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Behavioral adaptation to warm conditions via Lim1-mediated acceleration of neuronal clocks

Nature Neuroscience (2025)Cite this article

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Abstract

Temperature compensation stabilizes the speed of circadian clocks. Uncompensated molecular clock cycles would accelerate severalfold with each 10 °C increase, precluding reliable timekeeping. Despite such thermal buffering, some clock-controlled behavioral cycles complete by up to two hours earlier or later depending on environmental temperatures. We show that temperature-dependent changes in the speed of behavioral cycles can be explained by changes in the speed of the clock itself. Although the speed of all clocks is insensitive to thermal energy, we found that in neurons the clock speed is regulated by temperature information. When the threshold of ~26 °C is exceeded for ~24 h, a pathway mediated by the LIM-homeodomain transcription factor Lim1 instructs the clocks in the Drosophila brain to accelerate. Clock acceleration enables earlier morning awakening. This work suggests that modestly altering the clock speed enables behavioral thermoadaptation, via regulated steps that do not compromise the reliability of circadian timekeeping.

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Fig. 1: Temperature-dependent regulation of the clock speed requires Lim1.
Fig. 2: Temperature-dependent acceleration of locomotor rhythms is a generalizable phenomenon that requires Lim1 and TRPA1-expressing neurons.
Fig. 3: Lim1 levels increase in DN1p neurons with warmth.
Fig. 4: Warming promotes DN1p activity through Lim1.
Fig. 5: Lim1 accelerates the clock through transcriptional SK repression.
Fig. 6: DN1 neurons signal to LNv neurons via glutamate to accelerate the clock in warm conditions.
Fig. 7: Shortening of the circadian period allows earlier waking in warm conditions.

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Data availability

Source data are provided with this paper.

Code availability

MATLAB programs used to (1) quantify calcium transients and (2) parse DAM activity data are available online at https://github.com/CrickmoreRoguljaLabs.

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Acknowledgements

We thank D. Anderson (California Institute of Technology), J. Blau (New York University), J. Botas (Baylor College of Medicine), P. Dolph (Dartmouth College), P. Hardin (Texas A&M University), F. Rouyer (CNRS Université Paris-Saclay), M. Rosbash (Brandeis University), G. Rubin (Janelia Research Campus), R. Stanewsky (University of Münster), the Vienna Drosophila Resource Center, TsingHua Fly Center and the Bloomington Stock Center for providing reagents. Enhanced Neuroimaging Core at the Harvard Neurodiscovery Center provided access to their confocal microscope during the initial stages of the project. B. Sabatini provided support and equipment for two-photon microscopy. We thank the members of the Rogulja and Crickmore labs, as well as M. Rosbash, M. Young and C. Weitz for comments on the manuscript and helpful discussions. This work was supported by the Whitehall Foundation and the Klingenstein-Simons Foundation (to D.R.) and the National Science Foundation of China (32571159 to Z.L.). Z.L. was an Edward R. and Anne G. Lefler Postdoctoral Fellow. S.X.Z. was a Stuart H.Q. and Victoria Quan Fellow. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, China

    Zhihua Liu, Dapeng Xie & Hao Zhou

  2. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Zhihua Liu, Stephen X. Zhang, Wenji Cai & Dragana Rogulja

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  1. Zhihua Liu
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  2. Dapeng Xie
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Contributions

Z.L. and D.R. designed the study and wrote the manuscript with input from all authors. Z.L., D.X., S.X.Z., W.C. and H.Z. performed the experiments. All authors analyzed the data.

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Correspondence to Dragana Rogulja.

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Extended data

Extended Data Fig. 1 Pan-neuronal versus clock cell-specific Lim1 depletion leads to distinct sleep phenotypes.

a, Left, daily sleep profiles of controls (nSyb-Gal4 and UAS-lim1-RNAi) and flies with Lim1 depleted from the nervous system (nSyb>lim1-RNAi) at 25 °C in 12-h light/12-h dark conditions (denoted by white–black bars on the bottom). Right, quantification of total daily sleep. n = 19 for nSyb-Gal4; 15 for UAS-lim1-RNAi; 15 for nSyb>lim1-RNAi, one-way ANOVA followed by Dunnett’s multiple comparisons test. b, Immunostaining for Lim1 in controls and flies with Lim1 depleted from the nervous system. Two different lim1-RNAi lines produced the same result. Scale bar = 50 µm. c, Quantification of Lim1 levels in controls (tim-Gal4) and flies with Lim1 depleted from clock neurons (tim>lim1-RNAi), at 25 °C and 30 °C. Sample sizes ranged from n = 10 to n = 24, two-way ANOVA followed by Tukey’s multiple comparisons test. d, Daily sleep profiles in 12-h light/12-h dark conditions (denoted by white–black bars on the bottom). Right, total sleep during the last 4 h of darkness (black bars in left panels). Flies depleted of Lim1 in clock neurons (tim>lim1-RNAi) are compared to the parental controls (tim-Gal4 and UAS-lim1-RNAi). n (25 °C, 30 °C): tim-Gal4 (32, 32); UAS-lim1-RNAi (32, 18); tim>lim1-RNAi (32, 66), two-way ANOVA followed by Tukey’s multiple comparisons test. e, Daily averaged locomotor activity profiles (top left) and sleep profiles (bottom left) in 12-h light/12-h dark conditions (denoted by white–black bars on the bottom). Flies were depleted of Lim1 in circadian clock neurons using a second RNAi line (tim>lim1-RNAi 2) and compared to the parental controls (tim-Gal4 and UAS-lim1-RNAi 2) at 25 °C and 30 °C. Right, quantification of the total locomotor activity (top right) and sleep time (bottom right) during the 4 h preceding lights-on (black bars in left panels). n (25 °C, 30 °C): tim-Gal4 (29, 16); UAS-lim1-RNAi 2 (32, 11); tim>lim1-RNAi 2 (32, 23), two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m. ***P < 0.001.

Source data

Extended Data Fig. 2 Warming accelerates the clock via Lim1.

a, Percentage of rhythmic animals when clock proteins PER or TIMELESS (TIM) are depleted from s-LNvs (PDF-Gal4), LNds (Mai179-Gal4;pdf-Gal80)90, or both (Mai179-Gal4), using CRISPR. Disrupting the molecular clock in both populations renders flies arrhythmic. b, PER oscillations in the brains of control flies (tim-Gal4) and flies with Lim1 depleted from circadian clock neurons (tim>lim1-RNAi) on day 6 in darkness, extra sum‑of‑squares F test. Data are replotted from Fig. 1b. c, Quantification of PER oscillation in nonpacemaking clock neurons (clock neurons other than s-LNv and LNd), in controls (tim-Gal4) and flies with Lim1 depleted from clock neurons (tim>lim1-RNAi). In controls, DN1ps had an advanced phase of PER oscillations at 30 °C compared to 25 °C, consistent with previous findings that s-LNvs pace the molecular clocks in DN1ps47. Additionally, DN3s displayed an advanced PER oscillation phase at 30 °C compared to 25 °C, likely because s-LNvs also synchronize DN3 clocks. Depleting Lim1 from clock neurons abolished the temperature-induced phase shift. l-LNvs showed no daily PER rhythmicity, mirroring previous reports of rapid PER oscillation decay in darkness90. DN2 neurons exhibited a ~12-h phase shift in PER oscillations relative to pacemaking neurons, consistent with previous reports91. n (25 °C, 30 °C): tim-Gal4 (6–14); tim>lim1-RNAi (6–15), extra sum‑of‑squares F test. Quantification represents mean ± s.e.m. ***P < 0.001.

Source data

Extended Data Fig. 3 TRPA1 is required for clock acceleration in warm conditions.

a, Quantification of temperature-dependent PER oscillations in common fly strains. n (25 °C, 30 °C): Canton-S (5–14); w1118 (8–16), extra sum‑of‑squares F test. b, Locomotor rhythm periodicity in common strains at 20 °C, 25 °C and 30 °C. n = 16 for Hikone-A-W; 24 for Canton-S; 21 for Florida-9; 17 for Oregon-R; 18 for Berlin-K at 20 °C, two-way ANOVA followed by Tukey’s multiple comparisons test. The data for 25 °C and 30 °C are the same as plotted in Fig. 2b. c, Left, schematic of fly thermosensors. Right, quantification of locomotor rhythm periodicity in controls (w1118), TRPA11 mutants, nocte1 mutants, and w1118 flies with aristae surgically removed. n (25 °C, 30 °C): Canton-S (16, 16); TRPA11 (17, 21); nocte1 (20, 15); flies with no aristae (12, 32), two-way ANOVA followed by Tukey’s multiple comparisons test. d, Left, schematic of the four TRPA1 isoforms and the three isoform-specific TRPA1 mutants. Gray bar: UTR; black bar: exon. TRPA1AB: isoforms A and B are deleted. TRPA1CD: isoforms C and D are deleted. TRPA1ACD: isoform A is mutated and isoforms C and D are deleted. Right, quantification of locomotor rhythm periodicity of isoform-specific TRPA1 mutants. n (25 °C, 30 °C): TRPA1AB (9, 10); TRPA1CD (23, 22); TRPA1ACD (18, 21), two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m. **P < 0.01, ***P < 0.001. Fruit fly diagram in c created with BioRender.com.

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Extended Data Fig. 4 Warmth increases Lim1 levels in DN1ps in a TRPA1-dependent manner.

a, Levels of a pan-neuronal protein Osa (a subunit of the SWI/SNF chromatin remodeling complex) in DN1ps do not change with temperature. n = 11 and 10 at 25 °C and 30 °C, respectively, Student’s t-tests. b, Quantification of Lim1 levels in DN1p neurons of controls (w1118), TRPA11 and nocte1 mutants, and flies with aristae surgically removed. TRPA1 mutation, but not other manipulations, prevents Lim1 levels in DN1ps from increasing when temperature increases. n (25 °C, 30 °C): w1118 (32, 15); TRPA11 (32, 17); nocte1 (27, 17); flies with no aristae (33, 16), two-way ANOVA followed by Tukey’s multiple comparisons test. c, Immunostaining for Lim1 and CLK. Lim1 is expressed in DN1ps and DN2s. Lim1 signal is eliminated from DN1ps but not DN2s when lim1-RNAi is expressed with DN1-Gal4 during adulthood. Scale bar = 5 µm. d, Averaged daily locomotor activity (top) and sleep (bottom) profiles in 12-h light/12-h dark conditions (denoted by white–black bars on the bottom). Flies with Lim1 depleted from DN1 neurons during adulthood (DN1>lim1-RNAi;Tub-Gal80ts) are compared to the parental controls (DN1-Gal4 and UAS-lim1-RNAi;Tub-Gal80ts). Please note that in this experiment flies lost more sleep after shifting from 25 °C to 30 °C than in experiments shown in Figs. 1a and 7a, likely because they were raised at 18 °C; flies raised at lower temperature are more sensitive to warming (our observation). Although the combination of 18 °C rearing and 30 °C testing induced a significant baseline reduction in sleep, a statistically significant difference can be observed between Lim1 knockdown flies and controls during the predawn hours (black bars in left panels) within the 30 °C condition. n (25 °C, 30 °C): DN1-Gal4 (32, 35); UAS-lim1-RNAi;Tub-Gal80ts (13, 23); DN1>lim1-RNAi;Tub-Gal80ts (32, 28), two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m. ***P < 0.001.

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Extended Data Fig. 5 LPN clock neurons are not required for temperature-dependent adjustment of clock speed.

Top, averaged locomotor activity for control flies (LPN-Gal4) and flies with LPN neurons silenced (LPN>Kir2.1 and LPN > TNT), at 25 °C and 30 °C. Bottom, quantification. n (25 °C, 30 °C): LPN-Gal4 (15, 13); UAS-Kir2.1 (25, 22); LPN>Kir2.1 (16, 14); UAS-TNT (25, 25); LPN > TNT (14, 21), two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m. ***P < 0.001.

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Extended Data Fig. 6 Lim1 increases both the basal activity and the optogenetically induced calcium transients in DN1 neurons specifically during the morning.

a, DN1 neurons can be activated by blue light via Cryptochrome-mediated excitation84. We used two-photon laser-scanning microscopy for calcium imaging to confirm the results obtained using regular fluorescence microscopy (Fig. 4b), which might be affected by experimental activation of Cryptochrome. Two-photon images of DN1 projections in the SMPp showing GCaMP6s fluorescence when excited at 910 nm (calcium-dependent) and 820 nm (calcium-independent). Depletion of Lim1 from DN1 neurons (DN1>lim1-RNAi + GCaMP6s) specifically abolishes the morning (ZT0-2) calcium signal compared to controls (DN1-Gal4). Each image is a maximum projection spanning ~45 µm along the anterior-posterior axis. See Methods for details. Scale bar = 5 µm. b, Left, normalized DN1 > GCaMP6s fluorescence (F910 nm/F820 nm) in the SMPp is higher in the morning (ZT0-2) than evening (ZT10-12), a difference that is abolished when Lim1 is depleted. Right, no difference was seen in the calcium-independent fluorescence (F820 nm) among different groups. n = 8 for all conditions, two-way ANOVA followed by Tukey’s multiple comparisons test. c, Optically triggered calcium transients in DN1 projections (DN1>CsChrimson + GCaMP6s) have a higher amplitude in the subjective morning (left, CT0-2) than evening (middle, CT10-12), a difference that is abolished when Lim1 is depleted from these cells (DN1>lim1-RNAi + CsChrimson + GCaMP6s). The GCaMP6s transient peaks are quantified on the right. All experiments were performed at 25 °C. n = 8 for all conditions, two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m.

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Extended Data Fig. 7 Circadian period analysis and Lim1-dependent modulation of sleep timing under simulated seasonal light and temperature cycles.

a, Quantification of locomotor rhythm periodicity of Gal4 controls (DN1-Gal4, PDF-Gal4, and Mai179-Gal4;PDF-Gal80), UAS controls (UAS-VGlut-RNAi 2, UAS-mGluRA-RNAi 2), flies with VGlut depleted from DN1s using a second RNAi line (DN1>VGlut-RNAi 2), mGluRA depleted from LNvs (s-LNvs and l-LNvs) using a second RNAi line (PDF>mGluRA-RNAi 2), and mGluRA depleted from LNds using two independent RNAi lines (Mai179;pdf-Gal80>mGluRA-RNAi 1 and Mai179;pdf-Gal80>mGluRA-RNAi 2) in constant darkness at 25 °C or 30 °C. The data for DN1-Gal4 and PDF-Gal4 are the same as plotted in Fig. 6a. n (25 °C, 30 °C): DN1-Gal4 (27, 29); UAS-VGlut-RNAi 2 (31, 32); DN1>VGlut-RNAi 2 (27, 28); PDF-Gal4 (21, 31); UAS-mGluRA-RNAi 2 (14, 21); PDF>mGluRA-RNAi 2 (20, 26); Mai179-Gal4;PDF-Gal80 (20, 25); Mai179-Gal4;PDF-Gal80>mGluRA-RNAi 1 (8, 15); Mai179-Gal4;PDF-Gal80>mGluRA-RNAi 2 (8, 14), two-way ANOVA followed by Tukey’s multiple comparisons test. b, Left, averaged daily sleep profiles in 12-h light/12-h dark conditions (denoted by white–black bars on the bottom). Controls (DN1-Gal4) are compared to flies with Lim1 depleted from DN1 neurons (DN1>lim1-RNAi), and flies with Lim1 depleted and dominant-negative SK overexpressed in DN1 neurons (DN1>lim1-RNAi + SK-DN). Right, quantification of total sleep during the final 4 h of the dark phase (black bars in left panels), n (25 °C, 30 °C): DN1-Gal4 (18, 29); DN1>lim1-RNAi (15, 23); DN1>lim1-RNAi + SK-DN (32, 33), two-way ANOVA followed by Tukey’s multiple comparisons test. c, Locomotor activity in naturalistic conditions. Top, average daily locomotor activity profiles under spring-like (left) and summer-like conditions (right). Temperature fluctuations (background color gradient) and light cycle (color bar, peak ~380 lux) are indicated. Morning (M), afternoon (A), and evening (E) activity peaks are labeled. Bottom right, quantification of total locomotor activity within a 3-h window spanning the typical morning activity period (black bars in top panels; defined as 1 h before scheduled lights-on to 2 h after scheduled lights-on) to specifically assess changes in morning wake timing. n (spring, summer): DN1-Gal4 (27, 48); UAS-lim1-RNAi (32, 32); DN1>lim1-RNAi (48, 40), two-way ANOVA followed by Tukey’s multiple comparisons test. Quantification represents mean ± s.e.m. ***P < 0.001.

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Extended Data Fig. 8 Lim1 does not regulate entrainment of the clock phase to temperature cycles.

Circadian clocks can be reset not only by light-dark cycles, but also by temperature cycles with an amplitude as low as ~2 °C61. DN1ps mediate entrainment to low-amplitude temperature cycles31; we asked whether this requires Lim1. Double-plotted actograms of control (DN1-Gal4) and Lim1-depleted (DN1>lim1-RNAi) flies. a, Flies were entrained to 12-h light/12-h dark (LD) cycles for 3 days before shifting to the first temperature cycle (TC1, 21 °C/25 °C, constant darkness, 6 h delayed relative to the LD cycle) for 5 days, followed by 5 days in the second temperature cycle (TC2, 21 °C/25 °C, constant darkness, 6 h delayed relative to TC1), finishing with 5 days in constant temperature and darkness. n = 31 and 30 for DN1-Gal4 and DN1>lim1-RNAi, respectively, Student’s t-tests. b, As in a, but with TC1 and TC2 at 25 °C/29 °C. n = 31 and 31 for DN1-Gal4 and DN1>lim1-RNAi, respectively, Student’s t-tests. c, Flies were entrained to LD cycle at 25 °C for 3 days before shifting to the first temperature cycle (TC, 25 °C/27 °C, constant darkness, 8 h advanced relative to the LD cycle) for 6 days, finishing with 8 days in constant temperature and darkness. n = 28 and 31 for DN1-Gal4 and DN1>lim1-RNAi, respectively, Student’s t-tests. d, Flies were kept in constant light (LL) for 3 days before shifting to the first temperature cycle (TC1, 25 °C/29 °C, constant light, 8 h advanced relative to the LD cycle) for 6 days, followed by 8 days in the second temperature cycle (TC2, 25 °C/29 °C, constant light, 6 h delayed relative to TC1). n = 28 and 30 for DN1-Gal4 and DN1>lim1-RNAi, respectively, Student’s t-tests. Quantification represents mean ± s.e.m.

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Liu, Z., Xie, D., Zhang, S.X. et al. Behavioral adaptation to warm conditions via Lim1-mediated acceleration of neuronal clocks. Nat Neurosci (2025). https://doi.org/10.1038/s41593-025-02139-2

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