Research reportImmunohistochemical analyses of thyroid-specific enhancer-binding protein in the fetal and adult rat hypothalami and pituitary glands
Introduction
Thyroid-specific enhancer-binding protein (T/EBP) is a transcription factor containing a homeodomain that has 82% identity to the Drosophila NK-2 homeodomain [16], [10], and is also called NKX2.1. T/EBP/NKX2.1 was identified in functional studies on the upstream sequence of the human thyroid peroxidase (TPO) gene in which was found to mediate transcription of the gene by interacting with a thyroid-specific enhancer element located approximately 5.5 kb upstream of transcription start site [16], [13]. The same DNA-binding factor was independently identified as thyroid-specific transcription factor-1 (TTF-1), which interacts with the rat thyroglobulin (TG) gene promoter sequence between −120 and +1 relative to the transcription start site and confers thyroid-specificity to TG gene expression [10], [5], [22]. T/ebp messenger RNA was found to be expressed in the thyroid gland, lungs and a restricted area of the brain [25].
A T/ebp/Nkx2.1-null mutant mouse was stillborn but lacked the thyroid and pituitary glands, and abnormal formations were found in the lungs and hypothalamus [14]. In the brain of the null mutant mouse, extensive abnormalities were found in the ventral region of the forebrain from the septal area to the mammillary body of the hypothalamus. In the preoptic and hypothalamic area, abnormalities were extensively demonstrated in the ventral and medial regions and, to a lesser extent, in the dorsal and lateral regions. At the level of the tuberal hypothalamus, the ventromedial and dorsomedial hypothalamic nuclei were reduced in size and appeared to be fused in the midline. The premammillary nucleus and the arcuate nucleus were not developed. As described above, in the T/ebp/Nkx2.1-null mutant mouse, the ventral hypothalamic area was particularly abnormal, suggesting that T/EBP/NKX2.1 plays an important role in the formation of this area. Although it is well known that T/EBP/NKX2.1 stimulates TPO [13], [1], [9], TG [6], [22], [5], [12] and thyrotropin receptor [7], [21], [18] gene expression in the thyroid gland, and also regulates, in the lungs, the surfactant proteins and the Clara cell secretory protein gene [3], [32], [4] in a tissue-specific manner, its function in the brain remains unknown. However considering its tissue-specific function in these organs, it is possible that T/EBP/NKX2.1 regulates a brain-specific gene. On the other hand, the anterior, intermediate and posterior lobes of the pituitary gland, known to be derived from the oral epithelium and infundibulum of the diencephalon, respectively, had disappeared entirely in the T/ebp/Nkx2.1-null mutant newborn mouse [14].
Localization of T/EBP/NKX2.1 has been partially studied using in situ hybridization during development [26], [15], but detailed localization of the T/EBP/NKX2.1 protein has not been examined during development of the hypothalamus and pituitary gland. The present study was designed to determine the localization of T/EBP/NKX2.1 in the hypothalamus and pituitary gland of fetal and adult rats by immunohistochemistry as the first step toward understanding the function of T/EBP/NKX2.1 in the rat brain, particularly its role in the formation of the hypothalamus and pituitary gland.
Section snippets
Animals
Intact adult male Wistar rats (8 weeks of age) and fetuses were used in this study. The day on which spermatozoa were found in a vaginal smear was designated as embryonic day 0.5 (E0.5). The animals were maintained under a 12-h light–dark cycle (lights on between 8:00 and 20:00 h) with laboratory chow and water provided ad libitum.
Tissue preparation
At various gestational days from E11.5 to E19.5, pregnant rats were deeply anesthetized with sodium pentobarbital (15 mg/rat, i.p., Nembutal, Dainippon
Localization of T/EBP/NKX2.1 in the fetal rat hypothalamus during development
T/EBP/NKX2.1 immunoreactivities were found not only in the hypothalamic region but also in the presumptive thyroid gland and lungs in the E13.5 embryo (Fig. 1B) as was previously observed [15], suggesting that the antibody used in this study specifically recognized T/EBP/NKX2.1 protein. Intense T/EBP/NKX2.1 immunoreactivities were found in the cell nucleus (Fig. 1C), and we regarded this restricted staining in the cell nucleus as a specific reaction.
In the embryo at E11.5,
Discussion
There has been no detailed studies on localization of the T/EBP/NKX2.1 protein in the hypothalamus and pituitary gland, and this is the first report on detailed localization of this protein in the hypothalamus and pituitary gland in fetal and adult rats. The findings of the present study are therefore considered to be a significant contribution to an understanding of the functions of T/EBP/NKX2.1 in the hypothalamus and pituitary gland.
Acknowledgements
We are grateful to Dr. K. Suzuki (National Institutes of Health, USA), Dr. S. Hayashi (Tokyo Metropolitan Institute for Neuroscience, Japan) and Dr. K. Ohyama (Keio University, Japan) for their helpful discussion. We also thank Dr. Y. Nakazato (Gunma University, Japan) for the rabbit antisera against glial fibrillary acidic protein and Dr. K. Wakabayashi (Gunma University, Japan) for the goat anti-rabbit IgG serum. A part of this work was supported by the Sasakawa Scientific Research Grant from
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