Abstract
The transcription factor CLOCK is ubiquitously expressed and important for circadian rhythms, while its human-specific expression in neocortex suggests additional functions. Here, we generated a mouse model (HU) that recapitulates human cortical expression of CLOCK. The HU mice show enhanced cognitive flexibility, which might be associated with alteration in spatiotemporal expression of CLOCK. Cell-type-specific genomic profiling identified upregulated genes related to dendritic growth and spine formation in excitatory neurons of HU mice. We also found that excitatory neurons in HU mice have increased dendritic complexity and spine density, and a greater frequency of excitatory postsynaptic currents, suggesting a greater abundance of neural connectivity. In contrast, CLOCK knockout in human induced pluripotent stem cell-derived neurons showed reduced complexity of dendrites and lower density of presynaptic puncta. Together, our data demonstrate that CLOCK might have evolved brain-relevant gains of function via altered spatiotemporal gene expression and that these functions may underlie human brain specializations.
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Data availability
snRNA-seq data are deposited in NCBI Gene Expression Omnibus (GEO; GSE224759). We used reference mouse genome (MM10 GRCm38p6) for annotation. Other datasets used in this paper were snRNA-seq data from the Allen Brain Institute (RRID: SCR_016152; https://assets.nemoarchive.org/dat-net1412/), single-nuclei RNA-seq data of Caglayan et al.29 from NCBI GEO (GSE192774) and single-nuclei RNA-seq data of Khrameeva et al.28 from NCBI GEO (GSE127774). Source data are provided with this paper. Due to the amount of data, all other data will be made available upon request to the corresponding authors.
Code availability
We have deposited all original code to GitHub (https://github.com/konopkalab/CLOCK-humanized-mice/) and Zenodo (https://doi.org/10.5281/zenodo.15319773)157.
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Acknowledgements
We thank M. Dehnad and E. Oh for their critical comments on the manuscript, C. C. Sherwood for providing brain tissues from nonhuman primates, D. R. Weaver for helping with mouse genotyping protocols, E. Caglayan for help in analysis of snRNA-seq data, N. Park for verifying the antibody for CLOCK WB, F. Ferreira, L. Thomas and S. Dixon for analysis and collection of wheel-running data. We also thank S. Yamazaki and the UTSW Neuroscience Microscopy Facility for helping with imaging. G.K. is a Jon Heighten Scholar in Autism Research and Townsend Distinguished Chair in Research on Autism Spectrum Disorders at UT Southwestern. J.S.T. is an Investigator in the Howard Hughes Medical Institute. This work was supported by the James S. McDonnell Foundation 21st Century Science Initiative in Understanding Human Cognition Scholar Award (no. 22002046) and National Institutes of Health (HG011641, MH207672 and MH103517) to G.K., an American Heart Association Postdoctoral Fellowship (915654) to Y.L. and a National Institutes of Health F30 Predoctoral Fellowship (MH105158-01A1) to M.R.F.
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Contributions
Y.L., M.R.F., J.S.T. and G.K. conceptualized the project and designed experiments. Y.L. carried out most of the bench work, including mouse work, genotyping, behavior tests, AAV injection, brain dissection, brain slicing, IHC, RT–qPCR, snRNA-seq, iPS cell culture, CRIPSR–Cas9 and ICC. Y.L. collected and analyzed the data. M.R.F. generated the CLOCK humanized mouse model. A.K. and Y.L. analyzed the snRNA-seq data. N.K. and J.R.G. designed and performed electrophysiology experiments as well as data analysis. S.H.E.V.P. generated CLOCK KO iPS cell lines and helped in iPS cell culture and CRISPR–Cas9 experiments. C.C. and M.H. did mouse work, genotyping and helped with AAV injections. P.X. designed and helped with some behavior tests. N.G. contributed to brain slicing, IHC and image taking. N.K. and S.H.E.V.P. edited the manuscript. Y.L., J.S.T. and G.K. wrote the manuscript.
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J.S.T. is a cofounder of, a scientific advisory board member of and a paid consultant for Synchronicity Pharma, a biotechnology company aimed at discovering small-molecule therapies that modulate circadian activity for a variety of diseases. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Human-specific upregulation of CLOCK in neocortex.
(a) CLOCK expression among human, chimpanzee, and macaque across major neocortical cell types through reanalyzing recently published comparative snRNA-seq genomics datasets28,29,30. We applied a general linear mixed model (GLMM) with species as fixed factors, cell type as random factor nested with individual, and Tukey’s test for post-hoc analysis. EX: excitatory neuron; IN: inhibitory neuron; ASTRO: astrocyte; Oligos: oligodendrocyte and OPC; ENDO: endothelium; _MICRO: microglia. (b) Representative images of immunofluorescent staining for neurons (NEUN, yellow), CLOCK+ cells (CLOCK, red), oligodendrocytes (OLIG2, green), and all cells (DAPI, blue) in posterior cingulate cortex. The colored dash polygons labelled CLOCK- neurons (yellow) and oligodendrocytes (green) in the CLOCK channel, while solid polygons indicated the corresponding cells in each channel. (c-e) Quantification of (c) fluorescent intensity of CLOCK staining, CLOCK+ fraction in (d) neurons and (e) oligodendrocytes. For panels c-e, the boxplots are defined by the first quartile, mean, and third quartile for the box, and whiskers extending to the most extreme data points within 1.5 times the interquartile range. Each data point is a subarea of a section in the posterior cingulate cortex. We sampled 3 subareas in 2 sections per individual and 3 individuals per species. Points with the same color are from the same individual. We applied a general linear mixed model (GLMM) with species as fixed factors, subarea as random factor nested with section which nested with individual, and Tukey’s test for post-hoc analysis. HS: human, PT: chimpanzee, and MM: macaque. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. All statistics were two-sided tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Extended Data Fig. 2 Human CLOCK increases cell density in adult somatosensory cortex but not nucleus accumbens.
Quantification of cell density in adult somatosensory cortex (a-d) and nucleus accumbens (e-h). (a,e) Representative image of IHC staining for all cells (DAPI, blue), neurons (NEUN, green), and oligodendrocytes (OLIG2, red). (b-d and f-h) Quantification of cell number in (b,f) neurons, (c,g) oligodendrocytes, and (d,h) all cells. For all data panels, each data point is a section containing somatosensory cortex. We sampled 3-5 adult mice per genotype (HU: n = 5; WT: n = 3; KO: n = 3) and 4 sections per mouse. We did GLMM with genotype and sex as fixed factors, section as random factor nested with individual, and Tukey’s test for post-hoc analysis. The open circles represent female samples, while closed circles represents male samples. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. All statistics were two-sided tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Extended Data Fig. 3 Spatiotemporal expression of CLOCK/Clock across cell types in the neocortex.
(a,b) Comparison of CLOCK/Clock+ cells percentage in cell subtypes among HU, WT, and previous work on human neocortical development50 based on snRNA-seq data at (a) P07 mice and 12 years human and (b) P56 mice and 28 years human. (c,d) Cellular expression of CLOCK in each cell type at (c) P07 or (d) P56. (e,f) Feature plot of CLOCK expression in UMAP at (e) P07 or (f) P56.
Extended Data Fig. 4 Comparison of downstream genes regulated by human and mouse CLOCK in subtypes of excitatory neurons across developmental stages.
Number (bar plot) and Log2 fold change (violin plot) of DEGs in each subtype of excitatory neurons, and hypergeometric tests between DEGs from each pair of cell types (heatmap) at (a) P07 HU vs. WT comparison, (b) P07 KO vs. WT comparison, (c) P56 HU vs. WT comparison, and (d) P56 KO vs. WT comparison. The solid line in violin plot indicates the median, while the two dash lines mark the 25 and 75 percentiles. In EX2-3_IT neurons, overlap of DEGs from human and mouse CLOCK at (e) P07 or (f) P56. (g) Overlap of DEGs result from human CLOCK between P07 and P56. (h) Overlap of DEGs from mouse Clock between P07 and P56. The color of each cell represents the -Log10 q value of hypergeometric test in heatmaps. The number in each cell is the number of overlapped DEGs. The number in brackets are the number of DEGs. (i) Venn diagram to show overlap between DEGs of EX2/3_IT and human-specific open chromatin in neurons. (j) Representative images to compare TENM2 fluorescent intensity (green) between HU and WT mice in excitatory neurons (CAMKII, red) of frontal cortex at P07. (k) Quantification of TENM2 fluorescent intensity. Each data point is an excitatory neuron, and we sampled 3 mice per genotype and 5 neurons per mouse. (l) Representative images to compare SORCS2 fluorescent intensity (green) between HU and WT mice in the frontal cortex at P56. (m-n) Quantification of (m) absolute and (n) normalized SORCS2 fluorescent intensity. Each data point is an image of a frontal cortex section, and we sampled 3 mice per genotype and 5-6 images per mouse. We did GLMM with genotype and sex as fixed factors, image as random factor nested with individual, and Tukey’s test for post-hoc analysis. The open circles represent female samples, while closed circles represents male samples. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Extended Data Fig. 5 Neuronal morphology of layer 2-4 excitatory neurons in frontal cortex.
(a) AAV transduced (tdTomato, red) excitatory neurons, but not astrocytes (GFAP, green) and oligodendrocytes (OLIG2, magenta). (b) AAV transduced (tdTomato, red) excitatory neurons across cortical layers (layers 2-4: CUX1, green; layer 6: FOXP2, magenta) of adult frontal cortex. (c-f) In adult mice, (c) comparison of soma area. Each data point is a neuron, and we sampled 39 neurons from 4 HU mice, 46 neurons from 4 WT mice, and 20 neurons from 3 KO mice. We did GLMM with genotype and sex as fixed factors, neuron as random factor nested with individual, and Tukey’s test for post-hoc analysis. (d-f) Comparison of cumulative probability distribution of (d) head area, (e) spine length, and (f) neck width of spines across genotypes. Each data point is a spine, and we sampled all spines from 2-3 segments from 3-7 neurons per mouse. We did GLMM with genotype and sex as fixed factors, spine as random factor nested with segment which again nested with neuron which further nested with individual, and Tukey’s test for post-hoc analysis. (g-n) In P18 mice, (g) CLOCK does not alter soma area. Each data point is a neuron, and we sampled 4 mice per genotype and 6-19 neurons per mouse (total number of neurons: HU: n = 59; WT: n = 71; KO: n = 37). (h-i) Quantification of (h) number and (i) total length of branch in each genotype. For panel g-i, each data point is a neuron, and we sampled 4 mice per genotype and 7-17 neurons per mouse (total number of neurons: HU: n = 48; WT: n = 63; KO: n = 35). For panel g-i, we did GLMM with genotype and sex as fixed factors, neuron as random factor nested with individual, and Tukey’s test for post-hoc analysis. (j) Sholl analysis to quantify encountered intersections between neuron branches and concentric rings from soma for dendrite complexity. The data are from the same samples of panel h and i. We did repeated measures ANOVA with genotype and sex as between-subject factors, distance to soma as within-subject factor, and Tukey’s test for post-hoc analysis. (k) Cumulative probability distribution of spines density across genotypes. Each data point is a segment, and we sampled 2-3 segments from 3-5 neurons per mouse. (l-n) cumulative probability distribution of (l) head area, (m) spine length, and (n) neck width of spines across genotypes. Each data point is a spine, and we sampled all spines from 2-3 segments from 3-5 neurons per mouse. We did GLMM with genotype and sex as fixed factors, spine as random factor nested with segment which again nested with neuron which further nested with individual, and Tukey’s test for post-hoc analysis. The open circles represent female samples, while closed circles represents male samples. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. All statistics were two-sided tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Extended Data Fig. 6 Additional data to confirm CLOCK KO reduces dendritic complexity and spine density in iPSC-derived neurons.
Date collected from different CLOCK KO cell lines (KO#2 and KO#7) at different differentiation stages (Day 21, Day 28, and Day 35). Data of WT and KO#2 at Day 28 are replotted from Fig. 7, and date of Day 21 are replotted from Extended Data Fig. 12. (a) Total length of dendrites normalized to number of neurons. (b) Number of segments of dendrites per neuron. (c) Number of puncta per µm of dendrites. For all panels, each data point is an independent culture of neurons on a circle glass cover slip sitting on the bottom of a well in a 24-well plate. We did GLM with genotype/treatment as fixed factors with Tukey’s test for post-hoc analysis. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. All statistics were two-sided tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Extended Data Fig. 7 TENM2 partially rescued CLOCK KO mature neuronal features in iPSC-derived neurons.
(a) ICC images of iPSC-derived neurons on Day 21 with nuclei marker DAPI (blue), CRISPR-Cas9 transfection marker GFP (green), neuronal dendrite mark TUJ1 (red), and presynaptic marker VGLUT2 (magenta). (b) Total length of dendrites, which were quantified from TUJ1 staining, normalized by the number of nuclei overlapped with TUJ1 signals. (c) Number of TUJ1 segments normalized by the number of nuclei overlapped with TUJ1 signals as a quantification of dendrite complexity. (d) Number of VGLUT2 puncta overlapped with TUJ1 signal and normalized by length of dendrites as a quantification of presynaptic spine density. For all data panels, each data point is an independent culture of neurons on a circle glass cover slip sitting on the bottom of a well in a 24-well plate. We did GLM with genotype/treatment as fixed factors with Tukey’s test for post-hoc analysis. Details of statistical results can be found in Supplementary Table 1. All data are shown as means ± SEM. All statistics were two-sided tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Supplementary information
Supplementary Information (download PDF )
Supplementary Results, Supplementary Methods, Supplementary Figs. 1–6 and additional Supplementary Fig. 1.
Supplementary Table 1 (download XLSX )
Statistical results in figures.
Supplementary Table 2 (download XLSX )
Gene enrichment in clusters at P07 and P56.
Supplementary Table 3 (download XLSX )
List of DEGs between HU and WT by major cell types at P07.
Supplementary Table 4 (download XLSX )
List of DEGs between KO and WT by major cell types at P07.
Supplementary Table 5 (download XLSX )
List of DEGs between HU and WT by major cell types at P56.
Supplementary Table 6 (download XLSX )
List of DEGs between KO and WT by major cell types at P56.
Supplementary Table 7 (download XLSX )
List of DEGs between HU and WT by subtype of excitatory neuron at P07.
Supplementary Table 8 (download XLSX )
List of DEGs between KO and WT by subtype of excitatory neuron at P07.
Supplementary Table 9 (download XLSX )
List of DEGs between HU and WT by subtype of excitatory neuron at P56.
Supplementary Table 10 (download XLSX )
List of DEGs between KO and WT by subtype of excitatory neuron at P56.
Supplementary Table 11 (download XLSX )
GO term enrichment of EX2/3_IT DEGs.
Supplementary Table 12 (download XLSX )
Consistency between DEGs of human CLOCK in EX2/3_IT and human-specific chromatin status.
Supplementary Table 13 (download XLSX )
Enhancer regions of Tenm2/TENM2 and Sorcs2/SORCS2 containing CLOCK–BMAL1 binding motifs.
Supplementary Table 14 (download XLSX )
Examples of odor and medium combinations for the set-shifting tests.
Supplementary Data 1 (download ZIP )
Statistical source data for Supplementary Figs. 1–3 and 6. In Supplementary Fig. 6b, the expression of CLOCK and NR1D1 were log10 transformed.
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Unprocessed western blots. Panel a for Supplementary Fig. 1b; panel b for Supplementary Fig. 6c.
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Liu, Y., Fontenot, M.R., Kulkarni, A. et al. Human CLOCK enhances neocortical function. Nat Neurosci 28, 1716–1728 (2025). https://doi.org/10.1038/s41593-025-01993-4
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DOI: https://doi.org/10.1038/s41593-025-01993-4
