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

During eye lens development, regulation of Wnt/ß-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/ß-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/ß-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/ß-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/ß-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/ß-catenin signaling in lens epithelial cells.

Synthesis and in vitro evaluation of analogues of avocado-produced toxin (+)-(R)-persin in human breast cancer cells.

A structure-activity study of several new synthetic analogues of the avocado-produced toxin persin has been conducted, with compounds being evaluated for their cytostatic and pro-apoptotic effects in human breast cancer cells. A 4-pyridinyl derivative demonstrated activity comparable to that of the natural product, suggesting future directions for exploration of structure-activity relationships.

Non-essential role for cilia in coordinating precise alignment of lens fibres.

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet-Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

PURPOSE: The Fat family of atypical cadherins, originally identified in Drosophila, play diverse roles during embryogenesis and adult tissue maintenance. Among four mammalian members, Fat1 is essential for kidney and muscle organization, and is also essential for eye development; Fat1 knockout causes partial penetrant microphthalmia or anophthalmia. To account for the partial penetrance of the Fat1 phenotype, involvement of Fat4 in eye development was assessed. Lens phenotypes in Fat1 and 4 knockouts were also examined. METHODS: Fat1 and Fat4 mRNA expression was examined by in situ hybridization. Knockout phenotypes of Fat1 and Fat4 were analyzed by hematoxylin and eosin (H&E) and immunofluorescent staining. RESULTS: We found Fat4 knockout did not affect eye induction or enhance severity of Fat1 eye defects. Although Fat1 and Fat4 mRNAs are similarly expressed in the lens epithelial cells, only Fat1 knockout caused a fully penetrant lens epithelial cell defect, which was apparent at embryonic day 14.5 (E14.5). The columnar structure of the lens epithelial cells was disrupted and in some regions cell aggregates were formed. In these multilayered regions, apical cell junctions were fragmented and the apical-basal polarity was lost. EdU incorporation assay also showed enhanced proliferation in the lens epithelial cells. Interestingly, these defects were found mainly in the central zone of the epithelial layer. The lens epithelial cells of the germinative zone maintained their normal morphology and fiber differentiation occurred normally at the equator. CONCLUSIONS: These observations indicate that Fat1 is essential for lens epithelial cell polarity and proliferation but not for terminal differentiation.

Cone degeneration in aging and age-related macular degeneration.

OBJECTIVE: To examine the morphological features of macular photoreceptors in histologically normal retina from normal donor eyes and eyes with age-related macular degeneration (AMD). METHODS: The macular region was excised from 18 donor eyes (aged 22-96 years) and cryosectioned. Sections were stained with hematoxylin-eosin or double immunolabeled using opsin antibodies or synaptic markers. RESULTS: Three of 8 retinas studied in detail had AMD lesions; the remainder were histologically normal. Immunoreactivity to cone opsin was abnormal in parts of all retinas (3.5%-95.0% of each sample) and was associated with swelling of and altered immunoreactivity in the cone distal axon. In non-AMD retinas, the anomalies were mainly in nonfoveal macular locations. The nature of the anomalies was identical in non-AMD retinas and in parts of AMD retinas adjacent to overt degeneration. CONCLUSION: Redistribution of opsin and anomalies in the distal cone axon are common in the aging human macula and may indicate susceptibility to AMD. CLINICAL RELEVANCE: The findings are consistent with tests of cone function in aging and early AMD, which suggests that integrated cone functions--including contrast sensitivity, color matching, and short wavelength-sensitive cone sensitivity--are the most reliable prognostic indicators of progression in AMD.