Background

Among mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) promotes proliferation or differentiation, whereas c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) are thought to inhibit cell growth and induce apoptosis. MAPK phosphatase-1 (MKP-1) inactivates and modulates MAPKs. During renal development, large scale proliferation and apoptosis occur. We investigated the temporal and spatial expression patterns of MAPKs and MKP-1 in rat kidney during development.

Methods

Western blot analysis and immunohistochemistry were performed in the developing and mature kidney of the rat.

Results

The expression of ERK, p38, and MKP-1 were high in developing kidney. On the other hand, JNK was abundantly expressed in adult kidney. Active forms of ERK, p38, and JNK correlated with the protein expression levels. Immunohistochemical studies revealed that ERK was strongly expressed by blastema cells, mesenchymal cells, and ureteric bud tips in nephrogenic zone of embryonic kidney. In neonatal kidney, ERK was more abundant in the deep cortex and the medulla corresponding to tubule maturation. p38 and MKP-1 were detected uniformly in mesenchymal cells, mesangial cells, and ureteric bud epithelia of fetal kidney without an obvious correlation with the occurrence of apoptosis. JNK was expressed by tubular cells and podocytes of adult kidney.

Conclusions

ERK, p38, and MKP-1 are strongly expressed in developing kidney, and JNK is detected predominantly in adult kidney. Both the temporal and spatial expression of ERK coincides with the maturation of the kidney.

METHODS

Materials

Nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, N,N-dimethylformamide, and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO, USA). Anti-ERK (erk1/2-CT, rabbit polyclonal IgG) was from Upstate Biotechnology (Lake Placid, NY, USA). Anti-p38 (C-20), anti-JNK (FL), anti-MKP-1 (V-15), and MAP kinase p42 (FL) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-ERK (P-ERK), anti-phospho-p38 (P-p38), anti-phospho-JNK (P-JNK) were from New England Biolabs (Beverly, MA, USA). A monoclonal antibody specific for proliferating cell nuclear antigen (PCNA)/cyclin, peroxide-conjugated rabbit antimouse and swine antirabbit immunoglobulins, and DAKO^R Protein K Enzyme Digestion were from DAKO A/S (Glostrup Denmark). ApopTag^R Peroxides In Situ Apoptosis Detection Kit was from Intergen Company (Purchase, NY, USA).

Experimental animals and sampling

Sprague-Dawley rats were obtained from Saitama Experimental Animals Supply (Saitama, Japan). Pregnancy was determined by the detection of a vaginal plug. Before removal of the embryos, pregnant rats were sedated with an intraperitoneal injection of sodium pentobarbital. Embryos were removed and decapitated on day 14 and 18 of gestation (E14 and E18). E14 embryos were fixed with neutral buffered formalin. Kidneys from E18 were harvested and either fixed with neutral buffered formalin or homogenized in lysis buffer containing 20 mmol/L Hepes, pH 7.2, 1% Triton-100, 10% glycerol, 20 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 10 g/mL aprotinin, and 10 g/mL leupeptin. Kidneys from rats 1 (N1), 7 (N7), and 40 (A) days after birth were treated in the same manner.

Immunoblot analysis

Kidneys were homogenized, and insoluble material was removed by centrifugation (10,500 g, 10 min). The protein content in kidney lysates was measured using a DC protein assay (Bio-Rad Laboratories, Tokyo, Japan). Lysates (30 to 60 g) were resolved by SDS-PAGE, and transferred to PVDF membranes (Immobilon, Millipore Corp, Bedford, MA, USA). Nonspecific binding sites were blocked in TBS buffer (10 mmol/L Tris-Cl, pH 7.4, 0.15 mol/L NaCl) containing 5% BSA overnight at 4°C or for one hour at 25°C. Antibodies were added to TBS with 5% BSA in saturating titers and incubated with mixing for two hours at 25°C. Blots were washed two times, then incubated with goat anti-rabbit IgG/alkaline phosphatase conjugate for one hour with mixing and washed. Membranes were washed once in substrate buffer (0.1 mol/L Tris-Cl, pH 9.5, 0.1 mol/L NaCl, and 5 mmol/L MgCl₂), and then developed by the addition of fresh substrate (20 mg of nitroblue tetrazolium in 60 mL of substrate buffer, mixed immediately before blot exposure with 10 mg of 5-bromo-4-chloro-3-indolyl phosphate in 200 L of N,N dimethylformamide). Alternatively, bound antibodies were detected using the ECL Western blotting system (Amersham, Buckinghamshire, UK). At least three independent experiments were performed with similar results. Blots were scanned and quantitatively analyzed by NIH Image.

Immunohistochemistry

After fixation, embryos or kidneys were embedded in paraffin. Immunohistochemical staining was performed on serial sections 3 m thick, using the enzyme-labeled antibody method. Paraffin sections were deparaffinized and rehydrated. Endogenous peroxide activity was quenched by incubating sections in 0.3% H₂O₂/methanol for 15 minutes. To unmask antigens, slides were boiled 100°C for 10 minutes in 10% citrate buffer (pH 6.0)/methanol. Section were incubated with antibodies against ERK (dilution 1:20), p38 (1:100), JNK (1:20), MKP-1 (1:20), P-ERK (1:200), P-p38 (1:20), or P-JNK (1:200). The incubation time was 60 minutes at room temperature or overnight at 4°C. After incubating with secondary antibody at a concentration of 1:100, immunoreaction products were developed using 3,3'-diaminobenzidine (DAB) as the chromogen, with standardized development times. Sections were then counterstained with methyl green. Positive controls (brain for ERK, JNK, and MKP-1, and bone marrow for p38) were run simultaneously. Negative controls included adding saturating titers of antigen (for ERK), omitting the primary antibody or substitution of the primary antibody with rabbit serum.

Detection of apoptosis

Cells undergoing apoptosis were identified using an in situ DNA labeling method. Paraffin embedded sections were deparaffinized, and terminal deoxynucleotide transferase-mediated nick-end labeling (TUNEL) staining was performed using the Apoptag kit. Sections were counterstained with methyl green. TdT was omitted from the staining procedure in negative controls.

RESULTS

Immunoblot analysis

Immunoblot analysis was performed to examine protein levels of MAPKs and MKP-1 in rat kidneys at different stages of development. ERK was present in the kidney throughout the stages Figure 1a. The abundance of both p44 and p42 isoforms were highest at E18, and decreased gradually after birth. These bands were not detectable when immunoblotting was performed in the presence of the synthetic p42 ERK (not shown). The different molecular weight forms of p44 ERK were thought to reflect the different phosphorylation levels, with more phosphorylated forms in the fetal kidney. In support of this notion, p44 P-ERK was similar in molecular weight during development. p38 and MKP-1 were present in the embryonic kidney, with greatly diminished levels in N1 kidneys (Figure 1 b, d). They were undetectable in N7 and the adult kidney. The opposite pattern of expression was seen for JNK, which was strongly expressed in the adult kidney, was present with lower levels in neonatal kidneys, and was barely detectable in embryos Figure 1c. Phosphorylated and activated form of MAPKs correlated with the protein levels of MAPKs. Thus, P-ERK was highest in the fetal kidney with diminishing levels thereafter Figure 1a. P-p38 was detectable at E18 and with lower levels at N1 Figure 1b. P-JNK was abundant in the adult kidney and slightly detectable in N7 kidneys Figure 1c.

Figure 1.

Expression of mitogen-activated protein kinases (MAPKs) and MAPK phosphatase-1 (MKP-1) protein during kidney development. Lysates from fetal (E18), neonatal (N1, N7), and adult (A) rat kidney were examined by immunoblot analysis for ERK and P-ERK (A), p38 and P-p38 (B), JNK and P-JNK (C), and MKP-1 (D). Kidneys were treated as described in the Methods section. Equal amounts of lysates (30 to 60 g) were loaded and immunoblotted with each antibody. Quantitative analysis is shown below. Symbols in A are: (top) () p42; () p44; (bottom) () P-p42; () P-p44. Symbols in B are: () p38; () P-p38. Symbols in C are: () JNK; () P-JNK.

Full figure and legend (20K)

Immunohistochemistry

The localization of MAPKs and MKP-1 protein was assessed by immunohistochemistry. To examine the correlation between expression of these proteins and the occurrence of proliferation and apoptosis during kidney development, PCNA staining and TUNEL assay were performed.

PCNA

PCNA expression correlates with the S-phase of the cell cycle, and thus is used as a marker for proliferating cells. At E14, condensing mesenchyme surrounding the ureteric bud (blastema), and ureteric bud epithelial cells showed strong PCNA staining Figure 2a. In the E18 kidney, PCNA positive cells were most abundant in a zone of nephrogenesis immediately under the renal capsule, which includes undifferentiated mesenchymal cells, blastema cells, and vesicles Figure 2b. Ureteric bud branches and folding epithelial structures corresponding to the earliest stages of glomerulogenesis were variably stained. In the N1 kidney, PCNA-positive cells were also observed in the more differentiated middle cortical layer Figure 2c. Vesicles, S-shaped bodies, and podocytes in maturing glomeruli were commonly labeled Figure 2c. At N7, the PCNA-positive cells shift from subcapsular cortical layer to deep cortical layer and medullary region Figure 2d. Tubular epithelial cells were stained more intensely than glomeruli. In the adult kidney, PCNA staining was absent (not shown).

Figure 2.

Distribution of proliferating cells and apoptosis during kidney development. (A–D) Immunohistochemical localization of PCNA staining (200). At E14 (A), blastema cells (bl) and ureteric bud epithelial cells (ub) are intensely stained. At E18 (B) PCNA staining is most intense in mesenchymal cells and vesicles in nephrogenic zone (arrow). At N1 (C), proliferating cells shift from nephrogenic zone to more differentiated central region where S-shaped bodies (s), and maturing glomeruli (g) are observed. At N7 (D), medullary region is most intensely stained.

(E–G) Labeling of apoptotic cells using TUNEL assay. In the E14 kidney, uninduced mesenchymal cells surrounding blastema (bl) and ureteric bud (ub) are stained (arrow). Arrowheads are artifacts (E, 200). At E18 and N1 (F, 200, at top of p. 33), apoptotic cells are abundant in comma- and S-shaped bodies, and mesenchymal cells within nephrogenic zone. Occasional apoptotic epithelial cells within glomeruli and tubules are also observed (arrow) (G, 1000, at top of p. 33).

Full figure and legend (159K)

TUNEL

In the E14 kidney, TUNEL staining was found in uninduced cells surrounding condensations of metanephrogenic mesenchyme Figure 2e. In the E18 and N1 kidney, nick-end-labeling was most intense in comma- and S-shaped bodies, and mesenchymal cells surrounding them within the nephrogenic zone subjacent to the uninduced mesenchyme Figure 2f. TUNEL positive nuclei were also found less frequently in epithelial cells within maturing glomeruli and tubular epithelial structures Figure 2g. No apoptosis was evident in the N7 or adult kidney (not shown).

ERK

Location of ERK changed markedly during the kidney development. At E14, ERK was expressed in blastema cells, and ureteric bud epithelial cells Figure 3a. At higher magnification, the ERK staining was exclusively cytoplasmic in ureteric bud cells, and nuclear and cytoplasmic in mesenchymal cell Figure 3b. In the E18 kidney, ERK was abundantly expressed in nephrogenic zone with decreasing levels towards the maturing center Figure 3c. An intense signal was detected in mesenchymal cells, blastema, vesicles, and tip of ureteric buds Figure 3c, d. Comma- and S-shaped bodies in subnephrogenic zone showed faint staining. There was ERK expression in the vascular cleft of S-shaped bodies where endothelial precursors reside. While the tip of ureteric buds showed intense cytoplasmic staining, the majority of ureteric bud cells in nephrogenic zone were unstained. However, the staining that was present was entirely nuclear. In contrast, ureteric bud cells in stroma showed diffuse and strong staining of mostly cytoplasmic ERK protein Figure 3c. In maturing glomeruli, positive immunostaining was found in mesangial cells, endothelial cells, and scatteredly in parietal and visceral epithelial cells Figure 3e. Immunoreactivity was intense in nuclei in the glomerular epithelial cells and mesenchymal cells, although some cytoplasmic staining was also noted. In contrast to the embryonic kidney, ERK was more abundant in subcortical region and the medulla than the outer nephrogenic layer in neonatal kidneys Figure 3f. ERK localized to maturing glomeruli as well as the epithelial cells of tubules, collecting duct cells, and interstitium. Thus, the localization of ERK, in general, correlated with the distribution of proliferating cells during kidney development. In the adult kidney, ERK was expressed only by glomerular visceral epithelial cells, distal tubular cells, and collecting duct cells Figure 3h. The distribution of P-ERK was the same as that of ERK, and detected in nuclei as well as cytoplasm in fetal and neonatal kidneys (not shown). In the adult kidney, however, P-ERK was present only in distal tubules Figure 3i.

Figure 3.

Immunohistochemical localization of MAPKs and MKP-1 during kidney development. (A–F) ERK. In the rat kidney on day E14, ERK is strongly expressed in blastema cells (bl), and ureteric bud epithelial cells (ub) (A, 200). ERK staining is entirely cytoplasmic in ureteric bud cells and both nuclear and cytoplasmic in mesenchymal cells (B, 400). At E18, ERK is expressed by undifferentiated mesenchyme, blastema, and vesicles in nephrogenic zone (arrow), and ureteric bud branches (ub) (C, 100). Comma- (c) and S-shaped (s) bodies are faintly stained, whereas vascular clefts stained positive (empty arrow). Note that ERK expressed in ureteric bud cells in nephrogenic zone in nuclear (arrow), while that in the stroma is entirely cytoplasmic (arrowhead) (D, 400). (E) Higher power view of maturing glomeruli at E18 showing ERK expression in glomerular epithelial cells (arrowhead), mesangial cells (thick arrow), endothelial cells (thin arrow), and mesenchymal cells (1000). (F) In contrast to E18 kidney, ERK is expressed at higher levels in the sub-nephrogenic zone of N1 kidney (100). (Figure 3 continues on pp. 34–35.)

(G) Negative control of N1 kidney (100). ERK immunohistochemistry was performed in the presence of saturating titer of synthetic ERK. (H) In the adult kidney, ERK localized to glomerular visceral epithelial cells (arrowhead), parietal epithelial cells, distal tubules (dt), and collecting ducts (not shown) (200). (I) P-ERK is detected in distal tubules in the adult kidney. (J–L) p38. At E14, p38 was expressed in blastema (bl), and ureteric bud cells (ub) similarly to ERK (J, 200). In contrast to ERK, p38 does not show cortico-medullary gradient of expression in the E18 kidney (K, 200). Mesenchymal cells, maturing glomeruli, and ureteric bud cells were stained. Similarly to ERK, p38 in ureteric bud cells in nephrogenic zone is nuclear, while those in stroma is cytoplasmic. In glomeruli, mesangial cells (arrow), endothelial cells, and parietal and visceral epithelial cells exhibited varying expression (L, 1000). (M) JNK localizes to all segments of the tubules, and glomerular visceral epithelial cells (arrowhead) in the adult kidney (400). (N) P-JNK is detected only in distal tubular cells and podocytes (100).

(O, P) In the E14 kidney, MKP-1 is expressed in blastema (bl) and ureteric bud cells (ub) (0, 200). At E18, MKP-1 is expressed in mesenchymal cells, ureteric buds, and mesangial cells without a cortico-medullary gradient (P, 400).

(Q–S) Negative controls using normal rabbit serum. The E14 (Q), E18 (R), and adult (S) kidney.

Full figure and legend (352K)

P38

Immunohistochemically, p38 was abundant in E14, and was detected in E18 and N1 kidneys with decreasing expression. Similarly to ERK, p38 localized to blastema cells and ureteric bud cells at E14 Figure 3j. In the E18 kidney, however, there was no cortico-medullary gradient of expression observed Figure 3k. Immature glomeruli showed faint p38 staining. Ureteric bud branches in nephrogenic zone showed scattered nuclear staining, while those in stroma had a strong cytoplasmic immunoreactivity. Mesangial cells were clearly stained, and glomerular endothelial cells, epithelial cells, and mesenchymal cells exhibited varying expression Figure 3l. Tubules did not express p38 at E18. In the N1 kidney, the expression pattern was similar to that seen at E18 except that no mesangial staining was detected and that tubular cells showed weak staining (not shown). The distribution of P-p38 was similar to that of p38 (not shown).

JNK

Contrary to ERK and p38, JNK was detectable in the adult kidney Figure 3m, and with diminished intensity in the N7 kidney. JNK localized to all segments of the tubules and collecting ducts. In glomeruli, JNK was expressed only in glomerular visceral epithelial cells. In the tubular epithelial cells, JNK localized mostly to cytoplasm. Strikingly, P-JNK was detected only in distal tubular cells and podocytes Figure 3n.

MKP-1

MKP-1 was detected only in the fetal kidney. The E14 kidney exhibited most intense staining Figure 3o. The localization was similar to that of ERK or p38. At E18, there was widely scattered staining with no cortico-medullary gradient Figure 3p. Mesenchymal cell, mesangial cells, ureteric buds, and to a lesser extent glomerular epithelial cells exhibited expression of MKP-1.

Table 1 summarizes the immunohistochemical observations for the localization of MAPKs and MKP-1.

Table 1 - Expression of MAPKs and MKP-1 in embryonic, neonatal, and adult rat kidney.

Full table

DISCUSSION

The present data from immunoblot of kidney homogenates and immunohistochemistry indicate that ERK, p38, and MKP-1 are predominantly expressed in the developing kidney with the highest levels in the embryo. On the other hand, JNK was not detectable in the fetal kidney, became detectable at seven days after birth, and was most abundant in the adult kidney.

Kidney development is a two stage process¹². The first stage involves inductive interactions between ureteric bud and metanephric mesenchyme¹². The undifferentiated metanephric mesenchyme in nephrogenic zone is rescued by the ureteric bud from apoptosis and form condensations of epithelial cells, vesicles, which subsequently generate comma- and S-shaped bodies. As nephron are forming, ureteric bud branches form collecting ducts. A second stage is marked by the rapid division of tubular epithelial cells which takes place in the subnephrogenic zone. Various growth factors have been implicated in the sequence of inductive events that lead to the formation of nephrons¹¹. An essential mechanism in the signaling pathways of these growth factors involves the phosphorylation and dephosphorylation regulated by kinases and phosphatases. MAPK family cascades are highly conserved in all eukaryotic cells and are thought to be essential for a variety of cellular functions. Of these, ERK has been shown to be necessary and sufficient for mesoderm formation in Xenopus^13,14. Overexpression of either constitutively active ERK or ERK kinase induced mesoderm. Inactivation of ERK, on the other hand, by MKP-1 overexpression disrupted normal mesoderm induction. Mice lacking MEK1, an upstream kinase of ERK, have been demonstrated to die at 10.5 days of gestation¹⁵. These findings underscore the importance of MEK1/ERK pathway in embryogenesis. Since blockade of ERK leads lethality before renal organogenesis, however, methods inhibiting ERK at a later stage is needed to investigate the role of ERK in kidney development.

The role of JNK and p38 in embryogenesis has also been investigated. Targeted disruption of SEK1, an activator of p38 and JNK, has been reported to cause embryonic death before embryonic day 14^16,17. Examination of earlier embryos demonstrated a reduced size of the liver with increased number of apoptotic hepatocytes. Activation of JNK and p38 in cells from these mice were variably inhibited. Accordingly, SEK1/JNK and/or SEK1/p38 pathway seems to be necessary for normal early embryonic development. In this regard, our results are interesting in that JNK and p38 are differentially expressed during kidney development. p38 and JNK probably have different physiological functions in the kidney.

MKP-1 is a dual specificity phosphatase that has been implicated in the inactivation of MAPKs. MKP-1 has been shown to be highly expressed in the developing mouse embryo¹⁸. The present result that MKP-1 expression is most prominent in the fetal kidney is consistent with previous reports. Of interest, MKP-1 deficient mice have been generated that showed no phenotypic or histologic abnormalities¹⁹. Furthermore, ERK activity was shown to be unaltered in MKP-1 deficient embryo fibroblasts. These results suggest that MKP-1 is not essential for embryonic development. However, a role of MKP-1 in embryonic development could not be precluded. Most likely, the lack of MKP-1 activity can be compensated by other phosphatases.

Expression of MAPKs and MKP-1 protein are tightly regulated not only temporally, but also spatially during renal ontogeny. ERK was highly expressed in nephrogenic zone of fetal kidney where widespread proliferation of cells is present as shown in PCNA staining. ERK staining was most intense in mesenchymal cells, blastema, and vesicles. As vesicles matured to comma- and S-shaped bodies, ERK expression became lost. With vascularization, endothelial cells and mesangial cells became intensely positive. In the neonatal period when tubules develop and elongate, ERK is more abundantly detected in subnephrogenic zone. Thus, the temporal and spatial expression of ERK coincide with the development of kidney. In the adult kidney, on the other hand, ERK was expressed by glomerular visceral epithelial cells, distal tubules, and collecting ducts. Only distal tubules, however, stained positive for P-ERK. In tubule and collecting duct cells, ERK may be involved in signaling processes of transport and/or proliferation. Glomerular visceral epithelial cells, on the other hand, are known to have limited capacity of proliferation²⁰. They cease to proliferate when mature glomeruli are formed. In this regard, it is notable that glomerular visceral epithelial cells express ERK throughout the stages of development. Its function in these cells remains to be determined. Compared to ERK, p38 is more uniformly expressed in the fetal and neonatal kidneys. Several lines of evidence suggest that p38 and JNK are involved in the induction of apoptosis^2,21. To correlate the localization of p38 with the occurrence of apoptosis, the TUNEL method was used to visualize fragmented DNA. As reported previously, mesenchymal cells surrounding the newly formed epithelium showed positive labeling. The majority of labeled cells were within the nephrogenic zone. The distribution of p38, however, was diffuse, and not restricted to these specific areas. Furthermore, in the E14 kidney, p38 expression was found in blastema cells and ureteric bud epithelial cells where intense PCNA staining was observed. Recent evidence suggests that there are several isoforms of p38 present, some of which promote proliferation and hypertrophy²². The present results suggest that p38 promotes proliferation rather than apoptosis at least at E14. In contrast to p38, JNK was confined to tubular cells, collecting duct cells, and glomerular epithelial cells in N7 and the adult kidney. Thus, JNK may participate in the growth and differentiation of the tubules at a late stage of kidney development. JNK may also be involved in the hyperosmotic stress response in tubular cells. In this regard, P-JNK was detected only in the distal tubules of adult kidney. As stated earlier, glomerular visceral epithelial cells are highly differentiated and incapable to proliferate postnatally. It is tempting to speculate that JNK may be exerting a growth inhibitory action antagonizing ERK. Finally, MKP-1 showed a similar pattern of expression to p38. It was abundant in blastema cells and ureteric buds at E14, suggesting that it has a role in regulating ERK and/or p38. In the E18 kidney, MKP-1 was expressed predominantly in mesenchymal cells, mesangial cells, and ureteric bud cells. These cells express ERK as well as p38, again suggesting that MKP-1 may regulate activities of ERK and/or p38 in the embryonic kidney.

Subcellular localization of MAPKs has been shown to suggest the status of their activation. MAPKs locate to nuclei upon activation²³. Phosphorylated forms of MAPKs in general showed the same distribution as that of MAPKs. Of note, cells showing cytosolic staining of ERK or p38 also were found to be positive for the phosphorylated forms. This suggests that MAPKs target cytosolic proteins as well as nuclear proteins. In this regard, it is noteworthy that ureteric bud cells in stroma showed strong cytosolic staining of ERK and p38. In contrast, those in the nephrogenic zone where active branching is occurring showed nuclear staining. The physiologic significance of these MAPKs in ureteral development remains to be clarified.

In conclusion, we have demonstrated the temporospatially unique expression of MAPKs and MKP-1 in the rat kidney during development. The expression of ERK, p38, and MKP-1 was most prominent in the embryonic kidney, whereas that of JNK was high in the mature kidney. In general, expression of ERK correlated with the maturation processes of nephrogenesis and tubulogenesis. Further studies examining the functional role of each MAPK and MKP-1 in specific cell types may provide a better understanding of the mechanisms that lead to kidney malformations.

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Acknowledgments

This study was supported by grants from the Ministry of Education, Science, and Culture, Japan (C10670757), and Pharmacia-Upjohn Fund for Growth and Development Research. We thank Mr. Hiroshi Suzuki for the technical assistance with immunohistochemistry.