protein starts being expressed in the embryonic brain at stage 12, extending gradually to 160 neurons at stage 16; 130 of those also express Scer\GAL4tim.PE
-positive neurons are found throughout the brain but with more cells located rostrally. Around 20 cells co-express per
. These cells are located in the lateral protocerebrum, close to where the lateral and dorsomedial larval clock neurons are found. Expression of tim
in the ventral nerve cord starts at stage 12 in a few neurons in the anterior commissure. The number of midline and lateral labeled cells increases until stage 16, when per
is detected in 8 midline cells and 11 pairs of lateral cells per segment. The lateral clusters of neurons express tim
more strongly than the glial midline cells. More cells are detected in the thoracic segments than in the abdominal ones. Co-expression with en
shows that almost all of the tim
cells are anterior to the en
levels are seen to strongly deplete in pacemaker neurons after lights on.
At 1 hour before lights on in an LD cycle, tim
is predominantly nuclear localized, close to the nuclear membrane, but slightly centrifugal (lateral) to the per
protein levels are highest 3hrs before lights on and lowest 12hrs later.
Observed at ZT00, 06, 12 and 18, tim
protein shows oscillation in the LP neurons and DN1a neurons under a 12:12 LD cycle.
protein shows strong oscillations under LD conditions, with a peak in the night and a rapid decrease at lights on.
By ZT2 tim
is no longer seen in the nucleus of the l-LNvs. Significant levels of tim
are seen in the cytoplasm by ZT18, 6 hours after lights off, and starts to appear in the nucleus by ZT19, becoming predominantly nuclear by ZT21. In the s-LNvs, tim
was seen to be largely restricted to the cytoplasm through to ZT20, but becomes predominantly nuclear by ZT24.
Expression levels of tim
cycle in Malpighian tubules in a circadian pattern.
protein is coexpressed with Pdf
protein in larval lateral neurons.
Subcellular localization of tim
protein in the larval LNs, DN1s and DN2s is both nuclear and cytoplasmic at the peak-expression time point. tim
protein colocalizes with per
protein. In the DN2s, tim
expression was found to be in antiphase compared to the LNs and DN1.
In adult head sections, tim
protein is detected in the nuclei of photoreceptor cells. This nuclear localization does not occur in per01
flies, and is thus dependent on the presence of per
protein. Additional expression is detected in some lateral neurons of the protocerebrum, and glial cells in the lamina and medulla of the optic lobes. The peak of tim
protein expression lags behind that of tim
Rhythmic accumulation of tim
protein was detected in Western blots of adult head protein, whether the flies were kept in a LD 12:12 cycle, or in total darkness. In flies kept in a LD 12:12 cycle, there was an upward shift in the mobility of tim
protein late at night, and peak accumulation was seen at the 6th hour of darkness. Immunolocalization to adult head sections shows that tim
protein accumulates rhythmically in nuclei of photoreceptor cells and in nuclei and cytoplasm of pacemaker cells of the brain. The putative pacemakers, also called "lateral neurons", are clusters of neurons in the lateral protocerebrum. Lower levels of tim
protein are detected in cells dispersed throughout the optic lobes.