The HLH domain of a zebrafish hE12 homologue can partially substitute for functions of the HLH domain of Drosophiladaughterless

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Period gene expression in four neurons is sufficient for rhythmic activity of Drosophila melanogaster under dim light conditions.

International audienceThe clock gene expressing lateral neurons (LN) is crucial for Drosophila 's rhythmic locomotor activity under constant conditions. Among the LN, the PDF expressing small ventral lateral neurons (s-LN(v)) are thought to control the morning activity of the fly (M oscillators) and to drive rhythmic activity under constant darkness. In contrast, a 5th PDF-negative s-LN( v) and the dorsal lateral neurons (LN(d)) appeared to control the fly's evening activity (E oscillators) and to drive rhythmic activity under constant light. Here, the authors restricted period gene expression to 4 LN-the 5th s-LN(v) and 3 LN(d)- that are all thought to belong to the E oscillators and tested them in low light conditions. Interestingly, such flies showed rather normal bimodal activity patterns under light moonlight and constant moonlight conditions, except that the phase of M and E peaks was different. This suggests that these 4 neurons behave as ''M'' and ''E'' cells in these conditions. Indeed, they found by PER and TIM immunohistochemistry that 2 LN(d) advanced their phase upon moonlight as predicted for M oscillators, whereas the 5th s-LN(v) and 1 LN(d) delayed their activity upon moonlight as predicted for E oscillators. Their results suggest that the M or E characteristic of clock neurons is rather flexible. M and E oscillator function may not be restricted to certain anatomically defined groups of clock neurons but instead depends on the environmental conditions

The Novel Drosophila tim(blind) Mutation Affects Behavioral Rhythms but Not Periodic Eclosion

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, tim(blind), encodes a constitutively hypophosphorylated TIM protein. The mutant protein is not properly degraded by light, and tim(blind) flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TIM(BLIND) protein, which we observed in brain pacemaker neurons and photoreceptor cells of the compound eye. tim(blind) encodes two closely spaced amino acid changes compared to the wild-type TIM protein; one of them is within a putative nuclear export signal of TIM. Under constant conditions, tim(blind) flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that TIM—in addition to its well-established role as core clock factor—functions as a clock output factor, involved in determining the period length of adult locomotor rhythms

Daily p38 mRNA (A) and protein expression (B–D) in <i>Canton S</i> wildtype.

<p>A: Quantitative real-time PCR on head extracts revealed constant mRNA expression throughout the day with allover higher levels of <i>p38b</i> compared to <i>p38a</i> (p<0.001). B: Antibody staining with anti-p-p38 on adult brains displayed rhythmic phosphorylation of p38 in DN_1as in LD with significant higher p-p38 levels occurring during the night than in the day (p<0.05). C: A highly significant reduction of active p38 in DN_1as at CT6 compared to CT18 in DD indicates a clock-controlled activation of p38 (p<0.001) D: Only a 15 minute light pulse (LP) during subjective night (CT18) and not during the subjective day (CT6) leads to a reduction in active p38 in DN_1as, suggesting a clock-dependent photic reduction of active p38. The “C” in D indicates control brains without 15 minute light pulse (LP). Error bars show SEM. Significant differences (p<0.05) are indicated by *, highly significant differences (p<0.001) by **.</p

p38b promotes PER phosphorylation during the dark phase.

<p>To analyze daily phosphorylation of PER in flies that express the dominant-negative form of p38b in clock neurons and photoreceptor cells, we performed Western blots on head extracts after 4 days entrainment to LD 12∶12 cycles. According to our behavioral data, timing of PER accumulation was not affected in experimental flies (<i>UAS-p38b^DN-S;cry-Gal4/+;+</i>) in comparison with their respective control (A). However, regarding the degree of PER phosphorylation we observed differences at all time points when we compared both genotypes. For better comparison Western blots were repeated and samples of control and <i>UAS-p38b^DN-S;cry-Gal4/+;+</i> flies were plotted side by side for each ZT (B). Interestingly, flies with impaired p38 signaling indeed had less phosphorylated PER, showing the largest differences to the controls at the end of the night. Western blots were repeated 4 times and always gave similar results. Bars above the blots depict the light regime of the LD 12∶12. The “<i>C</i>” refers to respective control, <i>DNS</i> to <i>UAS-p38b^DN-S;cry-Gal4/+;+</i>.</p

Daily oscillations of nuclear PER in s-LN_vs and l-LN_vs of flies expressing a dominant negative form of p38b in these cells.

<p>Flies were entrained in LD 12∶12, dissected every one to two hours and staining intensity of nucleus and cell body was measured as described in Material and Methods. Nuclear PER staining intensities were normalized to total staining and tested for statistically significance. Expression of the dominant negative form of p38b phase delayed nuclear accumulation of PER in the s-LN_vs (A) and l-LN_vs (B). Arrows indicate the maxima of nuclear PER staining that occurred significantly later in <i>UAS-p38b^DN-S;Pdf-Gal4/+;+</i> flies than in control flies. This delay in nuclear PER accumulation in PDF-positive clock neurons is well consistent with the shifted evening activity in these flies. Bars above the graphs depict the light regime of the LD 12∶12.</p

Rhythmicity and period length of all investigated genotypes in constant darkness (DD) according to χ²-periodogram analysis.

<p><i>n</i> indicates the number of tested flies per genotype that survived locomotor recordings. Power and period values were averaged over all rhythmic flies for each genotype.</p>1<p>Flies with power values <20 were defined as arrhythmic.</p>2<p>Power is a measure of rhythmicity and is given in % of variance.</p

Locomotor activity rhythms of <i>p38b</i> and <i>p38a</i> null mutants and hypomorphic double mutant flies.

<p>Both <i>p38</i> null mutants, <i>p38b^Δ45</i> (upper panels in A) and <i>p38a^Δ1</i> (upper panels in B), displayed wildtype-like behavior with activity bouts around lights-on and lights-off when recorded in LD 12∶12. Even if evening activity onset of <i>p38a^Δ1</i>seems to be delayed compared to <i>w¹¹¹⁸</i>, this delay did not result in a longer free-running period under constant darkness (lower panels in B). Similarly, flies, lacking the <i>p38b</i> gene, also showed comparable free-running rhythms as their respective controls (lower panels in A). Activity data in C show two representative single actograms of a double mutant strain with a hypomorphic <i>p38b</i> allele (<i>p38b^Δ25;p38a^Δ1</i>). Since these flies are hardly viable and die within 3–6 days after emergence of the pupa, flies were already entrained to LD12∶12 during pupal stage and subsequently monitored in DD conditions after eclosion. Even if periodogram analysis was not possible due to the short recording period, <i>p38b^Δ25;p38a^Δ1</i> flies clearly showed a long free-running period when kept in constant darkness (C). For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p

Locomotor activity rhythms of flies expressing a dominant-negative form of p38b <i>(p38b^DN-S)</i> in <i>Drosophila</i> clock neurons and respective controls.

<p>Both, expression of a dominant negative form of p38b in either all clock neurons (<i>UAS-p38b^DN-S;tim(UAS)-Gal4/+;+</i>) or just in a subset of clock cells, the PDF-positive LN_vs (<i>UAS</i>-<i>p38b^DN-S;Pdf-Gal4/+;+</i>), resulted in a diurnal activity profile with a significantly delayed evening activity onset in comparison with respective controls (upper panels in A and B). This delay in evening activity is accompanied by a significantly prolonged free-running period in <i>UAS</i>-<i>p38b^DN-S;tim(UAS)-Gal4/+;+</i> (lower panels in A) as well as in <i>UAS</i>-<i>p38b^DN-S;Pdf-Gal4/+;+</i> flies(lower panels in B), when released into constant darkness. For recording and processing of activity data as well as for figure labeling see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004565#pgen-1004565-g003" target="_blank">Figure 3</a>.</p