Wagner M, Han B, Jessell T M
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Development. 1992 Sep;116(1):55-66. doi: 10.1242/dev.116.1.55.
Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.
维甲酸及相关类视黄醇被认为在脊椎动物胚胎发育过程中对细胞分化模式有影响。为了确定释放具有形态发生活性类视黄醇的细胞群，我们开发了一种报告基因检测方法，该方法利用维甲酸诱导反应元件（RARE）在稳定转染的细胞系中驱动lacZ或荧光素酶报告基因。这种报告基因检测方法能够检测胚胎组织释放的类视黄醇，其范围相当于飞摩尔量维甲酸所诱导的范围。我们首先使用该检测方法来确定底板（在神经管和肢芽分化中具有极化特性的细胞群）是否是脊髓内类视黄醇的局部来源。我们还研究了外源性给予维甲酸对发育中的中枢神经系统细胞前后模式形成的影响是否与前后神经组织类视黄醇释放的差异相关。我们发现，具有神经管或肢极化活性的背侧脊髓释放的形态发生活性类视黄醇仅约为底板的1.5倍。这些结果表明，脊髓组织类视黄醇释放的空间分布与神经和肢极化活性的分布不同。该检测方法还表明，胚胎脊髓释放的类视黄醇水平比前脑高得多。这一结果与先前观察到的低浓度维甲酸会抑制前脑结构发育的现象一起表明，前脑结构的正常发育依赖于胚胎轴前部区域低浓度类视黄醇的维持。该检测方法还提供了初步证据，表明体内具有极化特性的其他胚胎组织在体外也会释放类视黄醇。
