The roles of extracellular vesicles from the RPE in eye development and their regulation by primary cilia.

• Applicant: Professorin Helen May-Simera, Ph.D.
• Subject Area: Cell Biology ; Developmental Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 547810895

The transfer of extracellular vesicles (EVs) has recently been identified as an important mechanism for intercellular communication. Recent studies have shown that the primary cilium, an evolutionarily conserved signaling organelle, is important for coordinating the release of small EVs (smEVs). The current challenge is to determine the content and functional significance of smEVs during specific organ development and disease. We are studying these topics in the eye’s retinal pigmented epithelium (RPE), a monolayer of specialized, polarized epithelia essential for vision. Our preliminary data characterized small EVs secreted by human RPE generated from stem cells (hES-RPE). We further demonstrate an important role for primary cilia in smEV secretion that modulates WNT signaling in the kidney. Since ciliary regulation of WNT signaling is essential for RPE differentiation, these findings lead us to hypothesize that smEVs play a significant role in signaling between the RPE and surrounding tissues, and that primary cilia contribute to their release. The goal of this study is to determine the role of smEVs in communication between RPE and retina/choroid during development, with a particular focus on the role of ciliary smEVs. To this end we will address the following specific aims: • Aim 1 - Characterization and functional assessment of RPE-derived smEVs released from mammalian RPE. • Aim 2 - Identification of the role of cilia in regulating RPE smEVs. • Aim 3 - Functional studies in vivo into the role of smEVs and primary cilia in RPE development and maintenance. Our collaborative research strategy is based on an integrative approach involving ex vivo and in vivo state-of-the-art technologies. Our synergistic research plan includes the comprehensive characterization and functional assessment of RPE-derived small EVs released from rat (RPE-J) and human RPE generated from stem cells (hES-RPE), imaging, proteomics and transcriptomic approaches, and functional studies of the role of cilia in RPE smEV biogenesis. We will further conduct functional studies in vivo in mice using conditional mutagenesis followed by comprehensive phenotypic analyses. The latter will include examining retinal physiology and development and comprehensive transcriptomic analyses of changes in gene expression in the RPE, choroid and photoreceptors using geographical position sequencing (Geo-seq), already implemented in our laboratories. Our joint study will yield novel insights into the role of tissue-specific smEVs, the role of cilia in their biogenesis, and smEVs’ contribution to eye development and adult retinal function. The knowledge we will gain regarding intracellular signaling between the RPE and surrounding tissues is not only important for vision research, but is also applicable to other epithelial tissues.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professorin Dr. Ruth Ashery-Padan