Publications by authors named "Michael L Robinson"

75 Publications

Design and Characterization of Biomimetic Kerateine Aerogel-Electrospun Polycaprolactone Scaffolds for Retinal Cell Culture.

Ann Biomed Eng 2021 Apr 6. Epub 2021 Apr 6.

Department of Chemical, Paper and Biomedical Engineering, Miami University, 650 East High Street, Oxford, OH, 45056, USA.

Age-related macular degeneration (AMD) is a retinal disease that affects 196 million people and causes nearly 9% of blindness worldwide. While several pharmacological approaches slow the effects of AMD, in our opinion, cell-based strategies offer the most likely path to a cure. We describe the design and initial characterization of a kerateine (obtained by reductive extraction from keratin proteins) aerogel-electrospun polycaprolactone fiber scaffold system. The scaffolds mimic key features of the choroid and the Bruch's membrane, which is the basement membrane to which the cells of the retinal pigment epithelium (RPE) attach. The scaffolds had elastic moduli of 2-7.2 MPa, a similar range as native choroid and Bruch's membrane. ARPE-19 cells attached to the polycaprolactone fibers, remained viable for one week, and proliferated to form a monolayer reminiscent of that needed for retinal repair. These constructs could serve as a model system for testing cell and/or drug treatment strategies or directing ex vivo retinal tissue formation in the treatment of AMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10439-021-02756-5DOI Listing
April 2021

A missense allele of PEX5 is responsible for the defective import of PTS2 cargo proteins into peroxisomes.

Hum Genet 2021 Apr 2;140(4):649-666. Epub 2021 Jan 2.

The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore MD, USA.

Peroxisomes, single-membrane intracellular organelles, play an important role in various metabolic pathways. The translocation of proteins from the cytosol to peroxisomes depends on peroxisome import receptor proteins and defects in peroxisome transport result in a wide spectrum of peroxisomal disorders. Here, we report a large consanguineous family with autosomal recessive congenital cataracts and developmental defects. Genome-wide linkage analysis localized the critical interval to chromosome 12p with a maximum two-point LOD score of 4.2 (θ = 0). Next-generation exome sequencing identified a novel homozygous missense variant (c.653 T > C; p.F218S) in peroxisomal biogenesis factor 5 (PEX5), a peroxisome import receptor protein. This missense mutation was confirmed by bidirectional Sanger sequencing. It segregated with the disease phenotype in the family and was absent in ethnically matched control chromosomes. The lens-specific knockout mice of Pex5 recapitulated the cataractous phenotype. In vitro import assays revealed a normal capacity of the mutant PEX5 to enter the peroxisomal Docking/Translocation Module (DTM) in the presence of peroxisome targeting signal 1 (PTS1) cargo protein, be monoubiquitinated and exported back into the cytosol. Importantly, the mutant PEX5 protein was unable to form a stable trimeric complex with peroxisomal biogenesis factor 7 (PEX7) and a peroxisome targeting signal 2 (PTS2) cargo protein and, therefore, failed to promote the import of PTS2 cargo proteins into peroxisomes. In conclusion, we report a novel missense mutation in PEX5 responsible for the defective import of PTS2 cargo proteins into peroxisomes resulting in congenital cataracts and developmental defects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-020-02238-zDOI Listing
April 2021

Lens fiber cell differentiation occurs independently of fibroblast growth factor receptor signaling in the absence of Pten.

Dev Biol 2020 11 25;467(1-2):1-13. Epub 2020 Aug 25.

Department of Biology and Center for Visual Sciences, Miami University, Oxford, OH, USA. Electronic address:

Fibroblast growth factor receptor (FGFR) signaling patterns multiple tissues in both vertebrates and invertebrates, largely through the activation of intracellular kinases. Recent studies have demonstrated that the phosphatase, PTEN negatively regulates FGFR signaling, such that the loss of PTEN can compensate for reduced FGFR signaling to rescue aspects of normal development. In the developing mouse lens, FGFR signaling promotes cell survival and fiber cell differentiation, and the loss of Pten largely compensates for the loss of Fgfr2 during lens development. To explore this regulatory relationship further, we focused on the phenotypic consequences of Pten loss on lens development and fiber cell differentiation in the absence of all FGFR signaling, both in vivo and in lens epithelial explants. Pten deletion partially rescues primary fiber cell elongation and γ-crystallin accumulation in FGFR-deficient lenses in vivo but fails to rescue cell survival or proliferation. However, in lens epithelial explants, where cells survive without FGFR signaling, Pten deletion rescues vitreous humor-induced lens fiber cell differentiation in the combined absence of Fgfr1, Fgfr2 and Fgfr3. This represents the first evidence that vitreous-initiated signaling cascades, independent of FGFR signaling, can drive mammalian lens fiber cell differentiation, when freed from repression by PTEN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ydbio.2020.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572784PMC
November 2020

Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types.

Transl Vis Sci Technol 2020 02 18;9(3):21. Epub 2020 Feb 18.

Department of Biology and Center for Visual Sciences, Miami University, Oxford, OH, USA.

Purpose: Early in mammalian eye development, , , and expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist.

Methods: CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the (Progenitor), (Ganglion), and (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene.

Results: Retinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types.

Conclusions And Translational Relevance: The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/tvst.9.3.21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352077PMC
February 2020

Detection of Circulating and Disseminated Neuroblastoma Cells Using the ImageStream Flow Cytometer for Use as Predictive and Pharmacodynamic Biomarkers.

Clin Cancer Res 2020 01 25;26(1):122-134. Epub 2019 Nov 25.

Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom.

Purpose: Circulating tumor cells (CTCs) serve as noninvasive tumor biomarkers in many types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as predictive and pharmacodynamic biomarkers.

Experimental Design: We collected matched blood and bone marrow samples from 40 patients with neuroblastoma to detect GD/CD45 neuroblastoma CTCs from blood and disseminated tumor cells (DTCs) from bone marrow using the Imagestream Imaging flow cytometer (ISx). In six cases, circulating free DNA (cfDNA) extracted from plasma isolated from the CTC sample was analyzed by high-density single-nucleotide polymorphism (SNP) arrays.

Results: CTCs were detected in 26 of 42 blood samples (1-264/mL) and DTCs in 25 of 35 bone marrow samples (57-291,544/mL). Higher numbers of CTCs in patients with newly diagnosed, high-risk neuroblastoma correlated with failure to obtain a complete bone marrow (BM) metastatic response after induction chemotherapy ( < 0.01). Nutlin-3 (MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and DTCs compared with DMSO controls. In five of six cases, cfDNA analyzed by SNP arrays revealed copy number abnormalities associated with neuroblastoma.

Conclusions: This is the first study to show that CTCs and DTCs are detectable in neuroblastoma using the ISx, with concurrently extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic biomarkers in early-phase clinical trials. Further investigation is required to determine whether CTC numbers are predictive biomarkers of BM response to first-line induction chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-0656DOI Listing
January 2020

High-throughput transcriptome analysis reveals that the loss of Pten activates a novel NKX6-1/RASGRP1 regulatory module to rescue microphthalmia caused by Fgfr2-deficient lenses.

Hum Genet 2019 Dec 5;138(11-12):1391-1407. Epub 2019 Nov 5.

Department of Biology, Miami University, Oxford, OH, 45056, USA.

FGFR signaling is critical to development and disease pathogenesis, initiating phosphorylation-driven signaling cascades, notably the RAS-RAF-MEK-ERK and PI3 K-AKT cascades. PTEN antagonizes FGFR signaling by reducing AKT and ERK activation. Mouse lenses lacking FGFR2 exhibit microphakia and reduced ERK and AKT phosphorylation, widespread apoptosis, and defective lens fiber cell differentiation. In contrast, simultaneous deletion of both Fgfr2 and Pten restores ERK and AKT activation levels as well as lens size, cell survival and aspects of fiber cell differentiation; however, the molecular basis of this "rescue" remains undefined. We performed transcriptomic analysis by RNA sequencing of mouse lenses with conditional deletion of Fgfr2, Pten or both Fgfr2 and Pten, which reveal new molecular mechanisms that uncover how FGFR2 and PTEN signaling interact during development. The FGFR2-deficient lens transcriptome demonstrates overall loss of fiber cell identity with deregulated expression of 1448 genes. We find that ~ 60% of deregulated genes return to normal expression levels in lenses lacking both Fgfr2 and Pten. Further, application of customized filtering parameters to these RNA-seq data sets identified 68 high-priority candidate genes. Bioinformatics analyses showed that the cis-binding motif of a high-priority homeodomain transcription factor, NKX6-1, was present in the putative promoters of ~ 78% of these candidates. Finally, biochemical reporter assays demonstrate that NKX6-1 activated the expression of the high-priority candidate Rasgrp1, a RAS-activating protein. Together, these data define a novel regulatory module in which NKX6-1 directly activates Rasgrp1 expression to restore the balance of ERK and AKT activation, thus providing new insights into alternate regulation of FGFR downstream events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-019-02084-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020882PMC
December 2019

Loss-of-function variants in myocardin cause congenital megabladder in humans and mice.

J Clin Invest 2019 12;129(12):5374-5380

Department of Experimental Cardiology, Amsterdam UMC, Amsterdam, Netherlands.

Myocardin (MYOCD) is the founding member of a class of transcriptional coactivators that bind the serum-response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies, and to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by cosegregation of MYOCD variants with the phenotype in 4 unrelated families by in vitro transactivation studies in which pathogenic variants resulted in abrogated SM gene expression and by the finding of megabladder in 2 distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI128545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877301PMC
December 2019

Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens.

Hum Genomics 2019 02 15;13(1):10. Epub 2019 Feb 15.

Department of Biology, Miami University, Oxford, OH, 45056, USA.

Background: Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous.

Result: Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar expression in the developing eye and pancreas. However, lineage tracing revealed widespread non-ocular CRE reporter gene expression in the P0-3.9GFPCre transgenic mice that results from stochastic CRE expression in the P0-3.9GFPCre embryos prior to lens placode formation. Postnatal hemizygous Le-Cre transgenic lenses express higher levels of CRE transcript and protein than the hemizygous lenses of P0-3.9GFPCre mice. Transcriptome analysis revealed that Le-Cre hemizygous lenses deregulated the expression of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre hemizygous lenses only deregulated two murine genes. No known PAX6-responsive genes or genes directly associated with lens differentiation were deregulated in the hemizygous Le-Cre lenses.

Conclusions: Although P0-3.9GFPCre transgenic mice appear free from ocular abnormalities, extensive non-ocular CRE expression represents a potential problem for conditional gene deletion studies using this transgene. The higher level of CRE expression in Le-Cre lenses versus P0-3.9GFPCre lenses may explain abnormal lens development in homozygous Le-Cre mice. Given the lack of deregulation of PAX6-responsive transcripts, we suggest that abnormal eye development in Le-Cre transgenic mice stems from CRE toxicity. Our studies reinforce the requirement for appropriate CRE-only expressing controls when using CRE as a driver of conditional gene targeting strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40246-019-0192-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377743PMC
February 2019

N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis.

Dev Biol 2017 08 26;428(1):118-134. Epub 2017 May 26.

Thomas Jefferson University, Pathology Anatomy and Cell Biology, Philadelphia, PA, United States. Electronic address:

Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ydbio.2017.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524459PMC
August 2017

Lens development requires DNMT1 but takes place normally in the absence of both DNMT3A and DNMT3B activity.

Epigenetics 2017 01 8;12(1):27-40. Epub 2016 Nov 8.

a Department of Biology , Miami University , Oxford , OH , USA.

Despite the wealth of knowledge of transcription factors involved in lens development, little information exists about the role of DNA methylation in this process. Here, we investigated the role of DNA methylation in lens development and fiber cell differentiation using mice conditionally lacking maintenance or de novo methyltransferases in the lens lineage. We found that while Dnmt1 inactivation at the lens placode stage (via the Le-Cre transgene) led to lens DNA hypomethylation and severe lens epithelial apoptosis, lens fiber cell differentiation remained largely unaffected. The simultaneous deletion of phosphatase and tensin homolog (Pten) elevated the level of phosphorylated AKT and rescued many of the morphological defects and cell death in DNMT1-deficient lenses. With a different Cre driver (MLR10) we demonstrated that a small number of lens epithelial cells escaped Dnmt1-deletion and over-proliferated to compensate for the loss of Dnmt1-deleted cells, suggesting that lens epithelium possess a substantial capacity for self-renewal. Unlike lenses deficient for Dnmt1, inactivation of both Dnmt3a and Dnmt3b by either the Le-Cre or MLR10-Cre transgene did not result in any obvious lens phenotype prior to 10 months of age. Taken together, while lens epithelial cell survival requires DNMT1, morphologically normal lenses develop in the absence of both DNMT3A and DNMT3B.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15592294.2016.1253651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270636PMC
January 2017

Controlling molecularity and stability of hydrogen bonded G-quadruplexes by modulating the structure's periphery.

Chem Commun (Camb) 2016 Sep 22;52(74):11112-5. Epub 2016 Aug 22.

Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.

We report on self-assembly of guanosines with aromatic esters at their 5'-position. Depending on the basicity of the 5'-ester either discrete octamers G8·K(+)I(-) or hexadecamers G16·3K(+)3I(-) are formed. The thermodynamic and kinetic stabilities of the G-quadruplex can be controlled by interlayer hydrogen-bonding and by dispersion interactions on the assembly's periphery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6cc06271gDOI Listing
September 2016

CT-Finder: A Web Service for CRISPR Optimal Target Prediction and Visualization.

Sci Rep 2016 05 23;6:25516. Epub 2016 May 23.

Department of Biology, Miami University, Oxford, Ohio, 45056, USA.

The CRISPR system holds much promise for successful genome engineering, but therapeutic, industrial, and research applications will place high demand on improving the specificity and efficiency of this tool. CT-Finder (http://bioinfolab.miamioh.edu/ct-finder) is a web service to help users design guide RNAs (gRNAs) optimized for specificity. CT-Finder accommodates the original single-gRNA Cas9 system and two specificity-enhancing paired-gRNA systems: Cas9 D10A nickases (Cas9n) and dimeric RNA-guided FokI nucleases (RFNs). Optimal target candidates can be chosen based on the minimization of predicted off-target effects. Graphical visualization of on-target and off-target sites in the genome is provided for target validation. Major model organisms are covered by this web service.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep25516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876460PMC
May 2016

Extending Self-Organizing Optic Cups Into Functional Ciliary Epithelium.

Invest Ophthalmol Vis Sci 2016 Jan;57(1):162

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.15-18980DOI Listing
January 2016

FGFR and PTEN signaling interact during lens development to regulate cell survival.

Dev Biol 2016 Feb 5;410(2):150-163. Epub 2016 Jan 5.

Department of Biology, Cell Molecular and Structural Biology Graduate Program, Miami University, Oxford, OH, USA. Electronic address:

Lens epithelial cells express many receptor tyrosine kinases (RTKs) that stimulate PI3K-AKT and RAS-RAF-MEK-ERK intracellular signaling pathways. These pathways ultimately activate the phosphorylation of key cellular transcription factors and other proteins that control proliferation, survival, metabolism, and differentiation in virtually all cells. Among RTKs in the lens, only stimulation of fibroblast growth factor receptors (FGFRs) elicits a lens epithelial cell to fiber cell differentiation response in mammals. Moreover, although the lens expresses three different Fgfr genes, the isolated removal of Fgfr2 at the lens placode stage inhibits both lens cell survival and fiber cell differentiation. Phosphatase and tensin homolog (PTEN), commonly known as a tumor suppressor, inhibits ERK and AKT activation and initiates both apoptotic pathways, and cell cycle arrest. Here, we show that the combined deletion of Fgfr2 and Pten rescues the cell death phenotype associated with Fgfr2 loss alone. Additionally, Pten removal increased AKT and ERK activation, above the levels of controls, in the presence or absence of Fgfr2. However, isolated deletion of Pten failed to stimulate ectopic fiber cell differentiation, and the combined deletion of Pten and Fgfr2 failed to restore differentiation-specific Aquaporin0 and DnaseIIβ expression in the lens fiber cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ydbio.2015.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767529PMC
February 2016

CADBURE: A generic tool to evaluate the performance of spliced aligners on RNA-Seq data.

Sci Rep 2015 Aug 25;5:13443. Epub 2015 Aug 25.

Department of Biology, Miami University, Oxford, Ohio 45056, USA.

The fundamental task in RNA-Seq-based transcriptome analysis is alignment of millions of short reads to the reference genome or transcriptome. Choosing the right tool for the dataset in hand from many existent RNA-Seq alignment packages remains a critical challenge for downstream analysis. To facilitate this choice, we designed a novel tool for comparing alignment results of user data based on the relative reliability of uniquely aligned reads (CADBURE). CADBURE can easily evaluate different aligners, or different parameter sets using the same aligner, and selects the best alignment result for any RNA-Seq dataset. Strengths of CADBURE include the ability to compare alignment results without the need for synthetic data such as simulated genomes, alignment regeneration and randomly subsampled datasets. The benefit of a CADBURE selected alignment result was supported by differentially expressed gene (DEG) analysis. We demonstrated that the use of CADBURE to select the best alignment from a number of different alignment results could change the number of DEGs by as much as 10%. In particular, the CADBURE selected alignment result favors fewer false positives in the DEG analysis. We also verified differential expression of eighteen genes with RT-qPCR validation experiments. CADBURE is an open source tool (http://cadbure.sourceforge.net/).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep13443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548254PMC
August 2015

Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing.

Mol Vis 2014 4;20:1491-517. Epub 2014 Nov 4.

Department of Biology, Miami University, Oxford, OH.

Purpose: The ocular lens contains only two cell types: epithelial cells and fiber cells. The epithelial cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens fiber cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial cells and lens fiber cells was conducted to comprehensively compare the transcriptomes of these two cell types.

Methods: RNA from three biologic replicate samples of epithelial and fiber cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR).

Results: Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial cells into fiber cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial cells and lens fiber cells. To our knowledge, this is the first study identifying the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes upregulated in the epithelial cells were enriched for extracellular matrix production, cell division, migration, protein kinase activity, growth factor binding, and calcium ion binding. Genes upregulated in the fiber cells were enriched for proteosome complexes, unfolded protein responses, phosphatase activity, and ubiquitin binding. Differentially expressed genes involved in several important signaling pathways, lens structural components, organelle loss, and denucleation were also highlighted to provide insights into lens development and lens fiber differentiation.

Conclusions: RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of protein-coding and non-coding transcripts from lens epithelial cells and lens fiber cells. This information provides a valuable resource for studying lens development, nuclear degradation, and organelle loss during fiber differentiation, and associated diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225139PMC
June 2015

Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

Development 2014 Sep;141(17):3388-98

Laboratory for Nutrition and Vision Research, Human Nutrition Research Center on Aging, Nutrition &Vision Res-USDA-HNRCA, Tufts University, Boston 02111, MA, USA Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel

Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.106005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199135PMC
September 2014

AP-2α is required after lens vesicle formation to maintain lens integrity.

Dev Dyn 2014 Oct 30;243(10):1298-309. Epub 2014 Apr 30.

Department of Pathology and Molecular Medicine, McMaster University Health Science Centre, Hamilton, Ontario, Canada.

Background: Transcription factors are critical in regulating lens development. The AP-2 family of transcription factors functions in differentiation, cell growth and apoptosis, and in lens and eye development. AP-2α, in particular, is important in early lens development, and when conditionally deleted at the placode stage defective separation of the lens vesicle from the surface ectoderm results. AP-2α's role during later stages of lens development is unknown. To address this, the MLR10-Cre transgene was used to delete AP-2α from the lens epithelium beginning at embryonic day (E) 10.5.

Results: The loss of AP-2α after lens vesicle separation resulted in morphological defects beginning at E18.5. By P4, a small highly vacuolated lens with a multilayered epithelium was evident in the MLR10-AP-2α mutants. Epithelial cells appeared elongated and expressed fiber cell specific βB1 and γ-crystallins. Epithelial cell polarity and lens cell adhesion was disrupted and accompanied by the misexpression of ZO-1, N-Cadherin, and β-catenin. Cell death was observed in the mutant lens epithelium between postnatal day (P) 14 and P30, and correlated with altered arrangements of cells within the epithelium.

Conclusions: Our findings demonstrate that AP-2α continues to be required after lens vesicle separation to maintain a normal lens epithelial cell phenotype and overall lens integrity and to ensure correct fiber cell differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvdy.24141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962590PMC
October 2014

Integrin linked kinase (ILK) is required for lens epithelial cell survival, proliferation and differentiation.

Exp Eye Res 2014 Apr 25;121:130-42. Epub 2014 Jan 25.

Ocular Development Laboratory, Anatomy and Neuroscience, University of Melbourne, Victoria 3010, Australia. Electronic address:

While the role of growth factors in lens development has been investigated extensively, the role of extracellular matrix signalling is less well understood. The developing lens expresses predominantly laminin-binding integrins (such as α3β1, α6β1), which are cooperatively required in the lens epithelium during development. We investigated the role of ILK, a downstream mediator of integrin signalling in mice conditionally null for Ilk. Mutant lenses showed epithelial thinning at E17.5 with reduced proliferation and epithelial cell number and aberrant fibre differentiation. There was complete loss of the central epithelium from postnatal day (P) 2 due to cell death followed by fibre cell degeneration and death by P10 as well as rupture of the lens capsule between P10 and P21. At E17.5 there was significant inhibition (∼50%) of epithelial cell cycle progression, as shown by BrdU incorporation, cyclin D1/D2 and phospho-histone H3 immunostaining. The epithelial marker, E-cadherin, was decreased progressively from E17.5 to P2, in the central epithelium, but there was no significant change in Pax6 expression. Analyses of ERK and Akt phosphorylation indicated marked depression of MAPK and PI3K-Akt signalling, which correlated with decreased phosphorylation of FRS2α and Shp2, indicating altered activation of FGF receptors. At later postnatal stages there was reduced or delayed expression of fibre cell markers (β-crystallin and p57(kip2)). Loss of Ilk also affected deposition of extracellular matrix, with marked retention of collagen IV within differentiating fibre cells. By quantitative RT-PCR array there was significantly decreased expression of 19 genes associated with focal adhesions, actin filament stability and MAPK and PI3K/Akt signalling. Overall, these data indicate that ILK is required for complete activation of signalling cascades downstream of the FGF receptor in lens epithelium and fibre cells during development and thus is involved in epithelial proliferation, survival and subsequent fibre differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2014.01.013DOI Listing
April 2014

AKT activation promotes PTEN hamartoma tumor syndrome-associated cataract development.

J Clin Invest 2013 Dec 25;123(12):5401-9. Epub 2013 Nov 25.

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI70437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859396PMC
December 2013

Deletion of autophagy-related 5 (Atg5) and Pik3c3 genes in the lens causes cataract independent of programmed organelle degradation.

J Biol Chem 2013 Apr 11;288(16):11436-47. Epub 2013 Mar 11.

Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.

The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.437103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630873PMC
April 2013

A mechanism for the synergism of FGF- and Wnt-signaling during lens fiber cell differentiation.

Invest Ophthalmol Vis Sci 2013 Mar 1;54(3). Epub 2013 Mar 1.

Department of Zoology, Miami University, Oxford, Ohio, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.13-11818DOI Listing
March 2013

The LEGSKO mouse: a mouse model of age-related nuclear cataract based on genetic suppression of lens glutathione synthesis.

PLoS One 2012 30;7(11):e50832. Epub 2012 Nov 30.

Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.

Age-related nuclear cataracts are associated with progressive post-synthetic modifications of crystallins from various physical chemical and metabolic insults, of which oxidative stress is a major factor. The latter is normally suppressed by high concentrations of glutathione (GSH), which however are very low in the nucleus of the old lens. Here we generated a mouse model of oxidant stress by knocking out glutathione synthesis in the mouse in the hope of recapitulating some of the changes observed in human age-related nuclear cataract (ARNC). A floxed Gclc mouse was generated and crossed with a transgenic mouse expressing Cre in the lens to generate the LEGSKO mouse in which de novo GSH synthesis was completely abolished in the lens. Lens GSH levels were reduced up to 60% in homozygous LEGSKO mice, and a decreasing GSH gradient was noticed from cortical to nuclear region at 4 months of age. Oxidation of crystallin methionine and sulfhydryls into sulfoxides was dramatically increased, but methylglyoxal hydroimidazolones levels that are GSH/glyoxalase dependent were surprisingly normal. Homozygous LEGSKO mice developed nuclear opacities starting at 4 months that progressed into severe nuclear cataract by 9 months. We conclude that the LEGSKO mouse lens mimics several features of human ARNC and is thus expected to be a useful model for the development of anti-cataract agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0050832PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511334PMC
May 2013

Frs2α enhances fibroblast growth factor-mediated survival and differentiation in lens development.

Development 2012 Dec 7;139(24):4601-12. Epub 2012 Nov 7.

Department of Zoology, Miami University, Oxford, OH 45056, USA.

Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to lens fiber cell differentiation in the developing mammalian embryo. Additionally, within the lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of lens fiber cell differentiation, including Prox1, p57(KIP2), aquaporin 0 and β-crystallins. Although not normally expressed in the lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of lens fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of β-crystallins. All these characteristics of NT3-TrkC transgenic lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent lens cell survival and provides a mechanistic basis for the unique fiber-differentiating capacity of Fgfs on mammalian lens epithelial cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.081737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509723PMC
December 2012

Sprouty is a negative regulator of transforming growth factor β-induced epithelial-to-mesenchymal transition and cataract.

Mol Med 2012 Jul 18;18:861-73. Epub 2012 Jul 18.

Anatomy and Histology, Bosch Institute & Save Sight Institute, University of Sydney, Sydney, Australia.

Fibrosis affects an extensive range of organs and is increasingly acknowledged as a major component of many chronic disorders. It is now well accepted that the elevated expression of certain inflammatory cell-derived cytokines, especially transforming growth factor β (TGFβ), is involved in the epithelial-to-mesenchymal transition (EMT) leading to the pathogenesis of a diverse range of fibrotic diseases. In lens, aberrant TGFβ signaling has been shown to induce EMT leading to cataract formation. Sproutys (Sprys) are negative feedback regulators of receptor tyrosine kinase (RTK)-signaling pathways in many vertebrate systems, and in this study we showed that they are important in the murine lens for promoting the lens epithelial cell phenotype. Conditional deletion of Spry1 and Spry2 specifically from the lens leads to an aberrant increase in RTK-mediated extracellular signal-regulated kinase 1/2 phosphorylation and, surprisingly, elevated TGFβ-related signaling in lens epithelial cells, leading to an EMT and subsequent cataract formation. Conversely, increased Spry overexpression in lens cells can suppress not only TGFβ-induced signaling, but also the accompanying EMT and cataract formation. On the basis of these findings, we propose that a better understanding of the relationship between Spry and TGFβ signaling will not only elucidate the etiology of lens pathology, but will also lead to the development of treatments for other fibrotic-related diseases associated with TGFβ-induced EMT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2119/molmed.2012.00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409273PMC
July 2012

3-Dimensional morphometric analysis of murine bladder development and dysmorphogenesis.

Dev Dyn 2012 Mar 31;241(3):522-33. Epub 2012 Jan 31.

Center for Molecular and Human Genetics, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio 43205, USA.

Background: Disorders of the urinary tract represent a major cause of morbidity and impaired quality of life. To better understand the morphological events responsible for normal urinary tract development, we performed 3-D reconstructive analysis of developing mouse bladders in control, mgb-/-, and Fgfr2(Mes-/-) mice.

Results: Detrusor smooth muscle differentiation initiated in the bladder dome and progressed caudally with the leading edge extending down the right posterior surface of the bladder. Gender-specific differences in detrusor smooth muscle development were observed during early embryonic development. Bladder trigone morphology transitioned from an isosceles to equilateral triangle during development due to the preferential lengthening of the urethra to ureter distance. The primary defect observed in mgb-/- bladders was a significant reduction in detrusor smooth muscle differentiation throughout development. Deviations from normal trigone morphology correlated best with VUR development in Fgfr2(Mes-/-) mice, while alterations in intravesicular tunnel length did not.

Conclusions: Multivariate morphometric analysis provides a powerful tool to quantify and assess urinary tract development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvdy.23744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288201PMC
March 2012

Conversion of mouse fibroblasts to sphere cells induced by AlbuMAXI-containing medium.

Front Biosci (Elite Ed) 2012 Jan 1;4:1813-22. Epub 2012 Jan 1.

Department of Biology, and Center for Tissue Regeneration and Engineering, University of Dayton, Dayton, Ohio 45469, USA.

The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been successfully manipulated by ectopic expression of defined factors. We demonstrate that mouse fibroblasts can be converted into sphere cells by detaching the fibroblast cells by protease and then using the AlbuMAX I-containing culture medium without genetic alteration. AlbuMAX I is a lipid-rich albumin. Albumin-associated lipids arachidonic acid (AA) and pluronic F-68 were responsible for this effect. The converted colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) staining. Global gene expression analysis indicated that the sphere cells were in an intermediate state compared with mES cells and MEF cells. The sphere cells were able to differentiate into tissues representing all three embryonic germ layers following retinoic acid treatment, and differentiated into smooth muscle cells following treatment with vascular endothelial growth factor (VEGF). The study presented a potential novel approach to transdifferentiate mouse fibroblast cells into other cell lineages mediated by AlbuMAX I-containing culture medium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2741/502DOI Listing
January 2012

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

Dev Biol 2011 Dec 16;360(1):30-43. Epub 2011 Sep 16.

Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.

Morphogenesis 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ydbio.2011.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215831PMC
December 2011

The cell adhesion gene PVRL3 is associated with congenital ocular defects.

Hum Genet 2012 Feb 17;131(2):235-50. Epub 2011 Jul 17.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

We describe a male patient (patient DGAP113) with a balanced translocation, 46,XY,t(1;3)(q31.3;q13.13), severe bilateral congenital cataracts, CNS abnormalities and mild developmental delay. Fluorescence in situ hybridization (FISH) and suppression PCR demonstrated that the chromosome 3 breakpoint lies ~515 kb upstream of the PVRL3 gene, while the chromosome 1 breakpoint lies ~50 kb upstream of the NEK7 gene. Despite the fact that NEK7 is closer to a translocation breakpoint than PVRL3, NEK7 transcript levels are unaltered in patient DGAP113 lymphoblastoid cells and Nek7-deficient mice exhibit no detectable ocular phenotype. In contrast, the expression of PVRL3, which encodes the cell adhesion protein Nectin 3, is significantly reduced in patient DGAP113 lymphoblastoid cells, likely due to a position effect caused by the chromosomal translocation. Nectin 3 is expressed in the mouse embryonic ciliary body and lens. Moreover, Pvrl3 knockout mice as well as a spontaneous mouse mutant ari (anterior retinal inversion), that maps to the Pvrl3 locus, exhibit lens and other ocular defects involving the ciliary body. Collectively, these data identify PVRL3 as a critical gene involved in a Nectin-mediated cell-cell adhesion mechanism in human ocular development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-011-1064-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279124PMC
February 2012

Spry1 and Spry2 are necessary for lens vesicle separation and corneal differentiation.

Invest Ophthalmol Vis Sci 2011 Aug 29;52(9):6887-97. Epub 2011 Aug 29.

Department of Surgery, Creighton University, Omaha, Nebraska, USA.

Purpose: The studies reported here were performed to analyze the roles of Sproutys (Sprys), downstream targets and negative feedback regulators of the fibroblast growth factor (FGF) signaling pathway, in lens and corneal differentiation.

Methods: Spry1 and -2 were conditionally deleted in the lens and corneal epithelial precursors using the Le-Cre transgene and floxed alleles of Spry1 and -2. Alterations in lens and corneal development were assessed by hematoxylin and eosin staining, in situ hybridization, and immunohistochemistry.

Results: Spry1 and -2 were upregulated in the lens fibers at the onset of fiber differentiation. FGF signaling was both necessary and sufficient for induction of Spry1 and -2 in the lens fiber cells. Spry1 and -2 single- or double-null lenses failed to separate from the overlying ectoderm and showed persistent keratolenticular stalks. Apoptosis of stalk cells, normally seen during lens vesicle detachment from the ectoderm, was inhibited in Spry mutant lenses, with concomitant ERK activation. Prox1 and p57(KIP2), normally upregulated at the onset of fiber differentiation were prematurely induced in the Spry mutant lens epithelial cells. However, terminal differentiation markers such as β- or γ-crystallin were not induced. Corneal epithelial precursors in Spry1 and -2 double mutants showed increased proliferation with elevated expression of Erm and DUSP6 and decreased expression of the corneal differentiation marker K12.

Conclusions: Collectively, the results indicate that Spry1 and -2 (1) through negative modulation of ERKs allow lens vesicle separation, (2) are targets of FGF signaling in the lens during initiation of fiber differentiation and (3) function redundantly in the corneal epithelial cells to suppress proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.11-7531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3176024PMC
August 2011