Palm is expressed in both developing and adult mouse lens and retina

BACKGROUND: Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. METHODS: The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. RESULTS: In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. CONCLUSION: Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation

Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

AbstractMorphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell–cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover

Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation

These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens

Functional interactions between alternatively spliced forms of Pax6 in crystallin gene regulation and in haploinsufficiency

Pax6 is essential for development of the eye, olfactory system, brain and pancreas. Haploinsufficiency of Pax6 causes abnormal eye development. Two forms of Pax6 protein, PAX6 and PAX6(5a), differ in a 14 amino acid insertion encoded by an alternatively spliced exon 5a in the N-terminal DNA-binding paired domain (PD), and they are simultaneously expressed. Here, we show that PAX6 and PAX6(5a) together synergistically activate transcription from promoters recognized by Pax6 PD and PD5a, but not by their homeodomain. This synergism promotes activation of transcription by c-Maf and MafA on the αB-crystallin promoter, and is required for transcriptional co-activation by RARβ/RXRβ and PAX6/PAX6(5a) on the γF-crystallin promoter. To determine the role of this synergism in haploinsufficiency, we tested four human missense (G18W, R26G, G64V and R128C) and one nonsense (R317X) mutants, with reporters driven by Pax6 PD consensus binding sites and the αB-crystallin promoter. The simultaneous activity of Pax6 proteins [PAX6, mutated PAX6, PAX6(5a) and mutated PAX6(5a)] modeling haploinsufficiency yielded results not predicted by properties of individual PAX6 or PAX6(5a). Taken together, these results indicate that complex ocular phenotypes due to Pax6 haploinsufficiency originate, at least partially, from functional interactions between alternatively spliced PAX6 and PAX6(5a) variants and other factors, e.g. MafA/c-Maf

Case series: Pyramidal cataracts, intact irides and nystagmus from three novel PAX6 mutations

Purpose: To investigate the association between novel PAX6 mutations to bilateral anterior pyramidal congenital cataracts (APyC), complete and intact irides, and nystagmus. Observations: This is a retrospective observational case series in a multi-center setting with genetic testing. Three female patients were diagnosed with bilateral APyC, intact irides and nystagmus. Genetic testing identified the three patients had novel missense mutations in PAX6 – c.128C > T; p.Ser43Phe (S43F), c. 197T > A; p.Ile66Asn (I66N) and c.781C > G; p.Arg261Gly (R261G). Conclusions and importance: This study demonstrates a novel phenotype of bilateral APyC, intact irides, and nystagmus in whom genetic testing for PAX6 identified novel missense mutations (S43F, I66N, R261G) in highly conserved DNA-binding domains. Implications of understanding why the iris is present in these cases is discussed. Keywords: Pyramidal cataracts, Intact irides, Nystagmus, Novel PAX6 missense mutation

Spectrum and range of oxidative stress responses of human lens epithelial cells to H2O2 insult. Invest Ophthalmol Vis Sci.

PURPOSE. Oxidative stress (OS) is believed to be a major contributor to age-related cataract and other age-related diseases. METHODS. cDNA microarrays were used to identify the spectrum and range of genes with transcript levels that are altered in response to acute H 2 O 2 -induced OS in human lens epithelial (HLE) cells. HLE cells were treated with 50 M H 2 O 2 for 1 hour in the absence of serum, followed by a return to complete medium. RNAs were prepared from treated and untreated cells at 0, 1, 2, and 8 hours after H 2 O 2 treatment. RESULTS. The data showed 1171 genes that were significantly up-and downregulated in response to H 2 O 2 treatment. Several functional subcategories of genes were identified, including those encoding DNA repair proteins, antioxidant defense enzymes, molecular chaperones, protein biosynthesis enzymes, and trafficking and degradation proteins. Differential expression of selected genes was confirmed at the level of RNA and/or protein. Many of the identified genes (e.g., glutathione S-transferase [MGST2], thioredoxin reductase ␤, and peroxiredoxin 2) have been identified as participants in OS responses in the lens and other systems. Some genes induced by OS in the current study (e.g., oxygen regulated protein [ORP150] and heat shock protein [HSP40]) are better known to respond to other forms of stress. Two genes (receptor tyrosine kinase [AXL/ARK] and protein phosphatase 2A) are known to be differentially expressed in cataract. Most of the genes point to a novel pathways associated with OS. CONCLUSIONS. The present data provide a global perspective on those genes that respond to acute OS, point to novel genes and pathways associated with OS, and set the groundwork for understanding the functions of OS-related genes in lens protection and disease. (Invest Ophthalmol Vis Sci

Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination

The vertebrate lens provides an excellent model with which to study the mechanisms required for epithelial invagination. In the mouse, the lens forms from the head surface ectoderm. A domain of ectoderm first thickens to form the lens placode and then invaginates to form the lens pit. The epithelium of the lens placode remains in close apposition to the epithelium of the presumptive retina as these structures undergo a coordinated invagination. Here, we show that F-actin-rich basal filopodia that link adjacent presumptive lens and retinal epithelia function as physical tethers that coordinate invagination. The filopodia, most of which originate in the presumptive lens, form at E9.5 when presumptive lens and retinal epithelia first come into close contact, and have retracted by E11.5 when invagination is complete. At E10.5 - the lens pit stage - there is approximately one filopodium per epithelial cell. Formation of filopodia is dependent on the Rho family GTPase Cdc42 and the Cdc42 effector IRSp53 (Baiap2). Loss of filopodia results in reduced lens pit invagination. Pharmacological manipulation of the actin-myosin contraction pathway showed that the filopodia can respond rapidly in length to change inter-epithelial distance. These data suggest that the lens-retina inter-epithelial filopodia are a fine-tuning mechanism to assist in lens pit invagination by transmitting the forces between presumptive lens and retina. Although invagination of the archenteron in sea urchins and dorsal closure in Drosophila are known to be partly dependent on filopodia, this mechanism of morphogenesis has not previously been identified in vertebrates