MAPK1 is required for establishing the pattern of cell proliferation and for cell survival during lens development

MAPK1 and MAPK3 (also known as ERK2 and ERK1, respectively) are members of the mitogen-activated protein kinase family. The classic MAPK1/3 activation pathway involves the sequential activation of the serine/threonine kinase Raf, a dual-specificity MAPK kinase (MAPKK or MEK) and then the MAPK (Sebolt-Leopold and Herrera, 2004). Activation of the Raf-MAPKK-MAPK kinase pathway transmits signals to both cytoplasmic signaling complexes and nuclear transcription factors, including protein kinases and phosphatases, signaling effectors, transcriptional regulators and cytoskeletal proteins (Yoon and Seger, 2006). As such, MAPK1/3 can activate a broad spectrum of cellular responses, ranging from cell proliferation and differentiation to migration and apoptosis. This evolutionarily conserved kinase pathway is a central signaling module that participates in numerous physiological and pathological processes, such as embryonic development and cancer (Osborne et al., 2012; Santamaria and Nebreda, 2010).

MAPK1/3 proteins are 84% identical, have similar biochemical properties, recognize the same substrates and share similar biological functions. Gene knockout studies in mice imply that the roles of MAPK1 and MAPK3 in vivo are not entirely equivalent. For instance, Mapk1 deletion results in early embryonic lethality due to failure in trophoblast formation, mesodermal differentiation and placental development (Hatano et al., 2003; Saba-El-Leil et al., 2003; Yao et al., 2003). By contrast, Mapk3^-/- mice survive embryonic development with minor developmental defects in thymocyte and adipocyte differentiation (Bost et al., 2005; Pagès et al., 1999). Recent results from Mapk1 conditional deletion mice suggest that MAPK1 and MAPK3 are functionally redundant in certain tissues during development but distinct in others. For example, conditional deletion of Mapk1 in the CNS caused a mild phenotype in neurogenesis (Satoh et al., 2011b) and MAPK3 deficiency enhanced the phenotype in these mice, suggesting that the total MAPK activity is essential for normal CNS development. Interestingly, MAPK1-deficient mice exhibited marked abnormalities in social behaviors related to facets of autism-spectrum disorders in humans (Satoh et al., 2011a). Blocking MAPK3 activity in these mice with a pharmacological inhibitor did not cause additional psychological impairments, suggesting that MAPK1 has a unique role in the CNS in the control of social behavior. Overall, genetic data indicate two different scenarios: (1) the two MAPK isoforms are functionally interchangeable, and a sufficient threshold of total MAPK activity is important for normal tissue development and function; or (2) MAPK1 and MAPK3 have evolved to play unique roles in development and physiology. These two mechanistic models have not been examined extensively in developmental systems.

The ocular lens is a classic developmental system with which to study growth factor signaling in tissue induction and the regulation of cell proliferation and differentiation (Chow and Lang, 2001; Lovicu et al., 2011; Robinson, 2006). During lens morphogenesis, the presumptive lens ectoderm is induced by the underlying optic vesicle to form lens placode between mouse embryonic day (E) 9.0 and 9.5. The lens placode invaginates to form the lens vesicle between E10.5 and E11.5. Subsequently, the posterior lens vesicle cells elongate and differentiate into the primary fiber cells, which fill the lens vesicle at E12.5, whereas the anterior cells become the lens epithelial cells. After formation, lens growth is driven by cell proliferation and differentiation in a spatially restricted manner. Cell proliferation is limited to the anterior epithelial cells, with the greatest mitotic activity in a region just above the lens equator known as the lens germinative zone (Kallifatidis et al., 2011). The progeny cells that have migrated below the lens equator initiate the differentiation process and eventually form the secondary fiber cells. This coordinated proliferation and differentiation pattern is maintained throughout the lifespan of the animal.

Previous studies in transgenic and knockout mouse models demonstrated that FGF-Ras signaling is essential for cell proliferation, differentiation and survival during lens development (Burgess et al., 2010; Garcia et al., 2005; Govindarajan and Overbeek, 2001; Pan et al., 2010; Qu et al., 2011; Reneker et al., 2004; Xie et al., 2006; Zhao et al., 2008). Abolishing MAPK activity with the MAPKK (or MEK) inhibitor U0126 blocks the FGF-induced cell proliferation and elongation response in rat lens explants (Lovicu and McAvoy, 2001). However, the specific contributions of MAPK1 and MAPK3 cannot be simply extrapolated from these studies because both MAPK isoforms are equally affected. It is known that MAPK3 is not required for mouse lens development, but the importance of MAPK1 in the lens is still uncertain owing to early embryonic lethality (Hatano et al., 2003).

In this study, we generated conditional Mapk1 knockout mice using the presumptive lens driver Le-Cre, which is active at lens induction stage E9.0 (Ashery-Padan et al., 2000). In contrast to Mapk3^-/- mice, the lens of Mapk1 conditional deletion mice exhibited severely compromised cell proliferation and survival. In the MAPK1-deficient lens, cell proliferation was drastically reduced in the peripheral germinative zone but not in the central epithelium. Increase of MAPK3 activity in the epithelium cannot compensate for the loss of MAPK1 to re-establish the cell proliferation pattern in the lens, suggesting that MAPK1 has an essential and unique role in the control of cell proliferation. MAPK1 activity is also crucial for lens epithelial cell survival, but is not required for fiber cell differentiation during lens development.

Mice carrying the Mapk1 flox alleles and Mapk3^-/- mice were described previously (Hatano et al., 2003; Pagès et al., 1999). In Le-Cre mice, the transgene was driven by a 6.5 kb genomic fragment from the mouse Pax6 gene (Ashery-Padan et al., 2000). The Le-Cre;Mapk1^flox/flox mice (referred to as Mapk1^CKO) were heterozygous for the Cre transgene in all the experiments because homozygous mice are reported to have an ocular phenotype (personal communication from Dr Michael Robinson at Miami University, Oxford, OH, USA). The Mapk1^flox/flox mice are referred to here as wild type (WT). All of the animals were used in accordance with the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, and all experimental procedures were approved by the Animal Care and Use Committee at the University of Missouri.

The heads of mouse embryos or newborn pups were fixed in 4% paraformaldehyde overnight and processed for histological analysis as described previously (Xie et al., 2006). Nuclei were counterstained with Hematoxylin or DAPI for immunohistochemistry or immunofluorescence, respectively. The following primary antibodies were used: anti-MAPK1/3 (9102), anti-pMAPK1/3 (9101), anti-cleaved caspase 3 (9664) and anti-survivin (2808) (all from Cell Signaling Technology, Beverly, MA, USA); anti-phospho-histone H3 (sc8656), anti-cyclin D2 (sc593) and anti-cMAF (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-cyclin D1 (ab16663), anti-p57^Kip2 (ab4058) and anti-p53 (ab61256) (all from Abcam, Cambridge, MA, USA); anti-PAX6 (PRB-278P) and anti-PROX1 (PRB-238C) (both from Covance, Princeton, NJ, USA); anti-MAPK1 (05-157, Millipore, Billerica, MA, USA); and anti-Ki67 (M7249, Dako, Carpinteria, CA, USA). We did not find a reliable commercial anti-MAPK3 antibody that worked well for immunostaining. The anti-αA-crystallin antibody was a generous gift from Dr Richard Cenedella (Department of Biochemistry, College of Osteopathic Medicine, Kirksville, MO, USA) and anti-β;-crystallin and anti-γ-crystallin antibodies were from Dr Samuel Zigler (National Institutes of Health, Bethesda, MD, USA). For immunofluorescence, Alexa Fluor 488 (A11008) and 568 (A10042) conjugated secondary antibodies were from Invitrogen (Carlsbad, CA, USA). The signal enhancement TSA kit (NEL741B001KT, PerkinElmer, Boston, MA, USA) was used f or pMAPK1/3 and cleaved caspase 3 immunofluorescence. For immunohistochemistry, biotinylated secondary antibodies (BA1000 or BA4001) and Vectastain Elite ABC Reagent were from Vector Laboratories (Burlingame, CA, USA). Color development was performed using 3,3′-diaminobenzidine as a substrate (D4293, Sigma, St Louis, MO, USA).

Pregnant mice were injected intraperitoneally with 5-bromo-2′-deoxyuridine (BrdU) (B5002, Sigma) at 100 μg/g body weight and sacrificed after 1 hour or 2 days. The BrdU-labeled cells were detected by immunohistochemistry as described previously (Fromm et al., 1994). TUNEL assay was performed with an in situ apoptosis detection kit (S7165, Millipore) following the manufacturer’s instructions.

Newborn (P0) mouse lenses were homogenized in RIPA buffer for protein preparation and then subjected to western blot analysis (Xie et al., 2007). Each western blot was repeated at least twice with independent preparations of lens proteins. Radiographic films were scanned and band intensity was analyzed using Adobe Photoshop CS2 software. The anti-p38 (9212) and anti-phospho-p38 (9211) antibodies were from Cell Signaling Technology.

For quantitative analysis, serial sections across the entire lens were collected and central sections (judged by the size of the lens) from a minimum of three independent eyes for each genotype from the same litter were used (n, number of sections). Data are expressed as mean ± s.e.m. and P-values were calculated using Student’s t-test (P<0.05 considered significant).

The Mapk1^CKO mice were viable and fertile, but their eyes were microphthalmic and their lenses were slightly opaque and smaller than normal (Fig. 1A,B). To confirm MAPK1 deletion in Mapk1^CKO lenses, immunostaining was performed at E14.5. Intensive MAPK1 staining was detected in the epithelial cell nuclei and in the fiber cells of the WT lens (Fig. 1C). By contrast, MAPK1 staining was absent in the Mapk1^CKO lens (Fig. 1D). Immunohistochemistry against MAPK1/3 revealed uniform immune reactivity in the epithelial layer and in the fiber cells at a lower level in WT lenses (Fig. 1E). In comparison, the signal was significantly reduced in Mapk1^CKO lenses; however, weak staining was still detected resulting from the presence of MAPK3 proteins (Fig. 1F). Western blot analysis confirmed that MAPK1 was lost, whereas the MAPK3 level was unchanged in Mapk1^CKO lenses (Fig. 1G). Cre expression in the pancreas has been reported in Le-Cre mice (Ashery-Padan et al., 2000), but because the Mapk1^CKO mice appeared healthy the MAPK1 levels in this tissue were not examined.

The effect of MAPK1 deletion on lens development was analyzed by histology (Fig. 1H-M). In the E11.5 WT embryo, the lens vesicle had formed and cells in the posterior half of the vesicle had begun to elongate to form the primary fiber cells (Fig. 1H). At this stage, the Mapk1^CKO lenses were phenotypically indistinguishable from WT lenses (Fig. 1I), suggesting that MAPK1 deletion did not affect lens induction and formation. Although the E14.5 Mapk1^CKO lenses retained normal polarity and architecture, they appeared substantially smaller than normal (Fig. 1J,K). The epithelial layer was thinner and with a lower cell density in Mapk1^CKO compared with WT lenses (Fig. 1J,K, insets). By postnatal day (P) 8, Mapk1^CKO lenses were severely hypoplastic, vacuoles had formed in the cortex (Fig. 1M; supplementary material Fig. S1D,H) and fiber cells were disorganized (supplementary material Fig. S1A,B). However, the equatorial region, in which epithelial cells are induced to differentiate into fiber cells (arrows in Fig. 1L,M insets), could still be identified in Mapk1^CKO lenses. The Mapk1^CKO mice also exhibited multiple defects in the anterior segments, but early retinal development appeared to be normal (supplementary material Fig. S1C-H). We also confirmed that MAPK3 deficiency did not affect lens or eye development (supplementary material Fig. S1I,J).

To monitor the progressive thinning of the lens epithelium in Mapk1^CKO lenses, we compared the total number of epithelial cells with WT lenses (Fig. 2). At E11.5, the WT and Mapk1^CKO lens epithelium had about the same number of cells (57±3 for WT and 54±2 for Mapk1^CKO, P>0.5). During normal lens development, the epithelial cell number increases along with the expansion of the lens surface area and size. In WT lenses, epithelial cell numbers were 149.9±3.1, 209.7±3.6 and 235.7±2.3 per section at E14.5, E17.5 and P0, respectively. By contrast, epithelial cell numbers in Mapk1^CKO lenses at these stages were 98.8±1.9, 93.6±2.3 and 88±1.9, a slight decrease as the lens grew. These results indicated that lens growth was severely inhibited by the loss of MAPK1.

The epithelial defect in Mapk1^CKO lenses suggested that MAPK1 might play an important role in cell proliferation, survival, or both during lens development. We first assessed the changes in cell proliferation by examining the expression patterns of several cell cycle markers. Ki67 is a nuclear protein present in proliferating cells (in G1, S, G2 and M phase) but absent from resting (G0) cells (Scholzen and Gerdes, 2000); thus, it is often used as a marker for determining the growth fraction of a given cell population. Our results showed that Ki67 is expressed in the epithelial layer in both WT and Mapk1^CKO lenses at E14.5 (Fig. 3A,B). However, the region above the lens equator in Mapk1^CKO lenses contained fewer Ki67⁺ cells than the same region in WT lenses. To quantify the regional distribution of Ki67⁺ cells, we divided the lens epithelium into 10° sections, with 0° at the lens equator and 90° in the middle of the central epithelium (Fig. 3A,B). At the equator, cells with horizontally located nuclei were considered as epithelial cells. We found that the total number of Ki67⁺ cells was lower in the Mapk1^CKO than in the WT lens epithelium (Fig. 3C). When the Ki67 labeling index (ratio of Ki67⁺ cells over total epithelial cells) was measured, it was significantly reduced in the region between 10° and 30° in Mapk1^CKO lenses, which corresponds to the germinative zone, an area in close contact with the anterior margin of the developing retina (Fig. 3D). Interestingly, beyond the lens germinative zone, the Ki67 labeling index was not significantly altered in Mapk1^CKO lenses. Our data suggested that MAPK1 activity is required for cell proliferation in the germinative zone, whereas MAPK3 might be able to compensate for the loss of MAPK1 beyond this region.

To investigate cell cycle changes, we examined BrdU and phosphorylated histone H3 (phospho-H3) expression patterns in WT and Mapk1^CKO lenses (Fig. 3E-L). BrdU labels cells in S phase and phospho-H3 is present in cells at G2-M (Gratzner, 1982; Hendzel et al., 1997). Consistent with the Ki67 results in the germinative zone (10-30°), the number of BrdU⁺ and phospho-H3⁺ cells and their labeling indices were significantly lower in Mapk1^CKO lenses (Fig. 3E-L; supplementary material Fig. S2). Furthermore, the drastic reduction in the ratio of phospho-H3⁺ over Ki67⁺ cells suggested that G2-M phase progression was severely inhibited in the germinative zone in Mapk1^CKO lenses (supplementary material Fig. S2). In the central epithelium (the region from 60-90°), the labeling index was either slightly higher (for BrdU) or unaffected (for phospho-H3), suggesting that MAPK3 or other signaling activity is sufficient to maintain mitotic activity in this area. Overall, the results supported the conclusion that MAPK1 is essential for cell proliferation and cell cycle progression in the lens germinative zone.

To confirm the redundant roles of MAPK1 and MAPK3 in regulating cell proliferation in central lens epithelium, Mapk1/3 double-deletion (referred to as Mapk1/3^DKO) mice were generated and cell proliferation analyzed. It was known that lens development is normal in Mapk3^-/- mice (supplementary material Fig. S1). At E14.5, the Mapk1/3^DKO lenses were severely hypoplastic and BrdU⁺ cells were significantly reduced throughout the entire epithelial layer (Fig. 3S-U). By E16.5, BrdU⁺ cells were absent from Mapk1/3^DKO lenses (data not shown). (Further studies on Mapk1/3^DKO lenses will be presented in a separate report.)

It is known that mitogenic stimulation activates the D-type cyclins, which are essential for cell cycle entry and G1 phase progression (Cooper, 2000). The mouse lens expresses three isoforms of D-cyclins, with cyclin D2 as the major form (Chen et al., 2000; Fromm and Overbeek, 1996). We compared the expression patterns and levels of cyclin D1 and D2 in WT and Mapk1^CKO lenses (Fig. 3M-R). In E14.5 WT lenses, cyclin D1 was expressed in the lens epithelial layer and its expression was upregulated at the lens equator (Fig. 3M). In Mapk1^CKO lenses, cyclin D1 was visibly reduced in the epithelial layer, particularly in the germinative zone (indicated by brackets in Fig. 3N). For cyclin D2, the expression pattern appeared similar in the two genotypes (Fig. 3P,Q). Western blot analysis confirmed that the levels of cyclin D1 were decreased, whereas cyclin D2 levels were unchanged in Mapk1^CKO lenses (Fig. 3O,R).

We have shown that MAPK1 deletion severely reduces cell proliferation in the lens germinative zone, whereas the central zone was largely unaffected. Because the number of epithelial cells did not increase with age in Mapk1^CKO lenses (Fig. 2), we speculated that cell death could also contribute to the defect. TUNEL assays revealed that TUNEL⁺ nuclei were not detected in E14.5 WT lenses (Fig. 4A), whereas significant numbers of epithelial cells were undergoing apoptosis in Mapk1^CKO lenses (10.3±1.4 TUNEL⁺ cells per section; Fig. 4B,C). A small number of fiber nuclei were also TUNEL⁺ (1.8±0.4) in Mapk1^CKO lenses. Activation of apoptosis in Mapk1^CKO lenses was also demonstrated by cleaved (active) caspase 3 immunofluorescence (Fig. 4D,E). These results together suggest that, in addition to reduced cell proliferation in the germinative zone, apoptosis across the epithelial layer is also a contributing factor to the reduced cell number and stalled growth of Mapk1^CKO lenses.

Survivin (BIRC5 - Mouse Genome Informatics) is highly expressed during embryonic lens development (Uren et al., 2000; Weber and Menko, 2005). Survivin inhibits apoptosis by blocking the activities of caspase 3, 7 and 9 and also plays an important role in cell proliferation by regulating the G2/M phase of the cell cycle (Chandele et al., 2004; Li et al., 1998; Shin et al., 2001). In vitro studies have shown that the survivin level can be regulated by MAPK activity (Teh et al., 2004; Wang et al., 2010). Survivin was expressed in the epithelial cells of E14.5 WT lenses, and the level was higher in the germinative zone than in the central zone (Fig. 4F,H). Loss of MAPK1 reduced survivin expression only in the germinative zone (Fig. 4G,H), which could be responsible for the increased apoptosis in this region in Mapk1^CKO lenses. Other apoptosis-regulatory proteins, such as p53 (TRP53 - Mouse Genome Informatics) (Pan and Griep, 1995) and the anti-apoptotic protein XIAP (XAF1 - Mouse Genome Informatics) (Leonard et al., 2007), were also examined, but their expression patterns were not discernably altered in Mapk1^CKO lenses (supplementary material Fig. S3A-D).

During normal lens development, following cell division in the germinative zone the epithelial cells near the lens equator exit the cell cycle and differentiate into secondary fiber cells (Griep, 2006; Zhang et al., 1998). Fiber differentiation is coupled with upregulation of the cell cycle inhibitor p57^Kip2 (CDKN1C - Mouse Genome Informatics) (Fig. 5A). Deletion of atypical protein kinase C λ (aPKCλ; PRKCι - Mouse Genome Informatics) in mouse lens has been shown to reduce cell proliferation in the germinative zone, probably as a result of increased p57^Kip2 and premature cell cycle withdrawal in this region (Sugiyama et al., 2009). To determine whether a similar change also occurs in Mapk1^CKO lenses, we compared p57^Kip2 expression patterns in E14.5 lenses between the two genotypes (Fig. 5A,B). Although cell proliferation was drastically reduced in the germinative zone in Mapk1^CKO lenses, unlike aPKCλ-deficient lenses the p57^Kip2 expression level was not increased in this region (Fig. 5B). Furthermore, these cells still expressed the epithelial cell marker E-cadherin (data not shown). Therefore, our results suggest that lack of cell proliferation in the germinative zone in Mapk1^CKO lenses is not caused by dysregulation of p57^Kip2 expression.

Cell proliferation in the germinative zone is thought to be a critical step in priming the cells for secondary fiber differentiation (Sue Menko, 2002). To determine whether a cell proliferation defect in the germinative zone would prevent cells from differentiating into fiber cells, we followed lens cell fate using a BrdU incorporation and tracing assay (Kallifatidis et al., 2011). The proliferating cells were marked with BrdU at E12.5 or E15.5 and the fate of these cells was examined at E14.5 or E17.5, respectively (Fig. 5C-F). In WT lenses, the cell nuclei in the lens cortex were strongly labeled with BrdU, indicating that the initially BrdU-tagged epithelial cells had differentiated into fiber cells (Fig. 5C,E, arrows). In Mapk1^CKO lenses, BrdU-tagged cells had also differentiated, but had not moved as far into the lens cortex as in WT lenses (Fig. 5F,G). This suggests that epithelial-to-fiber differentiation still occurs despite the defective cell proliferation in the germinative zone.

Lens fiber cell differentiation is characterized by the temporal and spatially restricted expression of crystallin genes (Cvekl and Duncan, 2007). Our data showed that loss of MAPK1 did not affect crystallin gene expression (Fig. 5H-N). The expression patterns of transcription factors such as PAX6, PROX1 and cMAF, which are known to be crucial for crystallin expression, were also not discernibly altered in Mapk1^CKO lenses (supplementary material Fig. S3E-J). These data indicated that MAPK1 is not essential for secondary fiber differentiation during lens development.

Both MAPK1 and MAPK3 are downstream targets of MAPKK (or MEK) in the growth factor-stimulated receptor tyrosine kinase (RTK)-Ras-MAPK signaling pathway. MAPK1 deletion could potentially alter the phosphorylation level of MAPK3 in the lens. We therefore examined MAPK activity by immunofluorescence and western blotting against phosphorylated MAPK1/3 (pMAPK1/3) (Fig. 6A-F; supplementary material Fig. S4A,B). In WT lenses, pMAPK immunofluorescence was most visible in the equatorial region (Fig. 6A,C, arrows). In Mapk1^CKO lenses, pMAPK was still detectable at a level comparable to that in WT lenses, but was more anteriorly localized, probably as a result of the reduced lens size and subsequent anterior shift of the retinal margins (see Fig. 6H). Overall, the high intensity pMAPK staining area in Mapk1^CKO lenses was still positioned adjacent to the anterior margin of the retina, a similar spatial relationship to that seen in WT eyes. When pMAPK immunofluorescence was performed without signal enhancement, the pMAPK levels progressively increased in Mapk1^CKO lenses from E14.5 to E17.5 (data not shown). By P0, the pMAPK intensity was higher in Mapk1^CKO than in WT lenses (supplementary material Fig. S4A,B). The increase in pMAPK3 (but not MAPK3 protein) level in Mapk1^CKO lenses was confirmed by western blot analysis (Fig. 6E,F), suggesting that MAPK3 is hyperactive in Mapk1^CKO lenses, probably as an adaptive response to compensate for the loss of MAPK1 activity. We also compared the levels of other signaling molecules, including JNK, p38 and Akt (MAPK8, MAPK14 and AKT1 - Mouse Genome Informatics), between the two genotypes. We found that the phosphorylated p38 (p-p38) level was higher in Mapk1^CKO lenses (Fig. 6E,F) than in WT lenses. The other two kinases (JNK and Akt) were unchanged (data not shown). Loss of MAPK1 might trigger a stress response and activate p38 in Mapk1^CKO lenses.

To determine whether an increase of pMAPK3 in the epithelium can rescue the cell proliferation defect in the germinative zone in Mapk1^CKO lenses, phospho-H3 staining was performed on E17.5 and P0 lenses (Fig. 6G,H; supplementary material Fig. S4C,D). Although the overall ocular architecture of the mutant eye had altered as a result of a smaller lens, we could still identify the presumptive lens germinative zone as the area adjacent to the anterior margin of the retina (brackets in Fig. 6G,H). Phospho-H3⁺ cells in this area were still visibly reduced in Mapk1^CKO lenses, suggesting that an increase in pMAPK3 cannot compensate for the loss of MAPK1 function in the germinative zone.

To investigate whether MAPK1 is preferentially activated over MAPK3 in response to growth factor stimulation, we examined pMAPK levels in transgenic mouse lenses overexpressing human platelet-derived growth factor A (PDGF-A) (Reneker and Overbeek, 1996). The lens epithelial cells in PDGFA transgenic mice were hyperproliferative (supplementary material Fig. S4F). MAPK1/3 protein levels were not altered, but pMAPK1/3 levels were higher in PDGFA transgenic lenses than in WT lenses (supplementary material Fig. S4G,H). The increase in pMAPK1 was more substantial than that of pMAPK3, suggesting that MAPK1 is more responsive to PDGF-A stimulation, which led to hyperproliferation in the lens epithelium.

The FGFR-Ras-MAPK signaling pathway has been implicated in the control of lens cell proliferation, differentiation and survival during embryonic development (Lovicu and McAvoy, 2001; Xie et al., 2006; Zhao et al., 2008). The mouse lens expresses a higher level of MAPK3 than MAPK1 protein (Fig. 1G), but the pMAPK1 level is higher than that of pMAPK3 (Fig. 6E), suggesting that MAPK1 is preferentially stimulated in the lens. In this study, we used a lens ectodermal driver (Le-Cre) to delete Mapk1 in the lens from the placode stage (E9.0). We demonstrate that the growth of Mapk1^CKO lens is severely retarded in contrast to the growth of the MAPK3-deficient lens, which did not display any defects (supplementary material Fig. S1I,J). We showed that MAPK1 is not required for early lens development (before E11.5) and is dispensable for fiber differentiation (Fig. 5), but is essential for cell proliferation in the germinative zone during the lens growth phase. An increase in MAPK3 activity in this region cannot compensate for the loss of MAPK1 (Fig. 6; supplementary material Fig. S4), suggesting that MAPK1 plays a unique role in maintaining the cell proliferation pattern during lens development.

Cell proliferation analysis (Fig. 3; supplementary material Fig. S2) suggests that lens cells in the germinative zone are not actively dividing and they mostly resemble the cells at the quiescent (G0) state. Cells can enter a quiescent stage if completion of one phase of the cycle does not occur successfully. Mechanistically, reduction in the cyclin D1 level could restrict the cells from passing the G1-S phase checkpoint. More importantly, a drastic reduction in the phospho-H3 index in Mapk1^CKO lenses (supplementary material Fig. S2) affects G2/M phase progression. Despite the proliferation defect in the lens germinative zone, fiber differentiation markers were not prematurely switched on in this region, suggesting that loss of MAPK1 leads to uncoupling of cell cycle exit and fiber cell differentiation in the equatorial zone of the lens. Thus, the role of MAPK1 in the lens germinative zone is to enhance the cell division rate while preventing cells from premature withdrawal from the cell cycle.

In all vertebrates, the growth of the lens follows a distinct, polarized pattern. Proliferating cells are restricted to the anterior lens epithelium and differentiating fiber cells are confined to the posterior hemisphere (Fig. 7). In the mid 1960s, the lens rotation experiment in chicken eye performed by Coulombre and Coulombre demonstrated that the state of the lens cell, either proliferating or differentiating, depends on its position in the eye (Coulombre and Coulombre, 1963). Within the lens epithelial layer the mitotic activity differs by region, although all of the cells retain the ability to proliferate (Lang, 1997). The highly mitotic region, which is defined as the lens germinative zone, is in close proximity to the anterior neural retina, which later differentiates into the iris and the ciliary body (Fig. 7). Various growth factors have been shown to be expressed in the anterior retina and developing iris/ciliary body (Reneker and Overbeek, 1996; Wilkinson et al., 1989). Both in vitro and in vivo studies have demonstrated that these growth factors can act as mitogens to stimulate cell proliferation (Iyengar et al., 2006; Potts et al., 1994; Reneker and Overbeek, 1996). The endogenous inductive molecules have not yet been identified and they are likely to play redundant roles in stimulating cell proliferation. Based on the data from previous and current studies, we proposed the following model to explain the high mitotic activity in the germinative zone during lens development (Fig. 7). After the lens formation stage (>E12.5), growth factors released from the anterior tip of the retina bind to the nearby lens cells and activate the RTK-Ras-MAPK1 signal transduction pathway to enhance the mitotic activity in this region. The downstream targets known to play important roles in cell cycle progression identified in this study include cyclin D1, phospho-H3 and survivin. Our data imply that MAPK1 has a unique role in the control of the lens cell proliferation pattern and that MAPK1 and MAPK3 are not functionally interchangeable.

We do not have a clear answer as to why MAPK1 and not MAPK3 functions uniquely in stimulating cell proliferation in the lens germinative zone. One explanation could be that MAPK1 has a higher capacity than MAPK3 to interact with the upstream activator MAPKK and therefore would enhance the signaling output (Vantaggiato et al., 2006). However, this model does not fit well with the observation that an increase in pMAPK3 level failed to restore the lens germinative zone in Mapk1^CKO lens (Fig. 6; supplementary material Fig. S4). An alternative explanation could be the different levels of effectiveness of MAPK1 and MAPK3 in activating the downstream targets leading to nuclear signaling. It is known that the nuclear localization of active MAPK is necessary for controlling the gene expression program stimulated by growth factors. MAPK1 and MAPK3 have been shown to differ drastically in their capacity to cross the nuclear envelope, contributing to the observation that MAPK1 is more efficient than MAPK3 in signaling to the nucleus (Marchi et al., 2008). Such a difference could make the transcription-dependent processes, such as G1 phase entry during the cell cycle, more dependent on MAPK1 than on MAPK3 (Baldin et al., 1993). We showed that MAPK1 proteins are mostly localized in the nuclei of the lens epithelial cells (Fig. 1C). We also showed that the cyclin D1 level was dramatically reduced in the germinative zone in Mapk1^CKO lens. Thus, our findings appear to support this model. In the lens germinative zone, MAPK1 function is indispensable because high mitotic activity is required. Even when MAPK3 activity was upregulated in P0 Mapk1^CKO lens, it did not restore the mitotic pattern (supplementary material Fig. S4). By contrast, in the central region of the lens epithelium, where mitotic activity is relatively low, MAPK3 is sufficient to compensate for the loss of MAPK1 in Mapk1^CKO lens.

Our study also indicates that cell proliferation in Mapk1^CKO lenses is unaffected before E11.5 (Fig. 1H,I; Fig. 2) and in the central region of the epithelium at a later stage (Fig. 3). To clarify the redundant role of MAPK1 and MAPK3 in cell proliferation during lens development, we recently generated Mapk1/3 double-deletion mice. The preliminary results indicate that cell proliferation before E11.5 is likely to be mediated by MAPK1/3-independent pathways (data not shown). It has been shown that BMP/activin-activated signals are essential for cell proliferation during early lens development (Rajagopal et al., 2009). Additionally, our results indicated that loss of both MAPK1 and MAPK3 in the lens drastically reduced cell proliferation throughout the entire epithelial layer at E14.5 (Fig. 3S-U), suggesting that MAPK1 and MAPK3 play a redundant role in the control of cell proliferation in the central region of the lens epithelium.

In this study, we have shown that apoptosis is significantly increased throughout the entire epithelial layer in the absence of MAPK1 (Fig. 4). Previous studies showed that disruption of cell cycle regulation can trigger apoptosis in the lens (Chen et al., 2000; Pan and Griep, 1995). In our study, cell death was found across the entire lens epithelial layer and was not limited to just the germinative zone, where cell proliferation was severely affected. Therefore, we speculate that apoptosis in the lens epithelium (at least in the central zone) is a direct effect of the absence of MAPK1 activity and not an indirect effect resulting from abnormal cell cycle regulation. We conclude that cell survival in the lens epithelium is dependent on MAPK1 activity and that MAPK3 activity alone is not sufficient.

FGF signaling is known to be essential for lens cell survival. Deletion of Fgfr2 alone, Fgfr1 and Fgfr2 or Fgfr1-3, all induced apoptosis in mouse lens (Garcia et al., 2011; Garcia et al., 2005; Zhao et al., 2008). Loss of FGFRs resulted in reduced or absent MAPK activity in the lens. Our study suggests that the FGFR-mediated survival signal is, at least in part, mediated through MAPK1 activation. How does MAPK1 regulate cell survival? One possibility is by regulating the level of survivin, which is a member of the inhibitor of apoptosis (IAP) family (Fig. 4). The function of survivin is to inhibit caspase 3 activation and thereby negatively regulate apoptosis (Li et al., 1998; Shin et al., 2001). We have demonstrated that MAPK1 deficiency results in a decrease in the survivin level (Fig. 4F,G) and in an increase in cleaved caspase 3 (Fig. 4E) in the peripheral region of the lens epithelium. It has been shown that MAPK activity plays a role in survivin expression and stability (Teh et al., 2004; Wang et al., 2010). However, other MAPK1-dependent survival signals must exist in the central epithelium during lens development that are independent of survivin.

In summary, the data presented in this study provide novel evidence concerning the unique role of MAPK1 in the control of the cell proliferation pattern during mouse lens development. MAPK1 appears to play an essential role in the lens germinative zone, whereas its function in cell proliferation in the central epithelium is likely to be redundant with MAPK3. Our study also shows that the overall MAPK activity in the lens epithelial layer is crucial for cell survival. However, MAPK1 deficiency does not block the process of epithelial-to-fiber differentiation. Future studies will focus on the combined deletion of MAPK1 and MAPK3 in the lens to elucidate the role of total MAPK activity in lens fiber differentiation and lens development.

We thank Drs Ruth Ashery-Padan and Gilles Pagès for permission to use the Le-Cre and Mapk3^-/- mice, respectively; Dr Samuel Zigler for anti-crystallin antibodies; and Drs Venkatesh Govindarajan, Paul Overbeek and Michael Robinson for insightful comments and suggestions.