Conditional ablation of the Notch2 receptor in the ocular lens

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.     

Duration of ERK1/2 phosphorylation induced by FGF or ocular media determines lens cell fate

The ocular environment is important for the establishment and maintenance of lens growth patterns and polarity. In the anterior chamber of the eye, the aqueous humour regulates lens epithelial cell proliferation whereas in the posterior, the vitreous humour regulates the differentiation of the lens cells into fiber cells. Members of the fibroblast growth factor (FGF) growth factor family have been shown to induce lens epithelial cells to undergo cell division and differentiate into fibers, with a low dose of FGF able to induce cell proliferation (but not fiber differentiation), and higher doses required to induce fiber differentiation. Both these cellular events have been shown to be regulated by the MAPK/ERK1/2 signalling pathway. In the present study, to better understand the contribution of ERK1/2 signalling in regulating lens cell proliferation and differentiation, we characterized the ERK1/2 signalling profiles induced by different doses of FGF, and compared these to those induced by the different ocular media. Here, we show that FGF induced a dose-dependent sustained activation of ERK1/2, with both a high (fiber differentiating) dose of FGF and vitreous, stimulating and maintaining a prolonged (up to 18 hr) ERK1/2 phosphorylation profile. In contrast, a lower (proliferating) dose of FGF, and aqueous, stimulated ERK1/2 phosphorylation for only up to 6 hr. If we selectively reduce the 18 hr ERK1/2 phosphorylation profile induced by vitreous to 6 hr, by specifically blocking FGF receptor signalling, the vitreous now fails to induce lens fiber differentiation but retains the ability to induce lens cell proliferation. These findings not only provide insights into the important role that FGF plays in the different ocular media that bathe the lens, but enlighten us on some of the putative molecular mechanisms by which one specific growth factor, in this case FGF, can elicit a different cellular response in the same cell type.     

Essential role of BMPs in FGF-induced secondary lens fiber differentiation

It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.     

Genetic epistasis between heparan sulfate and FGF-Ras signaling controls lens development

Vertebrate lens development depends on a complex network of signaling molecules to coordinate cell proliferation, migration and differentiation. In this study, we have investigated the role of heparan sulfate in lens specific signaling by generating a conditional ablation of heparan sulfate modification genes, Ndst1 and Ndst2. In this mutant, N-sulfation of heparan sulfate was disrupted after the lens induction stage, resulting in reduced lens cell proliferation, increased cell death and defective lens fiber differentiation in later lens development. The loss of Ndst function also prevented the assembly of Fgf/Fgfr complexes on the lens cell surface and disrupted ERK signaling within the lens. We further demonstrated that Ndst mutation completely inhibited the FGF1 and Fgf3 overexpression phenotypes, but Kras reactivation was sufficient to reverse the Ndst deficient lens differentiation defect. The epistatic relationship between Ndst and FGF–Ras signaling demonstrates that FGF signaling is the predominant signaling pathway controlled by Ndst in lens development.     

FGF signals induce Caprin2 expression in the vertebrate lens

The lens of the eye is derived from the non-neural ectoderm situated next to the optic vesicle. Fibroblast growth factor (FGF) signals play a major role at various stages of vertebrate lens development ranging from induction and proliferation to differentiation. Less is however known about the identity of genes that are induced by FGF activity within the lens. We have isolated and characterized mouse cytoplasmic activation/proliferation-associated protein-2 (Caprin2), with domains belonging to both the Caprin family and the C1q and tumour necrosis factor (TNF) super-family. Here we show that Caprin2 is expressed in the developing vertebrate lens in mouse and chick, and that Caprin2 expression is up-regulated in primary lens fiber cells, after the induction of crystallins the earliest known markers for differentiated lens fiber cells. Caprin2 is subsequently down-regulated in the centre of the lens at the time and at the position of the first fiber cell denucleation and terminal differentiation. In vitro analyses of lens fiber cell differentiation provide evidence that FGF activity emanating from neighboring prospective retinal cells is required and that FGF8 activity is sufficient to induce Caprin2 in lens fiber cells. These results not only provide evidence that FGF signals induce the newly characterized protein Caprin2 in the lens, but also support the general idea that FGF signals are required for lens fiber cell differentiation.     

Notch signaling regulates growth and differentiation in the mammalian lens

The Notch signal transduction pathway regulates the decision to proliferate versus differentiate. Although there are a myriad of mouse models for the Notch pathway, surprisingly little is known about how these genes regulate early eye development, particularly in the anterior lens. We employed both gain-of-function and loss-of-function approaches to determine the role of Notch signaling in lens development. Here we analyzed mice containing conditional deletion of the Notch effector Rbpj or overexpression of the activated Notch1 intracellular domain during lens formation. We demonstrate distinct functions for Notch signaling in progenitor cell growth, fiber cell differentiation and maintenance of the transition zone. In particular, Notch signaling controls the timing of primary fiber cell differentiation and is essential for secondary fiber cell differentiation. Either gain or loss of Notch signaling leads to formation of a dysgenic lens, which in loss-of-function mice undergoes a profound postnatal degeneration. Our data suggest both Cyclin D1 and Cyclin D2, and the p27^Kip1 cyclin-dependent kinase inhibitor act downstream of Notch signaling, and define multiple critical functions for this pathway during lens development.     

FGF10 signaling maintains the pancreatic progenitor cell state revealing a novel role of Notch in organ development

FGF10 plays an important role in the morphogenesis of several tissues by control of mesenchymal-to-epithelial signaling. In the pancreas, mesenchymal FGF10 is required to maintain the Pdx1-expressing epithelial progenitor cell population, and in the absence of FGF10 signaling, these cells fail to proliferate. Ectopic expression of FGF10 in the pancreatic epithelium caused increased proliferation of pancreatic progenitor cells and abrogation of pancreatic cell differentiation of all cell types. A hyperplastic pancreas consisting of undifferentiated cells expressing Pdx1, Nkx6.1, and cell adhesion markers normally characterizing early pancreatic progenitor cells resulted. Differentiation was attenuated even as proliferation of the pancreatic cells slowed during late gestation, suggesting that the trophic effect of FGF10 was independent of its effects upon cell differentiation. The FGF10-positive pancreatic cells expressed Notch1 and Notch2, the Notch-ligand genes Jagged1 and Jagged2, as well as the Notch target gene Hes1. This activation of Notch is distinct from the previously recognized mechanism of lateral inhibition. These data suggest that FGF10 signaling serves to integrate cell growth and terminal differentiation at the level of Notch activation, revealing a novel second role of this key signaling system during pancreatic development.     

Kynurenine inhibits fibroblast growth factor 2-mediated expression of crystallins and MIP26 in lens epithelial cells

Fibroblast growth factor-2 (FGF2)-mediated signaling plays an important role in fiber cell differentiation in eye lens. We had previously shown that kynurenine (KYN) produced from the overexpression of indoleamine 2,3-dioxygenase (IDO) causes defects in the differentiation of fiber cells, induces fiber cell apoptosis and cataract formation in the mouse lens, and leads to cell cycle arrest in cultured mouse lens epithelial cells (mLEC). In this study, we demonstrate that exogenous KYN reduces FGF2-mediated expression of α-, β-, and γ-crystallin and MIP26 in mLEC. We show that endogenously produced KYN in mLEC of IDO transgenic animals causes similar defects in FGF2-induced protein expression and that a competitive inhibitor of IDO prevents such defects. Our data also show that KYN inhibits FGF2-induced Akt and ERK1/2 phosphorylation in mLEC, which are required for crystallin and MIP26 expression in the lens. KYN does not inhibit FGF2 binding to cells but inhibit phosphorylation of FGFR1in mLEC. Together our data suggest that KYN might inhibit FGF2-mediated fiber cell differentiation by preventing expression of crystallins and MIP26. Our studies provide a novel mechanism by which KYN can exert deleterious effects in cells.     

FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.     

Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27^kip1 and p57^kip2, increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and α-, β- and γ-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.     

Role of the FGF and MEK signaling pathway in the ascidian embryo

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.     

Notch signaling regulates the pattern of auditory hair cell differentiation in mammals

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the K?lliker's organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation.     

Notch and TGF-β pathways cooperatively regulate receptor protein tyrosine phosphatase-κ (PTPRK) gene expression in human primary keratinocytes

Receptor protein tyrosine phosphatase-κ (PTPRK) specifically and directly dephosphorylates epidermal growth factor receptor (EGFR), thereby limiting EGFR function in primary human keratinocytes. PTPRK expression is increased by the TGF-β/Smad3 pathway and cell–cell contact. Because the Notch receptor pathway is responsive to cell–cell contact and regulates keratinocyte growth and differentiation, we investigated the interplay between Notch and TGF-β pathways in regulation of PTPRK expression in human keratinocytes. Suppression of Notch signaling by γ-secretase inhibitors substantially reduced cell contact induction of PTPRK gene expression. In sparse keratinocyte cultures, addition of soluble Notch-activating ligand jagged one peptide (Jag1) induced PTPRK. Of interest, cell contact–induced expression of TGF-β1 and TGF-β receptor inhibitor SB431542 inhibited contact-induced expression of PTPRK. Furthermore, inhibition of Notch signaling, via knockdown of Notch1 or by γ-secretase inhibitors, significantly reduced TGF-β–induced PTPRK gene expression, indicating that Notch and TGF-β pathways function together to regulate PTPRK. Of importance, the combination of Jag1 plus TGF-β results in greater PTPRK expression and lower EGFR tyrosine phosphorylation than either ligand alone. These data indicate that Notch and TGF-β act in concert to stimulate induction of PTPRK, which suppresses EGFR activation in human keratinocytes.     

Equarin is involved as an FGF signaling modulator in chick lens differentiation

Lens growth involves the proliferation of epithelial cells, followed by their migration to the equator region and differentiation into secondary fiber cells. It is widely accepted that fibroblast growth factor (FGF) signaling is required for the differentiation of lens epithelial cells into crystallin-rich fibers, but this signaling is insufficient to induce full differentiation. To better understand lens development, investigatory and functional analyses of novel molecules are required. Here, we demonstrate that Equarin, which is a novel secreted molecule, was expressed exclusively in the lens equator region during chick lens development. Equarin upregulated the expression of fiber markers, as demonstrated using in ovo electroporation. In a primary lens cell culture, Equarin promoted the biochemical and morphological changes associated with the differentiation of lens epithelial cells to fibers. A loss-of-function analysis was performed using zinc-finger nucleases targeting the Equarin gene. Lens cell differentiation was markedly inhibited when endogenous Equarin was blocked, indicating that Equarin was essential for normal chick lens differentiation. Furthermore, biochemical analysis showed that Equarin directly bound to FGFs and heparan sulfate proteoglycan and thereby upregulated the expression of phospho-ERK1/2 (ERK-P) proteins, the downstream of the FGF signaling pathway, in vivo and in vitro. Conversely, the absence of endogenous Equarin clearly diminished FGF-induced fiber differentiation. Taken together, our results suggest that Equarin is involved as an FGF modulator in chick lens differentiation.     

The stability of the lens-specific Maf protein is regulated by fibroblast growth factor (FGF)/ERK signaling in lens fiber differentiation

Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation.     

Notch ligand-dependent gene expression in human endometrial stromal cells

The purpose of this study is to investigate the expression patterns and role of Notch signaling in human endometrial cells. Notch receptors, Notch 1-3 were expressed in both endometrial epithelial and stromal cells. Notch ligands, Jag1 and Dll4 and Notch target genes, Hes1 and Hey1 were predominantly expressed in endometrial epithelial cells and scarce in stromal cells. Increased de novo synthesis of Dll4 or Jag1 in stromal cells by retroviral delivery significantly induced Hes1 and Hey1. Evaluations of global gene expression by microarrays revealed that more than 400 genes in stromal cells were significantly regulated by Jag1. Gene annotation-based functional analysis classified these genes into biological processes of cell adhesion, cell structure and motility, cell communication, cell cycle, and angiogenesis. This study provides evidence that Notch ligands control the Notch gene activities and may enhance development of human endometrium.     

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.