Photoreceptor Development and Regeneration

Using stem cells to model the development and regeneration of retinal neurons

Photoreceptor (PR) transplantation is a promising treatment option for blindness caused by photoreceptor degeneration, including the most common cause of blindness in developed countries, Age-Related Macular Degeneration. While PR transplant research is ongoing and shows some promise, the success of this approach is hindered by a lack of knowledge about the basic mechanisms by which PR axons develop, extend, and are guided to their targets. By understanding the mechanisms that control PR neurite extension, we can better inform transplant therapies to ensure effective PR integration for functional sight restoration.  Using stem cells we are generating retina-like optic vesicles, which contain all cell types within the retina, including late-born PRs.  Currently we are using traditional methods to assess basic mechanisms of PR axon extension, combined with RNAseq data sets to identify molecular changes that may be responsible for age-dependent changes in PR axon outgrowth.

Current Open Questions:

  1. Do human Photoreceptors (PRs) generate axons by traditional cell autonomous terminal extension driven by motile growth cones?
  2. How does PR terminal growth change with developmental time and what molecular changes are responsible for loss of terminal motility?
  3. Does PR axon formation occur by similar developmental time-dependent changes within 3D retinal organoids?
  4. Can we influence young and old PR terminal extension behaviors through targeted molecular manipulations to promote PR regeneration?
F-actin content in PR terminals decreases with age. PRs dissociated at different ages were stained with phalloidin to visualize F-actin (blue in merge) and immunostained for G-actin (yellow in merge). Pseudo-colored image shows tdTomato labeling in red. Note a clear loss of both F-actin and G-actin labeling in older PR terminals, at a stage where terminals become less motile.
PR Development Model: PRs transition from a growth cone mediated autonomous extension phase to an adherent phase during human retina development. It was previously known that human PRs extend axons beyond their target layer, the Outer Plexiform Layer (OPL), by fetal week 10 and that these appear to retract back. Based on our in vitro data, we propose a model in which PRs have a motile growth cone that drives axon extension until PRs have projected to the Inner Plexiform Layer (IPL). PRs then transition to an adherent phase during which they retract back to the OPL and stay tightly adhered to form synapses before being stretched as the retina continues to develop.
PR development within 3D Organoids: 2-photon imaging of PRs labeled with tdTomato (Crx+/tdtomato iPSCs) over 3 weeks of development within retinal organoids. PRs migrate and have dynamic terminals early within developing retinal organoids. Retinal organoids fixed at different ages show PRs (which are born on the outer surface) localized internally and migrating to the outer surface of the RO from ages d50-d77.