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. 1A-D). While staining for cabp2a, cabp5a and cabp5b results in strong labeling of the INL (Fig. 1A,B1,C1,D1), cabp2b staining is only weak in the retina (Fig. 1B1), also when compared to strong cabp2b staining observed in the inner ear (Fig. 1B2). Expression in the inner ear can also be detected for cabp5a and cabp5b (Fig. 1C2,D2). In the adult retina, all four cabps examined show exclusive and strong staining in the INL (Fig. 1G-H). Based on the shape and location of labeled cell bodies, all four genes are expressed in bipolar cells (central and distal INL), and possibly in horizontal cells (distal INL). The proximal INL, harboring amacrine and displaced ganglion cells, is devoid of staining.
Expression patterns of cabp2a and cabp5b in developing retina and ear are consistent with the findings by Di Donato et al.. While expression of cabp2b has previously been reported only in sensory hair cells, we detected weak expression in the retinal INL, likely reflecting low abundance of cabp2b transcripts in this region. Here, we show for the first time expression of cabp5a expression, which is strongly expressed in the retinal INL and in the inner ear. Our finding that expression of cabp2a, cabp2b, cabp5a and cabp5b is maintained up to adulthood strongly suggests functions in the mature retinal INL.
So far, no reports on zebrafish CaBP function exist. Studies of CaBP5 in mammalian cell culture and mouse retina suggest an important modulatory role in retinal signaling, possibly by regulation of voltage-gated Ca2+ channels and a function in synaptic vesicle recruitment. 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 Ca2+ binding proteins expressed in the nervous system allows finely-tuned Ca2+ signaling through their different Ca2+ affinities, subcellular and cellular localizations and different binding partners, making them non-redundant regulators of neuronal signaling. This and other studies suggest that bipolar cells exploit a repertoire of CaBPs for decoding Ca2+ signals, laying out the framework for further functional studies.