Photoreceptor nanotubes mediate the in vivo exchange of intracellular material.

Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes.

The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed , that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis.

Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth.

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP).

ARS2 is required for retinal progenitor cell S-phase progression and Müller glial cell fate specification.

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression.

Material Exchange in Photoreceptor Transplantation: Updating Our Understanding of Donor/Host Communication and the Future of Cell Engraftment Science.

Considerable research effort has been invested into the transplantation of mammalian photoreceptors into healthy and degenerating mouse eyes. Several platforms of rod and cone fluorescent reporting have been central to refining the isolation, purification and transplantation of photoreceptors. The tracking of engrafted cells, including identifying the position, morphology and degree of donor cell integration post-transplant is highly dependent on the use of fluorescent protein reporters.

Exosomes Mediate Mobilization of Autocrine Wnt10b to Promote Axonal Regeneration in the Injured CNS.

Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes) enable neurite growth on CNS inhibitory proteins.

Sortilin regulates sorting and secretion of Sonic hedgehog.

Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor.

Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis.

ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants.

Live imaging and analysis of postnatal mouse retinal development.

Background: The explanted, developing rodent retina provides an efficient and accessible preparation for use in gene transfer and pharmacological experimentation. Many of the features of normal development are retained in the explanted retina, including retinal progenitor cell proliferation, heterochronic cell production, interkinetic nuclear migration, and connectivity. To date, live imaging in the developing retina has been reported in non-mammalian and mammalian whole-mount samples.

A transgenic mouse line expressing cre recombinase in undifferentiated postmitotic mouse retinal bipolar cell precursors.

Approaches for manipulating cell type-specific gene expression during development depend on the identification of novel genetic tools. Here, we report the generation of a transgenic mouse line that utilizes Vsx2 upstream sequences to direct Cre recombinase to developing retinal bipolar cells. In contrast to the endogenous Vsx2 expression pattern, transgene expression was not detected in proliferating retinal progenitor cells and was restricted to post-mitotic bipolar cells.

Requirement for the paired-like homeodomain transcription factor VSX1 in type 3a mouse retinal bipolar cell terminal differentiation.

Retinal bipolar cells make up a class of at least 11 distinct interneurons that have been classified through morphological and molecular approaches. Previous work has shown that the paired-like homeodomain transcription factor Vsx1 is essential for the proper development of a subset of these interneurons. In Vsx1-null mice, bipolar cells are properly specified but exhibit terminal differentiation defects characterized by reduced expression of OFF bipolar cell markers and defects in OFF visual signaling.

Proliferation and expression of progenitor and mature retinal phenotypes in the adult mammalian ciliary body after retinal ganglion cell injury.

Purpose: Despite the identification of a small population of cells residing in the ciliary body (CB) of the adult mammalian eye that have the capacity to generate retina-like cells in vitro, their activity in vivo remains quiescent. The authors sought to identify whether the predictable and time-dependent death of retinal ganglion cells (RGCs) results in activation of progenitor-like cells within the CB.

Methods: RGC injury was induced by optic nerve axotomy in adult mice.

Expression of Hsp27 in retinal ganglion cells of the rat during postnatal development.

The small heat shock protein Hsp27 has been shown to protect neurons from apoptosis. We have recently shown the expression of Hsp27 in a subset of injured adult retinal ganglion cells (RGCs), a response that is muted by the administration of brain-derived neurotrophic factor. This work has suggested a role for Hsp27 in the long-term survival of RGCs following injury.