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. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.

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 InVision, 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. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

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. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3'-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2-FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

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). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.

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. Improvements in imaging and analysis of transplant recipients have revealed that donor cell fluorescent reporters can transfer into host tissue though a process termed material exchange (ME). This recent discovery has chaperoned a new era of interpretation when reviewing the field's use of dissociated donor cell preparations, and has prompted scientists to re-examine how we use and interpret the information derived from fluorescence-based tracking tools. In this review, we describe the status of our understanding of ME in photoreceptor transplantation. In addition, we discuss the impact of this discovery on several aspects of historical rod and cone transplantation data, and provide insight into future standards and approaches to advance the field of cell engraftment.

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. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3ß and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS.

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. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.

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. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions.

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. An integrated approach to rodent retinal culture/transfection, live imaging, cell tracking, and analysis in structurally intact explants greatly improves our ability to assess the kinetics of cell production. RESULTS: In this report, we describe the assembly and maintenance of an in vitro, CO2-independent, live mouse retinal preparation that is accessible by both upright and inverted, 2-photon or confocal microscopes. The optics of this preparation permit high-quality and multi-channel imaging of retinal cells expressing fluorescent reporters for up to 48h. Tracking of interkinetic nuclear migration within individual cells, and changes in retinal progenitor cell morphology are described. Follow-up, hierarchical cluster screening revealed that several different dependent variable measures can be used to identify and group movement kinetics in experimental and control samples. CONCLUSIONS: Collectively, these methods provide a robust approach to assay multiple features of rodent retinal development using live imaging.

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. Here, we further examined the role of Vsx1 in OFF bipolar cells using recently identified cell-type-specific markers. In contrast to its previously characterized expression in type 2 OFF bipolar cells, Vsx1 expression was not detected in type 3 OFF bipolar cells, by either immunohistological or transgenic reporter labeling approaches. This observation was unexpected given previous findings that Cabp5 immunolabeling of type 3 bipolar cell axon terminals is reduced in Vsx1-null mice. However, we observed reduced levels of the type 3a bipolar cell marker hyperpolarization-activated and cyclic nucleotide-gated channel 4 (HCN4) in Vsx1-null mice, which is consistent with a requirement for Vsx1 in type 3 bipolar cell differentiation. In contrast, expression of the type 3b bipolar cell marker regulatory subunit RII-beta of protein kinase A was unchanged. Despite the absence of Vsx1 in mature type 3 bipolar cells, colabeling of Vsx1 and HCN4 was observed at postnatal stages. These findings reveal a role for Vsx1 in type 3a bipolar cells and suggest that Vsx1 function is required transiently in this cell type during the postnatal period.

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. Cre immunolabeling was detected in rod bipolar cells and a subset of ON and OFF cone bipolar cells. Expression was first observed at postnatal day 3 and was detectable between 24 hours and 36 hours after the last S-phase of the cell cycle. The appearance of Cre-immunolabeled cells preceded the expression of bipolar cell type-specific markers such as PKCα and Cabp5 suggesting that transgene expression is initiated prior to terminal differentiation. In the presence of a constitutive conditional reporter transgene, reporter fluorescence was detected in Cre-expressing bipolar cells in the mature retina as expected, but was also observed in Cre-negative Type 2 bipolar cells and occasionally in Cre-negative photoreceptor cells. Together these findings reveal a new transgenic tool for directing gene expression to post-mitotic retinal precursors that are mostly committed to a bipolar cell fate.

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. Thymidine-analogue lineage tracing and immunocytochemistry were used to identify dividing cells and the phenotype of newly generated progeny. RESULTS: Two populations of nestin-expressing cells are present in the CB of the uninjured eye. One population resides in periendothelial cells of blood vessels, and a second resides in the ciliary epithelium. Axotomy increases proliferation in the CB, a response that begins before the onset of RGC death and continues during a time that corresponds with the peak in RGC death. In addition, a subpopulation of nestin-positive cells in the CB upregulates the homeodomain protein Chx10. Finally, recoverin, the expression of which is normally restricted to photoreceptors and bipolar cells of the retina, is upregulated in the CB in a manner that is independent of proliferation. CONCLUSIONS: Together, these results suggest that progenitorlike cells of the CB respond to cues associated with the loss of a single retinal cell type and that a subpopulation of those cells may differentiate into a cell that bears phenotypic resemblance to those seen in the retina.

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. The purpose of this study was to investigate the expression of Hsp27 during postnatal retinal development, based on Hsp27's role as a neuronal survival factor and on its up-regulation in the adult injured retina. Expression of Hsp27 in the developing retina was examined at various times postnatally (between P0 and P24) by using immunohistochemical techniques. We report that Hsp27 expression peaks in the ganglion cell layer between P6 and P12 and is not detected at earlier (P0-P3) or later (P15-P24) times. Double labeling of the Hsp27-positive cells with Fluorogold applied to the superior colliculus confirmed that Hsp27-positive cells in the ganglion cell layer are RGCs. We have shown developmentally regulated expression of Hsp27 in RGCs of the postnatal rat. The retinal expression of Hsp27 correlates temporally with innervation of the tectum by late-born RGCs and with onset of spontaneous retinotectal activity. We propose that the expression of Hsp27 may play an important role in retinal development during a critical period of RGC functional connectivity with the superior colliculus.