Developmental and diurnal dynamics of Pax4 expression in the mammalian pineal gland: nocturnal down-regulation is mediated by adrenergic-cyclic adenosine 3',5'-monophosphate signaling

Abstract

Pax4 is a homeobox gene that is known to be involved in embryonic development of the endocrine pancreas. In this tissue, Pax4 counters the effects of the related protein, Pax6. Pax6 is essential for development of the pineal gland. In this study we report that Pax4 is strongly expressed in the pineal gland and retina of the rat. Pineal Pax4 transcripts are low in the fetus and increase postnatally; Pax6 exhibits an inverse pattern of expression, being more strongly expressed in the fetus. In the adult the abundance of Pax4 mRNA exhibits a diurnal rhythm in the pineal gland with maximal levels occurring late during the light period. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA. At night the pineal gland is adrenergically stimulated by release of norepinephrine from the sympathetic innervation; here, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. This suppression appears to be mediated by cAMP, a second messenger of norepinephrine in the pineal gland, based on the observation that treatment with a cAMP mimic reduces pineal Pax4 mRNA levels. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by the sympathetic neural pathway that controls pineal function acting via an adrenergic-cAMP mechanism. The daily changes in Pax4 expression may influence gene expression in the pineal gland.

Figure 1

Expression of Pax4 in the pineal gland and retina of the adult rat. A, Northern blot analysis of Pax4 expression in tissues removed from adult animals killed at ZT7. Arrows on the upper image indicate molecular weight markers. The lower image displays the same blot hybridized for detection of 18S rRNA. B, qRT-PCR analysis of Pax4 expression in the pineal gland, retina, and pancreatic islet of adult animals killed at ZT7. PCR products were subcloned and sequenced confirming the expression of Pax4 in all three tissues. Values on the bar graph represent the mean ± sem of four to six independent RNA preparations. Statistical comparison of Pax4 expression in the three tissues revealed a significant difference (one-way ANOVA, F_2,13 = 11.8; P = 0.0012); pair-wise comparison revealed significantly lower Pax4 mRNA levels in pancreatic islets compared with the pineal gland (two-tailed Student’s t test, t₅ = 4.4; P = 0.0071) and retina (two-tailed Student’s t test, t₅ = 6.6; P = 0.0012). **, P < 0.01.

Figure 2

Ontogenetic expression of Pax4 and Pax6 in the rat pineal gland. Autoradiographs of radiochemical in situ hybridization for detection of Pax4 and Pax6 mRNA in median sections of the brain in a developmental series ranging from E16 to P30. The pineal gland is marked by an arrow. Autoradiographs of sections hybridized with sense control probes are provided in supplemental Fig. S2. Scale bar, 1 mm.

Figure 3

Densitometric quantification of in situ hybridization autoradiographs of ontogenetic Pax4 and Pax6 gene expression in the pineal gland. In the earliest stages, a Pax4 signal above background was not observed (dotted line). Values on graphs represent the mean ± sem of three animals at each developmental stage examined. Differential expression levels during development were detected for Pax4 (one-way ANOVA, F_5,12 = 11.4; P = 0.0003); Pax4 mRNA levels at P12 and P18 were significantly higher than that of earlier stages (Tukey’s multiple comparison test, P values <0.05). Differential expression levels were also detected for Pax6 (one-way ANOVA, F_10,22 = 59.2; P < 0.0001); Pax6 mRNA levels at E17 and E18 were significantly higher than those of other stages (Tukey’s multiple comparison test, P values <0.05).

Figure 4

Quantitative radiochemical in situ hybridization analysis of a diurnal variation in expression of Pax4 in the pineal gland of the adult rat housed under a 12-h light, 12-h dark schedule. A, Autoradiograph of a median brain section from an animal killed at midday (ZT6). B, Autoradiograph of a median brain section from an animal killed at midnight (ZT18). C, Autoradiograph of a median brain section from a superior cervical ganglionectomized animal killed at midday (ZT6). D, Autoradiograph of a median brain section from a superior cervical ganglionectomized animal killed at midnight (ZT18). E, Densitometric quantification of Pax4 mRNA in the rat pineal gland. Values on the bar graph represent the mean ± sem of four to five animals in each experimental group. Two-way ANOVA analysis identified a significant effect of time of sampling (F_1,14 = 23.0; P = 0.0003) as well as SCGx (F_1,14 = 25.3; P = 0.0002). Pair-wise statistical comparison identified a significant day-night difference in pineal Pax4 transcript levels in intact rats (two-tailed Student’s t test, t₅ = 16.4; P < 0.0001); differences in pineal Pax4 transcript levels among intact daytime rats and SCGx rats were not detected (one-way ANOVA, F_2,10 = 1.2; P = 0.33). ***, P < 0.001. Scale bar, 1 mm.

Figure 5

Circadian expression of Pax4 in the pineal gland of the adult rat. A, qRT-PCR analysis of diurnal expression of Pax4, Aanat, and Pax6 in the pineal gland of the adult rat housed under a 14-h light, 10-h dark schedule. Nine animals were killed at each of six time points throughout the 24-h period. Values on graphs represent the mean ± sem of three different pools of three glands at each time point. Significant changes in mRNA levels during the 24-h period were detected for both Pax4 (one-way ANOVA, F_5,12 = 18.5; P < 0.0001) and Aanat (one-way ANOVA, F_5,12 = 17.8; P < 0.0001), but not in the case of Pax6 (one-way ANOVA, F_5,12 = 2.4; P = 0.097). B, Northern blot analysis of Pax4 expression in the pineal gland of adult rats housed under a 14-h light, 10-h dark schedule. Four to five animals were killed at each time point (ZT7, ZT13, ZT19, and ZT23). Arrows on the upper image indicate molecular weight markers. The lower image displays the same blot hybridized for detection of Gapdh mRNA.

Figure 6

Quantitative radiochemical in situ hybridization analysis of the effect of ip isoproterenol injection on the expression of Pax4 in the pineal gland of the adult rat. A, Autoradiograph of a median brain section from a rat injected with PBS at ZT5 and killed at ZT8. B, Autoradiograph of a median brain section from a rat injected with isoproterenol (10 mg/kg) at ZT5 and killed at ZT8. C, Densitometric quantification of Pax4 mRNA in the rat pineal gland. Values on the bar graph represent the mean ± sem of three to five animals in each experimental group. A significant difference between animals injected with isoproterenol and control animals injected with PBS was identified (two-tailed Student’s t test, t₄ = 5.7; P = 0.0047). **, P < 0.01. Scale bar, 1 mm.

Figure 7

qRT-PCR analysis of the effect of NE (1 μm) and DBcAMP (500 μm) in combination with actinomycin D (ActD) (30 μg/ml) and puromycin (Pur) (50 μg/ml) on the expression of Pax4 in cultured pineal glands. Values on the bar graph represent the mean ± sem of three different pools of glands in each experimental group. Two-way ANOVA analysis identified a significant effect of activation of the adrenergic system, e.g. NE and DBcAMP (F_2,18 = 15.8; P = 0.0001), as well as blocking of gene product synthesis, e.g. actinomycin D and puromycin (F_2,18 = 47.8; P < 0.0001); pair-wise comparison identified significant differences between the untreated control and the NE-treated glands (two-tailed Student’s t test, t₄ = 6.1; P = 0.0036), and between the untreated control and the DBcAMP treated glands (two-tailed Student’s t test t₄ = 6.7; P = 0.0026). Differences in levels of Pax4 mRNA were not detected among control glands and glands treated with actinomycin D or puromycin (one-way ANOVA, F_6,14 = 1.0; P = 0.46). **, P < 0.01.