Richard J.W. Stump

A role for Wnt/β-catenin signaling in lens epithelial differentiation

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/β-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the β-catenin pathway. To access the role of this signaling pathway in the lens, we analyzed mice with a null mutation of lrp6. These mice had small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. We also showed that multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, were detected in the lens. Expression of these molecules was generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, we also showed the expression of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells.

Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture

How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.

Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition.

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1–30 mM LiCl we also showed that cell proliferation is inhibited in a dose‐dependent manner. Confocal microscopy and immunohistochemistry for ZO‐1 and E‐cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl‐treated explants maintained the apical baso‐lateral polarity and cobblestone‐like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract‐promoting effects of transforming growth factor β (TGFβ) on lens epithelial cells. In TGFβ‐treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing α‐smooth muscle actin (α‐SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of α‐SMA and maintained the cells in a polarized, adherent, cobblestone‐packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract. Copyright

TGFβ promotes Wnt expression during cataract development

TGFbeta induces lens epithelial cells to undergo epithelial mesenchymal transition (EMT) and many changes with characteristics of fibrosis including posterior capsular opacification (PCO). Consequently much effort is directed at trying to block the damaging effects of TGFbeta in the lens. To do this effectively it is important to know the key signaling pathways regulated by TGFbeta that lead to EMT and PCO. Given that Wnt signaling is involved in TGFbeta-induced EMT in other systems, this study set out to determine if Wnt signaling has a role in regulating this process in the lens. Using RT-PCR, in situ hybridization and immunolocalization this study clearly shows that Wnts 5a, 5b, 7b, 8a, 8b and their Frizzled receptors are upregulated in association with TGFbeta-induced EMT and cataract development. Both rat in vitro and mouse in vivo cataract models show similar profiles for the Wnt and Frizzled mRNAs and proteins that were assessed. Currently it is not clear if the canonical beta-catenin/TCF signaling pathway, or a non-canonical pathway, is activated in this context. Overall, the results from the current study indicate that Wnt signaling is involved in TGFbeta-induced EMT and development of fibrotic plaques in the lens.

Sfrp1 and Sfrp2 are not involved in Wnt/β-catenin signal silencing during lens induction but are required for maintenance of Wnt/β-catenin signaling in lens epithelial cells

During eye lens development, regulation of Wnt/β-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/β-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/β-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/β-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/β-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/β-catenin signaling in lens epithelial cells.

Linear growth in spines from Acanthaster planci (L.) involving growth lines and periodic pigment bands

We report the first observations of a linear growth pattern in aboral spine ossicles of adult Acanthaster planci (L.). This is unlike the spine development of other echinoderms. Growth in aboral spine ossicles of A. planci is essentially by addition of stereom at the base and the spines growth history is preserved along its length. There are numerous growth lines perpendicular to the long axis of the ossicle. These are clearly evident in longitudinal spine sections and apparently caused by frequent growth episodes. There are periodic pigment bands which are parallel to the growth lines and evident on the surface of the ossicle. Basal growth of the spine ossicle and the nature of the growth lines were confirmed by tetracycline staining. Size/frequency analyses of a population of A. planci from Davies Reef (GBR) found spine ossicle growth, but not body diameter growth, over the six month period between sampling dates. The additional pigment banding in spine ossicles of 4 individuals recaptured after 6 months suggests that pigment bands are laid down seasonally. If pigment band cyclicity is validated, it offers a simple method for ageing adults of A. planci in field populations.

Age estimation and patterns of growth in Acanthaster planci: a reply to Souter et al. (1997)

[Extract] The conclusion by Souter et al. (1997) that spine pigment band count (SPBC) is not an appropriate method to estimate age in Acanthaster planci is not justified. They are incorrect to suggest that the ages derived from the SPBC method bear no relationship to the actual age of the specimen, because their study did not test the SPBC method proposed by Stump and Lucas (1990) and further confirmed by Stump (1994, 1996). Instead, Souter et al. (1997) sampled spines from all over the aboral surfaces of five specimens, including regenerated arms. They found that numbers of pigment bands declined progressively in spines away from the longest spines at the midline base of arms and were less than maximal in regenerating arms. By employing an indiscriminant sampling method, they showed a potential for spines from adult A. planci to have variation in banding patterns. However, the SPBC method recognizes spine banding variation within individuals and specifies that the longest spines must be carefully sampled from the proximal area of whole arms; this allows for possible effects from spine autotomy, sublethal predation and regeneration. In addition, the interpretation of their results is not well founded. Ultimately, the testing of the validity of age-determining methods can be achieved only through medium-term to long-term mark recapture studies or by identifying known-age individuals in populations (Beamish and McFarlane 1983).