Fig 2: SCN neuronal sub-population transcriptomes ASub-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 sub-populations 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.BViolin 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.CVisualisation 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.DVenn 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. Source data are available online for this figure.

Fig 3: Raw bioluminescent traces of Cry1-/- SCN or Cry1,2-/- SCN complemented with Cry1 in Prok2 + and/or ProkR2 + cells, Related to Figs 7 and 8 ARaw bioluminescent traces of all Cry1-/- SCN slices transfected with pCry.DIO.eGFP, pProk2.Cre.T2A.mCherry (red, n = 5) or pProkR2.Cre.T2A.Venus (green, n = 6), followed by the other pProk2.Cre or pProkR2.Cre-expressing AAV (yellow). The mean period, µ, measured after each AAV transfection is shown on each respective plot.BRaw bioluminescent traces of all Cry1-/-Cry2-/- SCN slices transfected with pCry.DIO.eGFP, pProk2.Cre.T2A.mCherry (red, n = 8) or pProkR2.Cre.T2A.Venus (green, n = 8), followed by the other pProk2.Cre or pProkR2.Cre-expressing AAV (yellow). Source data are available online for this figure.

Fig 4: Circadian functions of SCN Prok2 + and ProkR2 + cells ARepresentative, 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.BRaster 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.CThe 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).DSimilarly, the amplitude of Cry1-luc bioluminescence rhythms from Prok2 + SCN cells and ProkR2 + SCN cells does not differ between treatments (P = 0.67).ERepresentative raster plots of single-cell bioluminescence (yellow = minimum, blue = maximum) from Prok2Cry1-luc (left) or ProkR2Cry1-luc (right) cells in single slices.FWithin-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.GThe 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).HLeft: 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 = 500 µm). 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.IAfter 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 intra-cellular clocks of Prok2 + cells are phase advanced by ˜0.6 h to those of ProkR2 + cells (**P < 0.01) (n = 5 SCN slices per group). Data information: All values depicted as mean ± SEM. C, D, G, I unpaired two-tailed t-tests. Source data are available online for this figure.

Fig 5: Prok2 + and Prok2R + cells can initiate de novo SCN network rhythms ARepresentative 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).BAs in A but order of complementation reversed (green = Cry1 complementation in ProkR2 + cells) (cohort n = 8).CSCN period after Cry1 expression in both Prok2 + and ProkR2 + cells did not differ with order of transduction (P = 0.4).DBioluminescence rhythm amplitude did not differ with order of complementation (P = 0.96).EPaired 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).FBinary 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).GPhase 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).HInferred neuronal group interactions in relation to circuit-level contribution as “Distributor” or “Integrator” hubs. Data information: All values depicted as mean ± SEM. C, D unpaired two-tailed t-tests; E two-way ANOVA with Sidak’s multiple comparisons. Source data are available online for this figure.