Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.

The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species.

Magnetic field-guided cell delivery with nanoparticle-loaded human corneal endothelial cells.

To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion.

Rod photoreceptors protect from cone degeneration-induced retinal remodeling and restore visual responses in zebrafish.

Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry, and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second-order neurons, bipolar cells, and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6c(w59) larval retina but not rod degeneration in the Xops:mCFP(q13) line. In adults, rods and cones are present in approximately equal numbers, and in pde6c(w59) mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2b(p25bbtl) or six7 morpholino-injected larvae protect from pde6c(w59)-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes, provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease.

Tbx2b is required for ultraviolet photoreceptor cell specification during zebrafish retinal development.

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67(phox), also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function.

Purpose: Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods: HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors ("proliferative media") to media without those factors ("stabilizing media"). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results: Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions: HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies.

Novel Identity and Functional Markers for Human Corneal Endothelial Cells.

PURPOSE: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. METHODS: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. RESULTS: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. CONCLUSIONS: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.

The bHLH Transcription Factor NeuroD Governs Photoreceptor Genesis and Regeneration Through Delta-Notch Signaling.

PURPOSE: Photoreceptor genesis in the retina requires precise regulation of progenitor cell competence, cell cycle exit, and differentiation, although information around the mechanisms that govern these events currently is lacking. In zebrafish, the basic helix-loop-helix (bHLH) transcription factor NeuroD governs photoreceptor genesis, but the signaling pathways through which NeuroD functions are unknown. The purpose of this study was to identify these pathways, and during photoreceptor genesis, Notch signaling was investigated as the putative mediator of NeuroD function. METHODS: In embryos, genetic mosaic analysis was used to determine if NeuroD functions is cell- or non-cell-autonomous. Morpholino-induced NeuroD knockdown, CRISPR/Cas9 mutation, and pharmacologic and transgenic approaches were used, followed by in situ hybridization, immunocytochemistry, and quantitative RT-PCR (qRT-PCR), to identify mechanisms through which NeuroD functions. In adults, following photoreceptor ablation and NeuroD knockdown, similar methods as above were used to identify NeuroD function during photoreceptor regeneration. RESULTS: In embryos, NeuroD function is non-cell-autonomous, NeuroD knockdown increases Notch pathway gene expression, Notch inhibition rescues the NeuroD knockdown-induced deficiency in cell cycle exit but not photoreceptor maturation, and Notch activation and CRISPR/Cas9 mutation of neurod recapitulate NeuroD knockdown. In adults, NeuroD knockdown prevents cell cycle exit and photoreceptor regeneration and increases Notch pathway gene expression, and Notch inhibition rescues this phenotype. CONCLUSIONS: These data demonstrate that during embryonic development, NeuroD governs photoreceptor genesis via non-cell-autonomous mechanisms and that, during photoreceptor development and regeneration, Notch signaling is a mechanistic link between NeuroD and cell cycle exit. In contrast, during embryonic development, NeuroD governs photoreceptor maturation via mechanisms that are independent of Notch signaling.