Expression of CaBP transcripts in retinal bipolar cells of developing and adult zebrafish

: Ca2+-binding proteins play important roles in neuronal function by transducing Ca2+ signals and thereby regulating crucial processes like synaptic signaling and neuronal development, growth and survival. The Ca2+-binding protein (CaBP) subfamily is part of the vast EF-hand containing calmodulin superfamily. Eight genes encoding CaBPs have been identified in zebrafish, and many of them are expressed in specific subpopulations of retinal neurons during development. Among them, cabp2a and cabp5b have been shown to be expressed in the retinal inner nuclear layer (INL). Here, we demonstrate that their paralogues, cabp2b and cabp5a, are also specifically expressed in the INL of the developing retina. By extending expression analysis of cabp2a, cabp2b, cabp5a and cabp5b to the adult retina, we reveal exclusive expression of all four genes in the INL after retinal development is completed. Thus, our findings suggest functions of cabp2a, cabp2b, cabp5a and cabp5b in Ca2+ signaling in mature retinal neurons, besides a role in the developing retina. of Zürich, Institute of Molecular Life Sciences; Abstract Ca 2+ -binding proteins play important roles in neuronal function by transducing Ca 2+ signals and thereby regulating crucial processes like synaptic signaling and neuronal development, growth and survival. The Ca 2+ -binding protein (CaBP) subfamily is part of the vast EF-hand containing calmodulin superfamily. Eight genes encoding CaBPs have been identiﬁed in zebraﬁsh, and many of them are expressed in speciﬁc subpopulations of retinal neurons during development. Among them, cabp2a and cabp5b have been shown to be expressed in the retinal inner nuclear layer (INL). Here, we demonstrate that their paralogues, cabp2b and cabp5a , are also speciﬁcally expressed in the INL of the developing retina. By extending expression analysis of cabp2a, cabp2b, cabp5a and cabp5b to the adult retina, we reveal exclusive expression of all four genes in the INL after retinal development is completed. Thus, our ﬁndings suggest


Introduction
Ca 2+ signaling is required for many fundamental aspects of neuronal function, such as signaling at pre-and postsynaptic sites, gene activation, growth, development and survival [1]. Ca 2+ -binding proteins sense and transduce Ca 2+ signals by undergoing conformational changes upon Ca 2+ binding, and consequently regulating target proteins. The largest group of Ca 2+ -binding proteins are structurally conserved EF-hand containing proteins belonging to the calmodulin superfamily. Within this superfamily, the vertebrate-specific CaBP subfamily comprises CaBP1, CaBP2, CaBP4 and CaBP5 in mammals [2] [3] [4]. In zebrafish, the CaBP subfamily is expanded to 8 members encoded by cabp1a, cabp1b, cabp2a, cabp2b, cabp4a, cabp4b, cabp5a and cabp5b [5], most likely due to duplicate gene retention after teleost-specific whole genome duplication [6]. CaBPs are specifically expressed in parts of the nervous system, including the retina [2] [5] [7]. The role of CaBP4 in retinal function is currently best understood. CaBP4, being within the retina specifically expressed in photoreceptors, has an established role in retinal disease and photoreceptor synaptic function [7] [8] [9] [10] [11] [12] [13] [14]. Functions of CaBPs expressed in second-order retinal neurons (i.e. bipolar and horizontal cells) are less well understood [15] [16]. Bipolar cells of the mouse retina have been reported to express CaBP5 [2], and cabp2 transcripts have also been detected in this cell type [17]. Characterization of the CaBP family in zebrafish has revealed two orthologues for each mammalian CaBP-encoding gene, and expression analysis was performed in the developing embryo [18]. Similar to mammals zebrafish cabp4b is expressed in photoreceptors, while no specific expression pattern was detected for cabp4a. cabp1a and cabp1b are both expressed in amacrine cells. Like the mammalian orthologues, cabp2a and cabp5b are expressed in retinal bipolar cells during development, while cabp2b expression was reported exclusively in hair cells. cabp5a expression has not been detected at these stages [5].

Results & Discussion
In order to study expression of cabp2a, cabp2b, cabp5a and cabp5b in the retina, we performed RNA in situ hybridization on developing whole-mount zebrafish and on adult retinal sections. In 3 day-old zebrafish larvae, all four genes studied show clear expression in the retinal inner nuclear layer (INL) (Fig.1 A-D). While staining for cabp2a, cabp5a and cabp5b results in strong labeling of the INL (Fig.1 A,B1,C1,D1), cabp2b staining is only weak in the retina (Fig.1 B1), also when compared to strong cabp2b staining observed in the inner ear ( Fig.1 B2). Expression in the inner ear can also be detected for cabp5a and cabp5b (Fig.1 C2,D2). In the adult retina, all four cabps examined show exclusive and strong staining in the INL (Fig.1 G-H) [16], possibly by regulation of voltage-gated Ca 2+ channels [17] and a function in synaptic vesicle recruitment [18]. Since expression of cabp5 orthologues is conserved between mammals and zebrafish, it is well conceivable that cabp5a and cabp5b have similar tasks. The diversity of Ca 2+ -binding proteins expressed in the nervous system allows finelytuned Ca 2+ signaling through their different Ca 2+ affinities, subcellular and cellular localizations and different binding partners, making them non-redundant regulators of neuronal signaling [1]. This and other studies [2] [5] [15]  We show expression that strongly implies, but does not prove, function. The position of the cell bodies in the retina strongly suggests expression in bipolar and horizontal cells, but is not a formal proof.
The study implies a functional of these CaBPs in retinal processing. The obvious next step is to genetically manipulate these genes (e.g. by CRISPR/Cas9 genome editing) and assess altered retinal function in these fish. Due to overlapping expression patterns of paralogues in the INL, it might be necessary to generate double knockouts to observe a phenotype.

Additional Information
Methods and Supplementary Material Please see https://sciencematters.io/articles/201604000009. This work was supported by the Swiss Science Foundation (31003A_153289/1). We thank Ramon Maegert for assistance during an internship, and Kara Dannenhauer and Martin Walther for excellent animal care.

Ethics Statement
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local authorities (Veterinäramt Zürich TV4206).