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 hsf4^null 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 hsf4^null 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.

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.

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.