Bone morphogenetic protein 4 mediates apoptosis of capillary endothelial cells during rat pupillary membrane regression

Programmed capillary regression is essential for development, but little is known about the mechanism behind this phenomenon. In this study, we characterized the molecular determinants of capillary regression utilizing the pupillary membrane (PM) in the newborn rat's eye. We observed in the 1-day-culture system that apoptotic endothelial cells decrease in number with the addition of a natural antagonist, Noggin, strongly suggesting the involvement of the bone morphogenetic protein (BMP) family in PM regression. In addition, the lens-conditioned medium (Lens-CM) induced apoptosis of HUVE cells and inhibited endothelial tubulogenesis, which were completely blocked by both Noggin and the BMP4-specific neutralizing antibody. Activation of BMP4 pathway in endothelial cells was confirmed by both the up-regulation of Msx genes correlated with apoptosis and the translocation of Smad1 into the nucleus. We showed a transient expression of BMP4 in Lens-CM by immunoprecipitation assay. Furthermore, the transcorneal injection of BMP4 in rats enhanced the apoptosis of PMs, while that of Noggin attenuated it. These results indicate that BMP4 pathways play pivotal roles in capillary regression in a paracrine manner between lens and PMs.     

VEGF deprivation-induced apoptosis is a component of programmed capillary regression

The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis.     

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.     

Glucosaminidase and Dedifferentiation of Newt Iris Epithelium

To try to understand the mechanism of the dedifferentiation process which occurs during metaplastic transformation of iris epithelial cells into lens cells in newt lens regeneration, the activity of N -acetylglucosaminidase in iris and iris epithelium was studied as a function of time after lentectomy. The activity was found to increase during the dedifferentiation phase of the iris epithelium. The dorsal iris, where definite dedifferentiation occurs side by side with incomplete dedifferentiation, shows significantly greater enhancement of the activity than the ventral iris, where only incomplete dedifferentiation takes place. When the cells complete dedifferentiation and engage in redifferentiation into lens cells, the level of activity drops, approaching that of the normal lens. Evidence is also presented for release of the enzyme into the ocular fluid during dedifferentiation. The possibility that the enzyme is involved in surface alterations of iris epithelial ceils engaged in dedifferentiation is discussed.     

Apoptotic force: active mechanical function of cell death during morphogenesis

Apoptosis, or programmed cell death, is an essential process for the elimination of unnecessary cells during embryonic development, tissue homeostasis, and certain pathological conditions. Recently, an active mechanical function of apoptosis called apoptotic force has been demonstrated during a tissue fusion process of Drosophila embryogenesis. The mechanical force produced during apoptosis is used not only to force dying cells out from tissues in order to keep tissue integrity, but also to change the morphology of neighboring cells to fill the space originally occupied by the dying cell. Furthermore, the occurrence of apoptosis correlates with tissue movement and tension of the tissue. This finding suggests that apoptotic forces might be harnessed throughout cell death-related morphogenesis; however, this concept remains to be fully investigated. While the investigation of this active mechanical function of apoptosis has just begun, here we summarize the current understandings of this novel function of apoptosis, and discuss some possible developmental processes in which apoptosis may play a mechanical role. The concept of apoptotic force prompts a necessity to rethink the role of programmed cell death during morphogenesis.     

The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation

The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.     

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.     

Cellular analysis on localization of lens forming potency in the newt iris epithelium

The localization of a lens forming potency in the iris epithelium was studied by autoradiographic analysis of the distribution of ³H-thymidine labelled cells to be participated in lens regeneration in newts. DNA synthesis started from the dorsal portion of the iris epithelium around 4 days after lentectomy. 5 days after lentectomy, a large number of labelled cells were mostly found in the dorsal sector, showing strong contrast to the ventral and lateral sectors of iris, which contained a few labelled cells. The labelled index (the number of labelled cells/the number of cells in the definite pigmented area of the iris epithelium) of the dorsal sector attained the highest value, 29.7 ± 2.35, on day 7 after lentectomy, and dropped temporarily. This was followed by the second peak on day 12. The dorso-ventral ratio of the labelled index reached to the highest value, 6.87 ± 0.67, on day 5. This ratio decreased rapidly after the completion of a lens rudiment, and it became about 1. In “chase” experiments by diluting the radio-isotope with excess cold thymidine, it was obviously shown that most of the cells labelled with the radio-isotope and distributed in the dorsal marginal iris 5 days after lentectomy participated in the formation of a lens regenerate during the period of chasing. From these results, the following conclusion was drawn. The iris epithelium consists of at least 2 different cell populations; one is capable of transformation into lens cells and is distributed mostly in the dorsal portion of the iris epithelium, while the other has no potency for transformation and is able to grow to compensate a loss of the dorsal marginal cells which transformed into lens cells during the process of lens regeneration.     

A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens

The anterior segment of the vertebrate eye includes the cornea, iris, ciliary body, trabecular meshwork, and lens. Although malformations of these structures have been implicated in many human eye diseases, little is known about the molecular mechanisms that control their development. To identify genes involved in anterior segment formation, we developed a large-scale in situ hybridization screen and examined the spatial and temporal expression of over 1000 genes during eye development. This screen identified 62 genes with distinct expression patterns in specific eye structures, including several expressed in novel patterns in the anterior segment. Using these genes as developmental markers, we tested for the presence of inductive signals that control the differentiation of anterior segment tissues. Organ culture recombination experiments showed that a chick lens is capable of inducing the expression of markers of the presumptive iris and ciliary body in the developing mouse neural retina. The inducing activity from the lens acts only over short ranges and is present at multiple stages of eye development. These studies provide molecular evidence that an evolutionarily conserved signal from the lens controls tissue specification in the developing optic cup.     

Regulated lens regeneration from isolated pigmented epithelial cells of newt iris in culture in response to FGF2/4

When a lens is removed from the newt eye, a new lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish lens regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a lens with the synthesis of lens-specific crystallins, recapitulating the normal lens regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro lens regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in lens forming ability between them.     

Intracellular levels of adenosine 3':5'-cyclic monophosphate in the dorsal iris of the adult newt, during lens regeneration

The intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of cAMP. After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of cAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.     

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. Here, we show the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, and completed a full 360° rotation. This acceleration was impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that formed a ring shape and surrounded the male genitalia: the inner ring rotated with the genitalia and the outer ring rotated later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlated with the movement of the outer ring, and upregulation of the apoptotic signal increased the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame.     

Paracrine-mediated apoptosis in reproductive tract development

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions.     

Intracellular Levels of Adenosine 3':5'-Cyclic Monophosphate in the Dorsal Iris of the Adult Newt, During Lens Regeneration

The intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) were measured in the dorsal iris of the adult newt, during the first 20 days of lens regeneration. It was found that by day 2 after lens removal there is a significant drop in the levels of cAMP. After day 2 the levels of the nucleotide increase and by day 3 they are higher than those detected on day 0. The levels of cAMP remain high up to day 8. From day 8 to day 9 there is a second drop. From day 9 to day 20 the levels of cAMP did not differ significantly from the value obtained for day 0, except for days 10, 12, and 15. The period of high levels of cAMP coincides with the period of depigmentation of iris epithelial cells, the key event of lens regeneration.     

葛根素减轻部分由过氧亚硝基阴离子导致的糖尿病大鼠晶状体上皮细胞凋亡

本研究观察葛根素是否减轻部分由过氧亚硝基阴离子（peroxynitrite,ONOO^-）导致的糖尿病大鼠晶状体上皮细胞（lens epithelium cell,LEC）凋亡。采用大鼠腹腔注射链脲佐菌素（streptozotocin,STZ）的方法建立糖尿病动物模型。36只大鼠作为对照组,腹腔注射生理盐水;其他72只大鼠腹腔注射STZ（45mg/kg）后分为STZ组和STZ＋葛根素组,每组36只。STZ注射3d后,STZ＋葛根素组大鼠每天腹腔注射葛根素（140mg／kg）。于实验开始后第20、40和60天用裂隙灯检查晶状体的形态学变化后处死动物。用流式细胞仪检测LEC凋亡,用免疫组化方法检测晶状体中ONOO^-的标志物——硝基酪氨酸（nitrotyrosine,NT）的表达,用基因芯片分析技术检测LEC凋亡相关基因iNOS等的表达。结果发现,对照组大鼠晶状体均透明,各项指标基本正常;STZ组大鼠第20天时即出现晶状体混浊,40-60d期间混浊不断加重;STZ＋葛根素组大鼠20-40d时晶状体混浊呈加重趋势,但40-60d以后明显减轻。对照组LEC轻度凋亡,而STZ组凋亡细胞呈持续性增长,STZ＋葛根素组大鼠20-40d时细胞凋亡呈增长趋势,但40-60d以后明显下降。对照组大鼠晶状体NT未见明显表达;STZ组大鼠NT表达明显加强;STZ＋葛根素组大鼠20-40d时NT表达呈增长趋势,但40-60d以后明显下降。对照组凋亡相关基因未见明显变化,STZ组凋亡相关基因iNOS表达明显上调。其他凋亡相关基因如BCL-2、SOD表达明显下调,但NF-κB和TNFR1-FADD-caspase信号转导途径明显上调;STZ＋葛根素组凋亡相关基因表达则呈相反改变。上述结果表明,在糖尿病大鼠晶状体中有ONOO^-的标志物NT表达,证明糖尿病大鼠LEC凋亡部分由ONOO^-诱导,这可能是氧化损伤导致白内障形成的新途径。葛根素能够部分逆转ONOO^-对LEC的致凋亡作用,提示葛根素可能是治疗糖尿病性白内障的有效药物,其治疗机制可能与葛根素直接抑制凋亡和对抗ONOO^-对糖尿病大鼠LEC的损伤有关。     

Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals

Cataract is a major ocular disease that causes blindness in many developing countries of the world. It is well established that various factors such as oxidative stress, UV, and other toxic agents can induce both in vivo and in vitro cataract formation. However, a common cellular basis for this induction has not been previously recognized. The present study of lens epithelial cell viability suggests such a general mechanism. When lens epithelial cells from a group of 20 cataract patients 12 to 94 years old were analyzed by terminal deoxynucleotidyl transferase (TdT) labeling and DNA fragmentation assays, it was found that all of these patients had apoptotic epithelial cells ranging from 4.4 to 41.8%. By contrast, in eight normal human lenses of comparable age, very few apoptotic epithelial cells were observed. We suggest that cataract patients may have deficient defense systems against factors such as oxidative stress and UV at the onset of the disease. Such stress can trigger lens epithelial cell apoptosis that then may initiate cataract development. To test this hypothesis, it is also demonstrated here that hydrogen peroxide at concentrations previously found in some cataract patients induces both lens epithelial cell apoptosis and cortical opacity. Moreover, the temporal and spatial distribution of induced apoptotic lens epithelial cells precedes development of lens opacification. These results suggest that lens epithelial cell apoptosis may be a common cellular basis for initiation of noncongenital cataract formation.     

Pax-6 and Prox 1 expression during lens regeneration from Cynops iris and Xenopus cornea: evidence for a genetic program common to embryonic lens development

Lens regeneration from non-lens ocular tissues has been well documented in amphibians, from the dorsal iris in the newt and from the outer cornea in Xenopus. To understand the early molecular events which govern lens regeneration, we examined the expression of two early marker genes of normal lens development, Pax-6 and Prox 1. In both Cynops (newt) iris and Xenopus cornea, Pax-6 is expressed soon after lentectomy in a region broader than that giving rise to the regenerating lens, indicative of an important role for Pax-6 in determination of the regeneration potential. Then Prox 1 expression begins within the Pax-6-expressing tissue, and these Prox 1-expressing cells give rise to the regenerating lens. This sequence of events also takes place in the lens placode of the embryo, indicating that the presence of the same genetic program operates in both embryonic lens development and lens regeneration, at least partly. In the Cynops iris, Pax-6 expression occurs initially in the entire marginal region of the iris after lentectomy but then becomes restricted to the dorsal region. Further studies are expected to elucidate the mechanism of this long-standing problem of the dorsal-restriction of lens regeneration from the newt iris.     

Regulation of apoptotic signal by strontium in immunocytes

Exposure to heavy metals can initiate the development of negative effects in different organs and systems, including the immune system, and can be manifested as dysfunction of receptor systems and intracellular signaling. The participation of stable strontium (Sr²⁺) in the regulation of apoptotic signals in immunocompetent cells was analyzed. Various mechanisms of strontium-induced modulation of the apoptotic lymphocyte reaction were described, and the formation of intracellular signal transduction involving Sr²⁺ was defined. The flow cytometry method was used to study changes in membrane and intracellular markers of apoptosis in children who consumed drinking water with the elevated levels of strontium. It was shown that the content of strontium in blood in the range of 0.0129–0.173 mg/dm³ affected different ways of apoptosis regulation (CD95-, p53-, and TNF-induced apoptosis) and their integration sites, thereby reducing the transmission of apoptotic signal in immunocompetent cells, and formed an alternative pathway of the cell elimination based on the mechanism of necrosis.     

Tissue factor expression in newt iris coincides with thrombin activation and lens regeneration

Lens regeneration in adult salamanders occurs at the pupillary margin of the mid-dorsal iris where pigmented epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. It is not understood how the injury caused by removal of the lens (lentectomy) in one location is linked to initiating the response in a different spatial location (dorsal iris) and to this particular sector. We propose that the blood provides a link between the localised coagulation and signal transduction pathways that lead to regeneration. A transmembrane protein (tissue factor) is expressed in a striking patch-like domain in the dorsal iris of the newt that localises coagulation specifically to this location, but is not expressed in the axolotl, a related species that does not show thrombin activation after lentectomy and cannot regenerate its lens. Our hypothesis is that tissue factor expression localises the initiation of regeneration through the activation of thrombin and the recruitment of blood cells, leading to local growth factor release. This is the first example of gene expression in a patch of cells that prefigures the location of a regenerative response, and links the immune system with the initiation of a regenerative program.     

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.