Clock is necessary to maintain circadian behavior.

(A) Phylogenetic tree showing the evolutionary relationship of CLOCK orthologs across different animal species. (B) In situ hybridization of Clock in the WT juvenile with scale bars representing 0.1mm and 5μm. (C) Schematic representation of the Clock gene in grey, with the open reading frame (ORF) in orange and the conserved protein domains bHLH (green) and PAS1 and PAS2 (dark red). The CRISPR generated allele has a 20nt insertion after the PAS1 domain, represented by a black arrowhead. (D-G) Normalized locomotor activity (a.u), hourly binned over 72h, under different light conditions: 12h light:12h dark, continuous dark, continuous light, and 6h light:6h dark. The black line represents the WT and the red line represents the Cloc/- mutant. (D’-G’) Periodograms for each corresponding light condition. The significant period value (p<0.01) is indicated for each genotype in the top left corner of each graph.

Clock mutation causes a broad impact on the rhythmic transcriptome.

(A) Overview of the experimental design used to generate RNA-seq data. Polyps were entrained for 72 hours before sampling at 4-hour intervals over a 24-hour period (dark arrows) in both LD and DD cycles. (B) Venn diagram comparing the total number of rhythmic genes identified in WT and Clock-/-in LD and DD cycles. (C) Heatmap showing genotype-specific rhythmic transcripts in WT and Clock-/-. (D) Gene Ontology (GO) terms that were significantly enriched (Bonferroni corrected p < 0.05) for rhythmic genes in WT. (E) De novo sequence motif analysis of circadian transcriptional regulators detected in WT and Clock-/- using Homer/MEME. The 1000bp upstream ATG of each rhythmic gene was used for motif analysis.

Clock cooperates with light to drive the transcription rhythm of the core clock genes.

(A) Each core clock gene is individually plotted showing the read count over 24h in LD and DD in WT (black) and Clock-/- (red). The continuous line represents significant rhythmicity (RAIN p<0.05) while the dashed line indicates no rhythmicity. (B) The presence of Circadian E-box in all core clock gene promoters was detected using HOMER within 1000bp upstream ATG. (C) Conserved rhythmic genes were selected from our WT data and Leach et al., 2020. (D) The temporal expression pattern of Clock-downstream gene Myh7 is shown. (E) In situ hybridization of Myh7 in the WT juvenile with scale bars representing 0.1mm. Nuclei are DAPI-stained, colored in grey.

Clock regulates genetic pathways.

(A) Volcano plot showing the differential expression of genes between WT and Clock-/- in LD. The dashed lines indicate the threshold used to detect DEGs (Padj<0.05) and the WT and Clock-/- are represented by black and red dots, respectively. (B) Volcano plot showing the differential expression of genes between WT and Clock-/- in DD (C) Gene Ontology (GO) terms with significant fold-enrichment (Bonferroni corrected p-value < 0.05) are shown for DEG analysis in DD.

Generation of Clock mutant by CRISPR-Cas9-mediated mutagenesis.

Nucleotide and translated amino acid sequences of wild-type and mutant alleles. sgRNA target site is boxed in green, and PAM site are shown in green. Predicted translation termination site is boxed in black. Insertion is labeled in red. Predicted immature peptide sequences in red.

Spatial expression pattern of Clock reveals endodermis enrichment.

Max projection of N. vectensis at 4 tentacle stages, showing signal in Red from the two-channel (Clock-B1/Clock-B3 for Clock mRNA detection and Clock-B1/zfHcrt-B3 for the control) redundant detection. Nuclei are labeled with DAPI. Scale bar: 0.1 mm.

Clock is necessary to maintain circadian behavior.

(A-B) Normalized locomotor activity (a.u), hourly binned over 72h, in continuous dark. The black line represents the WT, and the red line represents the Clock-/+. (A’-B’) Periodograms for each corresponding light condition. The significant period value (p<0.01) is indicated for each genotype in the top left corner of each graph.

Clock causes a broad impact on the rhythmic transcriptome.

Venn diagram showing the overlap and unique sets of rhythmic genes identified in wild-type (WT) and Clock-/-mutant under light-dark (LD) and constant darkness (DD) conditions. The asterisk (*) indicates the core circadian genes that exhibit rhythmic expression in both WT and Clock-/- in both LD and DD conditions.