Expression of the Otx2 homeobox gene in the developing mammalian brain: embryonic and adult expression in the pineal gland

Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day-night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis.     

Lcg is a light-inducible and clock-controlled gene expressed in the chicken pineal gland

The circadian clock is an autonomous biological clock that is entrainable to environmental 24-h cycles by receiving time cues such as light. Generally, light given at early and late subjective night, respectively, delays and advances the phase of the circadian oscillator. We previously searched for the chicken pineal genes that are induced by light in a phase-dependent manner. The present study undertook cDNA cloning and characterization of a gene whose expression was remarkably up-regulated by light at late subjective night. The mRNA level of this gene exhibited robust diurnal change in the pineal gland, with a peak in the early (subjective) day under light-dark cycles and constant dark condition, and hence it was designated Lcg (Light-inducible and Clock-controlled Gene). Chicken Lcg encodes a coiled-coil protein composed of 560 amino acid residues. Among chicken tissues, the pineal gland and the retina exhibited relatively high expression levels of LCG. LCG was colocalized with gamma-tubulin, a centrosomal protein, when expressed in COS7 cells, and LCG is the first example of a clock-related molecule being accumulated at the centrosome. Coimmunoprecipitation of LCG with gamma-tubulin in the chicken pineal lysate suggests a link between the circadian oscillator and the centrosomal function.     

Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR

A second gene encoding a functional tryptophan hydroxylase activity has recently been described (TPH2), which is expressed abundantly in brainstem, the primary site of serotonergic neurons in the CNS. As serotonin (5-HT) has an important role as a precursor of the nocturnal synthesis of the pineal gland hormone, melatonin, it was of interest to determine the relative expression of TPH1 and 2 mRNA in the rat pineal during the light:dark (L:D) cycle using sensitive real-time RT-PCR assays which were developed for each TPH isoform. TPH1 mRNA expression was 105-fold more abundant in rat pineal than TPH2, and showed a significant approximately 4-fold nocturnal increase in expression which may contribute to the previously described nocturnal increase in pineal tryptophan hydroxylase activity. TPH2 expression within the gland showed no significant variation with time of day and was very low (approximately 300 copies/gland) indicating expression in the small proportion of "non-pinealocyte" cells in the gland.     

Proteomic analysis of day-night variations in protein levels in the rat pineal gland

The pineal gland secretes the hormone melatonin. This secretion exhibits a circadian rhythm with a zenith during night and a nadir during day. We have performed proteome analysis of the superficial pineal gland in rats during daytime and nighttime. The proteins were extracted and subjected to 2-DE. Of 1747 protein spots revealed by electrophoresis, densitometric analysis showed the up-regulation of 25 proteins during nighttime and of 35 proteins during daytime. Thirty-seven of the proteins were identified by MALDI-TOF MS. The proteins up-regulated during the night are involved in the Krebs cycle, energy transduction, calcium binding, and intracellular transport. During the daytime, enzymes involved in glycolysis, electron transport, and also the Krebs cycle were up-regulated as well as proteins taking part in RNA binding and RNA processing. Our data show a prominent day-night variation of the protein levels in the rat pineal gland. Some proteins are up-regulated during the night concomitant with the melatonin secretion of the gland. Other proteins are up-regulated during the day indicating a pineal metabolism not related to the melatonin synthesis.     

Zebrafish cone-rod (crx) homeobox gene promotes retinogenesis

The mammalian Cone-rod homeobox (Crx) gene is a divergent member of the Otx gene family known to be involved in differentiation and survival of retinal photoreceptors and photoentrainment of circadian rhythms. Zebrafish have two genes in the Otx5/crx orthology class, and we previously showed that crx can transactivate rhodopsin expression in vitro, and that otx5 (orthodenticle-related gene), but not crx, regulates expression of circadian genes in the pineal. Here, we show that zebrafish crx does not regulate expression of opsins and other photoreceptor-specific genes in the pineal. We further show that crx is expressed in proliferating retinal progenitors and may be involved in patterning the early optic primordium and in promoting the differentiation of retinal progenitors, including photoreceptors. These results suggest novel functions for zebrafish crx during retinal specification and differentiation.     

Overexpression of Hoxc13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia

Studying the roles of Hox genes in normal and pathological development of skin and hair requires identification of downstream target genes in genetically defined animal models. We show that transgenic mice overexpressing Hoxc13 in differentiating keratinocytes of hair follicles develop alopecia, accompanied by a progressive pathological skin condition that resembles ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified 16 previously unknown and 13 known genes as presumptive Hoxc13 targets. The majority of these targets are downregulated and belong to a subgroup of genes that encode hair-specific keratin-associated proteins (KAPs). Genomic mapping using a mouse hamster radiation hybrid panel showed these genes to reside in a novel KAP gene cluster on mouse chromosome 16 in a region of conserved linkage with human chromosome 21q22.11. Furthermore, data obtained by Hoxc13/lacZ reporter gene analysis in mice that overexpress Hoxc13 suggest negative autoregulatory feedback control of Hoxc13 expression levels, thus providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hox gene expression. Combined, these results provide a framework for understanding molecular mechanisms of Hoxc13 function in hair growth and development.     

大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响

探讨12h光照、12h黑暗交替(12h-light：12h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化。Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律。结果如下：(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P＜0．05)。(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P＜0．05)。(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P＞0．05)。结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调。