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Previous Article | Next Article 
Molecular and Cellular Biology, October 2007, p. 7236-7247, Vol. 27, No. 20
0270-7306/07/$08.00+0 doi:10.1128/MCB.00780-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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Department of Molecular Physiology and Biophysics,1 Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 770302
Received 3 May 2007/ Returned for modification 4 June 2007/ Accepted 7 August 2007
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[or RBP-J, also known as CSL for CBF1/Su(H)/Lag-1] and the coactivator Mastermind (Mam) and, at the same time, the domain releases RBP-J from a corepressor complex. The trimeric complex further recruits histone acetyltransferases (p300 and/or PCAF/GCN5) and chromatin-remodeling complexes (BRM, TRA1/TRRAP, and Dom) to form a transcriptional activator (9, 11, 14, 15). Genes activated by Notch signaling include the Hes family of transcription repressors, homologues of Drosophila Hairy/Enhancer of split (2, 6, 18). More recently, another family of transcription repressors, Herp (Hes-related repressor protein; also known as Hesr/Hey/Hrt/Chf/gridlock), was identified and shown to be activated by Notch (10, 26). However, few genes are known to be regulated by either the Hes or the Herp family of transcription factors. Ocular lens development can be divided into two stages (Fig. 1). The first stage results in the formation of a lens vesicle (Fig. 1A) and the primary lens. The lens vesicle is derived from the lens placode region of the head ectoderm. The optic vesicle (future retina) induces invagination of the lens placode, which eventually pinches off the head ectoderm to form a hollow sphere, the lens vesicle (Fig. 1A). Cells in the posterior portion of the lens vesicle, facing the optic vesicle or retina, differentiate into primary lens fiber cells (Fig. 1B) under the induction of a putative signal emitted from the retina. The anterior portion of the lens vesicle remains undifferentiated. During the second stage of lens development, the anterior epithelial cells continue to proliferate and their progeny differentiate into secondary lens fiber cells in the transition zone (or bow region), which is located around the lens equator where the epithelium terminates (Fig. 1C). Thus, the growth of the lens is realized through the addition of secondary fiber cells and is fueled by mitotic activities in the epithelium. The mechanism(s) that determines the boundary of differentiation during the lens vesicle stage and during the formation of secondary lens fiber cells remains unknown.
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MATERIALS AND METHODS |
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For LacZ staining, embryos were dissected in cold phosphate-buffered saline (PBS) and washed in cold 0.1 M phosphate buffer before being fixed in 0.24% glutaraldehyde, 5 mM EDTA, 2 mM MgCl2, and 10 mM phosphate buffer at 4°C while rocking for 45 min. After fixation, embryos were stained overnight in 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-galactoside at room temperature while rocking. Stained embryos were rinsed with PBS and photographed. Histological analyses of embryos were carried out as described previously (33).
Yeast one-hybrid screening. We used the yeast one-hybrid kit from Clontech (Palo Alto, CA). Conserved element E-I or E-II from the mouse p57Kip2 gene was subcloned into pHis1-1 and pLacZi-1 and integrated into the genome of the YM4271 yeast strain to generate YE-I and YE-II. An embryonic day 17.5 (E17.5) mouse cDNA library in pACT2 (Clontech) was screened in YE-I and YE-II. Approximately 2 x 106 clones were screened. Clones that survived 3-amino-1,2,3-triazole (3-AT) selection were tested for LacZ expression. Those without LacZ expression were eliminated. The clones that expressed LacZ were used to extract the library plasmid DNA. Extracted DNA was transformed into Escherichia coli and then transformed back into YE-I, YE-II, and YE-p53 (provided with the kit) to test for specificity.
Cell culture and CAT and chromatin immunoprecipitation (ChIP) assays. Cos7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The chloramphenicol acetyltransferase (CAT) assay was carried out 24 h after transfection using the CAT enzyme-linked immunosorbent assay kit (Roche). CAT expression levels were normalized to cotransfected ß-galactosidase.
The ChIP assay was carried out according to the protocol suggested by the manufacturer of ChIP-grade anti-His6 antibodies (catalog no. ab9108; Abcam). His6-tagged cyclin D1 and Herp2 were subcloned into pCDNA3.1 (Invitrogen). The expression vectors were transfected into NIH 3T3 cells by using Lipofectamine 2000 (Invitrogen). Twenty-four hours after the transfection, formaldehyde was added to medium to cross-link proteins with DNA and the cells were lysed in 50 mM HEPES, pH 7.5-140 mM NaCl-1 mM EDTA-1% Triton X-100-0.1% sodium deoxycholate-0.1% sodium dodecyl sulfate. Cell lysates were sonicated using a Microson ultrasonic cell disruptor (four cycles of 30 s on and 30 s off) to shear genomic DNA to an average fragment size of 500 to 1,000 bp and then immunoprecipitated with anti-His6 antibodies. The precipitated DNA was purified with a QIAquick PCR purification kit, followed by PCR amplification of two regions of the mouse p57Kip2 promoter. The primers were 5'-TACAAGGCAGGCCCTGTAATCGGA-3' and 5'-CCCCGCCGCCCCAGCAGTAAGCAG-3' for region a and 5'-CTCTGCAGGGCCTTTCAAGTATGT-3' and 5'-TTGGCTGGAAGTAGTTATGCTAGA-3' for region b.
Immunofluorescence analysis and in situ hybridization. Mouse embryos at the desired stages of development were harvested after a 2-h pulse of bromodeoxyuridine (BrdU) and were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin wax. Sections (5 mm) were cut and collected.
Immunofluorescence staining was performed according to the instructions provided by the antibody suppliers. In brief, dewaxed and rehydrated sections were heated in 10 mM sodium citrate (pH 6.0) in a microwave oven (700 W, 10 min) to retrieve the antigen. Slides were washed three times for 5 min in PBS. Primary antibodies were diluted in blocking buffer (PBS, 2% bovine serum albumin, 0.1% Triton X-100). We used 1:50 rabbit anti-p57 (catalog no. ab4058; Abcam Inc., Cambridge, MA) and 1:50 goat anti-Jagged 1 (C-20; Santa Cruz Biotechnology), 1:100 rabbit anti-cleaved caspase-3 (Cell Signaling), 1:100 mouse anti-E-cadherin (BD Bioscience), and 1:10 rabbit anti-ß-crystallin (a gift from P. Overbeek, Baylor College of Medicine). Tissue sections were incubated with antibody overnight at 4°C. After being washed with PBS three times, sections were incubated with the following secondary antibodies for 1 h at room temperature: Alexa 594-conjugated donkey anti-goat immunoglobulin G (IgG) (Invitrogen), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology), and FITC-conjugated rabbit anti-mouse IgG (Sigma). Nuclei were counterstained with 100 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Sigma) in PBS. Slides were viewed with a Zeiss LSM-510 META confocal microscope by using LSM 510 (version 3.2) software to acquire images, and the lens region was exported as the region of interest. BrdU incorporation was visualized with a BrdU staining kit (Amersham Biosciences).
In situ hybridization was performed as described previously (23, 27). The probe for Herp2 was a PCR clone of its open reading frame obtained from Deepak Srivastava (University of Texas Southwestern Medical Center). The probe for Notch3 was the SacII-to-XbaI fragment of the cDNA (a gift from U. Lendahl, Karolinska Institute) cloned into pBS.
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RESULTS |
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Herp2 is expressed in lens epithelium. To understand the molecular mechanism(s) underlying the small-lens phenotype in Rbp-J mutants, we first determined which of the Notch effectors is expressed in the lens. To that end, we used in situ hybridization to survey the expression of the Hes and Herp families of transcription repressors. At E17.5, Herp2 was expressed in lens epithelium (Fig. 3A) in a domain right above (but also overlapping with) that of p57Kip2 (Fig. 3B), a cyclin-dependent kinase (Cdk) inhibitor (19, 22) that is required for cell cycle withdrawal during lens fiber cell differentiation (32, 33). Herp2 expression was lost in Rbp-J mutants (Fig. 3C; also see Fig. 6C), demonstrating that Notch signaling is required for the expression of this transcriptional repressor. We also examined Jag1 expression by immunofluorescence. This Notch ligand was expressed in the differentiating fiber cells that express p57Kip2 (Fig. 3D). The expression of Jag1 is likely induced by the same signal that induces p57Kip2 expression. The Jag1 expression pattern places it in the right position to activate Notch signaling (and hence Herp2 expression) in adjacent epithelial cells. Jag1 expression was not detected in more mature, center-localized lens fiber cells.
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Herp2 can suppress the expression of p57Kip2.
The interaction of Herp2 with E-I in yeast suggests that this Notch effector can suppress p57 expression directly. To test this possibility, we generated two constructs, p57-CAT and p57
E-I-CAT, by using the 7-kb mouse p57Kip2 promoter region and its E-I-deleted version to drive CAT reporter expression (Fig. 5A). The CAT constructs were transfected into Cos7 cells, together with a Herp2 expression vector. As shown in Fig. 5B, Herp2 suppressed CAT expression in an E-I-dependent fashion. Herp3, another member of the Herp family of repressors, also suppressed the p57Kip2 promoter (Fig. 5B).
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Increased number of p57Kip2-expressing cells in Rbp-J mutants at primary lens stage. The ability of Herp2 to suppress p57Kip2 expression prompted us to ask whether there is an increase in the number of p57Kip2-expressing (thus nondividing) cells in the Rbp-J mutant that might account for the smaller-lens phenotype. We first examined the process of primary lens fiber cell differentiation in Rbp-J mutants. At this stage of development, the epithelial cells in the posterior half of the lens vesicle have begun to differentiate, expressing p57Kip2 and Jag1 (Fig. 6A), while the anterior half of the lens vesicle remains undifferentiated. The size and the cell number (see Fig. S2A in the supplemental material) of the lens vesicle in Rbp-J mutants are comparable to those in the control, indicating that the small lens size in the mutants is not a result of defective lens vesicle formation. However, the number of p57Kip2-positive cells in the mutants was much greater: p57Kip2 expression was detected not only in almost every cell in the posterior region but also in some cells in the more anterior region. More p57Kip2 expression in the posterior region indicates that Notch signaling also functions in the posterior part of the lens vesicle, and the expression in the anterior region indicates that the p57Kip2 expression domain in the mutants expands into a region that does not normally express p57Kip2. In support of the function of Notch signaling in the posterior region, Herp2 expression was detected by in situ hybridization in the posterior portion of control lens vesicles, but not in lens vesicles from Rbp-J mutants at E11.5 (Fig. 6C). Furthermore, Notch3 is expressed throughout the lens vesicle (and in other eye structures as well) as detected by in situ hybridization (see Fig. S1 in the supplemental material).
As a result of the increase of p57Kip2-expressing cells both anteriorly and posteriorly, the number of cells undergoing active DNA synthesis decreases in the mutant lens vesicle relative to control (Fig. 6B and see Fig. S3 in the supplemental material), indicating a decline in the proliferation potential of the mutant lens vesicle.
Enhanced differentiation of primary lens fiber cells in Rbp-J mutants. The overall differentiation of primary lens fiber cells also proceeded faster in the absence of Notch signaling. Primary fiber cell elongation was greater in the Rbp-J mutant than in the control (Fig. 6A, compare images for the control and mutant phases and Jag1 immunostaining; see Fig. S2B in the supplemental material), as was the expression of ß-crystallin, a marker of lens fiber cell differentiation (Fig. 6D). Furthermore, the number of p27Kip1-positive cells also increased slightly in the mutant (see Fig. S4 in the supplemental material). This result is consistent with the notion that Notch signaling could also suppress the expression of p27Kip1 (8, 24). p27Kip1 plays a minor role in lens fiber cell differentiation (33). Its protein levels (25), but not its message levels (33), are increased in differentiating fiber cells. Taken together, these results indicate that Notch signaling suppresses the differentiation of primary lens fiber cells.
Thinning of the lens epithelium in Rbp-J mutants. Next, we examined the process of secondary lens fiber cell formation, which lasts until adulthood in mice (16). During this stage of lens development, secondary fiber cells are generated in a region called the transition zone which is located where the epithelium terminates at the equator of the lens. Continued proliferation of anterior lens epithelial cells generates secondary fiber cells. In Rbp-J mutant mice, fewer epithelial cells are left due to the enhanced differentiation of primary lens fiber cells, which predicts that the mutant lens epithelium will be smaller and contain fewer cells. Indeed, we found that the epithelium in the mutant lens was thinner and contained fewer cells than the epithelium in the control did (Fig. 7A). No disruption of the epithelial structure was revealed by E-cadherin immunostaining (Fig. 7B). However, it is apparent from the E-cadherin staining that the size of the mutant lens epithelium is smaller than that in the control, as is the lens itself (Fig. 7B). To rule out the possibility that Notch signaling is required for the proliferation of lens epithelial cells, we analyzed BrdU incorporation in control and mutant lenses. The total number of BrdU-positive cells was significantly reduced in the mutants compared to that in the controls (Fig. 7C and see Fig. S5 in the supplemental material), and so was the total number of epithelial cells (Fig. 7D), resulting in similar BrdU indices in mutants and controls (Fig. 7E). We also asked whether an increase in the rate of apoptotic cell death could account for the apparent loss of cells in mutant lens epithelium. Activated caspase-3 was used as an indicator of apoptosis. No differences in activated caspase-3 staining were observed. In fact, no apoptotic cells were detected in either control or mutant lenses (data not shown). These data indicate that the Notch signaling pathway is not essential for the proliferation of lens epithelial cells and that the loss of Rbp-J does not induce apoptosis. Thus, the thinning of the mutant epithelium is not the result of an inability to proliferate or an increased tendency to die, but rather, it results from the early loss of epithelial cells to differentiation (Fig. 6A).
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The small-lens phenotype in Rbp-J mutants is rescued by simultaneous inactivation of p57Kip2. If the aberrant p57Kip2 expression pattern underlies the small-lens phenotype, we should be able to restore the lens size in Rbp-J mutants by genetically removing this Cdk inhibitor. To that end, we generated double-mutant embryos lacking p57Kip2 and Rbp-J in the lens. We previously showed that deleting p57Kip2 increases the lens size and reduces the levels of ß-crystalline expression (33). As expected, p57Kip2 single- and p57Kip2/Rbp-J double-mutant lenses were slightly bigger than wild-type lenses at E16.5 (Fig. 9A). Importantly, the double-mutant lens was as big as the p57Kip2 single-mutant lens and bigger than the Rbp-J mutant lens, demonstrating that the deletion of p57Kip2 restored the lens size in Rbp-J mutants. Furthermore, the removal of p57Kip2 normalized the number of Jag1-expressing lens fiber cells undergoing differentiation in the transition zone, as indicated by increased Jag1 expression in the double mutant relative to its expression in the Rbp-J single mutant (Fig. 9B). In p57Kip2/Rbp-J double-mutant lenses, the ß-crystallin expression is also reduced to the same level as that in the p57Kip2 single-mutant lens (Fig. 9C). These results strongly support the notion that the Notch signaling pathway protects lens epithelial cells by suppressing p57Kip2 expression.
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DISCUSSION |
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Our results indicate that the Notch signaling pathway also plays a role in defining the differentiation boundary, in addition to the proposed differentiation signal gradient. In the absence of Notch signaling, some more anteriorly localized, would-be epithelial cells in the lens vesicle prematurely start to differentiate, resulting in the shrinkage of the future lens epithelium, the precursor pool of lens fiber cells. Consequently, the production of secondary lens fiber cells is reduced and smaller lenses are generated in mutants with defective Notch signaling.
The expression of Jag1 by differentiating fiber cells suggests that Jag1 may be induced by the differentiation signal to activate Notch receptors on the adjacent epithelial cells, preventing them from differentiating. Thus, the lens fiber cell differentiation signal sets up a negative feedback loop at cellular level through the Notch signaling pathway to limit the number of epithelial cells undergoing differentiation (Fig. 10). The disruption of this feedback mechanism results in the apparent strengthening of the differentiation signal and premature differentiation.
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How does the Notch signaling suppress lens fiber cell differentiation? We found that the transcriptional repressor, Herp2, is expressed in epithelial cells in a Notch signaling-dependent way. We also found that p57Kip2 is a direct target of Herp2. Given the importance of this Cdk inhibitor in the differentiation of lens fiber cells (32, 33), by blocking its expression, Herp2 (and hence the Notch signaling) may indirectly inhibit lens fiber cell differentiation. However, Herp2 could suppress other genes directly involved in the differentiation (Fig. 10, dashed line), which will require further investigation. Since p57Kip2 is a Cdk inhibitor, the suppression of its expression by Notch signaling also helps maintain the proliferation potential of lens epithelial cells.
Notch signaling has been implicated in the development of various structures of the eye. A hypomorphic Notch2 allele results in an aberrant bulbous structure, retrolenticular hyperplasia, and microphthalmia (22a). The mild small-eye phenotype may be caused in part by the effect on lens development described here. Jag1-deficient mice have been generated (30a). Although heterozygous mice show defects in irises and corneas, no impairment in the lens was reported and Jag1-null mice died too early (before E10.5) for the analysis of the role of Jag1 on lens development. By deleting Rbp-J specifically in the lens, our work revealed a critical role for the Notch signaling pathway in balancing proliferation and differentiation during ocular lens development. Notch signaling performs this balance by controlling the number of cells undergoing differentiation without affecting the proliferation or differentiation processes themselves. Directly repressing the expression of p27Kip1 (8, 24) and p57Kip2 (this study and reference 7) or inducing their degradation (5, 28) is perhaps the general mechanism used by the Notch signaling to control tissue growth in a large number of developing systems.
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ACKNOWLEDGMENTS |
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This work was supported in part by NIH grant EY12825 and in part by research grant 6-FY00-812 from the March of Dimes Birth Defects Foundation.
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FOOTNOTES |
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Published ahead of print on 20 August 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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