dilution 1:50) or lotus lectin (Sigma, 1:50) for 1 h at 37°C. Slides were mounted in Vectashield mounting medium and viewed under a Nikon Microphot-SA microscope (Olympus Optical).
Hematoxylin and periodic acid-Schiff staining. Kidney sections were fixed, incubated with 0.5% periodic acid for 5 min, and treated with Schiff’s reagent (PAS). Slides were then stained with hematoxylin for 2 min and blued in tap water. Slides were viewed under a Nikon Microphot-SA microscope (Olympus Optical). These studies were approved by the institutional animal care committee, AC-IACUC protocol AAAA2035.
RESULTS
Expression of Hairy and Notch-related genes using RT-PCR. To characterize the expression of the Hairy family of genes, we initially performed a screening assay using RT-PCR in embryonic kidneys at different stages of development and in two cell lines derived from mouse kidneys. We found that the Hairy-related genes Hes1, Hes5, Hes7, Hey1, Hey2, and HeyL were all expressed. However, Hes7 was only expressed at a very low level and then transiently, and no signal could be detected by in situ hybridization (data not shown). Because Hairy-related genes are a target of Notch signaling, we also studied the expression of Notch and its ligands by RT-PCR. These results indicated that mRNAs for Dll1, Jagged1, and all Notch receptors were present in different stage of metanephric development. Jagged2 and Dlk were only expressed at a low level and then only transiently. Dll3 was not expressed in metanephroi, although it was expressed in the brain of the same mice. Identity of these products was confirmed by sequencing of the PCR products. No RT-PCR products were detected in the H2O or non-RT controls of any sample. The results are summarized in Table 2.
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To test whether cultured embryonic kidney cells maintain similar expression as in vivo kidneys, we examined Notch and Hairy-related gene expression in UB cell and stromal cell lines by RT-PCR. As shown in Table 3, Hes1, Hes7, Hey1, and Hey2 transcripts were detected in the renal stromal cell lines, but only Hes1 transcripts were observed in the UB cell lines. The stromal cell line also expressed Dll4, Dlk, Jagged1, Notch1, Notch2, and Notch3 mRNA. Jagged1, Notch1, and Notch2 were expressed in UB cells. However, Jagged1 was not detected in the ureteric branches by in situ hybridization (data not shown).
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Hes1 is expressed in both mesenchymal and epithelial structure. In E13.5 kidneys, Hes1 transcripts were present in most cells during the early stages of kidney development. The signal was prominent in both the condensed mesenchyme, UB tips, ureteric branches, primary vesicles, comma-shaped bodies, and S-shaped bodies. A weak signal was also observed in the capsule and subcapsular interstitium (Fig. 1A).
To identify segment-specific expression in developing nephrons, we stained E15.5 kidneys with lectins known to bind to specific segments. By colabeling ureteric branches and tips with DBA (in green), a marker of the UB (27), we confirmed that while high levels of expression were present in UB tips in E15.5 and the nephrogenic zone of newborn kidneys (data not shown), only faint expression was detected in more developing collecting ducts (Fig. 1B). In the nephrogenic zone, all regions of the S-shaped bodies expressed Hes1 mRNA, but in the more developing segments, the pattern changed such that the expression disappeared from the developing proximal tubules (LTA-positive tubules in green) but remained in the other segments of the developing tubules (Fig. 1, C-E). Expression of Hes1 mRNA also appeared to decrease with glomerular maturation. A strong signal was detected throughout the early capillary loop stage of glomeruli including podocyte progenitors (stained with PNA in red), mesangial cell progenitors, and vascular wall (Fig. 1, F-H), while expression was hardly detectable in more mature capillary loops (Fig. 1, I-K, arrows). No signal was detected in the corresponding section hybridized with sense cRNA probe. We also analyzed by RT-PCR an embryonic kidney stromal cell line and found that Hes1 was also present, suggesting that it might be expressed also in developing renal stromal cells. (Table 2).
Hes5. Hes5 was observed in comma- and S-shaped bodies. To precisely identify the boundary of the different segments, in situ hybridization sections of E13.5 kidneys were triple labeled with the podocyte marker PNA and the proximal tubule marker LTA (27). As can be seen in Fig. 2, A-C, there was no colocalization of the in situ signal with these tubular markers. At this stage of development, that leaves the loop of Henle and the distal tubule. We performed serial sections and stained adjacent ones by in situ hybridization or E-cadherin, a marker for distal and collecting tubules. We found that Hes5 was only expressed in the most proximal end of these E-cadherin-expressing segments (Fig. 2, D and E). Because the proximal end of E-cadherin staining was colocalized with anti-uromucoid antibody, a marker for the ascending limb of the loop of Henle (data not shown), we think Hes5 was expressed in the prospective loop of Henle segment not the proximal tubules as stated by others (30, 38). This is best seen in Fig. 2F where the in situ signal did not colocalize with either the distal tubular elements (long arrow) or the proximal tubular elements (short arrow). The strongest staining was thus seen in the segment that will develop into the loop of Henle. There was also some faint staining in an adjacent region of the prospective proximal tubules, but whether this was simply background staining or low level of expression is difficult to tell (Fig. 2, A-C).
As defined by Neiss (36), three distinct stages of developing loop of Henle were present in the developing nephron. Hes5 mRNA was only detected in the first stage (the anlage) of the developing loop in the nephrogenic zone and absent in primitive loop and immature loops (Fig. 2, G-J).
Hey1. Whole mount in situ hybridization analysis of cultured embryonic kidneys demonstrated that Hey1 first appeared at E11.5. Initially, Hey1 was expressed in regions surrounding the UB tips and in pretubular aggregates. The strongest signals were often located below the UB tips, an area that will eventually become the renal vesicle. Early nephron development progresses in an ordered manner from loose mesenchyme, to condensed mesenchyme, and finally to renal vesicle, a pattern that recurs after each generation of branches (Fig. 3). It is likely that after each branch of the UB the mesenchyme next to each tip has reached a similar level of induction. With that in mind, we found a surprising result that the expression of Hey1 in each mesenchyme surrounding the UB often was not identical (Fig. 3, top, compare 1 and 3 and 2 and 4). The signal could be categorized into four different patterns (1–4 in the figure): 1, no signal in mesenchyme; 2, signal in the mesenchyme surrounding the tip; 3, transcripts in the mesemchyme below the UB and partially surrounding the tip; and 4, signal only in the mesenchyme below the UB. This suggested that the Hey1 expression pattern might oscillate in mesenchyme. Similar oscillations in presomitic mesoderm during somite segmentation have attracted much attention. However, due to a large variation between left and right kidneys when cultured in vitro, we were unable to determine the precise timing of each cycle using the similar method as in the presomitic mesoderm. However, this unique expression pattern only occurred in the very early stage. After 2 days of in vitro culture, no signal was detected in condensed mesenchyme and pretubular aggregates (Fig. 3).
In situ hybridization showed that Hey1 mRNA was localized in renal vesicles, comma-shaped bodies, and S-shaped bodies. In the S-shaped bodies, it localized into the more proximal regions of the developing nephron, as shown by the lack of colocalization with E-cadherin in adjacent sections (Fig. 4, A and B). In early S-shaped bodies, the expression colocalized with the podocyte marker PNA (Fig. 4, C-E, arrows). As the nephrons matured and acquired a strong lectin-staining pattern, the expression of Hey1 was downregulated (Fig. 4, F-H). Similar to Hes5, Hey1 was only observed in the nephrogenic region (Fig. 4, F-H). Hey1 was also detected in vascular structures (not shown).
HeyL. HeyL mRNA was present in the developing glomerular cleft but not in the PNA-positive podocytes. Cells that expressed HeyL, presumably the vascular elements of primitive glomeruli, were located within the glomerulus (Fig. 5, A-C, long arrows), but we occasionally found cells that surrounded the pododcytes (Fig. 5, A-C, short arrows). It was only expressed in early capillary loop stage of glomeruli including both endothelial cells and mesangial cell progenitors but not in the maturing glomeruli (Fig. 5D, short arrows). HeyL mRNA was also present in primary vesicles (Fig. 5, A-C), comma-shaped body, and S-shaped body. Figure 5E indicated the structure of an S-shaped body located below the right side of F in E. It clearly showed that only the segment of the developing proximal tubule in the S-shaped body had a signal. However, its expression in proximal tubules was transient, disappearing in more mature stages (Fig. 5, H-J).
There was no HeyL expression in collecting ducts (cd) or distal tubules (db) as determined by the lack of colocalization with E-cadherin (performed in adjacent sections; Fig. 5, E and G). This is different from the findings of the Gessler group (30), who stated that they found HeyL expressed in the collecting duct; however, they did not use colocalization with markers of the collecting duct. We also found a strong signal in various vascular elements (Fig. 5, A and C, ). No specific signal was found for the sense probes of HeyL.
Hey2 and Hes7. In situ hybridization studies showed that Hey2 was localized in large blood vessels, probably smooth muscle cells. Hey2 expression initially appeared at late E12.0 kidneys (not shown). In vitro culture of E11 kidneys for 48 h showed no staining of Hey2 (not shown). No specific signal was found for the sense probe of Hey2. No Hes7 signal was detected by in situ hybridization (not shown).
Comparison of Notch receptor expression pattern. The renal expression pattern for Notch was examined in several recently published reports; unfortunately, they came to differing conclusions regarding the site of localization (30, 38). We reexamined the expression pattern with other markers including E-cadherin, PNA, or lotus lectin to clarify the precise segment of these transcripts.
We found that Notch1 was expressed inside PNA-positive regions, suggesting that it is expressed in the prospective mesangium but not in the podocytes (Fig. 6, A-C). There was good colocalization with E-cadherin in adjacent sections, suggesting that it was expressed by the prospective distal tubule and collecting ducts (Fig. 6, D and E, arrows). However, prospective proximal tubules had no expression (Fig. 6F).
Notch2 was present in the primitive proximal tubule (Fig. 6, G-L, arrows) with very faint colocalization with podocyte progenitors. It was expressed in the early capillary loop stage of glomerular podocytes with faint staining in the mesangial region probably in both endothelial and mesangial cells (Fig. 6J, arrow). More mature podocytes had no expression of Notch2 (Fig. 6J, star). There was good colocalization with E-cadherin as judged by E-cadherin staining in adjacent sections (Fig. 6, K and L, arrows).
Notch3 was expressed inside PNA-positive regions (Fig. 6, M and N, long arrows), suggesting that it is expressed in the capillary loop vascular wall including the prospective mesangium and endothelial precursors. Notch3 was also detected in UB tips and large blood vessels (Fig. 6, N and O). However, the prospective podocytes did not express Notch3.
Expression of the Notch ligands Delta and Jagged. Both Dll1 and Hes5 first appeared at E12.75 kidneys. As shown in Fig. 7, A-C, the tissue distribution patterns were quite similar for Hes5 and Dll1 transcripts, with expression in the prospective loop of Henle and proximal tubules in the nephrogenic zone. In these adjacent sections, Hes5 was expressed at a very faint level in proximal tubules, only appearing after prolonged color development, raising the question of whether it reflected background staining rather than actual mRNA expression. In contrast, a Dll1 signal was strong in both the prospective proximal tubules and loop of Henle. We also noted that the Hes5 signal was ubiquitously distributed in the prospective loop of Henle, whereas a Dll1 signal seems not.
Jagged1 was expressed at a faint level in primary vesicles, comma-shaped bodies, and S-shaped bodies. In more advanced nephrons, it was observed in developing distal tubules and prospective loop of Henle but absent in collecting ducts (Fig. 7, G and H). Its signal was also detected in proximal tubules; however, no expression was observed in podocytes (Fig. 7, H and I). Jagged1 signals were located in the apical region of the tubular cells.
Hes1 and Hes5 null mice have grossly normal developing kidney. We examined embryonic mouse kidneys using hematoxylin/PAS staining and found that both Hes1 and Hes5 knockout mice have normal development of nephrogenesis (Fig. 8). We also stained sections with LTA for the development of proximal tubules and with PNA staining for podocytes formation in both Hes1 and Hes5 mutant mice. We did not detect any significant difference between wild-type and Hes1 or Hes5 knockout mice (data not shown). To test whether Hes5 mutants had defects in the loop of Henle, we stained Hes5 knockout and wild-type mouse kidneys with anti-Tamm-Horsfall protein antibody to examine them for a differentiated marker of the loop of Henle. Positive staining was first detected in wild-type E16.5 kidneys. However, no difference was detected in both embryonic kidneys (E15.5, E16.5, and E17.5) and postnatal kidneys (newborn, 2 wk, and 1 mo). Hes1 mutant and wild-type kidneys were also stained with E-cadherin for distal tubules and collecting ducts and with Pax-2 for early nephron formation. Again, no difference was found (data not shown).
We then crossbred Hes1 and Hes5 mice. We were unable to get Hes1 –/– Hes5 –/– mice because the double mutants die around E11.5 before nephron formation. We examined Hes1 +/– Hes5 –/– and Hes1 –/– Hes5 +/– and wild-type mice using HE or hematoxylin/PAS sections. No obvious defect was detected in these compound mutant embryonic kidneys (Fig. 8). However, we noticed that some of the mutant kidneys (Hes5 –/–, Hes5 +/– Hes1 +/–, Hes1 +/– Hes5 –/–) were smaller.
DISCUSSION
The Notch signaling pathway controls cell identity in a variety of systems. Initially described in the fruit fly, mutations in Notch resulted in an excess of neuroblasts at the expense of epidermal cells (20). Since then, Notch signaling in vertebrates has been implicated in the control of cell identity in a variety of organs derived from all three germ layers (15). Mutations in Notch pathway genes lead to anomalies in blood vessel development, hematopoiesis, thymus, central nervous system, heart, somites, ribs, craniofacial development, and the kidney (7, 10–12, 21, 24, 32, 42, 43). Because the Notch pathway is involved in specifying cell identity in different organs, it was possible that it might also mediate such an effect in the kidney.
Detailed functional and morphological analysis shows that the kidney and the epithelium of the nephron in particular are composed of a variety (as many as 15) specific cell types (40). The determination of cell identity during this process is unknown. It is likely that these cell types are specified early in development (2). Examination of the morphology of the developing nephron shows that after conversion of the mesenchyme to an epithelium, all the cells of the S-shaped body resemble each other. However, lectin staining and immunocytochemistry showed that at that stage the primitive nephron had begun to differentiate as LTA lectins bound to the future proximal tubule (27), PNA lectins bound to the future podocyte (27), and E-cadherin began to be expressed in the future distal tubule. Hence if a cell identity gene were going to be expressed, it would have to appear at that stage or earlier in the specific segment.
It is well known that Hairy-related genes are Notch downstream effectors. Previous studies examined their renal localization (30–32, 38). However, in both studies (32, 38), the segmental boundaries for those genes were not definitively identified. To more precisely define the segmental expression of these genes, we performed a survey of the expression pattern of members of the Hairy-related genes and used triple staining or serial sections with segment-specific markers. Dynamic and segmental expression patterns were observed at critical stages of nephron development. Figure 9 provides a summary cartoon of the expression of Notch effectors HES and HEY genes, whereas Fig. 10 shows the same for Notch receptors and ligands expressed in the kidney. Our results also show that HeyL was not expressed in collecting ducts as reported (30) and Hes5 was expressed in the prospective loop of Henle instead of proximal tubules as reported before (38) by staining serial sections with E-cadherin, a distal tubule and collecting duct marker.
As one can see, Hairy-related genes show segment-specific expression in nephrogenesis. Hes5 is only expressed in the region of the S-shaped body destined to become the loop of Henle with very low expression in the prospective proximal tubules. Hence, we suggest that Hes5 might contribute to the identity of the loop of Henle differentiation. Hes1 was present throughout the metanephros, including the ureteric epithelium and S-shaped bodies and the developing podocytes, indicating that it might have a broader role in kidney development.
HeyL was expressed in the glomerular cleft, renal vesicle, and endothelial cells surrounding the early developing glomerulus and also in the segment of prospective proximal tubules, suggesting that HeyL might function in regulating glomerular vascular wall formation. Hey1 was expressed in pretubular aggregates and renal vesicles, suggesting the possible role of Hey1 in early nephron formation. The repeated Hey1 expression pattern in each cycle of nephron formation in the cultured kidneys resembled its oscillating expression pattern found during somite formation. However, we were unable to determine the timing of one cycle due to the large variation between left and right kidneys in the culture system. The mechanism and role of this specific expression pattern in early nephron segmentation are unknown. Considering that each cycle of gene expression in the presomitic mesoderm is associated with an intrinsic segmentation clock that coordinates somite segmentation, Hey1 might play a similar role in determining the precise timing of the transformation of the renal vesicle to the comma-shaped bodies. Hey1 was also abundantly expressed in the S-shaped body from the region that will form the loop of Henle to the developing podocytes, suggesting it might also contribute to glomerular formation.
Recent studies showed that presenilins and Notch1 were required for the progression from renal vesicles to comma- and S-shaped bodies (9, 41). A hypomorphic allele of Notch2 was found to express defects in vascular development of the glomerulus (32). These data suggest that Notch pathway genes likely have a critical role in various stages of nephron development. The study of deletion mutants in the hairy-related genes suffers from the fact that many such mutations are embryonic lethal. We examined Hes5 knockout mice and found that they survived through nephrogenesis but we were unable to detect any defects in the loop of Henle or in other segments. One expectation was that, similar to studies in neurogenesis, the expression of differentiation markers of the loop of Henle would be accelerated in these mutant mice. However, using expression of Tamm-Horsfall protein, we did not find an earlier expression in mutant mice. Similarly, we did not find any gross anomalies in the Hes1 mutant mice and Hey1 mice had already been reported to have no nephron defect (16).
Hairy-related genes function by forming homo- or heteromultimers with other bHLH transcription factors (23, 28). Notch signaling thus may rely on both homodimer and heterodimer formation between different hairy genes to suppress the cell identity genes. Because the compound homozygotes for both Hes1and Hes5 died before kidney development, we are unable to study the double knockout mice to test this hypothesis. Furthermore, Hey1 was also detected in the prospective loop of Henle; hence, we could not exclude the possibility that Hey1 might compensate for Hes5 function in the Hes5 null kidneys. Hence, the lack of defect in early nephrogenesis is most likely due to overlapping expression of these genes.
In summary, our survey provides a comprehensive expression map of Notch signaling molecules during early nephron development. Future evaluation of targeted disruption of these genes using conditional knockout mice and misexpression of various components of Hairy signaling will help determine the functional role of these genes in renal segmentation.
GRANTS
This work was supported by National Institutes of Health Grant DK-55388.
ACKNOWLEDGMENTS
We are grateful to Dr. R. Kageyama from the Institute for Virus Research, Kyoto University, Japan, for a generous gift of Hes5 and Hes1 mutant mice.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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