per 01 and Quasimodo

3.3.6.1 Behavior

Animals with a mutated or lacking period gene loose their circadian rhythm under light/dark cycles and constant conditions (Konopka and Benzer, 1971). A subtle small peak in activity before „„lights on‟‟ is described for per⁰¹ animals, indicating some remaining clock function (Helfrich-Förster, 2001). But when the per⁰¹ mutation is combined with a mutation of the blue-light photoreceptor cryptochrome a residual rhythmicity was restored (Collins et al., 2005). per⁰¹;cry^b animals show a clear anticipation of the lights-off in LD environment. The RNAi mediated knock down of qsm in clock cells shows a similar phenotype like the cry^b mutation. Hence we wanted to investigate the combined effect of qsm and per⁰¹ mutations.

per⁰¹;;qsmRNAi(16) per⁰¹ qsmRNAi(16)

In Light/Dark cycles (12:12) per⁰¹ animals only reacted to the light off or light on change with elevated activity, but did not exhibit a circadian rhythm (Figure 3-20).

per⁰¹;;qsmRNAi(16) flies on the other hand showed a clear anticipation of the lights-off transition (Figure 3-20). This phenotype obviously resembles that of per⁰¹;;cry^b animals (Collins et al., 2005). A similar anticipation of „lights-off‟ could be revealed, when instead of L/D cycles the flies were kept in constant light and temperature cycles 25:18 °C (data lost). But is this anticipation really circadian entrainment caused by an oscillator or is it generated by a so called „hourglass effect‟ (Collins et al., 2005;

Pregueiro et al., 2005)? In the hourglass mechanism the activity peak always occurs

Figure 3-20 Daily average of qsmRNAi(16) animals in a per⁰¹ background

The flies were kept in 12:12 LD cycles. An average of 8 flies is displayed per genotype.

per⁰¹ flies have lost the ability to anticipate the change of the environmental light conditions.

per⁰¹;;qsmRNAi(16) animals on the other hand are still able to anticipate „lights-off‟. But here, compared to the control animals, a clear phase advance of the activity peak is visible.

after a set time, for example always nine hours after lights-on. So we repeated the experiment but this time changed the Light/Dark ratio from 12:12 to 18:6. Once more the per⁰¹ mutants showed only a reaction to the changed light setting (Figure 3-21).

The control animals exhibited a shifted evening activity peak – the evening activity

peak is in LD 12:12 after about 12 hours, while in LD 18:6 the maximum evening activity can be detected after about 15 hours. The per⁰¹;;qsmRNAi(16) animals though displayed no or only a very subtle shift in their evening activity – here the maximum activity can be found after nine hours (Figure 3-21).

This observation is evidence that we are looking at an “hourglass effect‟. Another proof should come from an additional experiment. A circadian rhythm should be endogenous and should function in the absence of environmental cues. So we investigated the per⁰¹;;qsmRNAi(16) animals under constant light or darkness and temperature condition. As expected the flies lost their rhythmicity under constant

Figure 3-22 Flies with per⁰¹ mutations in constant conditions (LL or DD)

Flies were kept for several days in 12:12 LD conditions and were than released in constant conditions. The animals on the left side of the graph were kept in LL conditions; the flies on the right were kept in DD. The genotype is indicated above the picture.

per⁰¹ per⁰¹ per⁰¹;;qsmRNAi(16) per⁰¹;;qsmRNAi(16)

per⁰¹;;qsmRNAi(16) per⁰¹ qsmRNAi(16)

Evening peak after About 9h

Evening peak after About 14.5h

Figure 3-21 Daily average of qsmRNAi(16) animals in a per⁰¹ background in a different photoperiod

Flies were kept in 18:6 Light/Dark cycles. An average of 8 flies is displayed per genotype.

While the control flies exhibit a shift of about 3 hours in their evening activity, the per⁰¹ display no evening activity at all. per⁰¹;;qsmRNAi(16) animals show no or only a very subtle change in their evening activity time.

darkness conditions (Figure 3-22). Also as expected constant light conditions rendered the per⁰¹ animals arrhythmic. The per⁰¹;;qsmRNAi(16) flies also loose their rhythmicity after transfer into LL conditions, but after one to three days they started to exhibit a rhythmic locomotor activity again (Figure 3-22). The period of the rhythm was not 24 hours, but looked rather ultradian (Table 3-2).

3.3.6.2 Tim Amount in per⁰¹

Next we tried to answer the question, what is causing this rhythmicity and/or evening anticipation in per⁰¹;;qsmRNAi(16). First we investigated if the Period lacking animals still showed rhythmic protein expression. A Western blot from whole head extracts revealed that the Timeless protein in per⁰¹ animals still showed light dependent degradation (Figure 3-23) but no sustained rhythmicity in constant conditions (Zeng et al., 1996). Less protein is detected during day time, when the light is on. At night Tim is not degraded and starts to accumulate again. A similar pattern can be observed in per⁰¹;;qsmRNAi(16) . Only here the trough Tim level is reached at ZT9, while in per⁰¹ flies the trough lies at ZT3. All in all slightly more Tim is visible in per⁰¹;;qsmRNAi(16) animals. Keeping in mind, that only four time points were

Genotype n Rhythmic Autocorrelation (with SEM) (hrs with SEM)

Mesa (hrs with SEM)

per⁰¹ 16 0 - -

per⁰¹;;qsmRNAi(16) 10 60 % 19.4 ± 1.769746 11.7125 ± 0.974588

Figure 3-23 Western blot of animals with per⁰¹ mutations in Light/Dark cycles

20 heads per lane were used to investigate the Tim protein amount. The animals were sacrificed at the indicated Zeitgeber times (3,9,15,21). The per⁰¹ mutants on the left show increasing Tim protein level in the dark timepoints (15, 21). The per⁰¹;;qsmRNAi(16) animals on the right blot half display the same phenomenon, but here we could detect more Tim protein at ZT 3 compared to ZT9.

Table 3-2 per⁰¹ and qsmRNAi(16) in LL

While the per⁰¹ animals were all arrhythmic in LL some per⁰¹;;qsmRNAi(16) animals showed rhythmicity. As one can see from the via autocorrelation or Mesa calculated period the assignment of the period is difficult because of the ultradian rhythm.

3 9 15 21 3 9 15 21

per

per

;qsmRNAi(16)

TIM

investigated and only a single experiment was performed those results should be handled with care (Figure 3-23).

Next we investigated the Tim level in the circadian clock neurons in per⁰¹ or

per⁰¹;;qsmRNAi(16) flies. Adult animals were kept in 12:12 LD cycles for three days.

Wholemount stainings of those animals were then investigated at ZT 3, 9, 15, 21. PDF was used to identify the LNvs. A first very obvious result was that Tim can be found almost exclusively in the cytoplasm. The results obtained from the Western blot experiments correlated well with those obtained by ICC. per⁰¹ animals show the trough Tim level at ZT3. Here hardly any Tim is visible. Tim levels then increase by the end of the day. The rise of Tim levels continues after “light off”, so that it reaches its peak by the end of the night. per⁰¹;;qsmRNAi(16) brains on the other hand still show many Tim containing cells by the beginning of the day. The trough level can be seen at ZT9. In the night Tim accumulates, but in the LN_ds a decreased Tim level can be found already at ZT21. Furthermore at ZT3 Tim can be detected in additional cells, whereas it is uncertain if those cells normally express Period in wild-type animals (Figure 3-25). Those cells most likely correlate with the additional Tim positive cells under LD conditions in qsmRNAi(16). per⁰¹;;qsmRNAi(16) animals behave in a rhythmic manner in LL. But not every per⁰¹;;qsmRNAi(16) fly exhibits this abnormal behavior and not with the same constant period (Table 3-2). For this reason it is very difficult to investigate this in the clock neurons. Hence we investigated this effect only by Western blot, because here we could investigate an average of several flies.

Figure 3-24 Timeless stainings in per⁰¹ wholemounts

Displayed to the left side is a quantification of Tim staining in the different clock neurons in per⁰¹ animals. The y-axis shows the intensity, while the x-axis displays the different ZTs. Only three brains (6 hemispheres) per timepoint were investigated. On the right side is an example of a per⁰¹ brain from ZT3. The only visible staining is in the dorsal neurons.

0 0.5 1 1.5 2 2.5 3 3.5

3 9 15 21

DN1+DN2 DN3 LNd l-LNv s-LNv

per

DN₃

TIM TIM+PDF

0

Animals were collected at ZT9 and ZT21 or CT9 and CT21 (first day in LL) and the Tim amount and phosphorylation level investigated. Phosphorylated Tim can be observed on the Western blot by a slower migrating form (Martinek et al., 2001).

Whilst the y w and qsmRNAi(16) showed a very clear increase in Tim protein and phosphorylation when ZT9 and ZT21 were compared, the per⁰¹ or per⁰¹;;qsmRNAi(16) mutants showed only a slight increase in total amount of Tim and no change in terms of phosphorylation. In constant light on the other hand there is hardly any Tim protein visible in y w animals at CT9 and CT22. qsmRNAi(16) flies have more Tim protein at CT9 compared to CT22. The two period mutated fly strains both still exhibit more Tim protein, but again no cycling in the protein or phosphorylation level is detectable. In total the protein level is diminished in LL conditions compared to LD (Figure 3-26).

per01

Figure 3-25 Timeless stainings in per⁰¹;;qsmRNAi(16) wholemounts

Displayed to the left side is the quantification of Tim staining in the different clock neurons in per⁰¹;;qsmRNAi(16) animals. The y-axis shows the intensity, while the x-axis displays the different ZTs. Only three brains (6 hemispheres) per timepoint were investigated. On the right side is an example of a per⁰¹;;qsmRNAi(16) brain from ZT3. Clearly one can see Tim staining in the cytoplasm of the LN_ds and in the LN_vs. In addition many more cells in close proximity to the clock neurons are positive for Timeless staining.

Figure 3-26 Western blot of per⁰¹ mutants in constant light

20 fly heads were investigated per lane. The left half of the blot shows different genotypes in LD cycles, while the right side of the blot exhibits animals from constant light conditions. The ZTs and CTs are as indicated. The upper rows display the Tim staining, while the second row shows the period staining as a control.