Dreyfus C F, Markey K A, Goldstein M, Black I B
Dev Biol. 1983 May;97(1):48-58. doi: 10.1016/0012-1606(83)90062-3.
While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. To approach this problem, we initiated parallel in vivo and in vitro studies of the mouse locus coeruleus (l.c.), a brainstem noradrenergic nucleus. The catecholaminergic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were used to monitor phenotype expression and development. TH catalytic activity and immunocytochemical reactivity were initially detectable on gestational Day 13 (E-13) in vivo, and adult levels of activity were approximately by the third postnatal week. Immunotitration studies indicated that the developmental increase was due to accumulation of enzyme molecules and not enzyme activation. The in vivo developmental profile of DBH approximated that of TH. To begin defining regulatory mechanisms, explants of embryonic brainstem were placed in culture. Explantation on E-12, prior to expression of TH or DBH, resulted in the de novo appearance of these phenotypic characters after 4 days. Explantation on E-18, after the enzymes are already expressed, was followed by a striking sixfold rise in TH activity. Immunotitration studies revealed that the increase in TH activity in E-18 cultures was attributable to increased molecule number, reproducing the in vivo results. Moreover, the E-18 explants, cultured for 3 weeks, attained higher plateau levels of TH activity than E-12 cultures, and this differences was due to increased molecule number. Morphometric analysis indicated that 3-week E-12 cultures actually had more l.c. cells than E-18 cultures, indicating that differences in TH were not due to increased cells in the E-18 l.c. Finally, systemic study revealed that the development of TH activity in culture increased progressively from E-11 to E-12 to E-13, suggesting that critical regulatory events occur at this time. Our studies suggest that the l.c. is an excellent model for the study of brain development in vivo and in vitro. Initial phenotypic expression and dramatic development occur in culture in the absence of normal targets, normal afferent innervation and, presumably, normal humoural milieu.
虽然大量研究已开始阐明外周神经系统的个体发生，但大脑发育的分子机制仍不清楚。为了解决这个问题，我们对小鼠蓝斑核（l.c.），即脑干去甲肾上腺素能核，开展了体内和体外平行研究。儿茶酚胺能酶酪氨酸羟化酶（TH）和多巴胺-β-羟化酶（DBH）被用于监测表型表达和发育情况。TH催化活性和免疫细胞化学反应最初在体内妊娠第13天（E-13）可检测到，成年水平的活性大约在出生后第三周达到。免疫滴定研究表明，发育过程中的增加是由于酶分子的积累而非酶的激活。DBH在体内的发育情况与TH相似。为了开始确定调控机制，将胚胎脑干外植体进行培养。在TH或DBH表达之前的E-12进行外植，4天后这些表型特征会重新出现。在酶已经表达后的E-18进行外植，随后TH活性显著升高了六倍。免疫滴定研究表明，E-18培养物中TH活性的增加归因于分子数量的增加，这与体内结果一致。此外，培养3周的E-18外植体比E-12培养物达到更高的TH活性平台水平，这种差异是由于分子数量增加。形态计量分析表明，培养3周的E-12培养物中的l.c.细胞实际上比E-18培养物中的更多，这表明TH的差异不是由于E-18的l.c.细胞增加所致。最后，系统研究表明，培养物中TH活性的发育从E-11到E-12再到E-13逐渐增加，表明关键调控事件在这个时期发生。我们的研究表明，l.c.是体内和体外研究大脑发育的优秀模型。在没有正常靶标、正常传入神经支配以及大概正常体液环境的情况下，培养物中会出现初始表型表达和显著发育。
