EMBO/soda-nel huggingface dataset:
Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network (2021)
Figure 3

Figure 3. SCN neuronal sub-population transcriptomes

A. Sub-clustering of eight neuronal populations using unsupervised graph-based clustering within the transcriptomes of CT7.5 (top) and CT15.5 (bottom) SCN cells. The heatmaps show the respective up-regulation of three representative marker genes in each sub-population. When compared, three subpopulations showed strong transcriptional similarity between the time-points of sequencing (Groups A, B and C), two showed partial similarity (Groups D and E) and three were transcriptionally distinct to either CT7.5 or CT15.5 sequenced cells, giving rise to the identification of eleven SCN neuronal sub-populations.

B. Violin plots depicting the distribution of the quantified average cellular expression of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr in each sub-cluster in CT7.5 (left) and CT15.5 (right) datasets.

C. Visualisation of the location within the pooled t-SNE plot of cells positively expressing >90% of the overall expression range of Prok2, ProkR2, Vip, Vipr2, Avp, Avpr1a, Grp and Grpr. This shows a contrasting expression pattern of the respective genes in cells collected at CT7.5 versus CT15.5.

D. Venn diagram summarising the degree of non-/overlapping signalling axes: Prok2 (purple) - ProkR2 (brown), Vip (green) - Vipr2 (red), Avp (blue) - Avpr1a (orange) and Grp (pink) - Grpr (grey). The number SCN neurons collected at CT7.5 positively expressing each gene, which is associated to the respective signalling axis, is further annotated within the population circles.
Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network (2021)
Figure 5

Figure 5. Circadian functions of SCN Prok2+ and ProkR2+ cells

A. Representative, detrended Cry1-luc traces of emitted bioluminescence (photon count values) from 4 SCN slices, pre-transfected with AAV pCry1-DIO-Luciferase after addition of (left) pProk2.Cre.T2A.mCherry (red, cohort n=10) or (right) pProkR2.Cre.T2A.Venus (green, cohort n=9) transfection.

B. Raster plots (yellow=trough, blue=peak) of Cry1-luc bioluminescence oscillations from a representative slice of each cohort. The black arrow marks the time-point of pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus AAV transfection, thus resulting in the evolution of rhythmic Cry1-luc bioluminescence.

C. The period of Cry1-luc bioluminescence rhythms in SCN slices transfected with either pProk2.Cre.T2A.mCherry or pProkR2.Cre.T2A.Venus does not differ between treatments (p=0.71, n= 10, 9 SCN slices per group, respectively).

D. Similarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2+ SCN cells and ProkR2+ SCN cells, does not differ between treatments (p=0.67).

E. Representative raster plots of single-cell bioluminescence (yellow=minimum, blue=maximum) from Prok2Cry1-luc (left) or ProkR2Cry1-luc (right) cells in single slices.

F. Within-slice cellular synchrony depicted in representative Rayleigh plots of the phases of peak Cry1-luc bioluminescence for Prok2Cry1-luc (red) and ProkR2Cry1-luc (green) cells. The Rayleigh plots can be read as the face of a clock with CT0 marking the top and CT12 the bottom of the plot circle. The black arrow depicts the mean peak phase across the individual oscillators.

G. The mean Rayleigh vector lengths for Prok2Cry1-Luc and ProkR2Cry1-luc (p>0.99) do not differ between the cohorts (n=5 SCN slices per group).

H. Left: confocal images of representative SCN slices dual-transfected with pProk2.Cre.T2A.mCherry and Syn.GCaMP6f (top), or pProkR2.Cre.T2A.Venus and Syn.jRCaMP1a (below) (scale bar=500µm). Right, above: representative detrended plots of signal from GCaMP6f (green) traces and pCry1-DIO-Luc, emitted from Prok2+ SCN cells (red). Right, below: representative detrended plots of jRCaMP1a (purple) and pCry1-DIO-Luc emitted from ProkR2+ cells (light green). Orientation bars depict the horizontal axis of the optic chiasm and the vertical axis of the third ventricle for each SCN.

I. After registering the respective peaks of [Ca2+]i fluorescence to the peaks of Cry1-luc bioluminescence in each respective cohort, phase-alignment between the peaks of Prok2Cry1-luc and ProkR2Cry1-luc bioluminescence shows that the intracellular clocks of Prok2+ cells are phase advanced by ~0.6h to those of ProkR2+ cells (**p<0.01) (n=5 SCN slices per group).

Data information: All values depicted as mean±S.E.M. C, D, G, I unpaired two-tailed t-tests.
Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network (2021)
Figure 7

Figure 7. SCN rhythm amplitude and period are determined by endogenous Prok2 signalling

A. Representative Per2::Luciferase traces before (black) and after vehicle (grey) or Prok2RA (pink) topical application of SCN slices.

B. Comparison of the Per2::Luciferase rhythm amplitude of the cycle preceding and the first and second cycles succeeding vehicle (applied CT23-20 dark grey n=12 SCN slices, CT-20-23 light grey n=6 SCN slices) or Prok2RA (applied CT23-20 purple n=13 SCN slices, CT20-23 pink n=11 SCN slices) treatment (interaction, p<0.0001). A significant decline in amplitude occurred when Prok2RA signalling was blocked between CT20-23 (****p<0.0001).

C. Change in SCN period after treatment with vehicle (grey) or Prok2RA (pink) between CT23-20 and CT20-23 (vehicle CT23-20 vs. Prok2RA CT20-23, **p=0.009; vehicle CT20-23 vs. Prok2RA CT20-23, **p=0.001) (n = 6, 12, 13 and 11 independent SCN slices, respectively).

D. Prok2RA application resulted in a significant period lengthening effect of ongoing SCN ensemble Per2::Luciferase rhythms, which was reversed upon drug washout (p=0.0002, ***p=0.0009, *p=0.01) (n = 22 independent SCN slices with repeated measure).

Data information: All values depicted as mean ±S.E.M. B two-way ANOVA with Sidak's multiple comparisons; C,D one-way ANOVA with Tukey's multiple comparisons.
Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network (2021)
Figure 8

Figure 8. Prok2+ and Prok2R+ cells control SCN circadian period

A. Representative Per2::Luciferase trace of Cry1-/- SCN slice at baseline (black) and after AAV-mediated (arrows) Cry1 complementation in Prok2+ cells (red) or ProkR2+ cells (yellow).

B. Corresponding peak-to-peak period plots for baseline (black), Cry1-expressed in Prok2+ (red), and then in ProkR2+ cells (yellow). Individual slices are depicted as grey traces, filled points are their mean±S.E.M.

C. Group data (n=5) across three Per2::Luciferase cycles as shown in B. (p=0.006; baseline vs. Prok2.Cre *p=0.04, baseline vs. Prok2.Cre and ProkR2.Cre *p=0.02, Prok2.Cre vs. Prok2.Cre and ProkR2.Cre ns. p=0.95).

D. As in A but with order of transfection reversed; green =Cry1 only in ProkR2+ cells (baseline vs. ProkR2.Cre *p=0.04).

E, F. As in B and C but with transfection order reversed (green =Cry1 expression in ProkR2+ cells only, n=6) (p=0.006; baseline vs. ProkR2.Cre *p=0.03, baseline vs. ProkR2.Cre and Prok2.Cre **p=0.009, ProkR2.Cre vs. ProkR2.Cre and Prok2.Cre ns. p=0.1).

G. The final circadian periods of Cry1-/- SCN slice double-transduced in complementary reverse orders did not differ between each other, p=0.7.

H. Period lengthening occurred at the same rate when Prok2.Cre or ProkR2.Cre was the first AAV to be applied (p=0.1; Prok2.Cre - baseline vs. 1 AAV, *p=0.01; baseline vs. both AAVs, *p=0.04; ProkR2.Cre - baseline vs. 1 AAV, *p=0.03; baseline vs. both AAVs, **p=0.01.

Data information: All values depicted as mean±S.E.M. C, D, E, F one-way ANOVA with Tukey's multiple comparisons; G unpaired two-tailed t-test; H two-way ANOVA with Sidak's multiple comparisons. In histograms individual points are individual slices, bars are their mean±S.E.M.
Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network (2021)
Figure 9

Figure 9. Prok2+ and Prok2R+ cells can initiate de novo SCN network rhythms

A. Representative Per2::Luciferase traces of Cry1,2-/- SCN slices at baseline (black, arrhythmic), after Cry1 complementation in Prok2+ cells (red) and further complementation in ProkR2+ cells (yellow, rhythmic) (cohort n=8).

B. as in A but order of complementation reversed (green =Cry1 complementation in ProkR2+ cells) (cohort n=8).

C. SCN period after Cry1 expression in both Prok2+ and ProkR2+ cells did not differ with order of transduction (p=0.4).

D. Bioluminescence rhythm amplitude did not differ with order of complementation (p=0.96).

E. Paired goodness-of-fit values for Per2::Luciferase oscillations of baseline, single-transfection and double-transfection SCN (p=0.02, Prok2.Cre before and after, ns. p=0.17; ProkR2.Cre before and after, ns. p=0.99; both Prok2.Cre and ProkR2.Cre applied before and after ****p<0.0001).

F. Binary representation of initiated slice elements of a representative SCN slice (CCD recorded bioluminescence pixels) - black indicates non-initiated and pink indicates initiated, i.e., rhythmic elements (cohort n=12).

G. Phase map of Per2::Luciferase bioluminescence in a representative Prok2+/ProkR2+ Cry1-initiated SCN slice showing spatial phase synchronisation across the slices (phases colour-coded relative to the mean phase of each slice).

H. Inferred neuronal group interactions in relation to circuit-level contribution as "Distributor" or "Integrator" hubs.

Data information: All values depicted as mean±S.E.M. C, D unpaired two-tailed t-tests; E two-way ANOVA with Sidak's multiple comparisons.

Expanded View Figure Legends.
A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint (2020)
Figure 1

Figure 1. The mps1-3 mutant is defective in chromosome biorientation.

A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

B: Mps1 sequence alignment around S. cerevisiae Ser635. Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Mm: Mus musculus: Hs: Homo sapiens; Dm: Drosophila melanogaster.

C-D: Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C) and for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples.

E: Frequencies of chromosome loss were quantified at permissive temperature (25°C).

F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm.

G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.
A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint (2020)
Figure 2

Figure 2. The mps1-3 mutant is defective in SAC signalling and does not localise Mps1 at kinetochores.

A: Wild type and mps1 mutant cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.

B: Cells were treated as in (A) and after 2 hours from the release α-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells.

C: Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from E. coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue-stained gel (right). Bottom right: 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays.

D: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S2). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.

E: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.
A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint (2020)
Figure 3

Figure 3. Characterisation of the extragenic suppressors of mps1-3 mutant cells.

A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at 25°C and 34°C.

B: List of extragenic suppressors found in the unbiased genetic screen.

C: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.

E: Cells were treated as in (D) and collected at the indicated time points for western blot analysis of Mad1-3HA. Cyc: cycling cells.

F: Cells with the indicated genotypes were synchronised at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.

G-H: Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for one hour before filming. Cells were filmed at 34°C every 2 or 4 minutes by time lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild type and mps1-3 cells are shown in Fig. 1F. DIC: differential interference contrast. Scale bar: 5 μm.

I: Wild type, mps1-3, mps1-3 spc105-18, and mps1-3 GLC7-24 cells were synchronised in G1 with α-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 slightly decreases the maximal temperature of suppression). Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S5). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.
A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint (2020)
Figure 4

Figure 4. Spc105 phosphorylation and Bub1 kinetochore recruitment are impaired in mps1-3 cells.

A-B: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (A) or absence (B) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.

C-D: Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 hour before filming; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 minutes to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 before imaging in the same medium. Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 μm. GFP signals (Bub1) co-localizing with Tomato signals (kinetochores) were scored on the movies (D).

E-H: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (E-F) or absence (G-H) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins (E, G) and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.
A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint (2020)
Figure 5

Figure 5. Artificial recruitment of Bub1 to Spc105 is sufficient to suppress the chromosome biorientation defects of mps1-3 cells.

A: SPC105-GBD cells expressing Bub1-GFP were grown at 30°C and filmed every 4 minutes at 30°C by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.

B: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.

C: cells with the indicated genotypes were grown at 25°C and then shifted at 34°C (t=0). Every hour after the temperature shift the same number of cells was plated on YEPD for each strain and incubated at 25°C to determine the number of colony-forming units. Percentages of viable cells have been calculated for each strain relative to t=0. Error bars: SD. N=3.

D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.
Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana (2019)
Figure 1

Figure 1.

TPS1 is required for high temperature-induced hypocotyl growth

A Hypocotyl lengths of wild-type and suc2 seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium with or without 3% sucrose. Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). In the box plots (A-D), the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.

B Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under the same growth conditions as (A). Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C).

C Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under continuous white light at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium containing either mock or 20 µM of dexamethasone (DEX). ns, not significant (Student's t-test P ≥ 0.05; n > 10). **P < 0.01 (Student's t-test); n > 10. Number indicates the ratio of hypocotyl lengths (Dex/Mock).

D Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown in the dark at 20 °C for 7 days. Representative seedlings are shown in the left panel. ns, not significant (Student's t-test P ≥ 0.05; n > 10).

E Diagram showing plant growth conditions used for the qRT-PCR analyses in (F-I). Seedlings were maintained under 12-h light/12-h dark cycles at 20 °C for 4 days and then transferred to continuous light on the 5th day. The growth temperature was increased to 28 °C or kept at 20 °C for 4 hours at Zeitgeber Time (ZT) 20-24 before harvesting for total RNA extraction.

F-I The qRT-PCR analyses of the transcript levels of YUC8, IAA19, IAA29, and PIF4. Transcript levels of each gene were normalized to those of PP2A and presented as values relative to those of wild-type seedlings at 20 °C. Error bars indicate s.d. (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). Numbers indicate the ratio of transcript levels in seedlings grown at two different temperatures (28 °C/20 °C).
Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana (2019)
Figure 2

Figure 2.

The stability of PIF4 protein is dependent on TPS1

A Western blotting with anti-PIF4 antibody shows that PIF4 protein levels are decreased in the tps1-2;GVG::TPS1 mutants compared to wild-type plants. Seedlings were maintained under 12-h light/12-h dark cycles at 20 °C for 4 days and then transferred under continuous light on the 5th day. The growth temperature was increased to 28 °C or kept at 20°C for 4 hours at ZT 20-24 before harvesting for total protein extraction. Equal loading of samples is shown by Ponceau S staining.

B Western blotting showing the levels of PIF4-Myc protein in PIF4-OX and PIF4-OX;tps1-2;GVG::TPS1 seedlings. Total protein was extracted from the seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Western blotting was probed using an anti-Myc antibody. Ponceau S staining is shown for equal loading.

C, D Hypocotyl lengths in seedlings of different genotypes. Seedlings were grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (C). Numbers in (D) indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 12). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.