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Brain Res Mol Brain Res
1996 Apr 01;371-2:157-65. doi: 10.1016/0169-328x(95)00307-e.
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Use of a high stringency differential display screen for identification of retinal mRNAs that are regulated by a circadian clock.
Green CB
,
Besharse JC
.
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We report here the initiation of a systematic screen to identify clock-controlled mRNAs from the retina of Xenopus laevis using mRNA differential display. Xenopus retina contains an endogenous circadian clock located within the photoreceptor layer. The retinal block controls many aspects of physiology, including gene transcription. This screen uses differential display, a PCR based procedure, to compare retinal mRNA populations at different times of day in constant darkness, for identification of messages that exhibit rhythmic expression. Out of approx. 2000 mRNAs that we have screened to date, we have identified four candidates for clock-controlled mRNAs. Initial characterization of one of these PCR products shows that it recognizes a pair of mRNA bands on Northern blots that exhibit high amplitude rhythms. This pair of messages is called RM1 and shows peak levels of expression in the subjective night. In situ hybridization shows that this clock-controlled message is specifically localized to the clock containing photoreceptor cell layer within the retina. Identification of new messages that are under the control of the circadian clock has broad relevance in retinal physiology and provides an opportunity to gain insight into the molecular mechanism of vertebrate circadian control.
Fig. I. Experimental paradigm for high stringency differential display. A:
Eyecups (including the retina, pigment epithelium, choroid and sclera)
were prepared late in the afternoon and cultured in constant darkness,
beginning at the time of nornlal dark onset (ZT 12). Beginning the next
morning (ZT 0), retinas were harvested and culture medium was collected
under infrared light, at 6-h intervals, for 2 days. Harvested retinas were
frozen quickly on dry ice for subsequent RNA isolation and differential
display analysis. White bar indicates light period (normal daytime).
Hatched bars indicate subjective daytime (in constant darkness), while
black bars indicate subjective night. Numbers above bars indicate time of
harvest. All times are specified according to the animals normal light
cycle, where light onset was defined as Zeitgeber time [I (ZT 0) and dark
onset was ZT 12. B: Melatonin release from the cultured eyecups is
rhythmic over the entire culture period. The times indicated on the graph
represent the time of collection of the media samples: therefore the
melatonin measurement actually reflects an average for the 6 h preceding
that time. Thus, compared to continuous plots of melatonin release [24],
the rhythm is shifted to the right. Each line on the graph represents media
samples from one set of 10 eyecups. Because one set was harvested at
each time point, each subsequent time point contains one less group.
Fig. 2. Most retinal mRNAs do not change throughout the day in constant
darkness. Differential display analysis was performed simultaneously on
8 RNA samples prepared from retinas harvested at 6-h intervals over 2
days in constant darkness and separated on a 6% sequencing gel. The
primers used to generate this display were 5'-TAGCAAGTGC-3' and
5'-(T)12 MC-3', where the position marked by M is degenerate, consisting
of A, C and G. In most cases, the banding pattern is extremely consistent
between all samples, indicating that the vast majority of mRNAs do not
change as a function of time of day. Some bands do vary between
samples, but even most of these bands do not exhibit a consistent pattern
when the first 4 time points (day 1) are compared with the second 4 time
points (day 2). These differences may be due to PCR artifacts or other
variability between the retinas in these samples. The numbers above the
lanes correspond to times of harvest (Zeitgeber time). Dark bars represent
subjective night, light hatched bars represent subjective day. Bands
shown represent a size range from about 100 bp to about 400 bp.
Fig. 3. Candidates for clock-controlled mRNAs exhibit the same differential
display pattern on both days in culture. Sections of differential
displays that contain candidate clock-controlled mRNAs are shown. The
arrows mark the position of the candidate bands. The size of the T71
band is 445 bp, A72 is 145 bp, C13 is 360 bp, and Cll is 275 bp. The 4
bands shown here fit the criterion for further analysis based on their
consistent temporal pattern. The primers used to generate these displays
are as follows: (T71) 5'-CCGAAGGAAT-3' and 5'-(T)12MT-3'; (A72)
5'-CCGAAGGAAT-3' and 5'-(T)I2MA-3'; (C13) 5'-AGTFAGGCAC-3'
and 5'-(T)n2 MC-3'; (C 11) 5'-CAGACCGTTC-3' and 5'-(T) 12 MC-3'. The
position marked by M is degenerate, consisting of A, C, and G. Dark bars
represent subjective night, light hatched bars represent subjective day.
Fig. 4. T71 identifies a pair of mRNAs, named RM1, that are rhythmic in
constant darkness. A Northern blot of retinal RNA isolated at 6-h
intervals over 2 days in constant darkness was probed with the random
prime labeled differential display product T71, using previously described
methods [16]. This probe identifies a pair of bands that is expressed
during the subjective night, but not during subjective day. The pair of
bands (marked by the arrows) were named RM1. Reprobing of the blot
with an 18S rRNA riboprobe verifies consistent loading. Each lane in this
blot contains 3 /zg total retinal RNA, isolated at the times indicated from
eyecups cultured in constant darkness. Numbers to the right of the blot
indicate the approximate size of the mRNAs in kilobases. Dark bars
represent subjective night, light hatched bars represent subjective day.
Exposure time was 5 days for the T71 probe, i0 min for the 18S rRNA
probe.
Fig. 5. RM1 is a retina-specific message. Northern blot analysis of
various Xenopus tissues hybridized with radiolabeled T71 riboprobe
reveals that only retina contains detectable RM1 message. All tissues
were collected at ZT12 and used for RNA isolation. Each lane contains 5
~g of total RNA except for the retinal lane, which contains only 0.5 /xg
total RNA. The arrows mark the positions of the 2 RM1 bands. Positions
of the 28S and 18S rRNA bands are indicated. (1) heart; (2) kidney; (3)
brain; (4) liver; (5) skeletal muscle; (6) retina. Exposure time was 2 days.
Fig. 6. RM1 is localized to the photoreceptor layer within the Xenopus retina. In situ hybridizations were carried out to determine which retinal cell types expressed RM1. 35S-labeled riboprobes were
synthesized from cloned T7 1 and were hybridized to 5 /xm sections prepared from retinas fixed at ZTI2. PE, pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; INL,
inner nuclear layer; GC, ganglion cell layer. Scale bar represents l0 /xm. A: Phase contrast view of retina hybridized with anti-sense T-/1 riboprobe. B: Dark-field view of A. Note the position of the white
silver grains corresponding to specific signal over the photoreceptor inner segments. The pigment epithelium appears bright in dark-field images due to the refractory nature of the pigment granules, not due
to the presence of silver grains. C: Dark-field view of retina hybridized with sense RMI riboprobe. No specific hybridization can be seen
