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In the Xenopus laevis retina, a principal model for retinal circadian organization, photoreceptors have all the properties of circadian oscillators. However, rhythmic oscillations of Per1 gene expression in the inner retina (but not photoreceptors) have been reported in mice with the suggestion that mice and frogs have a different retinal circadian organization. Although it is known that two period genes (xPer1 and xPer2) exhibit different temporal patterns of expression in the Xenopus retina, and that one (xPer2) is directly responsive to light and dopamine, it is not known whether this reflects the properties of period genes within photoreceptor oscillators or among distinct retinal cell populations. We addressed this by determining the cellular site of light and dopamine regulated xPer2 expression, and the diurnal expression of both xPer1 and xPer2 using in situ hybridization. Our data show that both xPer1 and xPer2 are expressed in most cell types in the retina, including inner nuclear neurons and ganglion cells. However, light and quinpirole, a dopamine agonist, increase xPer2 levels specifically in photoreceptors, and the effect of quinpirole, but not light, is blocked by pCPT-cAMP. Furthermore, antiphasic diurnal expression of xPer1 and xPer2 also occurs in photoreceptors. Our analysis does not provide insight into the near constitutive expression of period genes in the inner retina, but supports a model in which light- and dopamine regulated-xPer2 and rhythmic xPer1 play critical roles in entrainment and circadian oscillations within photoreceptors.
Fig. 1. xPer2 in situ hybridization of the Xenopus laevis retina with the
digoxigenin technique at ZT2.5 shows labelling over multiple cell types with
the antisense (A) but not with the sense (B) probes. Photoreceptors (both rod
and cone) and cells at the margin of the inner nuclear and inner plexiform layers
(black arrow) and ganglion cell layers (white arrow) were prominently labelled.
However, virtually all cells within the retina also exhibited significant labelling
above background. RPE, retinal pigment epithelium; OS, outer segments; ON,
outer nuclear layer; IN, inner nuclear layer; IPL, inner plexiform layer; GC,
ganglion cell layer.
Fig. 2. Northern analysis of the effects of light, quinpirole, and pCPT-cAMP
on retinal xPer2 mRNA level. (A) xPer2 in darkness, light, light (L) + cAMP,
and dark (D) + cAMP; each group includes three adjacent lanes representing
three retinas from different animals all taken from the same X-ray film.
B. xPer2 in darkness, light, quinpirole in darkness and quinpirole
(Q) + cAMP in darkness; each group includes three adjacent lanes representing
three retinas from different animals all taken from the same X-ray film.
(C) Quantitative analysis using phosphor-imaging of the experiment illustrated
in A with five retinas per treatment group. Relative mRNA is the ratio of
xPer2 mRNA to xActin mRNA on the same blot. Data are standardized so that
the dark group equals 1. Light and L + cAMP groups are different from
darkness (P ¼ 0.0001); the D + cAMP group also differs (P ¼ 0.005) from
darkness. (D) Quantitative analysis (as in C) of the experiment illustrated in B
with five retinas per treatment group. Light and quinpirole groups are different
from darkness (P ¼ 0.0001); the Q + cAMP group is significantly different
(P ¼ 0.005) from quinpirole alone.
Fig. 3. In situ hybridization with 33P-labled RNA probes showing that light and quinpirole induce xPer2 mRNA specifically in photoreceptors and the effect of
quinpirole is blocked by pCPT-cAMP. Shown are phase contrast images of unstained retinal sections from eyecups treated 3 h in darkness (A), light (B), light plus
pCPT-cAMP (C), darkness plus pCPT-cAMP (D), darkness plus quinpirole (E), or darkness plus quinpirole and pCPT-cAMP (F). At this magnification black
silver grains are small and are most readily seen over photoreceptor inner segments in B, C and E. OS, outer segments; IS, photoreceptor inner segments; IN, inner
nuclear layer; GC, ganglion cell layer.
Fig. 4. Quantitative analysis of in situ hybridization sections for xPer2
(A) and xPer1 (B) mRNA from retinas fixed as in Figure 3. The Y-axis
depicts silver grain density (grains ⁄ lm2) over photoreceptor inner segments
(IS), cells along the border of the inner nuclear and inner plexiform layers
(INL), and cells in the ganglion cell layer (GC). The X-axis depicts categories
identical to the treatments illustrated in Figure 3.
Fig. 5. In situ hybridization with 33P-labelled RNA probes showing that xPer1 mRNA is found in multiple cell types throughout the retina. (A and B) Unstained
sections imaged with Hoffman differential contrast optics. (C) Brightfield image of a haematoxylin and eosin-stained section. (A)Section through the optic nerve
(ON) showing a low level of hybridization in the optic nerve and plexiform layers and a high level of hybridization over inner segments, inner nuclear layer and
ganglion cell layer. The arrow in A indicates a red blood cell. (B and C) Show patches of hybridization corresponding to cell bodies in the INL and GC layer.
Although silver grains are found over nuclei, most are found immediately around the periphery of the nuclei. Scale bars, 50 lm (A–C).
Fig. 6. Quantitative analysis of in situ hybridization sections for xPer2
(A) and xPer1 (B) mRNA in retinas fixed at ZT0 and at ZT8 (X-axis). The Yaxis
depicts silver grain density (grains ⁄ lm2) over photoreceptor inner
segments (IS), cells along the border of the INL and IPL and cells in the GC
layer.
