Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish

Authors

Warlen P. Piedade, University of KentuckyFollow
Kayla F. Titialii-Torres, University of KentuckyFollow
Ann C. Morris, University of KentuckyFollow
Jakub K. Famulski, University of KentuckyFollow

Abstract

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS.

Document Type

Article

Publication Date

11-23-2020

Notes/Citation Information

Published in Frontiers in Cell and Developmental Biology, v. 8, article 594290.

© 2020 Piedade, Titialii-Torres, Morris and Famulski

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Digital Object Identifier (DOI)

https://doi.org/10.3389/fcell.2020.594290

Funding Information

This work was supported by grants from the National Institutes of Health (R01EY021769, to ACM and EY027805-01 to JKF). WP was supported by Brazilian National Council for Scientific and Technological Development (CNPq) under grant number 202970/2014-0 and the Morgan Fellowship from the department of Biology, University of Kentucky. KT-T was supported by the Lyman T Johnson scholarship and department of Biology Merit Fellowship, University of Kentucky. This project was supported by the department of Biology, University of Kentucky startup funds awarded to JKF.

Related Content

The datasets presented in this study can be found in the supplementary material, which is available for download as the additional file listed at the end of this record.

Descriptions of Supplementary Materials:

Supplementary Figure 1 | Siah1 overexpression decreases the number of rods and cones. Confocal stacks of heat shocked (HS) Tg[XOPS:GFP] (wildtype), Tg[hsp70:siah1]/Tg[XOPS:GFP] (siah1), and Tg[hsp70:siah1ΔRING]/Tg[XOPS:GFP], (siah1ΔRING) embryos were collected analyzed in 3D for GFP fluorescence (A–C). Region analyzed and presented is outlined in yellow. Overexpression of Siah1 resulted in significantly fewer GFP + rod cells (G). Confocal stacks of heat shocked (HS) Tg[TαC:eGFP] (wildtype), Tg[hsp70:siah1]/Tg[TαC:eGFP] (siah1), and Tg[hsp70:siah1ΔRING]/Tg[TαC:eGFP], (siah1ΔRING) embryos were analyzed in 3D for GFP fluorescence (D–F). Region analyzed and presented is outlined in yellow. Overexpression of Siah1 resulted in significantly fewer GFP + cone cells (H). Scale bar = 50 μm.

Supplementary Figure 2 | Inner retinal neurons are not affected by Siah1 overexpression. Retinal cryosections from wildtype, Tg[hsp70:siah1] (siah1), and Tg[hsp70:siah1ΔRING] (siah1ΔRING) embryos heat shocked (HS) and analyzed for effects on retinal inner neurons using IHC. Retinal ganglion and amacrine cells were visualized and quantified using Huc/D staining (A–D). Bipolar cells were visualized and quantified using PKCα (E–H). Horizontal cells were observed and quantified using prox1 staining (I–L). DNA was stained with DAPI (blue). Scale bar = 50 μm. L, lens; ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; ON, optic nerve; D, Dorsal; and V, Ventral.

Supplementary Figure 3 | Siah1 does not affect retinal cell proliferation or cell cycle progression. Retinal cryosections from wildtype, Tg[hsp70:siah1] (siah1), and Tg[hsp70:siah1ΔRING] (siah1ΔRING) embryos heat shocked (HS) and analyzed for cell cycle status using PCNA (A–C) and PH3 (D–F) IHC staining. Number of PCNA or PH3 positive cells did not significantly change upon Siah1 overexpression (G,H). DNA was stained with DAPI (blue). Scale bar = 50 μm. L, lens; ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer; ON, optic nerve; D, Dorsal; and V, Ventral.

Supplementary Figure 4 | Proteasome inhibition rescues Siah1 overexpression phenotype. Confocal stacks of heat shocked (HS) Tg[XOPS:GFP] (wildtype), Tg[hsp70:siah1]/Tg[XOPS:GFP] (siah1), and Tg[hsp70:siah1ΔRING]/Tg[XOPS: GFP], (siah1ΔRING) embryos treated with DMSO or MG132 were collected analyzed in 3D for GFP fluorescence. Quantification was restricted to the ventral retina (A–D). Treatment with MG132 prevented a significant decrease in GFP + rod cells compared to DMSO in siah1 HS embryos (E). Confocal stacks of heat shocked (HS) Tg[TαC:eGFP] (wildtype), Tg[hsp70:siah1]/Tg[TαC:eGFP] (siah1), and Tg[hsp70:siah1ΔRING]/Tg[TαC:eGFP], (siah1ΔRING) embryos treated with DMSO or MG132 were analyzed in 3D for GFP fluorescence. Quantification was restricted to the ventral retina (F–I). Treatment with MG132 prevented a significant decrease in GFP + cone cells compared to DMSO in siah1 HS embryos (J). Scale bar = 50 μm.

Supplementary Figure 5 | Time course of retinal apoptosis following siah1 or siah1ΔRING heat shock. Retinal cryosections from heat shocked Tg[hsp70: siah1] (siah1), and Tg[hsp70:siah1ΔRING] (siah1ΔRING) embryos were analyzed using IHC for apoptosis using TUNEL staining (red) at various time points (A–E’). DNA was visualized using DAPI (blue). Heat shock induction of siah1ΔRING expression had little effect on induction of retinal apoptosis between 52–72 hpf (A–F). Heat shock induction of siah1 resulted in a significant increase of apoptotic cells in the retina beginning at 60 hpf and up to 72 hpf compared to siah1ΔRING (A’–E’, F). Each point on the graph represents counts from individual embryos (F).

Supplementary Figure 6 | Genomic PCR screen for CRISPR induced insertion or deletion. Genomic DNA collected from injected embryos was used to amplify ∼250 bp amplicon from cdhr1a coding sequence. Golden crRNA control injected embryos all display a strong 250 bp band (embryos 1–3, 14–16). When examining PCR products from cdhr1a crRNA injected embryos various combinations of insertion or deletion products were amplified (embryos 4–13 and 17–26.

The preprint of this article is available from bioRxiv at https://doi.org/10.1101/2020.05.15.098350.

Repository Citation

Piedade, Warlen P.; Titialii-Torres, Kayla F.; Morris, Ann C.; and Famulski, Jakub K., "Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish" (2020). Biology Faculty Publications. 201.
https://uknowledge.uky.edu/biology_facpub/201