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High throughput sequencing has generated an enormous amount of information about the genes expressed in various cell types and tissues throughout the body, and about how gene expression changes over time and in diseased conditions. This knowledge has made targeted gene knockdowns an important tool in screening and identifying the roles of genes that are differentially expressed among specific cells of interest. While many approaches are available and optimized in mammalian models, there are still several limitations in the zebrafish model. In this article, we describe two approaches to target specific genes in the retina for knockdown: cell-penetrating, translation-blocking Vivo-Morpholino oligonucleotides and commercially available lipid nanoparticle reagents to deliver siRNA. We targeted expression of the PCNA gene in the retina of a P23H rhodopsin transgenic zebrafish model, in which rapidly proliferating progenitor cells replace degenerated rod photoreceptors. Retinas collected 48 h after intravitreal injections in adult zebrafish reveal that both Vivo-Morpholinos and lipid encapsulated siRNAs were able to successfully knock down expression of PCNA. However, only retinas injected with Vivo-Morpholinos showed a significant decrease in the formation of P23H rhodopsin-expressing rods, a downstream effect of PCNA inhibition. Surprisingly, Vivo-Morpholinos were able to exit the injected eye and enter the contralateral non-injected eye to inhibit PCNA expression. In this article we describe the techniques, concentrations, and considerations we found necessary to successfully target and inhibit genes through Vivo-Morpholinos and lipid encapsulated siRNAs.

IntroductionUnderstanding how photoreceptor genes are regulated is important for investigating retinal development and disease. While much is known about gene regulation in cones, the mechanism by which tandemly-replicated opsins, such as human long wavelength-sensitive and middle wavelength-sensitive opsins, are differentially regulated remains elusive. In this study, we aimed to further our understanding of transcriptional heterogeneity in cones that express tandemly-replicated opsins and the regulation of such differential expression using zebrafish, which express the tandemly-replicated opsins lws1 and lws2.MethodsWe performed bulk and single cell RNA-Seq of LWS1 and LWS2 cones, evaluated expression patterns of selected genes of interest using multiplex fluorescence in situ hybridization, and used exogenous thyroid hormone (TH) treatments to test selected genes for potential control by thyroid hormone: a potent, endogenous regulator of lws1 and lws2 expression.ResultsOur studies indicate that additional transcriptional differences beyond opsin expression exist between LWS1 and LWS2 cones. Bulk RNA-Seq results showed 95 transcripts enriched in LWS1 cones and 186 transcripts enriched in LWS2 cones (FC > 2, FDR < 0.05). In situ hybridization results also reveal underlying heterogeneity within the lws1- and lws2-expressing populations. This heterogeneity is evident in cones of mature zebrafish, and further heterogeneity is revealed in transcriptional responses to TH treatments.DiscussionWe found some evidence of coordinate regulation of lws opsins and other genes by exogenous TH in LWS1 vs. LWS2 cones, as well as evidence of gene regulation not mediated by TH. The transcriptional differences between LWS1 and LWS2 cones are likely controlled by multiple signals, including TH.

Objective: Systemic inflammation after subarachnoid hemorrhage (SAH) is implicated in delayed cerebral ischemia (DCI) and adverse clinical outcomes. We hypothesize that early changes in peripheral leukocytes will be associated with outcomes after SAH.Methods: SAH patients admitted between January 2009 and December 2016 were enrolled into a prospective observational study and were assessed for Hunt Hess Scale (HHS) at admission, DCI, and modified Ranked Scale (mRS) at discharge. Total white blood cell (WBC) counts and each component of the differential cell count were determined on the day of admission (day 0) to 8 days after bleed (day 8). Global cerebral edema (GCE) was assessed on admission CT, and presence of any infection was determined. Statistical tests included student's t-test, Chi-square test, and multivariate logistic regression (MLR) models.Results: A total of 451 subjects were analyzed. Total WBCs and neutrophils decreased initially reaching a minimum at day 4–5 after SAH. Monocyte count increased gradually after SAH and peaked between day 6–8, while basophils and lymphocytes decreased initially from day 0 to 1 and steadily increased thereafter. Neutrophil to lymphocyte ratio (NLR) reached a peak on day 1 and decreased thereafter. WBCs, neutrophils, monocytes, and NLR were higher in patients with DCI and poor functional outcomes. WBCs, neutrophils, and NLR were higher in subjects who developed infections. In MLR models, neutrophils and monocytes were associated with DCI and worse functional outcomes, while NLR was only associated with worse functional outcomes. Occurrence of infection was associated with poor outcome. Neutrophils and NLR were associated with infection, while monocytes were not. Monocytes were higher in males, and ROC curve analysis revealed improved ability of monocytes to predict DCI and poor functional outcomes in male subjects.Conclusions: Monocytosis was associated with DCI and poor functional outcomes after SAH. The association between neutrophils and NLR and infection may impact outcomes. Early elevation in monocytes had an improved ability to predict DCI and poor functional outcomes in males, which was independent of the occurrence of infection.

Synaptic signaling complexes are held together by scaffold proteins, each of which is selectively capable of interacting with a number of other proteins. In previous studies of rabbit retina, we found Synapse-Associated Protein-102 (SAP102) and Channel Associated Protein of Synapse-110 (Chapsyn110) selectively localized in the tips of horizontal cell processes at contacts with rod and cone photoreceptors, along with several interacting ion channels. We have examined the equivalent suites of proteins in mouse retina and found similarities and differences. In the mouse retina we identified Chapsyn110 as the scaffold selectively localized in the tips of horizontal cells contacting photoreceptors, with Sap102 more diffusely present. As in rabbit, the inward rectifier potassium channel Kir2.1 was present with Chapsyn110 on the tips of horizontal cell dendrites within photoreceptor invaginations, where it could provide a hyperpolarization-activated current that could contribute to ephaptic signaling in the photoreceptor synapses. Pannexin 1 and Pannexin 2, thought to play a role in ephaptic and/or pH mediated signaling, were present in the outer plexiform layer, but likely not in the horizontal cells. Polyamines regulate many ion channels and control the degree of rectification of Kir2.1 by imposing a voltage-dependent block. During the day polyamine immunolabeling was unexpectedly high in photoreceptor terminals compared to other areas of the retina. This content was significantly lower at night, when polyamine content was predominantly in Müller glia, indicating daily rhythms of polyamine content. Both rod and cone terminals displayed the same rhythm. While polyamine content was not prominent in horizontal cells, if polyamines are released, they may regulate the activity of Kir2.1 channels located in the tips of HCs. The rhythmic change in polyamine content of photoreceptor terminals suggests that a daily rhythm tunes the behavior of suites of ion channels within the photoreceptor synapses.

More than 1.5 million people suffer from Retinitis Pigmentosa, with many experiencing partial to complete vision loss. Regenerative therapies offer some hope, but their development is challenged by the limited regenerative capacity of mammalian model systems. As a step toward investigating regenerative therapies, we developed a zebrafish model of Retinitis Pigmentosa that displays ongoing regeneration. We used Tol2 transgenesis to express mouse rhodopsin carrying the P23H mutation and an epitope tag in zebrafish rod photoreceptors. Adult and juvenile fish were examined by immunofluorescence, TUNEL and BrdU incorporation assays. P23H transgenic fish expressed the transgene in rods from 3 days post fertilization onward. Rods expressing the mutant rhodopsin formed very small or no outer segments and the mutant protein was delocalized over the entire cell. Adult fish displayed thinning of the outer nuclear layer (ONL) and loss of rod outer segments, but retained a single, sparse row of rods. Adult fish displayed ongoing apoptotic cell death in the ONL and an abundance of proliferating cells, predominantly in the ONL. There was a modest remodeling of bipolar and Müller glial cells. This transgenic fish will provide a useful model system to study rod photoreceptor regeneration and integration.