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. 2022 Jan;208(1):69-81.
doi: 10.1007/s00359-021-01506-1. Epub 2021 Oct 22.

Localisation of cryptochrome 2 in the avian retina

Angelika Einwich  1 , Pranav Kumar Seth  1 , Rabea Bartölke  1 , Petra Bolte  1 , Regina Feederle  2 , Karin Dedek  1   3 , Henrik Mouritsen  4   5
Affiliations

Localisation of cryptochrome 2 in the avian retina

Angelika Einwich et al. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2022 Jan.

Abstract

Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception.

Keywords: Chicken; Circadian clock; Erithacus rubecula; Magnetic compass; Migratory orientation.

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Conflict of interest statement

The authors declare no conflicts of interests.

Figures

Fig. 1
Fig. 1
Alignment of the amino acid sequences of erCry2a, clCry2a, clCry2b, ggCry2a and mmCry2. The 14 amino acid long peptide from the European robin that the antibody erCry2-26E11 was raised against is shown in green (amino acid position 544 to 557 in erCry2), the 51 amino acid long peptide for mmCry2-A20 is shown in yellow (amino acid position 520 to 570 in mmCry2). All five Cry2 proteins possess a nuclear localisation signal (blue, bold). The amino acid sequences of clCry2a, clCry2b and ggCry2a have two mismatches to the peptide that the antibody erCry2-26E11 was raised against (marked in red). The multiple sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/)
Fig. 2
Fig. 2
Cry2 localisation in the retina of the European robin. In the European robin retina (a), Cry2 was detected in the photoreceptor inner segments, outer nuclear layer, inner nuclear layer and ganglion cell layer (b–d). Whereas the erCry2-26E11 antibody labelled the ellipsoid of the inner segments as well as the cytoplasm of cells in the outer, inner and ganglion cell layers (b), mmCry2-A20 recognised more prominently the myoid of the photoreceptor inner segments as well as the nuclei of outer, inner and ganglion cell layers (c). Image a is a transmission image, images b–d are maximum projections of confocal stacks (z-size 1.27 µm, 7 sections). Scale bar: 50 µm. OS photoreceptor outer segments, PE retinal pigment epithelium, IS photoreceptor inner segments, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, NFL nerve fibre layer
Fig. 3
Fig. 3
Cry2 location was confirmed in two additional bird species. The antibodies erCry2-26E11 and mmCry2-A20 stained the photoreceptor inner segments as well as the cytoplasm of cells in the inner nuclear layer and ganglion cell layer in the day-flying pigeon (a–d) and the domestic chicken (e–h). mmCry2-A20 was also labelling the outer limiting membrane in the pigeon (c) and the chicken (g). In contrast to the immunosignals observed in the European robin retina, mmCry2-A20 did not label the nuclei but the cytoplasm in pigeon and chicken. Images a and e are bright-field images, images b–d and f–g are maximum projections of confocal stacks (b, c: z-size 4 µm, 20 sections; f–h: z-size 2 µm, 10 sections). Scale bars: d, h 50 µm. PE retinal pigment epithelium, OS photoreceptor outer segments, IS photoreceptor inner segments, OLM outer limiting membrane, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer, NFL nerve fibre layer
Fig. 4
Fig. 4
Controls for the antibodies. Staining of N2A cells (a-a’’) and HEK293 cells (e-e’’) expressing erCry1a-GFP (b, f), erCry2a-GFP (b’, f’) or erCry4a-GFP (b’’, f’’) protein with either our custom-made erCry2-26E11 antibody (c–c’’) or the commercially available mmCry2-A20 (g-g’’), respectively, indicates that both antibodies detected erCry2a-GFP (d’, h’) but not the other erCry-GFP proteins (d, d’’, h, h’’). Since all images are scanned and processed with the same parameters, some of the antibody signals are not visible in the resulting figure image. Immunoblot on recombinantly expressed erCry2a- and erCry4a-GFP fusion proteins (Ponceau staining, i incubated with the erCry2-26E11 antibody showed one single band only in the erCry2a-GFP lane (j). When omitting the primary antibodies in immunohistochemistry, no immunosignal was detected (k, l). Images a-a’’, e-e’’ and k are bright-field images; images b-d’’, f–h’’ and l are single confocal scans. Scale bars: 50 µm. PE retinal pigment epithelium, OS photoreceptor outer segments, OD oil droplets, IS photoreceptor inner segments, ONL outer nuclear layer, OPL outer plexiform layer, INL inner nuclear layer, IPL inner plexiform layer, GCL ganglion cell layer. For the immunoblot, the total homogenate of the protein samples was used
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