A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development.

The cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27-) that abrogates all protein function and a knockin allele (p27CK-) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (~0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK-/CK- and p27-/- cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27-/- cells being highly restricted to their site of production and p27CK-/CK- cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.

3D map of the human corneal endothelial cell.

Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions.

Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area.

PURPOSE: The corneal endothelium is widely believed to consist of geometrically regular cells interconnected by junctional complexes. However, while en face visualization of the endothelial apical surface reveals characteristic polygonal borders, the overall form of the component cells has rarely been observed. METHODS: To visualize the shape of individual endothelial cells within the native monolayer, two independent Cre/LoxP-based cell labeling approaches were used. In the first, a P0-Cre mouse driver strain was bred to an R26-tdTomato reporter line to map neural crest-derived endothelial cells with cytosolic red fluorescent protein. In the second, HPRT-Cre induction of small numbers of green and red fluorescent protein-filled cells within a background of unlabeled cells was achieved using a dual-color reporter system, mosaic analysis with double markers (MADM). Selective imaging of the endothelial lateral membranes at different apicobasal levels was accomplished after staining with antibodies to ZO-1 and the neural cell adhesion molecule (NCAM). RESULTS: When viewed in their entirety in whole-mount preparations, fluorescent protein-filled cells appear star-shaped, extending multiple dendritic processes that radiate outward in the plane of the monolayer. Examination of rare cases where cells expressing different fluorescent proteins lie directly adjacent to one another reveals that these long processes undergo extensive interdigitation. The resulting overlap allows individual cells to extend over a greater area than if the cell boundaries were mutually exclusive. Anti-NCAM staining of these interlocking peripheral cell extensions reveals an elaborate system of lateral membrane folds that, when viewed in optical sections, increase in complexity from the apical to the basal pole. This not only produces a substantial increase in the basolateral, relative to the apical, membrane but also greatly extends the paracellular pathway as a highly convoluted space. CONCLUSIONS: Our analysis indicates that, far from being simple polygonal prisms, endothelial cells possess an elaborate multipolar shape. Their unusual geometry may be essential for the endothelium to carry out its role as the principal regulator of corneal extracellular fluid flux, and thus ultimately of tissue clarity.

Effect of neurturin deficiency on cholinergic and catecholaminergic innervation of the murine eye.

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123-4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4-6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 µm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1.

BACKGROUND: Mammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene. RESULTS: Histological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1-null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types. CONCLUSIONS: p27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia and tissue disruption seen in certain developing p27Kip1-null sensory organs, and may reflect a compensatory capability inherent in the regenerative taste system.

Defects in retinal pigment epithelium cell proliferation and retinal attachment in mutant mice with p27(Kip1) gene ablation.

PURPOSE: Little is known about the mechanisms that regulate cell cycle withdrawal of the retinal pigment epithelium (RPE) during development, or about the mechanisms maintaining epithelial cell quiescence in adult retinas. The present study examines the potential role of the negative cell cycle regulator p27(Kip1) in controlling RPE proliferation, using mice with targeted ablation of the p27(Kip1) gene. METHODS: Ocular tissues were obtained from wild-type and p27(Kip1)-null mice at several postnatal ages. Following aldehyde fixation, eyes were processed intact for JB-4 histology and electron microscopy. Alternatively, tissues were removed by manual or enzymatic dissection in order to obtain flat-mounts of the RPE attached to either the choroid-sclera or neural retina, respectively. Epithelial flat-mounts were either left unlabeled, in which case melanin pigment provided internal contrast, or labeled with Alexa Fluor 488-phalloidin and propidium iodide to visualize cell boundaries and nuclei, respectively. RESULTS: Morphometric analysis using transverse plastic sections revealed a 96% increase in nuclear density and a 12% increase in thickness (apical to basal) for mutant vs. normal epithelia at postnatal day 35 (P35). These changes were not restricted to central or peripheral regions, and were uncorrelated with focal areas of dysplasia seen in the mutant neural retina. When similar tissues were viewed as flat-mounts, an observed 100% increase in nuclear density was accompanied by only a 46% enhancement in cellular density. This resulted in a larger proportion of multinucleated cells in the nullizygous RPE as compared with the wild-type epithelium (91 versus 47%). Such a pattern was achieved relatively early in development since, at P7 when the increase in RPE nuclear density was essentially complete, cellular density was augmented by only 39%. In addition to these proliferative changes, individual epithelial cells sometimes exhibited structural abnormalities, including an altered cortical actin cytoskeleton and displacement of nuclei from their normal central position. Surprisingly, while the RPE cells of null animals were similar ultrastructurally to those of the wild-type, interdigitation of their microvillous processes with outer segments was incomplete. Quantitative analysis revealed that such areas of detachment characterize, on average, 42% of the nullizygous retina, and that there is little correlation between detachment and neural retina dysplasia from one eye to another. Together with parallel evidence demonstrating a substantial decline in the apparent adhesiveness of mutant retinas relative to the normal tissue, the data is strongly indicative of an altered epithelium-photoreceptor interaction following gene ablation. CONCLUSIONS: The absence of a functional p27(Kip1) gene results in enhanced RPE nuclear division, without a commensurate increase in cell division. Although the mutant epithelium as a whole appears structurally normal, individual cells exhibit cytoskeletal changes and their interaction with the neural retina is compromised.

Epidermal growth factor stimulation of RPE cell survival: contribution of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways.

Epidermal growth factor (EGF) previously has been shown to stimulate short-term survival in vitro of cells derived from the native amphibian retinal pigment epithelium (RPE). In the present experiments, we have examined intracellular signaling pathways responsible for mediating these survival-specific growth factor effects, distinct from proliferative effects, using the human epithelial cell line RPE D407. When maintained as single cells in suspension culture in the absence of serum and exogenous survival factors, RPE D407 cell viability gradually declined over a 3-4 day period as a result of apoptotic cell death, a pattern similar to that seen for eye-derived RPE cells. Exposure to EGF (50 ng ml(-1)) enhanced cell survival by nearly 40% and caused a parallel increase in the tyrosine phosphate content of the EGF receptor (EGFR), as determined by immunoprecipitation and Western blotting. Both effects were completely blocked by 1 microm AG1478, an EGFR-selective tyrosine kinase inhibitor. EGF also stimulated phosphorylation of the phosphatidylinositol 3'-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). Furthermore, EGF-induced protection was substantially reduced by either the PI3K inhibitor LY294002 (25 microm) or the MEK inhibitor U0126 (10 microm), under conditions in which phosphorylation of Akt and ERK1/2, respectively, was blocked. Our results indicate that EGF-stimulated survival of RPE D407 cells takes place as a result of signaling through both PI3K and ERK/MAPK pathways. Further, residual anti-apoptotic activity stimulated by EGF in the presence of both blockers suggests that additional as yet unidentified growth factor-dependent survival pathways exist.

Expression of the cyclin-dependent kinase inhibitor p27Kip1 by developing retinal pigment epithelium.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.