The Vision Health Research Network Annual Research Day

The Vision Health Research Network(VHRN)was established in 1995 to increase research capacity,productivity,and visibility of vision science carried out in Quebec,Canada.The VHRN is funded by the Fonds de la recherche du Québec en santé(FRQS),a Quebec government agency,and the Foundation Antoine-Turmel,a private charity organization.In the past decades,the VHRN has fostered productive collaborations and helped improve access to specialized infrastructure and tissue banks,thereby enhancing competitivity of Quebec vision research on the international stage.The VHRN also supports vision research directly by providing seed funding for national and international collaborative projects.The VHRN is also dedicated to support the training of the next generation of vision researchers by funding scholarships and awards for the best trainees,which not only help them carry out their projects but also make them more competitive to apply for funding at national and international granting agencies.The VHRN strongly believe that these research building initiatives will help us achieve our ultimate goal,which is to improve care and develop treatments for patients living with various vision impairments.

AB040.Pou2f1/2 are required for the specification of cone photoreceptors in the developing retina

Background:Rods and cones are critical for light detection.Although there has been considerable work done in elucidating the molecular mechanisms involved in rod development,not much is known about how the cone cell fate decision is made by the multipotent retinal progenitor cells during development.Analysis of the promoter regions of Nrl and trβ2,rod and cone differentiation factors respectively,revealed DNA binding motifs of two POU-domain containing transcription factors,Pou2f1 and Pou2f2.Preliminary experiments showed that Pou2f1/2 are expressed during the peak of cone genesis in the embryonic retina.Therefore,we hypothesize that Pou2f1/2 specify cone cell fate in the developing retina.Methods:We used immunofluorescence and in situ hybridization to establish the spatiotemporal expression of Pou2f1/2 during retinogenesis.We performed in vivo electroporation in post-natal mice to misexpress Pou2f1/2 and used antibodies specific to proteins expressed in cones such as Rxrγand S-opsin to count cones.Using ex vivo electroporation of embryonic retinal explants,we knocked out Pou2f1 and Pou2f2 using CRISPR/Cas9 gRNAs at the peak of cone production window.Finally,we transfected post-natal retinal explants with a combination of regulatory elements of Nrl or thrb with control backbone vector,Pou2f1 or Pou2f2 using electroporation.Results:We found that Pou2f1/2 are expressed in retinal progenitor cells in the developing retina and subsequently in the differentiated cones.Pou2f1/2 misexpression outside the cone genesis window led to an increase in cones at the expense of rods.Pou2f1/2 indel knockouts generated by CRISPR/Cas9 gRNAs led to a decrease in cones and a converse increase in rods.Finally,we found that Pou2f1/2 activate the cis-regulatory module(CRM)of the thrb gene and repress the activity of the CRM of Nrl.Conclusions:These results uncover novel players that establish the complex gene regulatory network for cone photoreceptor fate specification in the retinal progenitor cells.We anticipate that this work should help us devise improved replacement therapies in the future utilizing stem cells for retinal degenerative diseases such as aged-related macular degeneration(AMD)and Stargardt’s disease.

AB046.The Endocytic Adaptor Protein Numb Functions in Müller Glia to Maintain Retinal Polarity and Photoreceptor Survival through the Polarity Determinant Crumbs

Background:The loss of cell polarity plays a key part in retinal dystrophies such as retinitis pigmentosa(RP)and Leber congenital amaurosis(LCA),resulting in photoreceptor(PR)degeneration and vision loss.Despite not knowing the direct genotype-to-phenotype correlation,many disease-causing mutations in the polarity determinant Crumbs(Crb1),have been identified.Indeed,the loss of Crb1 in mice was shown to cause PR death,due to the loss of adhesions between PR and Müller cells at the apical surface of the retina.Unfortunately,although the role of Crb1 in neuron polarity and survival is well established,little is known about how its intracellular trafficking is regulated.With future treatments for retinal degenerative diseases in mind,the goal of this project is to understand the mechanism by which Crb1 is regulated and how it maintains retinal integrity.Previous work in our laboratory showed that Numb,an endocytic adaptor protein,is an important regulator of protein trafficking in retinal cells.We therefore hypothesized that Numb might function as regulator of Crb1 in Müller glia.Methods:To study Numb function in Müller cells,we generated a conditional knockout(cKO)mouse line to inactivate Numb specifically in Müller cells by crossing a Glast-CreERT2 mouse line with a Numb-floxed line.At 30 days,mice were administered tamoxifen to trigger inactivation of Numb and retinas were then collected at time points varying from 2 weeks to 17 months for analysis.Firstly,we studied the retinal morphology and outer limiting membrane integrity by histology and immunohistochemistry.Using electron microscopy(EM),adhesions between Müller glia and photoreceptors were analysed and retinal function was assayed in live mice by electroretinography(ERG).To detect protein expression levels,protein extracts were prepared from cKO and control retinas for immunoblotting.To test for the presence of a biochemical interaction,Hek-293 cells were transfected with Numb and Crb1 vectors,and protein extracts were processed for co-immunoprecipitation.Results:When Numb was deleted in Müller cells,we observed a similar retinal phenotype than what was reported in the Crb1 KO.In 3-month-old animals,we found a disruption of the outer limiting membrane and an ingression of photoreceptor cells in the inner layers of the retina.In older animals(17 months),we observed a clear thinning of the photoreceptor layer and reduced ERG responses.Immunoblotting of retinal lysates revealed that Numb cKO retinas had significantly lower expression of Crb1,suggesting that Numb function in Müller cells is critical to maintain Crb1 levels and thereby outer limiting membrane integrity.Interestingly,we found that Numb can interact with Crb1 both in vitro and in vivo,suggesting that Numb might function as an adaptor protein regulating Crb1 trafficking.Conclusions:Based on these results,we suggest that,in the absence of Numb,Crb1 cannot be trafficked to the apical membrane of Müller cells,and is instead degraded.This ruptures the adhesion between Müller and photoreceptor cells,leading to photoreceptor degeneration.We anticipate that understanding the mechanisms by which Crb1 maintains the structural integrity of the retina will lead to new possibilities for target-based therapies against retinal dystrophies.