Circadian rhythms depend on culture density

As described above the SCN exhibits synchronized and robust tissue rhythmicity due to intercellular coupling of heterogenous single cell oscillators within the tissue network. Whether peripheral circadian oscillators couple with each other is still debated. However, Noguchi et al. (2013) demonstrated that primary fibroblasts display weakened circadian rhythmicity on single cell level when cultured at low-densities [61].

Moreover, rhythmicity could be enhanced by substitution of low-density cultures with conditioned medium from high-density cultures [61]. This suggested that peripheral circadian oscillators required secreted signals from other cells to express normal circadian rhythms.

Thus, to test whether circadian rhythmicity of U-2 OS cells, as peripheral oscillator model, displays dependency on culture density, oscillations of Per2:Luc circadian reporter cells were imaged at varying culture densities. Comparable to fibroblasts, U-2 OS oscillations (on population level) showed a reduction of amplitude and increased damping with decreasing cell density (Figure 3-1 A-C). Note that these results were replicated in two independent sets of experiments: (i) performing the same experiment by an independent researcher [293] and (ii) by culturing U-2 OS cells at decreasing densities on membrane inserts (Figure 6-1 A-C). Impaired circadian rhythmicity may be a consequence of weakened single cell rhythms (as shown in [61]), desynchronization among single cell oscillators, or both. Nevertheless, according to theoretical concepts of intercellular coupling, decreased network amplitudes and

increased damping may be explained by dephasing and non-resonating single cell oscillators within incoherent networks (for details see 1.4).

Figure 3-1: U-2 OS circadian rhythmicity depends on culture density

To assess effects of culture density on circadian dynamics, U-2 OS cells harboring a Per2:Luc reporter gene were seeded into 35-mm culture dishes in increasing densities, synchronized, and luciferase activity was continuously monitored. (A) Detrended time series of a representative culture density experiment. (B,C) Quantification of amplitudes (B) and damping (C) of circadian oscillations (n=1 repeat experiment with 2 technical replicates, individual values and connecting line displayed, linear regression test: **p<0.01).

To further test whether impaired circadian rhythmicity is reflected on the molecular level, expression levels of core clock genes were determined for sparse (0.3 x10⁵ cells/35-mm dish) and dense (3.0 x10⁵ cells/35-mm dish) cultures of U-2 OS cells.

Consistent with weakened rhythms of U-2 OS Per2:Luc reporter cells, low-culture density resulted in reduced transcript levels of most of the core clock genes (Figure 3-2 A). This may indicate that low culture density and lack of contact to adjacent oscillators results in increased transcriptional repression or lack of transcriptional activation of the molecular core clock machinery. Yagita et al. (2010) reported that the emergence of circadian rhythmicity during cellular differentiation depends on threshold expression levels of certain core clock components driving functional molecular TTFLs [353]. Thus, assuming that reduced expression of core clocks genes is directly related to weakened rhythmicity, findings may suggest that peripheral circadian oscillators require signals from neighboring cells to enhance circadian rhythmicity on both, molecular and phenotypic level.

To gain a better understanding of how culture density dependent changes may be related to global transcriptional regulation, culture density dependent changes to the

0.2 0.4 0.8 1.5 3.0

U-2 OS transcriptome were assessed by RNA sequencing and differential gene expression (DGE) analysis. Indeed, on a global level culture density could be associated with specific gene expression profiles of dense and sparse cultures (Figure 3-2 B). In contrast to the core clock machinery, no trend towards significant transcriptional suppression was observed globally. Up- and downregulated transcripts were distributed equally (Figure 3-2 C), i.e. 47% of transcripts with padj < 0.01 displayed log2-fold changes > 0 and 53% Log2FC < 0. Moreover, gene expression changes of core clock genes resembled those detected by RT-qPCR (Figure 3-2 A), displaying transcriptional suppression of most of the core clock genes (Figure 3-2 C).

Gene ontology (GO) analysis showed that DGE profiles of sparse versus densely cultured U-2 OS cells are associated with distinct biological functions. Significantly downregulated transcripts were associated with extracellular matrix (ECM) structure and extracellular signaling activity, significantly upregulated transcripts with nucleic acid binding and regulation (Figure 3-2 D). The top 20 differentially regulated transcripts (sparse versus dense cultures) reflected these global changes (Figure 3-2 E,F). Downregulated transcripts included a large number of genes involved in ECM remodeling and function, e.g. extracellular peptidases/proteases (MMP7, KLK3, CFI) and enzymes (PPBP, ENPP3), as well as filament (KRT71, MYL10) and glycoproteins (CHI3L1, PRB1/2). Upregulated transcripts included mainly long noncoding RNAs, which have been described as regulators of gene expression by controlling chromatin landscape, transcription, RNA turnover, as well as translational and post-translational processes in the cytoplasm [354]. RNA sequencing results may suggest that reducing culture density and thereby proximity to neighboring cells drives peripheral oscillators into a desynchronized state, characterized by reduced responsiveness to extracellular (paracrine) signals, as well as of increased cellular replication. This is in agreement with published studies, showing that peripheral oscillators require paracrine signals from adjacent cells to maintain normal circadian rhythmicity [61], as well as that increased cell division reduces coherence among single cell oscillators [355].

Figure 3-2: U-2 OS transcriptional profiles depend on culture density

To investigate density dependent transcriptional changes, U-2 OS cells were seeded at high (3.0 x10⁵ cells/well) or low (0.3 x10⁵ cells/well) culture density into 6-well plates, synchronized, and RNA was harvested 18 hours after synchronization. RT-qPCR, RNA sequencing (RNAseq) and bioinformatic analysis was performed as described. (A) Quantification of mRNA expression changes for sparse versus dense cultures determined by RT-qPCR (n=6 repeat experiments with 1-3 technical replicates each, measured in triplicates, normalized to GAPDH, mean ± SD, individual values displayed, Unpaired

A B

distance clustering of rlog-transformed RNAseq read counts (all RNAseq data are from n=3 repeat experiments with 1 technical replicate each). (C) Magnitude Average (MA)-plot of log2-transformed expression changes for sparse versus dense cultures, expression changes of core clock genes analyzed in (A) are highlighted in black (red dots=padj<0.01). (D) Top 5 GO terms (biological function) associated with significantly (padj<0.01) up- or downregulated transcripts tested against all expressed genes (E,F) Histograms of top 20 up- (E) and downregulated (F) transcripts in sparse versus densely cultured cells with padj<0.01 (Euclidean distance clustering of log2-transformed expression changes).

Overall, weakened rhythmicity, as well as transcriptional suppression of the core clock machinery and of genes associated with intercellular signaling activity in sparse cultures indicate that proximity to and communication among neighboring oscillators is able to enhance circadian rhythms. We suspect that amplitude increases and reduced damping are a reflection of both, strengthened single cell rhythmicity and enhanced network synchrony due to intercellular coupling.