Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception - PubMed (original) (raw)

Abnormal development of the locus coeruleus in Ear2(Nr2f6)-deficient mice impairs the functionality of the forebrain clock and affects nociception

Marei Warnecke et al. Genes Dev. 2005.

Abstract

The orphan nuclear receptor Ear2 (Nr2f6) is transiently expressed in the rostral part of the rhombic lip in which the locus coeruleus (LC) arises. LC development, regulated by a signaling cascade (Mash1 --> Phox2b --> Phox2a), is disrupted in Ear2-/- embryos as revealed by an approximately threefold reduction in the number of Phox2a- and Phox2b-expressing LC progenitor cells. Mash1 expression in the rhombic lip, however, is unaffected, placing Ear2 in between Mash1 and Phox2a/b. Dopamine-beta-hydroxylase and tyrosine hydroxylase staining demonstrate that >70% of LC neurons are absent in the adult with agenesis affecting primarily the dorsal division of the LC. Normally, this division projects noradrenergic efferents to the cortex that appear to be diminished in Ear2-/- since the cortical concentration of noradrenaline is four times lower in these mice. The rostral region of the cortex is known to contain a circadian pacemaker regulating adaptability to light- and restricted food-driven entrainment. In situ hybridization establishes that the circadian expression pattern of the clock gene Period1 is abolished in the Ear2-/- forebrain. Behavioral experiments reveal that Ear2 mutants have a delayed entrainment to shifted light-dark cycles and adapt less efficiently to daytime feeding schedules. We propose that neurons in the dorsal division of LC contribute to the regulation of the forebrain clock, at least in part, through targeted release of noradrenaline into the cortical area.

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Figures

Figure 1.

Figure 1.

Targeted mutagenesis of Ear2 in mice. (A) Targeting strategy for the Ear2 gene. Exons 1 and 2 encode the DNA-binding domain, and exons 3 and 4 encode the putative ligand-binding domain. A part of exon 1 and all of exon2 are replaced by a LacZ/Neo cassette. (B) Southern blots: wild-type (16 kb) and mutant (6.8 kb) alleles are revealed by HindIII digestion and hybridization with a 595-bp 5′ external probe (see A). (C) PCR genotyping using primers producing a 532-bp (wild type) or a 478-bp (mutant) fragment. (D) Northern blot analysis of adult liver and kidney reveal a 2.5-kb mRNA in wild-type tissue. EF1α serves as a loading control.

Figure 2.

Figure 2.

Expression of Ear2 in the embryo and brain anatomy of the adult _Ear2_-/- mouse. (A) Ear2 expression revealed by LacZ staining at E8.5 in prerhombomers A and B and in mesencephalon and adjoining hindbrain tissue. (B) In a 9.25-d-old embryo LacZ staining is observed in rhombomeres 3 and 5, at the midbrain-hindbrain junction, and the rostral part of rhombomere 1. (C) A transverse section through rhombomere 1 of a 9-d-old _LacZ_-stained embryo shows strong LacZ staining in the rhombic lip. (_D_-_E_′) Nissl-stained coronal sections through an adult wild-type (D) and _Ear2_-/- (_D_′) brain. No morphological defects are seen in the mutant cerebellum, but partial agenesis of the LC (outlined with a black line in E and _E_′) is observed in _Ear2_-/- mice. The mesencephalic nucleus of the fifth cranial nerve is an anatomical landmark. (LC) Locus coeruleus; (mes) mesencephalon; (MH) midbrain-hindbrain boundary; (MeV) mesencephalic nucleus of the fifth cranial nerve; (prA, prB) prerhombomeres A and B; (r1, r3, r5) rhombomeres 1, 3, and 5; (rl) rhombic lip.

Figure 3.

Figure 3.

Anatomical changes of the LC in adult _Ear2_-/- mice documented by ISH (_A_-_H_′) and immunohistochemistry (_J_-_L_′). (A) Tyrosine hydroxylase (Th)-positive cell bodies were counted in serial coronal sections through a wild-type (open bars) and _Ear2_-/- (black bars) LC. All section levels show a significant reduction of the number of LC neurons in _Ear2_-/- mice. (B) Cell counts of _Th_-positive LC neurons from four wild-type and mutant brains, corrected by the Abercrombie method. (_C_-_H_′) Comparison of the neuro-anatomy of wild-type and _Ear2_-/- LC revealed by Th and Dbh staining. The mesencephalic nucleus and fourth ventricle serve as anatomical landmarks. Note the reduction in neuronal numbers in the dorsal division (black arrowheads), central core (arrows), and ventral division (open arrowheads) in Ear2 mutant brains. (I) Boundaries of the LC of wild-type and mutant mice were stacked to illustrate size and shape differences. (_J_-_L_′) Differences in cytoarchitecture between wild-type and mutant LC. The boxes in overview J and _J_′ lie within the regions characterized by fusiform (_K,K_′) and multipolar (_L,L_′) neurons and are enlarged in _K_-_L_′. Densely packed fusiform neurons characteristic for the dorsal division (K) are absent in the LC of _Ear2_-/- mice (_K_′), while large multipolar neurons found in the ventral division (L) are retained in the mutant (_L_′). (d) Dorsal; (m) medial; (MeV) mesencephalic nucleus; (p) posterior; (IV) fourth ventricle.

Figure 4.

Figure 4.

Expression of LC-determination and differentiation genes in the LC anlage at E9.5 and E14.5. (_A,A_′) Mash1 expression in rhombomere 1 is unchanged (arrowhead) in mutant embryos. (_B,B_′) Phox2a is expressed in cells of the LC anlage (enlarged in inset) in wild type, but in mutant embryos the number of _Phox2a_-expressing cells is greatly diminished. (_C,C_′) Phox2b is expressed in cells of the LC anlage of wild type (enlarged in inset), but _Phox2b_-positive cells are not detected in _Ear2_-/- embryos. Note that the intensity of expression of Phox2a/b in the third and fourth cranial ganglion is similar for both genotypes, indicating that whole mounts were stained identically. (_D,D_′) Sagittal sections of the LC at E14.5 hybridized with Phox2a reveal agenesis in dorsal division (arrow) and core (arrowhead). (_E_-_F_′) Identical results are observed with LC neuronal markers Dbh and Th. (r1) Rhombomere 1; (3rd and 4th) oculomotor and trigeminal ganglia; (IV) fourth ventricle.

Figure 5.

Figure 5.

Impairment of the circadian forebrain oscillator in _Ear2_-/- mice. (_A_-D) Circadian expression profiles of Per1 (A), Per2 (B), Bmal1 (C), and Npas2 (D) in the frontal cortex of wild-type and _Ear2_-/- mice in DD. Upper panels show representative peak and trough autoradiographs for each genotype. Lower panels depict densitometric quantification of ISH hybridization signals on X-ray film (n = 3). (E) Effect of an 8-h phase advance on activity onset in wild-type and _Ear2_-/- mice. Shown is the mean daily advance in activity onset over 7 d. Asterisks indicate significant differences in onset time for the genotypes on a particular day (p < 0.05, Student's _t_-test, n = 6). (F) NA levels in forebrain cortical slices of wild-type and _Ear2_-/- mice measured in the middle of the subjective night (CT18, DD; p = 0.0096, Student's _t_-test, n = 3). (_G_-I) Behavioral adaptation of wild-type and _Ear2_-/- mice to a 3-h restricted feeding schedule in LD. (G,H) Representative double-plotted activity profiles of a wild-type (G) and an _Ear2_-/- mouse (H). Black and white bars on top indicate light schedule; food access was restricted from 10 a.m. to 2 p.m. (I) Anticipatory activity (AA) normalized to total activity during 19 d of restricted feeding (asterisks indicate significant differences between wild-type and mutant animals; p < 0.05; Student's _t_-test, n = 6).

Figure 6.

Figure 6.

The accuracy and stability of circadian wheel-running behavior is affected in _Ear2_-/- mice. (A) Representative locomotor activity records of wild-type and _Ear2_-/- mice (right panel) in DD. Activity is represented by vertical black bars. Red lines represent least-squares-fit regressions through the onsets of activity as calculated by the ClockLab software. (B) Onset variability of wild-type and _Ear2_-/- mice calculated as mean deviation of real onset from a least-squares-fit regression line over a period of 10 consecutive days in the given lighting conditions ([★] p < 0.05; [★★] p < 0.01; [★★★] p < 0.001; Student's _t_-test, n = 12). (C) Representative locomotor activity records of wild-type and _Ear2_-/- mice in LD and LL of increasing intensity as indicated on the right side of the actograms. (D) Internal period lengths (τ) of wild-type and _Ear2_-/- mice in LL of different intensities. All τ values were significantly smaller in _Ear2_-/- animals for any tested LL condition (p < 0.05; Student's _t_-test, n = 6). (E) Percentage of rhythmic animals in LL for wild-type and _Ear2_-/- mice (n = 6). Animals were kept in each condition for at least 2 wk, and rhythmicity was determined by χ2 periodogram analysis.

Figure 7.

Figure 7.

Nociception is increased in Ear2 mutant mice. (A) Response latency of wild-type and mutant animals in a hot plate test using three different temperatures. Male littermates (n = 10) were used for both genotypes. (B) Response latency of wild-type and _Ear2_-/- mice assessed at 44°C with and without prior application of allylisothiocyanate to the paws (n = 10). (C) NA content in the spinal cord of wild-type and _Ear2_-/- males (n = 6). ([★] p < 0.05; [★★] p < 0.01, Student's _t_-test).

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