No abstract

Octamer-binding transcription factor 4 (Oct4, also known as Pou5F3) is an essential pluripotency-inducing factor, governing a plethora of biological functions during cellular reprogramming. Retina regeneration in zebrafish involves reprogramming of Müller glia (MG) into a proliferating population of progenitors (MGPCs) with stem cell–like characteristics, along with up-regulation of pluripotency-inducing factors. However, the significance of Oct4 during retina regeneration remains elusive. In this study, we show an early panretinal induction of Oct4, which is essential for MG reprogramming through the regulation of several regeneration-associated factors such as Ascl1a, Lin28a, Sox2, Zeb, E-cadherin, and various miRNAs, namely, let-7a, miR-200a/miR-200b, and miR-143/miR-145. We also show the crucial roles played by Oct4 during cell cycle exit of MGPCs in collaboration with members of nucleosome remodeling and deacetylase complex such as Hdac1. Notably, Oct4 regulates Tgf-β signaling negatively during MG reprogramming, and positively to cause cycle exit of MGPCs. Our study reveals unique mechanistic involvement of Oct4, during MG reprogramming and cell cycle exit in zebrafish, which may also account for the inefficient retina regeneration in mammals.

Highlights d CTCF insulates the domains of assorted mono-and biallelically expressed genes d The dosage of mono-allelically expressed genes is more sensitive to CTCF depletion d Active and inactive alleles of mono-allelically expressed genes conform distinctly d CTCF-depletion de-represses inactive alleles of monoallelically expressed genes

Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.

Purpose: To report the association of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 ( PLOD2 ) mutations with bilateral primary congenital glaucoma (PCG) in monozygotic twins and with nondominant juvenile-onset primary open-angle glaucoma (JOAG). Methods: We utilized family-based whole-exome sequencing to detect disease-causing mutations in a pair of monozygotic twins with de-novo PCG and compared its existence in 50 nonfamilial cases of JOAG and 30 healthy controls. To validate the identified mutations, direct Sanger sequencing was performed. For further evaluation of gene expression in the ocular tissues, we performed whole-mount in situ hybridization in zebrafish embryos. Results: We identified a novel missense mutation (c.1925A>G, p.Tyr642Cys) in the PLOD2 gene in the monozygotic twin pair with PCG and another missense mutation (c.1880G>A, p.Arg627Gln) in one JOAG patient. Both mutations identified were heterozygous. Neither the parents of the twins nor the parents of the JOAG patient harbored the mutation and it was probably a de-novo change. The zebrafish in situ hybridization revealed expression of the PLOD2 gene during embryogenesis of the eye. Conclusion: We observed an association of PLOD2 mutations with PCG and with nonfamilial JOAG. This new gene needs to be further investigated for its role in pathways associated with glaucoma pathogenesis.