SUMMARY

The Crumbs Complex in the retina: From animal models to function

Retinal cell generation and differentiation in the mouse occurs from embryonic day 11 to postnatal day 10. Six major neuronal and one glia cell types are generated from multipotent retinal progenitors in a characteristic sequence during development. In this temporally fine-tuned process, ganglion cells are generated first, followed by horizontal cells, cone photoreceptors and early born amacrine cells, rod photoreceptors and late born amacrine cells, and finally bipolar cells and Müller glial cells. Retinal progenitor cells are elongated and polarized cells that extend along the apicobasal axis and connect to adjoining cells by adherens junctions via their apical processes. Cell adhesion and cell polarity protein complexes, such as the Crumbs (CRB), PAR and adherens junctions complexes, play a critical role in maintenance of the proliferation of the progenitor cells. Changes in these complexes disturb the spatiotemporal aspects of retinogenesis, leading to retinal degeneration resulting in mild or severe impairment of retinal function and vision. A SUMMARY of the available mouse models, for the different members of the apical polarity complexes, as well as the main features of their phenotype is described in chapter 1.
The apical CRB complex is located at the subapical region adjacent to adherens junctions between the retinal progenitor cells in the developing retina and, after differentiation, at the subapical regions of Müller glia and photoreceptor cells. In mammals, the CRB family consists of CRB1, CRB2, CRB3A and CRB3B.
In humans, mutations in the CRB1 gene are responsible for retinopathies such as Leber congenital amaurosis and retinitis pigmentosa. The lack of a clear genotype–phenotype correlation suggests that other components of the CRB complex have a function influencing the severity of the retinal disease. To date no other CRB gene has been identified that is related with any human retinal disease, however we cannot exclude that some transcript variants may play a role in CRB1 retinal diseases.
We hypothesized that Pals1 and Crb2 have a function in retina development and maintenance. To test this hypothesis a Pals1 conditional knockdown and Crb2 conditional knockout mutant mice were generated and the retinal phenotype of these animals were analyzed. The data, presented in chapter 2, showed that PALS1 has an important role in the neuroretina as well as in the retinal pigment epithelium. Loss of PALS1 in these tissues led to retinal degeneration and vision impairment.
In chapter 3, we demonstrated that conditional deletion of Crb2, specifically in early progenitors, resulted in retinal disorganization during late retinal development leading to severe and progressive retinal degeneration with concomitant visual loss that mimics retinitis pigmentosa due to mutations in the CRB1 gene.
Variation in the genetic background may influence the severity of the retinal phenotype, as described before for the Crb1 knockout mice. Indeed, backcrossing Crb1 mutant retinas from mixed to C57BL/6J genetic background strongly suppressed the morphological phenotype. In chapter 4, we showed that backcrossing Crb2 null retinas from mixed (50% 129/Ola and 50% C57BL/6J) to 99.9% C57BL/6J background did not suppress the severe morphological phenotype.
Recent studies demonstrated that the CRB complex members are able to regulate several important signalling pathways including the Notch1, mechanistic target of rapamycin complex 1 (mTORC1), Wingless (Wnt) and the Hippo pathway (see chapter 1 for more details). To elucidate the molecular events that precede and lead to the morphological phenotype in the Crb2 retina-specific conditional knockout mice on 99.9% C57BL/6J background, we applied microarray-based mRNA profiling in retinal tissue at postnatal stages P0, P3, P6, and P10. Surprisingly/Unexpectedly, the data presented in chapter 4, showed that the morphological phenotype observed in these retinas did however not result in a significantly altered transcriptome at any stage of retinal development analysed. These findings may be explained in part by the mosaic genetic nature of the mutant retinas which contain mutant next to wild type cells due to insufficient levels of Cre expression in a subset of cells. Removal of Crb2 might be also compensated in part at the transcriptome level by other CRB proteins such as CRB1 and CRB3.
All the studies and observations so far suggest that, in the mice, the main and most important function of the CRB proteins resides in either the Müller glia cell or photoreceptors. In order to clarify this we decided to study the consequences of CRB2 specific removal from Müller glia or from photoreceptor cells, using different approaches: cell type specific Cre mouse lines and adeno associated viral vextors (AAVs) expressing Cre or shRNAs against Crb2. The results presented in chapter 5, showed that CRB2 has essential roles in the photoreceptor cells and redundant roles in the Müller glia cells.
In the mouse retina, CRB2 seems to be more important for retinal development and maintenance than CRB1, however the data suggested a possible overlap of function between these two proteins. To address this hypothesis we analyzed the retinal phenotype of the Crb1Crb2 double knockout mice (chapter 6). Loss of CRB1 and CRB2 from the retinal progenitor cells and their differentiated cell types led to a thicker retina containing all retinal cell types, but lacking a separate layer of photoreceptor cells. The eyes showed loss of retinal function mimicking the phenotype observed in human Leber congenital amaurosis patients with CRB1 mutations. We also demonstrated that CRB1 and CRB2 control the retina size by preventing the overproliferation of late progenitor cells during retinal development potentially through regulation of P120-catenin-Kaiso signaling pathways.
In conclusion, we described the generation and analysis of several mutant mouse retinas for different CRB complex members: Pals1 (chapter 2), Crb2 (chapter 3, 4 and 5) and Crb1Crb2 double mutant eyes (chapter 6) and the analysis of their retinal phenotype. The knowledge obtained in the analysis of their retinal phenotype helped us to better understand the function of these proteins in retinal development and maintenance. Mutations in some of these genes lead to human retinal diseases, as is the case for mutations in CRB1 that cause Leber congenital amaurosis and retinitis pigmentosa. Thus, these animals became important not only to answer fundamental questions, but they also may have pre-clinical value to study retinal degeneration and to test new therapies.