The use of in situ transcription and anti-sense RNA amplification to investigate gene expression in the developing murine embryo

Abstract

In situ transcription and anti-sense RNA amplification (RT/aRNA), new techniques in molecular biology, were used to examine developmental expression of a panel of candidate genes in two separate studies. (1) Cranial and caudal neural tube tissue was isolated from LM/Bc embryos, which were collected from dams sacrificed on gestational day (GD) 9:12 or 1 0:0. Retinoic acid receptors (RAR-a), the potassium channel gene (Kvl), and the nicotinic receptor subunit al (NACHR) were developmentally regulated The level of expression for these genes changed significantly between GD 9:12 and 1 0:0. The expression of RAR-A increased in the neural tube with advancing gestational age while Kvl and the nicotinic receptor expression were significantly decreased over the same time period. Kvl and the sodium channel gene were expressed at higher levels in the cranial region of the neural tube on GD 9:12, while expression of NACHR was higher cranially at GD 1 0:0. Although relatively few statistically significant changes in the expression patterns between cranial and caudal neural tube regions were observed, these changes may represent fundamental differences in molecular processes which are responsible for neural tube development among different regions of the neural tube. (2) LM/Bc females were treated with 5 mg/kg all-trans retinoic acid (RA) on GD 8:12. On GD 10:0, 10:1 2, or 12:0, untreated and RA-treated dams were sacrificed, and the embryos were recovered. First and second bronchial arches from each embryo were utilized for RT/aRNA studies to explore differences in gene expression which may lead to congenital malformations in second branchial arch derivatives of RA exposed fetuses. Significant differences in the level of expression between the first and second bronchial arches were observed. The expression of the type-1 cellular retinoid binding protein, the CAMP response element-binding protein, NACHR, RAR-Y, and transforming growth factor P2 were increased following RA exposure. The application of RT/aRNA techniques have allowed for the investigation of coordinate changes in gene expression which occurred within specific embryonic structures during development.