2009 Midwest Eye Research Symposium
Sponsored by the University of Iowa Department of Ophthalmology and Visual Sciences
MERS 2009 took place on Friday July 10, 2009 at
The University of Iowa Athletic Hall of Fame
Keynote Speaker / Session Chairs
Abbot Clark, PhD - Keynote Speaker
John H. Fingert, MD, PhD - Session I Chair
Brian A. Link, PhD - Session II Chair
Donald S. Sakaguchi, PhD - Session III Chair
Session I - Session Chair John H. Fingert, MD, PhD
Session II - Session Chair Brian A. Link, PhD
Session III - Session Chair Donald S. Sakaguchi, PhD
Brian S. Clark and Brian A. Link
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI
Purpose: To examine the relationship of nuclear position and endosome polarization in the developing zebrafish retina for study of intrinsic cellular properties on retinal neurogenesis.
Methods:Transgenic zebrafish embryos were generated expressing an N-terminal EGFP-Rab5c fusion. To assess endosome polarization, genetic mosaics were created through blastula transplants of EGFP-Rab5c/H2A-mCherry positive donor cells into wild-type embryo. Clonal retinal integrations were imaged, and both polarization of endosomes and nuclear position were measured using the Metamorph software.
Results:The fluorescent intensities of EGFP-Rab5c endosomes indicate that Rab5c positive endosomes concentrate within the apical most 50% of the apical/basal axis. Examination of the effect of nuclear position on endosome polarization indicates that when the nucleus is within 10%-50% of the distance to the apical surface, there is an increased concentration of Rab5c endosomes at the apical region as compared to cells with nuclei located more basally (>50% of distance of the apical/basal axis).
Conclusions:These results support the hypothesis that early endosomes (Rab5c positive) concentrate at the apical surface in a nuclear position dependent manner. This suggests that as the nucleus of retinal neuroepithelial cells migrates towards the apical surface, the concentrated population of endosomes may facilitate increased signaling. This hypothesis is consistent with the recent observations in which Notch activity as assessed by the her4:dRED transgene increased as the nucleus migrates towards the apical surface.
Investigation of the Role of Bone Morphogenic Proteins and their Antagonists in Anterior Segment Development
Ross F. Collery and Brian A. Link
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI
Purpose: Abnormal development of the eye can significantly impact the quality of vision and in severe cases may be associated with total blindness. Proper development of the anterior segment of the eye is required for correct aqueous humor flow and clear, unperturbed vision. Members of the TGFß superfamily, notably bone morphogenic proteins (BMPs), and their antagonists, are expressed in cells of the anterior segment, and have been implicated in eye development and ocular disease. Our goal is to investigate the contribution and subcellular regulation of the BMP pathway during anterior segment development and maintenance.
Methods:We will take advantage of the favorable experimental qualities of zebrafish, including amenability to genetic techniques and live imaging in embryos, to examine the expression profiles of BMPs and their antagonists in the eye. We will also investigate the effects of knockdown and overexpression of these factors using antisense morpholino technology and inducible transgenic constructs respectively.
Results:We have generated BMP response element promoter constructs expressing eGFP in zebrafish following BMP signaling. We have also generated eGFP-mature BMP fusion protein constructs.
Conclusions:Our initial work has demonstrated the usefulness of eGFP constructs that report BMP activity in vivo, and of tagged BMP proteins that allow live imaging of intracellular trafficking of these proteins. In future experiments we will explore the mechanisms and regulation of the BMP pathway in anterior segment development and maintenance.
Increased RGC dendrite branching is an early step in experimental glaucoma
Alina V Dumitrescu and Markus H. Kuehn
The University of Iowa, Department of Ophthalmology and Visual Sciences
Purpose: Elevated intraocular pressure (IOP) is one of the most important risk factors for the development of glaucoma. A number of studies have suggested that morphological changes of retinal ganglion cell (RGC) dendrites occur prior to RGC apoptosis. Here we sought to characterize the timecourse and morphological features of RGC changes in response to elevated IOP in a mouse model of glaucoma.
Methods: Elevated IOP was induced in one eye of transgenic mice expressing cyan fluorescent protein (CFP) under the control of the Thy1 promoter through injections of a hyaluronic acid solution into the anterior chamber. After four and nine days of elevated IOP mice were euthanized, enucleated and their retinas were flat mounted for confocal microscopy evaluation. Confocal image stacks of individual retinal ganglion cells were collected and analyzed using ImageJ software. Parameters evaluated included cell body size, dendritic field area, number of primary dendrites, and degree of dendrite branching.
Results: Hyaluronic acid injections increased the IOP on average to 16.2 mmHg (control eyes = 9.4 mmHg). Our findings demonstrate that the degree of dendrite branching increases in response to elevated IOP. The changes reach statistical significance as early as four days after induction of elevated IOP. We did not observe significant changes in RGC soma size, dendritic field size, or the number of primary dendrites. The number of RGC was not reduced after four and nine days of elevated IOP.
Conclusions: The Thy1-CFP mouse is an excellent model to evaluate morphological changes of individual RGC. Elevated IOP leads to rapid changes in RGC morphology that precede significant RGC loss. In particular we observed an increase in the degree of dendrite branching. These findings suggest that aberrant dendrite structure may be an early indicator of IOP induced retinal damage.
Cytokeratin Expression in Human Corneal Endothelium
Juan P. Fernandez de Castro MD and Nasreen A. Syed MD
The University of Iowa, Department of Ophthalmology and Visual Sciences
Purpose: Aberrant expression of cytokeratins in corneal endothelial cells has been described in several corneal conditions including posterior polymorphous dystrophy (PPMD) and the iridocorneal endothelial (ICE) syndromes. The expression of cytokeratins has been used to distinguish these diseases from other conditions histopathologically. However, recent studies suggest that cytokeratin expression may be a more general manifestation of corneal endothelial dysfunction and that it may not be disease specific. The purpose of this study is to characterize the spectrum of cytokeratin expression in human corneal endothelium in Fuchs endothelial dystrophy (FED), pseudophakic bullous keratopathy (PBK), PPMD, epithelial downgrowth (ED) and ICE in order to determine if patterns of expression are disease specific.
Methods: Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of twenty corneas with the diagnosis of FED, ten with PBK, four with ICE, six with PPMD, eight with ED and eight control corneas. Specimens were stained with monoclonal mouse antihuman antibodies CK 3/12, CK 5/6, CK 7, ZYM 5.2 (8 and 18), CK19, AE1/AE3 (CK 10,14,15,16,19 and CK 1,2,3,4,5,6,7,8), high molecular weight cytokeratin (HMWC) clone 34?E12 (CK 1, 5, 10 and 14) and vimentin, an intermediate filament found in mesenchymal cells. The sections were then analyzed using light microscopy and cells were graded as either positive or negative by two independent observers.
Results: CK 3/12 is consistently expressed in the endothelium of normal controls and all of the endothelial diseases. CK 5/6 is only positive in PPMD and ED. CK 7 appears widely expressed in all the diseases but not the controls. ZYM 5.2 is expressed only in a few cases of PPMD, PBK and FED. Vimentin is expressed uniformly in the control endothelial cells but is not present in some cases of ED and PPMD. The 34?E12 is only expressed in ED and PPMD after the cells lose their normal vimentin expression. AE1/AE3 and CK 19 are positive in all PBK but just some FED, suggesting a different mechanism of disease.
Conclusions: Cytokeratin expression, normally exclusive to epithelial cells, is present in normal endothelium and in various pathological states of the endothelium. HMWCs are expressed in ED and PPMD while low molecular weight cytokeratins are expressed in ICE, PBK and FED implying two distinct pathways resulting in endothelial dysfunction.
Cochlin - Association with elevated IOP and glaucoma
B.T. Gabelt1, E. Lee1,2, D.M. Peters1, C.R. Brandt1, S.K. Bhattacharya3, P.L. Kaufman1
1Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI; 2Yonsei University College of Medicine, Seoul, Republic of Korea; 3Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: Cochlin protein is detected in human glaucomatous trabecular meshwork tissue but not in controls. We determined the effects of cochlin transgene expression on outflow facility (OF) and intraocular pressure (IOP) in monkey organ cultured anterior segments (MOCAS). Transforming growth factor ?2 is elevated in the aqueous humor in human glaucoma. We determined if TGFβ-2 induces IOP elevation and cochlin expression in MOCAS.
Methods: An adenoviral (Ad) vector expressing cochlin was used to transduce MOCAS at 2.8E10 VP. OF and IOP were measured at 0, 3, 6, 9 and 12 days. Cochlin expression in the tissue and secretion into the media was monitored. Similar measurements were done in MOCAS treated with 10ng/ml TGFβ-2.
Results: Cochlin expression was detectable in media by 3 days after transduction. Reduction in OF was also detected after 3 days and continued to decrease further (70-75% decrease, p<0.001) for the 12-day duration of the study. IOP was more than doubled in segments injected with Ad-Coch-GFP when compared to control segments injected with Ad-GFP. In MOCAS treated with TGFβ-2 but not in controls, IOP began to increase after 3 days; OF was decreased approximately 40% after 5-9 days of treatment (p<0.005); and cochlin secretion into the media increased in a time dependent manner.
Conclusions: Cochlin expression alone was effective in decreasing OF and increasing IOP in MOCAS suggesting possible involvement of cochlin in IOP elevation. Cochlin may play a role in either the onset or maintenance of the TGFβ-2-induced IOP elevation and outflow facility decrease.
Comparison of Visual Preservation After Transplantation of BDNF or GDNF Secreting Mesenchymal Stem Cells in Glaucomatous Rat Eyes
Matthew M. Harper1, Donald S. Sakaguchi1, Markus H. Kuehn2, Young H. Kwon2, Randy H. Kardon2, Alexis Bemelmans3, Corinne Kostic3, Yvan Arsenijevic3, Sinisa D. Grozdanic1
1 Iowa State University, Ames, IA; 2 University Iowa and Department of Veterans Affairs; 3Jules-Gonin Eye Hosp., University of Lausanne, Switzerland
Purpose: To functionally and morphologically characterize the retina and optic nerve after transplantation of Brain-Derived Neurotrophic Factor (BDNF) and Glial-Derived Neurotrophic Factor (GDNF) secreting mesenchymal stem cells (MSCs) into glaucomatous rat eyes.
Methods: Chronic ocular hypertension (COH) was induced in Brown Norway rats. Lentiviral constructs were used to transduce rat MSCs to produce BDNF, GDNF, or green fluorescent protein (GFP). The fellow eyes served as internal controls. Two days following COH induction, eyes received intravitreal injections of transduced MSCs. Electroretinography was performed to assess retinal function. Tonometry was performed throughout the experiment to monitor intraocular pressure (IOP). 42 days after MSC transplantation, rats were euthanized and the eyes and optic nerves were prepared for analysis.
Results: Increased expression and secretion of BDNF and GDNF from lentiviral-transduced MSCs was verified using ELISA, and a bioactivity assay. Ratio metric analysis (COH eye/ Internal control eye response) of the Max combined response A-Wave showed animals with BDNF-MSCs (23.35 ± 5.15%, p=0.021) and GDNF-MSCs (28.73 ± 3.61%, p=0.025) preserved significantly more visual function than GFP-MSC treated eyes MSCs (18.05 ± 5.51%). Animals receiving BDNF-MSCs also had significantly better B-wave (33.80 ± 7.19%) and flicker ERG responses (28.52 ± 10.43%) than GFP-MSC treated animals (14.06 ± 12.67%; 3.52 ± 0.07%, respectively). Animals receiving GDNF-MSC transplants did not have significantly better B-wave function than animals with GFP-MSC transplants (p=0.057 and p=0.0639).
Conclusions: Mesenchymal stem cells are an excellent source of cells for autologous transplantation for the treatment of neurodegenerative diseases. We have demonstrated that lentiviral- transduced MSCs can survive following transplantation and preserve visual function in glaucomatous eyes. These results suggest that MSCs may be an ideal cellular vehicle for delivery of specific neurotrophic factors to the retina.
Identification of the modifier of retinoschisis 1 (Rs1) 1 (Mor1) gene
Britt Johnson, Natsuyo Aoyama, Sakae Ikeda, and Akihiro Ikeda
Department of Medical Genetics, University of Wisconsin-Madison, Madison, WI
Purpose: X-linked retinoschisis (XLRS) is an inherited form of macular degeneration that is caused by mutations in the retinoschisis 1 (RS1) gene. Previously, we reported the identification of a major QTL, named modifier of Rs1 1 (Mor1), which modifies the schisis phenotype in a mouse model of XLRS, the Rs1tmgc1 mouse. The purpose of this study was to identify the Mor1 gene.
Methods:The F2 intercross was made by mating (44TNJ-Rs1h tmgc1/Rs1h tmgc1x AKR) F1 mice. In order to narrow the Mor1 locus, F2 animals carrying the cross over within the minimal region of the Mor1 locus were collected and progeny tested. We compared the sequence of candidate genes between C57BL6/C3H and AKR. In order to test tyrosinase (Tyr) as a candidate gene for the Mor1 gene, we crossed female C57BL/6- Tyrc/Tyrc albino mice with Rs1tmgc1/y male mice. F1 mice were intercrossed to generate Rs1tmgc1/y; Tyrc/Tyrc mice. We tested whether the schisis phenotype caused by the Rs1 mutation is rescued in these mice.
Results:We have completed 7 lines of progeny testing, which narrowed the Mor1 genetic region down to 6.2Mb between markers D7Mit299 (90.0Mb) and D7Mit123 (96.2Mb). In the Mor1 minimal region, the AKR strain carries a mutation in the (Tyr gene. We observed no schisis in the retina of Rs1tmgc1/y; Tyrc/Tyrc mice.
Conclusions:Our results demonstrate that Tyr is the Mor1 gene.
Conditional ablation of Srf in the corneal epithelium rescues abnormalities in Dstncorn1 mice
Angela M. Verdoni1, Akihiro Ikeda1, Winston W. Kao2, and Sakae Ikeda1
1Medical Genetics, University of Wisconsin-Madison, Madison, WI; 2Ophthalmology, University of Cincinnati Medical Center, Cincinnati, OH
Purpose: Corneal disease-1 (Dstncorn1) mice are deficient for destrin, which depolymerizes filamentous actin (F-actin) into its monomeric form. Dstncorn1 mice exhibit accumulation of F-actin, epithelial cell hyperproliferation, autoinflammation, and neovascularization in the cornea. Previously, we observed upregulation of serum response factor (SRF) and its target genes in the Dstncorn1 cornea. The purpose of this study was to determine the role of SRF in the development of Dstncorn1 phenotypes through tissue-specific gene ablation.
Methods: Mice carrying the Dstncorn1 allele, an Srf allele flanked by loxP sites (Srff), a corneal epithelial specific reverse tetracycline transcriptional activator (Krt12rtTA), and cre recombinase downstream of a tetracycline responsive element (TetO-cre) were generated. Doxycycline was injected intraperitoneally every other day to induce cre recombinase expression. Immunofluorescent analyses were performed to assess the levels of F-actin and neovascularization, and to identify proliferating cells in the epithelium and inflammatory cells in the stroma.
Results: Administration of Doxycycline for 30 days demonstrated that conditional ablation of SRF expression in the corneal epithelium rescues the abnormalities observed in Dstncorn1 mice. The level of F-actin is greatly reduced, as is the level of inflammatory cell infiltration. The number of proliferating cells is comparable to that of a normal cornea. Neovascularization is also less evident.
Conclusions: This study demonstrated that abnormal activation of SRF in the corneal epithelium is primarily responsible for the corneal abnormalities observed in Dstncorn1 mice. It also demonstrated that the stromal phenotypes occur due to altered gene expression in the corneal epithelium of Dstncorn1 mice.
Elucidating the role of ALCAM in the eye: Identifying binding partners and assaying the effects of ALCAM modulation on melanoma motility
Karry Jannie and Joshua Weiner
Department of Biological Sciences, The University of Iowa
Purpose:ALCAM is an immunoglobulin superfamily member that typically localizes to cellular junctions in epithelial tissue, likely playing a role in adhesive complexes that preserve tissue architecture. Studies performed in Weiner et al., 2004 demonstrated the presence of retinal dysplasias and folds in ALCAM-null mice. Pigmented tissue containing melanocytes was observed to invade retinal space in these dysplastic regions. In the eye, ALCAM is highly expressed in the choroid; therefore, we hypothesize that dysplasias result from misregulation of adhesive processes therein. Our goal is to determine the role of ALCAM in the development and maintenance of retinal architecture.
Methods:We have assayed a panel of melanoma cell lines for motility and for expression of ALCAM and other adhesion molecules. We plan to modulate ALCAM levels in these cell lines and examine changes in motility (via wound-closure assay) and in expression of adhesion molecules. Candidate binding partners will be initially identified by peptide pulldown, and confirmed by immunoprecipitation.
Results:Analysis of the five melanoma cell lines revealed that ALCAM expression level positively correlates with motility in culture. Stable ALCAM knockdown cell lines have been confirmed by western blot, and are being analyzed for motility and expression of adhesion molecules. Four cytoplasmic candidate binding partners have been identified using the approaches outlined above.
Conclusions:This research will lead to a better understanding of the biology of cell adhesion within and between the choroid and retina, as well as provide insight into normal development of the eye and processes involved in ocular disease.
Photophobia-like behavior in a transgenic mouse sensitive to CGRP
Eric Kaiser1, Ana Recober2, Adisa Kubaras1, and Andrew Russo1
Departments of 1Molecular Physiology and Biophysics and 2Neurology, The University of Iowa
Purpose: Migraine is a neurological disorder affecting 11% of Americans; moreover, photophobia is a common migraine symptom affecting 66%-88% of migraineurs. Patients avoid intense lights, especially during an attack when photophobia is most intense. Photophobia reflects an allodynic response where non-painful light intensity is noxious. Calcitonin gene-related peptide has been implicated as a key neuromodulator in migraine based on its role in the trigeminovascular system, which is likely involved in photophobia as well. To study the effects of heightened CGRP sensitivity, we generated transgenic mice (nestin/hRAMP1) that overexpress human RAMP1, an obligate subunit of the CGRP receptor, in the nervous system.
Methods: As a correlate to photophobia, we have developed an assay to objectively study light-aversion in mice. nestin/hRAMP1mice were placed in a light-dark box for 10 minutes where they were free to move between the two zones. Since CGRP levels are elevated during a migraine attack, we tested the effect of exogenously delivered CGRP to the CNS on light aversion and the effect of a co-administered rizatriptan, a common antimigraine drug.
Results: nestin/hRAMP1 mice spent less time in the light after CGRP in comparison to control littermates and vehicle. This behavior could be prevented by rizatriptan administration. In response to CGRP, both control and nestin/hRAMP1 mice demonstrated reduced exploration and motility in the dark, which was also prevented by rizatriptan.
Conclusions: nestin/hRAMP1 mice appear to demonstrate photophobia-like behavior in response to centrally administered CGRP, which may serve as a novel behavioral animal model for both migraine and photophobia.
Validation of the TonoVet® Tonometer in Cats
Jeremy P. Kemmerling, Julie A. Kiland, and Gillian J. McLellan
Dept. of Ophthalmology and Visual Sciences, University of Wisconsin-Madison
Purpose: To test the validity of intraocular pressure readings obtained with the TonoVet® tonometer through comparison with manometry, while also taking readings with the widely used Tono-Pen XL® tonometer for further comparison.
Methods: The anterior chamber of each eye was cannulated through the cornea in three adult cats. The pressure was varied manometrically using a vertically adjustable reservoir and voltmeter, first increasing from 5 to 70 mm Hg in 5 mm Hg increments, then decreased from 70 to 10 mm Hg in 10 mm Hg decrements. At each increment/decrement, each of two observers each obtained three readings from both eyes, using both the TonoVet® and the Tono-Pen XL®. The data was then analyzed through linear regression for both tonometers.
Results: The resulting line for all of the TonoVet® data showed a slope of 1.0322, a y-intercept of 2.2338, and an r2 value of 0.9192. The line for the Tono-Pen XL® data showed a slope of 0.6222, a y-intercept of 1.4177, and an r2 value of 0.9382. The r2 values indicated that precision was acceptable for both tonometers. However, the Tono-Pen XL® readings correlated poorly with the actual values, as the slope was much less than one in value. The data showed no significant differences when comparing observers, incremental versus decremental readings; or right versus left eyes.
Conclusions: While both the TonoVet® and Tono-Pen XL® provide reproducible intraocular pressure measurements, the TonoVet® provides readings much closer to the true intraocular pressure than the Tono-Pen XL®.
Genomic Fragment Capture and Pyrophosphate Sequencing for Retinitis Pigmentosa Gene Discovery
Tyson R. Kinnick 2,3, A. Jason Grundstad4,5, Kevin Knudtson6, Todd E. Scheetz2,3,4,5, Terry A. Braun2,3,4,5, Val C. Sheffield1,7,and Edwin M. Stone1,2,3
1Howard Hughes Medical Institute, 2Carver Family Center for Macular Degeneration, 3Department of Ophthalmology and Visual Sciences, 4Biomedical Engineering, 5Center for Bioinformatics and Computational Biology, 6DNA Core Facility, Holden Comprehensive Cancer Center, 7Department of Pediatrics; The University of Iowa, Iowa City, IA
Purpose: Retinitis Pigmentosa (RP) is an inherited disease of photoreceptors that is likely to be caused by more than 100 different genes. Dozens of these genes remain to be discovered and many of these will be responsible for less than one percent of RP cases. This experiment was designed to use Roche/NimbleGen genomic fragment capture followed by pyrophosphate sequencing to identify disease-causing mutations in known RP genes as well as in genes not previously associated with this disease.
Methods: Custom Roche/NimbleGen sequence capture arrays were generated with sequence corresponding to the coding sequences and proximal promoter regions of 215 genes. One hundred twenty-three of these genes have been previously associated with human retinal disease while the remainder were judged to be good candidates for involvement in RP because of their retinal expression, their homology to known disease genes, or both. The sequence data were analyzed using custom software written by the authors. Plausible disease-causing mutations were confirmed with conventional automated DNA sequencing.
Results: Four RP patients were screened for mutations with this approach. An average of 84,657 reads of 231 bp in length were obtained from each patient in this experiment. Out of the 2914 exons included in the array design, 2878 were represented in the resulting sequence data. Plausible disease-causing mutations were identified in all four patients. In one patient, two correctly segregating disease-causing mutations were found in a known RP gene. The putative mutations observed in the other three patients were found in the heterozygous state and additional evaluation of these genes is underway in an attempt to identify a second disease allele.
Conclusions: Genomic sequence capture and pyrophosphate sequencing enable one to evaluate several million nucleotides of genomic sequence in a single experiment which is a significant advantage for extensively heterogeneous diseases like RP. A disadvantage of this broad approach is that as the number of genes sampled extends into the hundreds, the likelihood of observing a clinically irrelevant heterozygous carrier state of a true disease-causing allele becomes very high.
Disease Mechanism and Genetic Risk Factors of Visual Loss Associated with Increased Intracranial Pressure
Christopher Kirkpatrick and Markus H. Kuehn
Dept. Ophthalmology and Visual Sciences, The University of Iowa
Purpose: Idiopathic Intracranial Hypertension (IIH) currently has an annual incidence of 0.9 cases per 100,000. This number increases to 3.5 per 100,000 in women aged 15 to 44 years and to 19 per 100,000 in obese (at least 20% over ideal weight) women. Along with obesity and female gender, hypervitaminosis A, and steroid withdrawal are also known risk factors for the development of IIH. The purpose of these studies is to develop a mouse model of IIH to investigate environmental and genetic factors that may play a role in the development of IIH.
Methods: We developed a method to measure intracranial pressure (ICP) and are currently conducting a strain survey consisting of five different mouse strains (C57BL/6J, 129X1/SvJ, A/J, FVB/NJ, and DBA/2J) to investigate differences in ICP that can be attributed to genetic risk factors. We further developed a surgical approach in a mouse model to mimic IIH and artificially increase resistance to cerebral venous outflow through bilateral ligation of cerebral vena cavae and internal jugular veins.
Results: Our data obtained thus far (n=40, with n=5 for each strain) suggest that there are minor differences in ICP amongst the strains evaluated. Furthermore, it appears that our surgical approach may increase ICP to levels that will result in vision loss due to intracranial hypertension. Further study including OCT imaging of papilledema and immunohistochemistry of the optic nerve, will be conducted to confirm the outcome of this surgical approach and to evaluate its success in mimicking IIH.
Conclusions: There is little known about the pathophysiological effects of IIH on the optic disc, on the optic nerve, and on the retinal cells that leads to the visual loss. Our preliminary data suggest that the creation of an inducible mouse model of intracranial hypertension is possible. In addition, differences in ICP between mouse strains appear to exist which may be useful in the determination of genetic factors that govern regulation of ICP.
Voted Outstanding Poster Presentation
Mapping of the blind sterile 2 (bs2) locus in mice
R.P. Liegel1, B. Chang2, and D.J. Sidjanin1,3
1The Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 532262The Jackson Laboratories, Bar Harbor, ME, 04609; 3The Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 53226;
Purpose:Blind sterile locus 2 (bs2) is a novel autosomal recessive mouse mutant with a phenotype of congenital cataracts and male-specific sterility. Our goal was to identify the gene containing the mutation responsible for the bs2 phenotype, and to investigate the human ortholog of the bs2 locus.
Methods:bs2 homozygotes were outcrossed to the Cast/Ei strain followed by F1 (bs2 X Cast/Ei) intercross to generate F2 progeny, which were phenotyped at 3 weeks of age. Microsattelite markers were identified throughout Chr.2 and recombinant haplotypes were used to narrow the region containing the mutation until a candidate gene approach could be utilized for sequencing.
Results:Clinically, bs2 homozygote mice exhibit bilateral nuclear cataracts and micropthalmia. Histological evaluation shows that bs2 mice have disrupted lens epithelial and fibroblast cells resulting in mature cataracts. Bs2 testes are smaller than WT controls and do not contain mature sperm. Initial linkage analysis mapped bs2 to mouse chromosome 2, approximately 45cM from the centromere. Fine mapping narrowed the region to 3.1Mb, containing 19 genes. Sequencing of alkyl-dihydroxyacetonephosphate synthase (Agps), a gene within this region, revealed a G→A substitution at the +5 position of intron 14.
Conclusions:The identified mutation, located at a conserved nucleotide in the intron 14 splice-donor site, results in improper splicing of Agps mRNA and formation of two alternatively-spliced transcripts in the bs2 mouse. Agps encodes a ubiquitously expressed peroxisomal protein which catalyzes an early step in the synthesis of ether lipids. Further studies are needed to determine the functional consequences of this mutation on Agps protein.
Mutation in Sh3pxd2b Causes Congenital Glaucoma in Mice
Mao Mao1 and Michael G. Anderson1,2
1Department of Molecular Physiology and Biophysics, 2Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA
Using fine scale mapping and bioinformatics-driven sequencing strategies, we have identified the cause of the nm2702 phenotype as a 1 bp deletion within the Sh3pxd2b gene. Sh3pxd2b is a newly annotated gene about which little is known. SH3PXD2B contains one PX domain and four SH3 domains. The mutation is predicted to cause a frameshift and a protein truncation eliminating the last two SH3 domains.
Interestingly, a closely related homolog of SH3PXD2B, SH3PXD2A, has previously been shown to bind to members of ADAMs (a disintegrin and metallopeptidase) and to be required for podosome formation. Therefore, we have evaluated the ability of SH3PXD2B to form protein-protein interactions and to influence podosomes. Using GST pull-down, we found that SH3PXD2B is associated with ADAM15 through its last SH3 domain. In addition, we have shown that SH3PXD2B localizes to podosomes in cSrc-transfected fibroblasts.
These results suggest that Sh3pxd2b might be involved in the function of podosomes. In the eye, we speculate that SH3PXD2B may regulate podosomes of the trabecular meshwork, influencing intraocular pressure and contributing to the glaucoma phenotype.
Voted Outstanding Oral Presentation
Positional cloning of the lens opacity 13 (lop13) locus in mice
K.M. Merath1, L. Jackson1, J. Owens1, R.R. Dubielzig3, B. Chang3, and D.J. Sidjanin1
1Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226; 2School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI , 53706; 3The Jackson Laboratories, Bar Harbor, ME, 04609
Purpose: Cataracts are the leading cause of blindness worldwide and currently, the only treatment option for cataracts is the surgical removal of the lens. In order to develop novel therapies, the identification of the underlying molecular events participating in the development of cataracts is essential. The focus of this research is on the identification of the gene and associated molecular pathways that lead to autosomal recessive cataracts in the lens opacity 13 (lop13) mice.
Methods: A traditional positional cloning strategy was applied in order to identify the lop13 critical region. The lop13 mice were backcrossed to wild type C3H/HeJ mice and 144 F2 progeny were generated. Microsatellite markers were selected to evenly cover the mouse genome. Genotypes were determined by PCR amplification of genomic DNA and analysis of the alleles.
Results: The lop13 locus was mapped to a 520 Kb region between Csdc2 and Cyp2d9 on mouse chromosome 15. All of the genes within this region were sequenced and a single CàT base pair substitution was identified in nucleotide 3112 of Srebp2. This mutation leads to the substitution of a cysteine for an arginine in a highly conserved regulatory region of Srebp2. Complementation breeding schemes provided further genetic validation of the mutation.
Conclusions: Srebp2 plays an essential role in activating the transcription of several genes involved in the cholesterol biosynthesis pathway. This gene has not been previously linked to maintenance of lens transparency. Studies are currently in progress to evaluate the role of Srebp-2Arg1038Cys in the homeostasis of cholesterol within the lens.
Voted Outstanding Oral Presentation
Information theory in retinal maturation
Eric Nylen1, and Stephen Stasheff2
The University of Iowa, 1Department of Biomedical Engineering and 2Department of Pediatrics
Voted Outstanding Poster Presentation
Vision specific function for the newly identified Bardet-Biedl Syndrome 3 (ARL6) long isoform
Pamela R. Pretorius1, Lisa M. Baye2, Robert F. Mullins3, Charles C. Searby4, D.Y. Nishimura5, Kevin Bugge6, B. Yang5, Edwin M. Stone3,6, Diane C. Slusarski2, and Val C. Sheffield4,6
The University of Iowa 1Interdisciplinary Program in Genetics, 2Dept. Biological Sciences, 3Dept. Ophthalmology and Visual Sciences, 4Dept. Pediatrics, 5Dept. Obstetrics and Gynecology, 6 Howard Hughes Medical Institute
Purpose: We are interested in understanding the mechanism underlying retinal degeneration in Bardet-Biedl Syndrome (BBS) patients. We have identified a second longer transcript of BBS3, BBS3L, which is enriched in the eye and highly conserved between human, mouse and zebrafish. To gain insight into the mechanism underlying retinal degeneration associated with BBS, we have established mouse and zebrafish models of BBS3.
Methods: To evaluate the effects of bbs3L deficiency in the zebrafish, antisense morpholinos were utilized to knockdown bbs3 gene expression in the zebrafish. Rescue experiments were performed by co-injecting either human BBS3 or BBS3L RNA. A visual startle response and immunohistochemistry were used to assess bbs3 morphant and rescued zebrafish phenotypes. Additionally, a Bbs3L knockout mouse was generated and histologically evaluated.
Results: Consistent with an eye specific role, knockdown of bbs3L leads to an attenuation of vision function in the zebrafish. Rescue experiments demonstrate that BBS3L RNA, but not BBS3 RNA, is sufficient to rescue the vision defect. Moreover, in bbs3L morphants, green opsin was not restricted to the photoreceptor outer segment, but was also found in cell bodies of the outer nuclear layer. This mislocalization was rescued with BBS3L RNA. Histological analysis of Bbs3L-/- mice at 9 months revealed disorganization of the inner segments, indicative of retinal degeneration.
Conclusions: These data provide strong evidence that the BBS3L transcript is required for proper retinal function and organization. Moreover, the eye-specific expression of BBS3L will facilitate the dissection of BBS function in the retina and vision.
Analysis of functional properties across distinct morphological subpopulations of intrinsically photosensitive retinal ganglion cells
Tiffany M. Schmidt and Paulo Kofuji
The University of Minnesota Graduate Program in Neuroscience
Purpose: A subset of ganglion cells in the mammalian retina express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). These cells are implicated in non-image forming visual responses to environmental light such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness and activity. Morphological studies have identified least three distinct subpopulations of ipRGCs: those with monostratified dendritic arbors in either the outer (M1) or inner (M2) inner plexiform layer (IPL), and those with dendritic arbors bistratifying in both the inner and outer IPL. Research has also indicated that these subtypes receive synaptic inputs from the outer retina and may differentially and specifically project to various visual centers in the brain. The goal of the current study was to analyze and compare the functional properties of the various morphological subpopulations of ipRGC within the retina.
Methods: We utilized a transgenic mouse line in which ipRGCs are labeled in vivo with EGFP to target EGFP positive ganglion cells for whole cell recording and dye-filling.
Results: We observed functional differences between morphological ipRGC subpopulations, with M1 cells displaying larger intrinsic light responses, higher sensitivity to light, and higher input resistance compared to M2 cells. We also observed synaptic input from the ON pathway to both M1 and M2 cells.
Conclusions: These data indicate diversity of ipRGCs that goes beyond dendritic stratification in the IPL, and implies functionally distinct roles for these ipRGC subtypes in signaling light information to the brain.
Waved with open eyes (woe) phenotype is a hypomorphic mutation in Adam17
D.J. Sidjanin1,2, R.R. Dubielzig3, C. Zeiss4, B. Chang5, C. Blobel6, and E.L. Hassemer1,2
1The Department of Cell Biology, Neurobiology, and Anatomy 2Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 53226; 3School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706; 4School of Medicine, Yale, New Haven, CT 06520, 5The Jackson Laboratory, Bar Harbor, ME, 04609, 6Weill Medical College, Cornell University, New York, NY 10021
Purpose:Waved with open eyes (woe) is an autosomal recessive mutant that arose spontaneously on the C57BL/6J background. Phenotypically, the woe mice exhibit a wavy coat, open eyelids at birth, microphthalmia/anophthalmia and corneal opacities.
Methods:In order to map the woe locus, homozygote woe mice were backcrossed to C3H mice to generate 138 F2 progeny. Candidate gene analysis was performed via direct sequencing. Functional analysis of Adam17Thr265Met included evaluation of protein stability, trafficking and enzymatic activity.
Results:A genome wide scan mapped the woe locus to the proximal arm of mouse chromosome 12. Evaluation of the woe critical region identified Adam17 as a candidate gene. The Adam17 sequence analysis in woe mice revealed a C794T substitution that leads to a Thr265Met change in an evolutionary conserved residue. To unequivocally prove that the Adam17Thr265Met substitution is responsible for the woe phenotype, a complementation breeding was set up with an Adam17+/ - mouse. The Adam17- allele failed to complement confirming Adam17Thr265Met as the woe causing mutation. Adam17Thr265Met did not differ from wtAdam17 in protein stability and subcellular trafficking. Enzymatic activity revealed that Adam17Thr265Met exhibits severely reduced constitutive, but not PMA stimulated shedding activity.
Conclusions:Genetic and functional analysis revealed that Adam17Thr265Met is responsible for the woe phenotype. The role of Adam17 is in shedding the soluble forms of a variety of membrane-bound precursors essential for paracrine signaling. Adam17Thr265Met shows a defect in constitutive shedding activity. The molecular mechanism associated with Adam17 activation and substrate selectivity requires further investigation.
Voted Outstanding Poster Presentation
Complement System Components C3a and C5a in AMD
Jessica M. Skeie, Adam Hedberg-Buenz, Elizabeth A. Faidley, and Robert F. Mullins
The University of Iowa, Department of Ophthalmology and Visual Sciences
Purpose: Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the western world. Previous studies have shown that the complement system plays an important role in the progression of this disease. C5a and C3a are bioactive fragments of complement components C5 and C3. These peptides may be capable of binding to and activating choroidal endothelial cells during the pathogenesis of AMD.
Methods: Human RPE/choroid RNA was assayed for the presence of C3aR and C5aR using isoform-specific RT-PCR. Human tissue sections including retina, RPE, and choroid were immunolabeled with antibodies directed against C3aR and C5aR. Punches of human choroid from four different donor eyes were incubated for 24 hours in the presence of 1 ug/mL recombinant human C5a protein or the control protein, bovine serum albumin (BSA). Punches were immunolabeled with an anti-ICAM-1 antibody and choriocapillary labeling intensities were quantified and compared.
Results: C5aR, but not C3aR, is present in human choroid. By immunohistochemistry, C5aR is present in the choriocapillaris. Choroid punches incubated with C5a protein had an average 12% increase in ICAM-1 intensity (p-value < 0.001) in comparison to those incubated with BSA.
Conclusions: Endothelial cells in the choroid express C5aR, and may therefore be capable of binding to C5 peptides, which may activate these cells. More specifically, if C5a causes an increase of ICAM-1 on the surface of endothelial cells in the macula, it may help increase local leukocyte infiltration. C5a may therefore play a role in AMD pathogenesis progression by increasing ICAM-1 production by choroidal endothelial cells.
Spontaneous hyperactivity emerges before light responsiveness is lost during retinal degeneration in rd10 mice
Steve F. Stasheff and Michael P. Andrews
University of Iowa Children’s Hospital & Carver College of Medicine, Iowa City, IA
Purpose: Spontaneous and light-evoked ganglion cell activity alter substantially as photoreceptors degenerate in the retinal degeneration (rd1) mouse, suggesting significant reorganization of inner retinal circuitry. In the closely related rd10 mouse, degeneration begins only after a peak of developmental plasticity in retinal circuitry. We compared these two models to determine whether changes in spontaneous versus light-evoked activity stem from distinct mechanisms, and whether they reflect perturbations of retinal development.
Methods: Action potentials were recorded in vitro from wild type (wt), rd1 or rd10 retinal ganglion cells, using a multielectrode array. Spontaneous activity and responses to full field light flashes were monitored at various developmental ages.
Results: Spontaneous “waves” of correlated ganglion cell activity, comparable to those in wt mice, are present in rd1 and rd10 retinas prior to eye opening (P7-8). In rd1 and rd10 mice, spontaneous firing increases by postnatal day 14-15 (P14-15) and accelerates further by P28. By then light responses have disappeared in rd1 but remain vigorous in rd10, disappearing completely only after P48.
Conclusions: Despite a similar genetic defect as in rd1 mice, in rd10 retinas normal developmental processes form and maintain light-evoked activity for an extended period, even though spontaneous hyperactivity emerges as in rd1. Thus, developmental mechanisms do not contribute substantially to the emergence of hyperactivity, but mechanisms underlying the hyperactivity appear separable from those that alter light-driven responses. This raises hope that normal visual function might be preserved or restored even in the face of ganglion cell hyperactivity seen in inherited retinal degenerations.
Determining the effectiveness of the pupil light response (PLR) in estimating optic nerve damage
Michael Stefl and Markus H. Kuehn
Dept. Ophthalmology and Visual Sciences, The University of Iowa
Purpose: The pupil light response (PLR) is a reflex that constricts the pupils of both eyes, regardless of which eye light is shined upon. This reflex begins to fail when optic nerve damage or retinal ganglion cell death occurs. The purpose of these studies is determine if measurements of the PLR in mice with ocular hypertension is an accurate tool for evaluating the degree of optic nerve damage and retinal ganglion cell death.
Methods: In order to raise the intraocular pressure (IOP) groups of C57BL/6J mice received injections of adenovirus expressing a mutant form of myocilin into the anterior chamber. A control group was injected with empty virus. The IOP of these mice is measured weekly, and the mice also undergo weekly electroretinogram (ERG) and PLR measurements. Mice are euthanized at weekly intervals and morphological measurements are taken to determine damage to the optic nerve and retinal ganglion cells. By comparing the damage to the PLR results, I will be able to determine how effective PLR can be in estimating the levels of damage. This will also determine both the baseline level of damage required to see a measurable PLR effect and the deterioration of PLR as damage progresses.
Results: After 3 weeks, the IOP has increased in treated eyes and has not changed noticeably in control eyes. Measurements of PLR have also shown a slight decrease in function after two weeks.
Conclusions: The myocilin virus vector has been successful at raising the IOP of treated eyes. This approach seems to be a valid method of measuring several factors that are correlated with other in treated and control groups. The correlations between morphological data, PLR and ERG measurements, and IOP will allow us to determine if PLR measurements are closely correlated to damage of optic nerve and/or retinal ganglion cells.
Chaperonin-like Genes Underlying Bardet-Biedl Syndrome
Svetha Swaminathan1, Lisa M. Baye1, Edwin M. Stone2,3, Val C. Sheffield3,4, and Diane C. Slusarski1
1Dept. of Biological Sciences, The University of Iowa, Iowa City, IA; 2Dept of Ophthalmology, University of Iowa, Iowa City, IA; 3Howard Hughes Medical Institute; 4Dept of Pediatrics, University of Iowa, Iowa City, IA
Purpose: Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, autosomal recessive disease characterized by obesity, retinal degeneration, polydactyly, kidney malformations, hypogenitalism and learning disabilities. Among the twelve BBS genes, BBS6, 10 and 12 are chaperonin-like members of the CCT gene family which are known to interact as complexes. The zebrafish model system was used to characterize the phenotypes of these three BBS genes which underlie 25% of the known BBS cases.
Methods: Antisense oligonucleotides (morpholinos) were utilized to knockdown bbs6, 10 and 12 in the developing zebrafish embryo. Analysis of embryonic phenotypes included examining Kupffer’s Vesicle (KV) size, epinephrine treatment to stimulate and time melanosome transport, as well as a visual startle response assay to assess vision.
Results: The cardinal features of BBS gene knockdown in zebrafish were observed for bbs6, 10 and 12. Specifically, defects in KV formation, a transient ciliated structure, and delays in melanosome transport were seen. Interestingly, pair wise co-injections of low dose BBS morpholinos suggest that bbs6 and bbs10 may be interacting genetically to repress the KV defect. Additionally, to examine the molecular and cellular pathways underlying vision loss in BBS, vision screening assays specific to zebrafish were also used. Vision analysis on morpholino injected embryos suggests reduced visual function with knockdown of bbs12.
Conclusions: The chaperonin-like BBS genes have the cardinal BBS features along with an additional vision defect in bbs12. By understanding the normal function of these genes we will gain in site into common ailments in society including vision loss.
Voted Outstanding Oral Presentation
The role of ON bipolar cell mediated photoreceptor input to irradiance responses
Stewart Thompson1,2 and Edwin M. Stone1,2
1Dept. of Ophthalmology, University of Iowa, Iowa City, IA; 2Howard Hughes Medical Institute
Purpose: Irradiance information transmitted to central nuclei via intrinsically photosensitive melanopsin expressing ganglion cells (ipRGCs) regulates many aspects of behavior and physiology. The response of ipRGCs to light is a composite of the synaptically mediated (rod/cone) and endogenously generated (melanopsin) activity. The aim of this study was to identify the role of metabotropic-depolarizing bipolar cells (MBCs) in mediating rod/cone photoreceptor input to irradiance coding responses.
Methods: Negative masking responses and the pupil light reflex (PLR) were compared between mice with selective absence of MBC function (Grm6Nob4), and mice lacking rod/cone generated input to retinal pathways (rd1).
Results: Absent photoreceptor input in rd1 mice caused characteristic enhanced negative masking sensitivity, but the same increased sensitivity was also apparent in Grm6Nob4. In contrast, the sensitivity of the PLR was less severely reduced in Grm6Nob4 than rd1 mice.
Conclusions: MBCs are critical for synaptically mediated photoreceptor input to negative masking, but both MBCs and MBC independent pathways mediate significant photoreceptor input to the PLR. Together these findings show that the role of MBCs is different across irradiance responses, and begins to explain why the effect of retinal disease varies between irradiance responses. From these and previous tract-tracing findings we predict that retinal input to negative masking is via M1 ipRGCs, having unusual ON-MBC input in the OFF inner plexiform layer A of the retina.>
Positional Cloning and Functional Analysis of the Waved with Open Eye Lids 2 (woe2) Locus
J. Toonen1, L. Liang1, L. Jackson1, J. Besharse1, and D.J. Sidjanin1,2
1The Department of Cell Biology, Neurobiology & Anatomy, and 2Human Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI
Purpose: Waved with open eyelids 2 (woe2) is an autosomal recessive mouse mutation that arose spontaneously on the C57BL/6JX129 F1 background. Phenotypically, woe2 mice exhibit open eyelids at birth, wavy fur, microphthalmia/anophthalmia, and heart defects. The aims of this study are to identify the causative mutation and elucidate the mechanism of the protein in proper eye development.
Methods: The woe2 mice were outcrossed onto C3A.BLiA-Pde6b+/J strain and subsequently backcrossed for linkage mapping. 168 F2 progeny were utilized for isolation of DNA and RNA for genetic analysis. In situ hybridiazation was performed with a P33 labelled riboprobe and incubated for 6 weeks. TUNEL assay was performed on E15.5 sections using a kit from Millipore.
Results: Histology of woe2 eyes showed corneal neovascularization, anterior synechiae, retinal rosettes, and optic nerve hypoplasia. The woe2 locus was mapped to a 6.8 cM region on mouse chromosome 7. Protein Phosphatase 1, Regulatory (inhibitor) Subunit 13 Like (Ppp1r13l) as a candidate gene. Sequening of woe2 gDNA identified a 1308 bp deletion from intron 8-11. cDNA analysis revealed an alternatively spliced transcript in the woe2 mouse. Expression appears to be restricted to the periderm, eyelid dermis and epidermis, cornea and optic nerve. Recently, an increased apoptosis was detected in the migrating eyelids and optic nerves of woe2 mice in prenatal tissue sections.
Conclusions: Ppp1r13l has been shown to interact with Nuclear Factor-kB (NF- kB). Results suggest a novel role for Ppp1r13l in NF-kB mediated apoptosis in eyelid closure. We are further probing defects in differentiation, proliferation, and migration in woe2 mice.
Voted Outstanding Poster Presentation
Utilizing B6-Lystbg-J Mice to Extend Genetic Pathways of Exfoliation Syndrome
Colleen M. Trantow1 and Michael G. Anderson1,2
1Molecular Physiology and Biophysics, 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA
Purpose: Exfoliation syndrome (XFS) is a common age-related disorder recognized by the pathological accumulations of a fibrillar exfoliative material in the eye. Human eyes with XFS, and mice containing the Lystbg-J mutation, both exhibit a striking pattern of iris transillumination defects. The goal of these experiments was to capitalize on this resource by using mice to study the molecular pathways of XFS.
Methods: B6-Lystbg-J mice were identified from a screen of coat color variants assayed by slit-lamp exam. B6-Lystbg-J mice and age-matched C57BL/6J control mice were analyzed by clinical, histological, and physiological assays.
Results: The iris transillumination defect in both XFS patients and B6-Lystbg-J mice correlates to an unusual “saw-tooth” morphology of the iris pigment epithelium. At all ages clinically examined, B6-Lystbg-J mice exhibited significant iris defects. Similar to human XFS, B6-Lystbg-J mice produce an exfoliative-like material and exhibit pronounced pigment dispersion. The molecular basis of the bg-J mutation is a result of a three base pair deletion in the WD40 encoding region of the Lyst gene. Previously it was shown that the LYST WD40 domain interacts with CSNK2B. We confirmed this interaction with a GST-pulldown experiment. Interestingly, LYSTbg-J disrupts the interaction with CSNK2B. CSNK2B function in regulating E-cadherin and ß-catenin binding is subsequently disrupted.
Conclusions: These results lead to a working hypothesis that aspects of the XFS phenotype involve LYST and CSNK2B pathways, likely influencing cell-cell adherens junctions. Our results suggest that LYST, or a component of the LYST genetic pathway, are excellent candidates for contributing to human XFS.
Characterizing retinal phenotypes of an adult-onset large eyed zebrafish mutant
Kerry N. Veth, Matthew P. Gray, and Brian A. Link
Dept. of Cell Biology, Medical College of Wisconsin
Purpose: To describe the eye phenotypes of lrp2.1 mutant zebrafish and probe the molecular mechanisms contributing to the adult-onset large eyes.
Methods: Histology was used to compare the number of cells in mutants to wild type fish at ages prior to (1 month) and at the onset (2 – 3 months) of large eyes. Immunofluorescence against the cell cycle protein MCM5 was used to investigate retinal proliferation. Finally, microarray analysis was employed to compare the gene expression profiles of 36 hour and 1 month mutant eyes against age matched wild type eyes.
Results: There was no difference in estimated total cells in the central retinas of mutants versus wild type fish at 1 month, although there was a trend towards decreased cell density that reaches significance in the ganglion cell and photoreceptor layers. There was no difference in cell proliferation at 1 month. At 2-3 months, there was a significant decrease in retinal cell density, but not in estimated total cells. By 6 months, total retinal cell numbers were diminished, presumably through degeneration caused by prolonged stretch-stress. Pathways of interest from the microarray include retinoid trafficking, extracellular matrix stability, and cytoskeletal interaction.
Conclusions: Lrp2.1 mutant zebrafish develop large eyes over a prolonged period of time, but not as the result of increased retinal proliferation. After eyes become visibly enlarged, there is measurable loss of retinal neurons. Because lrp2.1 mutant fish also show elevated intraocular pressure, this strain provides a tractable model for investigating the relationships between high myopia and glaucoma.
Analysis of a potential role of forkhead transcription factor foxd3 in eye development
Bethany A. Volkmann and Elena V. Semina
Medical College of Wisconsin, Dept of Cell Biology, Neurobiology and Anatomy and Children’s Research Institute, Division of Developmental Biology. Milwaukee, Wisconsin
Purpose: The foxd3 gene encodes a fork head-domain transcription factor that is expressed in neural crest precursors and in early migratory neural crest cells in different species. Neural crest-derived periocular mesenchyme makes a major contribution to the developing anterior segment of the eye, but the molecular mechanisms involved in normal development of the anterior segment structures are not clear at this time. A role for foxd3 in eye development has not been evaluated. We decided to examine potential role of foxd3 in ocular development through analysis of a new foxd3 -deficient zebrafish line as well as foxd3 morphants.
Methods: A zebrafish line containing a viral insertion into foxd3 was obtained from Znomics, Inc. Presence of the viral insertion was confirmed and heterozygous carriers identified through sequencing and PCR genotyping. Heterozygous carriers were crossed and progeny were genotyped and analyzed for phenotypic defects at 24-120 hours post-fertilization (hpf). Histology, in situ hybridization and alcian blue staining were performed using standard protocols.
Results: The insert incorporation site was confirmed and found to disrupt sequence encoding the foxd3 forkhead domain. In addition to this, analysis of the foxd3 transcript by RT-PCR demonstrated a significant decrease in transcript level in mutant embryos. Since the remaining mutant transcript is predicted to encode nonfunctional protein, the identified foxd3viral allele appears to result a complete loss-of-function phenotype. Homozygous foxd3viral zebrafish display an obvious phenotype that includes a protruding jaw, a loss of posterior pharyngeal arches, cardiac edema and embryonic lethality by 7 days post-fertilization while heterozygous animals appear to be normal. To analyze ocular development in foxd3viral homozygous embryos, histological analysis was performed and revealed a notably disorganized lens and retina in 80-hpf embryos. A partial recovery of this phenotype was observed at later stages, 96-120hpf. To further evaluate the ocular phenotype associated with foxd3 deficiency, expression of pitx2, lmx1b, foxe3, sox2, grem2, prox1 and other genes that are known to play role in development of different ocular structures will be analyzed. In addition to foxd3viralmutants, foxd3 morphants generated through injection of foxd3 -specific morpholino will be examined.
Conclusions: The data suggest that foxd3 plays a role in eye development. Further characterization of a phenotype of foxd3viralmutants and morphants will help to reveal specific defects associated with foxd3 deficiency.
Does ccne2 affect proliferation and neurogenesis?
Haijie Xiao, Lisa M. Baye, and Brian A. Link
Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin
Purpose: In several contexts, cell cycle kinetics has been shown to be important for neurogenesis. We have observed that in zebrafish retina the sibling cell with a shorter cell cycle length is highly biased to exit the cell cycle and become a neuron. Our purpose for this study is to characterize the relationship of neurogenesis and cell cycle kinetics.
Methods: To study the relationship between neurogenesis and cell cycle kinetics, we chose to manipulate Ccne2 which is the dominantly expressed G1 cyclin in the retina. Mophilino knock-down of Ccne2 slowed the G1-S phase transition of retinal cells as judged by DNA content analysis. We study the function of gain and loss of Ccne2 by using Edu assay, proneuron gene promoter and neuron specific marker to check proliferation and neurogenesis of zebrafish retina.
Results: In ccne2 morphants, we found reduced neurogenesis as measured by GFP expression from the neurogenic atho7 promoter. Consistent with this, we also observed that when Ccne2 was transiently overexpressed, retinal progenitor cells tended to exit cell cycle earlier and resulted in less total cells within the retina as compared to control embryos. We observed that cells in the photoreceptor layer were the ones primarily affected.
Conclusions: Our data is consistent with an influence of G1 phase kinetics on regulating cell cycle exit of retinal progenitor cells. We are currently exploring whether alterations to cell cycle kinetics differentially affects neurogenesis in distinct populations of retinal progenitors.
Transgenic Mice Overexpressing Mutated Human Myocilin Develop Primary Open-Angle Glaucoma
Gulab S. Zode1, Alina V. Dumitrescu2, Charles C. Searby1, Markus H. Kuehn2, Val C. Sheffield1
The University of Iowa, 1Department of Pediatrics and 2Department of Ophthalmology and Visual Sciences
Purpose: Myocilin mutations have been associated with congenital glaucoma and 2–4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. The objective of this study was to examine the glaucoma phenotype of the Tyr437His mutation of human myocilin in transgenic mice (Tg)
Methods: Transgenic mice were created overexpressing the Tyr437His mutant form of human myocilin under the control of the CMV promoter. Immunohistochemistry and western blot analysis was used to examine myocilin expression in anterior chamber of Tg mice (age 3-8 month). Aqueous outflow pathway function was examined by measuring intraocular pressure using rebound tonometer. Gamma synuclein staining of whole mount retina was used to evaluate retinal ganglion cell (RGCs) death.
Results: Tg mice demonstrate higher myocilin expression in irodocorneal angle of transgenic mice compared to wild type (Wt) mice. Starting at 2-month age, Tg mice exhibit significantly higher intraocular pressure (12 mmHg in wt vs 16 mmHg in Tg, n=40). In addition, water-drinking test reveals abnormal outflow in Tg mice (n=4). In addition, Tg mice loose ~ 40% RGCs death in the retina (n=2). Interestingly, expression of alpha, beta, and gamma crystallins, a family of heat shock proteins is significantly increased in the anterior chamber of transgenic mice, indicating their anti-stress role in transgenic mice.
Conclusions: Our observations indicate that Tg mice exhibit pathological changes similar to those in glaucoma patients. In addition, induction of crystallins in transgenic mice may be response to misfolded myocilin in Tg mice.
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