Lhx1 controls terminal differentiation and circadian function of the suprachiasmatic nucleus

Abstract

Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition.

Figure 1. Lhx1 Effects on SCN Neuropeptides

(A–F’) ISH of SCN from adult Lhx1^lox/lox control (A–F) and Six3-Cre;Lhx1^lox/lox mutant (A’–F’) mice for Vip (A, A’), Prok2 (B, B’), Grp (C, C’), Avp (D, D’), Enk (E, E’), and Nms (F, F’). Insets show the neocortex (A and C), preoptic area (B, E, and F), and paraventricular nucleus (D). Scale bar represents 100 μm. (G) Mutants had fewer neuropeptide ISH-labeled SCN cells than controls (n = 3, paired two-tailed t tests, *p < 0.05, **p < 0.01; graph depicts mean ± SEM). (H) MOPAT bioinformatics analysis showed more putative Lhx1-binding sites in enhancers of an SCN-enriched gene pool compared to a randomly selected pool (pool shown in Table S2; method described in Hu et al., 2008; binomial distribution, *p < 0.05).

Figure 2. SCN Regional Identity Is Pre-served in Six3-Cre;Lhx1^lox/lox Mice, but Cell Death Is Elevated in Neonatal SCN

(A–D) ISH of SCN from Lhx1^lox/lox control (A–D) and Six3-Cre;Lhx1^lox/lox adult mutant (A⁰–D⁰) mice for Gad67 (A, A’), Calb1 (B, B’), Calb2 (C, C’), and Vipr2 (D, D’). (E) Mutants had fewer SCN neurons than controls at P4 and P7 (n = 3, 3, 3, 3, ANOVA and Tukey post hoc tests, *p < 0.05, ***p < 0.0005; graph depicts mean ± SEM). Mutant SCN neuron number in adults was significantly lower than controls when initially analyzed on its own by t test (p < 0.05) but fell just short of significance in the context of the time course (p = 0.07). (F) Mutants had increased SCN cell death in the aggregate period between P0 and P3 compared to controls, but the age(s) within this range with elevated cell death could not be pinned down (n = 3, 4, 4, 3, ANOVA with post hoc Tukey tests, **p < 0.005; graph depicts mean ± SEM). (G) Mutants had fewer Vip, Grp, and Avp, but not Prok2, expressing SCN cells than controls at P0 (n = 3, 3, 3, 3, paired two-tailed t tests, *p < 0.05; graph depicts mean ± SEM).

Figure 3. Lhx1 Effects on SCN Clock Gene Rhythms

(A) Adult Lhx1^lox/lox (left) and Six3-Cre;Lhx1^lox/lox (right) mouse SCN explants showed near-24 hr cycling of Per2::Luc bioluminescence over 10 days of recording, with moderately damped amplitude in the mutant explants. Traces show mean (black line) and SD (gray) of bioluminescence collected at 1 min intervals from 8 Lhx1^lox/lox (left) and 9 Six3-Cre; Lhx1^lox/lox (right) explants. (B) Raster plots demonstrate variations in synchrony of circadian rhythms across regions of interest (ROIs) from one representative control (left) and one Six3Cre;Lhx1^lox/lox SCN explant (right). White bars cover the region where data were lost while recording (40–46 hr). (C) Loss of Lhx1 caused greater variance of circadian period in Six3-Cre;Lhx1^lox/lox SCN explants (n = 5) compared to control littermates (n = 8). (D–F) Circadian variation in expression levels of SCN clock genes Per2 (D), Per2 (E), Bmal1 (C), and Avp (F). ISH labeling intensity was present in adult Lhx1^lox/lox control (D–F) and Six3-Cre;Lhx1^lox/lox mutant (D’–F’) mice, with genotype and interaction effects for some genes (n = 3, ANOVA).

Figure 4. Loss of Lhx1 in the SCN Influences Photoentrainment and Leads to Loss of Free-Running Circadian Rhythms

(A and A’) Representative actograms of wheel-running activity of Lhx1^lox/lox (A) and Six3-Cre; Lhx1^lox/lox (A’) housed in a 12 hr:12 hr light:dark cycle and subject to a 6 hr advance in the light/dark cycle. Gray shading represents lights off. (B) Six3-Cre; Lhx1^lox/lox mice show a decreased percentage of activity confined to the dark portion of the light/dark cycle (n = 10,13; p = 0.0002). (C) Six3-Cre; Lhx1^lox/lox mice show an advanced phase angle in 12:12 LD (n = 10, 11; p = 0.02). Data represent mean ± SEM. ***p < 0.001, *p < 0.05. (D and D’) Representative actograms of wheel-running activity of Lhx1^lox/lox (D) and Six3-Cre; Lhx1^lox/lox (D’) mice housed in constant darkness. Six3-Cre; Lhx1^lox/lox mice showed either arrhythmicitiy or split rhythms under DD. (E) Quantification of free-running circadian period lengths in DD. Period lengths for Six3-Cre; Lhx1^lox/lox are those found in mice with split rhythms. The multiple period lengths measured within a single animal are indicated by the same symbol in the mutant graph. (F) Distribution of circadian phenotypes observed in Six3-Cre;Lhx1^lox/lox mice. (G) Lhx1^lox/lox activity is decreased in constant light as compared to constant darkness. This difference was not observed in Six3-Cre; Lhx1^lox/lox mice (n = 9–11 per group; two-way ANOVA p_light = 0.02, p_genotype = 0.02, Bonferroni posttest Lhx1^lox/lox DD versus LL, p < 0.05). Data represent mean ± SEM. *p < 0.05.

Figure 5. The Lhx1-Deficient Circadian System Is Hypersensitive to Cannulation of the SCN Output Molecule Prok2

(A–C) Representative actograms of wheel-running activity of Lhx1^lox/lox (A–C) and Six3-Cre;Lhx1^lox/lox (A’–C’) mice before and after an i.c.v. injection (red asterisk) of saline (A), Grp (B), or Prok2 (C) in DD. (D and E) Mutants had higher-magnitude phase shifts (D) and period shifts (E) in response to Prok2 than controls (left) (n = 8, two-tailed heteroscedastic t tests, *p < 0.05; bar graphs depict mean ± SEM). Both phase and period shifts in mutants were gated by injection CT (right) (n = 5, linear regression, scatterplots depict CT versus phase or period shift of individual injections). (F) A model of SCN neuropeptide dynamics. Synchronizing factors are shown in green, desynchronizing factors are shown in red, and factors without clear implications for synchrony are shown in black. The following insights gained from our study are highlighted: (i) synchronizing and desynchronizing effects of SCN signals like Grp can be contingent on interpretation by the broader network, and (ii) core neuropeptides may buffer circadian period length, stabilizing it in the face of feedback signaling from SCN outputs like Prok2 that would otherwise alter it.