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The adaptive significance of adjusting behavioral activities to the right time of the day seems obvious. Laboratory studies implicated an important role of circadian clocks in behavioral timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under seminatural zeitgeber conditions. We here report evidence that proper timing of eclosion, a vital behavior of the fruit fly Drosophila melanogaster, requires a functional molecular clock under quasi-natural conditions. In contrast to wild-type flies, period01 mutants with a defective molecular clock showed impaired rhythmicity and gating in a temperate environment even in the presence of a full complement of abiotic zeitgebers. Although period01 mutants still eclosed during a certain time window during the day, this time window was much broader and loosely defined, and rhythmicity was lower or lost as classified by various statistical measures. Moreover, peak eclosion time became more susceptible to variable day-to-day changes of light. In contrast, flies with impaired peptidergic interclock signaling ( Pdf01 and han5304 PDF receptor mutants) eclosed mostly rhythmically with normal gate sizes, similar to wild-type controls. Our results suggest that the presence of natural zeitgebers is not sufficient, and a functional molecular clock is required to induce stable temporal eclosion patterns in flies under temperate conditions with considerable day-to-day variation in light intensity and temperature. Temperate zeitgebers are, however, sufficient to functionally rescue a loss of PDF-mediated clock-internal and -output signaling

The adaptive significance of adjusting behavioural activities to the right time of the day is intuitive. Laboratory studies have implicated an important role of circadian clocks in behavioural timing and rhythmicity. Yet, recent studies on clock-mutant animals questioned this importance under more naturalistic settings, as various clock mutants showed nearly normal diel activity rhythms under semi-natural Zeitgeber conditions.We here report evidence that proper timing of eclosion, a vital behaviour of the fruit fly Drosophila melanogaster, requires a functional molecular clock even under quasi-natural conditions. In contrast to wildtype flies, period01 mutants with a defective molecular clock eclose mostly arrhythmically in a temperate environment even in the presence of a full complement of abiotic Zeitgebers. Moreover, period01 mutants eclose during a much larger portion of the day, and peak eclosion time becomes more susceptible to variable day-to-day changes of light and temperature. Under the same conditions, flies with impaired peptidergic inter-clock signalling (pdf01 and han5304 mutants) stayed largely rhythmic with normal gate sizes. Our results suggest that the presence of natural Zeitgebers can mitigate a loss of peptide-mediated phasing between central clock neuron groups, but cannot substitute for the lack of a functional molecular clock under natural temperate conditions.

Summary Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage− SCA-1+ KIT+ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34− CD48− CD150+ LSK (CD34− SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34− SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27Kip1 expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity., Graphical Abstract, Highlights • LSK cells induce the expression of CTGF in stromal cells • Stromal CTGF drives cell-cycle progression and proliferation of CD34− LSK cells • Decreased stromal CTGF activates TGFB signaling to decrease LSK cell cycle • Stromal CTGF improves maintenance of HSCs with long-term repopulating activity, Oostendorp and colleagues show that hematopoietic stem cells (HSCs) induce expression of connective tissue growth factor (CTGF) in stromal cells. A decrease in stromal CTGF impairs the maintenance of HSC activity in co-culture by rapid modulation of HSC signaling and cell-cycle entry. Thus, HSCs regulate their own cell cycle by modulating gene expression in stromal cells.

Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems. We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.

Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit+ Sca-1+ (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2−/− mice, or replicative stress in regeneration of HSCs in Sfrp2−/− recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of β-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2−/− mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation.