Localization of Quasimodo

Preliminary data from RNA in situ stainings indicate that qsm is expressed in the adult brain, in cells close to clock neurons or in clock neurons (Peschel, 2004). In order to confirm this data and to investigate where the Qsm protein is expressed in the adult brain two different methods were approached. In the first approach a polyclonal antibody against two different parts of Qsm was used, in a second approach a P-element (that contained a β-Galactosidase reporter) in the qsm locus (Figure 3-5) was used to detect the Qsm positive cells.

3.3.5.1 Qsm Antibody

In his Ph. D. work Thomas Stempfl generated a polyclonal Qsm antibody (Stempfl, 2002). For the generation of this antibody the two following Qsm peptides were chosen (see Figure 3-3):

1) : H₂N - SQD GQK FTR DLT VK C - CONH₂ 2) : H₂N - QVG FGR RKR EIS SAN C - CONH₂ Localization of Qsm

In the first immunostainings of adult Drosophila brains with this antibody we used anti-PDF co-immunolabeling to differentiate between the different clock neurons.

Here we could see a very intense staining of the l-LNvs and the s-LNvs (data not shown). But this set of experiments could not be analyzed properly as the Qsm antibody or the secondary anti-rat antibody unspecifically recognized the PDF labeled cells. Immunostainings with Qsm antibody alone revealed very weak staining of the

cytoplasm or membrane of cells that are in close vicinity to the clock neurons or of the clock neurons. Only occasionally (The experiment was repeated three times. Two times there was intense staining at ZT16, one time this was not the case) at certain time points, like ZT16, a very intense staining could be detected (Figure 3-17). But here the Qsm protein seems to be localized either in the cell nucleus, in the extracellular matrix or in different smaller cells (Figure 3-17). Three different regions were observed, where the Qsm positive cells are near clock neurons. A dorsal cluster of mostly three cells (qDNs), a ventral cluster (qLN_vs) close to the s-LN_vs and l-LN_vs and a second ventral cluster (qLNvs 2) that is even further ventral than the first lateral cluster. The lateral clusters were much more heterogeneous in number and position than the DNs. The qLNvs cells consists of about 10 to 15 cells, the qLNv 2 cluster of about 5 cells. In addition two more clusters, not in close vicinity to clock neurons can be observed in the brain (1 and 2). The largest accumulation of Qsm positive cells is located dorsal-posterior-medial (1). A cluster of 5-8 cells can be found

ventral-posterior-medial (2) (Figure 3-17). Neuronal projections could not be detected, except for a connection from the q-LNvs to the optic lobe (arrow) (Figure 3-17 B). Based on its location, this connection resembles the connection from the extraretinal eyelet, the so called HB-eyelet to the s-LNvs (Hofbauer and Buchner, 1989).

3 qDNs

q-LN_vs

q-LN_vs 2

3 qDNs

q-LN_vs

q-LN_vs 2

A QSM B QSM

1

2

Figure 3-17 Drosophila wholemount staining with an anti-Qsm antibody

Adult Drosophila brains were treated with anti-Qsm antibody. The flies were sacrificed at ZT16. (A) displays an overview of one brain. The stainings were not consistent – not always (see above) could we reveal this distinct staining pattern, even if the dilutions/settings and genotype of the investigated animals were the same. In (B) we can see a close up of one of a brain hemisphere. Here we could reveal a projection (marked with an arrow) from cells with a similar position in the brain, like the s-LN_vs to the optic lobe. Based on its location, this connection resembles the connection from the extraretinal eyelet to the s-LN_vs.

Temporal Expression of Qsm

The mRNA of qsm is rhythmically expressed and under the control of the circadian clock. This suggests that the Qsm protein might be expressed in a rhythmic manner as well. We immunostained adult Drosophila brains of flies that were kept for three consecutive days in a 12:12 hour L/D regime in order to investigate rhythmic protein expression. Besides the Qsm antibody an anti-Per antibody was used to distinguish between clock and non-clock cells. In the beginning of the day (ZT0) Qsm is almost exclusively found in the cytoplasm or at the membran. Only in some q-LNv 2 cells

Figure 3-18 Staining series of y w animals in LD cycles with anti-Qsm and anti-Per

The lack of a marker protein that is throughout expressed in the clock neurons complicates the mapping of the Qsm positive cells to the clock neurons. The same is true for the assignment if the protein is nuclear or not.

PER QSM Merge

Qsm might be nuclear. The q-LN_vs all show cytoplasmic signals and in two or three cells Qsm might colocalize with Per in the l-LNvs (see asterisks). In close vicinity to the LN_ds one prominent stained Qsm positive cell can be observed (see circle). At ZT4 we see a similar picture, but the overall anti-Qsm staining is weaker. The 3 qDNs are still prominently stained. At ZT8 a trough level of overall staining can be observed. ZT12 represents the trough level of Period, but Qsm starts to accumulate again. Qsm signals in the q-LN_vs are located now in the nucleus or in smaller cells in close vicinity to the l-LN_vs. At ZT16 the peak level of nuclear (or extracellular) Qsm accumulation is reached. All q-LNvs are now nuclear (or extracellular) and stained very brightly. The same intensity can be observed for the 3 qDNs. Only the cells in close vicinity to the LNds are not stained at all or display a weak staining of the cytoplasm. AT ZT20 all q-LNvs are cytoplasmatic again, but the overall staining is stronger than at ZT 0. Again in approximately two q-LNvs Qsm colocalizes with Period (see asterisks). Those cells are most probably l-LNvs (Figure 3-18).

3.3.5.2 Reporter Gene Expression in P (PZ)l(2)05510⁰⁵⁵¹⁰Animals

Work with the Qsm antibody was problematic and very difficult because the obtained staining patterns were not always reproducible (see above and discussion). Another approach to detect Qsm positive cells was the use of a P-element insertion line, the fly strain P(PZ)l(2)05510⁰⁵⁵¹⁰ (Spradling et al., 1999). Here a P-element with the lacZ gene was inserted in the first intron of qsm (Figure 3-5). Theoretically this lacZ gene should be expressed under the control of the qsm promoter and thus reveal Qsm expression (Bier et al., 1989). So we investigated adult Drosophila brains of flies that were kept for three consecutive days in a 12:12 hour Light/Dark regime. The β-Galactosidase protein is stable for several hours in the adult fly (Helfand and Naprta, 1996; Stanewsky et al., 1997); nevertheless we investigated a time point with high qsm mRNA level. Besides the anti-β-Galactosidase antibody an anti-Per antibody was used to distinguish between clock and non-clock cells.

The overview in Figure 3-19 (A) shows a similar expression pattern as the pattern of the Qsm antibody in Figure 3-17. The largest accumulation of Qsm positive cells is located dorsal-posterior-medial (1). Weaker staining can be detected in cells that resemble from their position the qDNs, q-LNvs 1 and 2. One cell might be in close

vicinity to the LN_ds (see circle). Investigation of the DN₃ region revealed that the β-Gal protein is co-expressed in several DN3 neurons and in some neurons in close vicinity to the DN₃ cluster (B). A closer look to the l-LN_vs and s-LN_vs revealed, that β-Gal is coexpressed in the l-LN_vs and s-LN_vs and is expressed in cells nearby. Here a PDF antibody was used to discriminate between the large and small LN_vs (C). A close up on one brain hemisphere (D) verified the staining from the overview (A).

The assignement of the Qsm positive cells is still very vague. The bad quality of the anti-Qsm antibody and the therewith connected inconsistence of the staining in one experiment aggravate the interpretation of the results. The similar staining patterns of the lacZ experiment make it likely that Qsm is expressed in the s-LNvs, l-LNvs, partly

in the DN₃s and in many other cells in close vicinity to the clock neurons. Qsm is expressed in the cytoplasm or membrane of the cells. Interestingly Qsm can be found at some timepoints of the day, for example at ZT16, in either very small cells or in the nucleus of the cells where it was cytoplasmically expressed at ZT0.

The β-galactosidase staining should be mainly in the cell nucleus, because the beta-Gal protein carries a nuclear localization sequence.

(A) Overview of an adult brain. The fly was sacrificed at ZT0.

(B) A close up of the DN₃ region of the brain.

(C) A close up of the large and small LNvs.

(D) A close up on only one brain hemisphere.