Mutations in EYS are associated with autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD) however, the function of EYS and the molecular mechanisms of how these mutations cause retinal degeneration are still unclear. Because EYS is absent in mouse and rat, and the structure of the retina differs substantially between humans and Drosophila, we utilised zebrafish as a model organism to study the function of EYS in the retina. We constructed an EYS-knockout zebrafish-line by TALEN technology which showed visual impairment at an early age, while the histological and immunofluorescence assays indicated the presence of progressive retinal degeneration with a cone predominately affected pattern. These phenotypes recapitulate the clinical manifestations of arCRD patients. Furthermore, the EYS−/− zebrafish also showed mislocalisation of certain outer segment proteins (rhodopsin, opn1lw, opn1sw1, GNB3 and PRPH2), and disruption of actin filaments in photoreceptors. Protein mislocalisation may, therefore, disrupt the function of cones and rods in these zebrafish and cause photoreceptor death. Collectively, these results point to a novel role for EYS in maintaining the morphological structure of F-actin and in protein transport, loss of this function might be the trigger for the resultant cellular events that ultimately lead to photoreceptor death.

Macroautophagy/autophagy is an important intracellular mechanism for the maintenance of cellular homeostasis. Here we show that the CERKL (ceramide kinase like) gene, a retinal degeneration (RD) pathogenic gene, plays a critical role in regulating autophagy by stabilizing SIRT1. In vitro and in vivo, suppressing CERKL results in impaired autophagy. SIRT1 is one of the main regulators of acetylation/deacetylation in autophagy. In CERKL-depleted retinas and cells, SIRT1 is downregulated. ATG5 and ATG7, 2 essential components of autophagy, show a higher degree of acetylation in CERKL-depleted cells. Overexpression of SIRT1 rescues autophagy in CERKL-depleted cells, whereas CERKL loses its function of regulating autophagy in SIRT1-depleted cells, and overexpression of CERKL upregulates SIRT1. Finally, we show that CERKL directly interacts with SIRT1, and may regulate its phosphorylation at Ser27 to stabilize SIRT1. These results show that CERKL is an important regulator of autophagy and it plays this role by stabilizing the deacetylase SIRT1.

No abstract

Cataract refers to opacities of the lens that impede the passage of light. Mutations in heat shock transcription factor 4 (HSF4) have been associated with cataract; however, the mechanisms regarding how mutations in HSF4 cause cataract are still obscure. In this study, we generated an hsf4 knockout zebrafish model using TALEN technology. The mutant zebrafish developed an early-onset cataract with multiple developmental defects in lens. The epithelial cells of the lens were overproliferated, resulting in the overabundance of lens fiber cells in hsf4null zebrafish lens. Consequently, the arrangement of the lens fiber cells became more disordered and irregular with age. More importantly, the terminal differentiation of the lens fiber cell was interrupted as the organelles cannot be cleaved in due time. In the cultured human lens epithelial cells, HSF4 could stabilize and retain p53 in the nucleus to activate its target genes such as fas cell surface death receptor (Fas) and Bcl-2-associated X apoptosis regulator (Bax). In the hsf4null fish, both p53 and activated-caspase3 were significantly decreased. Combined with the finding that the denucleation defect could be partially rescued through microinjection of p53, fas and bax mRNA into the mutant embryos, we directly proved that HSF4 promotes lens fiber cell differentiation by activating p53 and its downstream regulators. The data we presented suggest that apoptosis-related genes are involved in the lens fiber cell differentiation. Our finding that HSF4 functions in the upstream to activate these genes highlighted the new regulatory modes of HSF4 in the terminal differentiation of lens fiber cell.

Disseminated superficial porokeratosis (DSP) is a rare keratinization disorder of the epidermis. It is characterized by keratotic lesions with an atrophic center encircled by a prominent peripheral ridge. We investigated the genetic basis of DSP in two five-generation Chinese families with members diagnosed with DSP. By whole-exome sequencing, we sequencing identified a nonsense variation c.412C > T (p.Arg138*) in the phosphomevalonate kinase gene (PMVK), which encodes a cytoplasmic enzyme catalyzing the conversion of mevalonate 5-phosphate to mevalonate 5-diphosphate in the mevalonate pathway. By co-segregation and haplotype analyses as well as exclusion testing of 500 normal control subjects, we demonstrated that this genetic variant was involved in the development of DSP in both families. We obtained further evidence from studies using HaCaT cells as models that this variant disturbed subcellular localization, expression and solubility of PMVK. We also observed apparent apoptosis in and under the cornoid lamella of PMVK-deficient lesional tissues, with incomplete differentiation of keratinocytes. Our findings suggest that PMVK is a potential novel gene involved in the pathogenesis of DSP and PMVK deficiency or abnormal keratinocyte apoptosis could lead to porokeratosis.

M 2019, 'Knockout of Nr2e3 prevents rod photoreceptor differentiation and leads to selective L-/M-cone photoreceptor degeneration in zebrafish', AbstractMutations in the photoreceptor cell-specific nuclear receptor gene Nr2e3 increased the number of S-cone photoreceptors in human and murine retinas and led to retinal degeneration that involved photoreceptor and nonphotoreceptor cells. The mechanisms underlying these complex phenotypes remain unclear. In the hope of understanding the precise role of Nr2e3 in photoreceptor cell fate determination and differentiation, we generated a line of Nr2e3 knockout zebrafish using CRISPR technology. In these Nr2e3-null animals, rod precursors undergo terminal mitoses but fail to differentiate as rods. Rod-specific genes are not expressed and the outer segment (OS) fails to form. Formation and differentiation of cone photoreceptors is normal. Specifically, there is no increase in the number of UV-cone or S-cone photoreceptors. Laminated retinal structure is maintained. After normal development, L-/M-cones selectively degenerate, with progressive shortening of OS that starts at age 1 month. The amount of cone phototransduction proteins is concomitantly reduced, whereas UV-and S-cones have normal OS lengths even at age 10 months. In vitro studies show Nr2e3 synergizes with Crx and Nrl to enhance rhodopsin gene expression. Nr2e3 does not affect cone opsin expression. Our results extend the knowledge of Nr2e3's roles and have specific implications for the interpretation of the phenotypes observed in human and murine retinas. Furthermore, our model may offer new opportunities in finding treatments for enhanced S-cone syndrome (ESCS) and other retinal degenerative diseases.

In humans, CERKL mutations cause widespread retinal degeneration: early dysfunction and loss of rod and cone photoreceptors in the outer retina and, progressively, death of cells in the inner retina. Despite intensive efforts, the function of CERKL remains obscure and studies in animal models have failed to clarify the disease mechanism of CERKL mutations. To address this gap in knowledge, we have generated a stable CERKL knockout zebrafish model by TALEN technology and a 7bp deletion in CERKL cDNA that caused the premature termination of CERKL. These CERKL-/- animals showed progressive degeneration of photoreceptor outer segments (OSs) and increased apoptosis of retinal cells, including those in the outer and inner retinal layers. Additionally, we confirmed by immunofluorescence and western-blot that rod degeneration in CERKL-/- zebrafish occurred earlier and was more significant than that in cone cells. Accumulation of shed OSs in the interphotoreceptor matrix was observed by transmission election microscopy (TEM). This suggested that CERKL may regulate the phagocytosis of OSs by the retinal pigment epithelium (RPE). We further found that the phagocytosis-associated protein MERTK was significantly reduced in CERKL-/- zebrafish. Additionally, in ARPE-19 cell lines, knockdown of CERKL also decreased the mRNA and protein level of MERTK, as well as the ox-POS phagocytosis. We conclude that CERKL deficiency in zebrafish may cause rod-cone dystrophy, but not cone-rod dystrophy, while interfering with the phagocytosis function of RPE associated with down-regulation of the expression of MERTK.

Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator–like effector nucleases. The bcas2−/− zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2−/− zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.