Figure 1.
Phylogenetic position of the lamprey and hagfish melanopsins.
Both the lamprey and hagfish melanopsins belong to the Opn4m group. The bootstrap probabilities >80% were indicated. Scale bar, 0.1 substitutions per site. The accession numbers of the sequences are as follows: amphioxus Opn4, AB205400; chicken OPN4M, AY882944; chicken OPN4X, AY036061; clawed frog opn4m, XP002937616; clawed frog opn4x, AF014797; hagfish Opn4m, AB932627; human OPN4, AF147788; lamprey Opn4m, AB932626; mouse Opn4, AF147789; pufferfish Opn4m-1, XP_003963814; pufferfish Opn4m-2, XP_003976773; pufferfish Opn4x-1, XP_003965597; pufferfish Opn4x-2, XP_003974868; zebrafish opn4m-1, GQ925715; zebrafish opn4m-2, AY078161; zebrafish opn4m-3, GQ925717; zebrafish opn4x-1, GQ925718; zebrafish opn4x-2, GQ925719.
Figure 2.
Molecular properties of the lamprey and hagfish melanopsins.
A, An absorption spectrum of the dark-state lamprey melanopsin. The absorption maximum is at approximately 480 nm. B, Light-induced transient Ca2+ increases in lamprey (blue solid triangles), hagfish (red solid circles), amphioxus (gray solid circles) and mouse (gray hollow circles) melanopsin-expressing cells were determined by using the Ca2+ indicator dye Fura-2. The cells were irradiated with blue light for 500 ms immediately after the first measurement point. The mock-transfected cells exhibited no responses (gray solid triangles). Full-length melanopsins of lamprey and hagfish and C-terminal-truncated melanopsins of amphioxus [8] and mouse [35] were used (see materials and methods section for details). Each error bar represents the average value of 12 cells and indicates the standard deviation.
Figure 3.
Localization of melanopsin in the lamprey retina.
Localization of melanopsin visualized by in situ hybridization with an antisense probe for melanopsin (A) and immunohistochemistry with an anti-melanopsin antibody (B) in the lamprey retina. Melanopsin expression is observed in the inner horizontal cells of the INL and in other cells in the proximal region of the INL (white arrows) and IPL (yellow arrows). Ph, photoreceptor cell; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bar, 50 µm.
Figure 4.
Immunohistochemical localization of melanopsin to ganglion cells identified by retrograde tracing in the lamprey retina.
Melanopsin-expressing cells were immunohistochemically stained with the anti-melanopsin antibody (A, green) and ganglion cells were labeled by retrograde tracing with neurobiotin (B, magenta). A merged image reveals that melanopsin is expressed in ganglion cells (C, white arrow). Ph, photoreceptor cell; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bar, 25 µm.
Figure 5.
Melanopsin-expressing ganglion cells in the hagfish retina.
(A) The central region of the hagfish retina stained with Hoechst consists of 2 layers, the photoreceptor (Ph) and inner layers (IL), which are further divided into two regions, the proximal region (p) and the distal region of the inner layer (d). The ganglion cells were stained by retrograde tracing (B, green), and melanopsin-expressing cells were stained by in situ hybridization (C, black arrowheads). Approximately 80% of melanopsin-expressing cells (75%, p<0.05, Wilcoxon t-test) were stained by retrograde tracing in the merged image (D, black arrowheads). Scale bar, 25 µm.
Figure 6.
Immunohistochemical characterization of the connection between melanopsin-expressing horizontal cells and photoreceptor cells in the lamprey retina.
Melanopsin-expressing horizontal cells (A, green) and two types of photoreceptor cells (B, magenta) are stained with the anti-melanopsin antibody and anti-transducin antibody (TF15), respectively. The terminals of short photoreceptor cells (B, yellow arrowheads) and long photoreceptor cells (B, white arrows) are shown. The connections between melanopsin-expressing horizontal cell dendrites and photoreceptor cell terminals are at the end of the short photoreceptor cell (C, yellow arrowheads). Ph, photoreceptor cell; INL, inner nuclear layer; OPL, outer plexiform layer. Scale bar, 10 µm.