Banker D E, Bigler J, Eisenman R N
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Mol Cell Biol. 1991 Oct;11(10):5079-89. doi: 10.1128/mcb.11.10.5079-5089.1991.
The c-erbA proto-oncogene encodes the thyroid hormone receptor, a ligand-dependent transcription factor which plays an important role in vertebrate growth and development. To define the role of the thyroid hormone receptor in developmental processes, we have begun studying c-erbA gene expression during the ontogeny of Xenopus laevis, an organism in which thyroid hormone has well-documented effects on morphogenesis. Using polymerase chain reactions (PCR) as a sensitive assay of specific gene expression, we found that polyadenylated erbA alpha RNA is present in Xenopus cells at early developmental stages, including the fertilized egg, blastula, gastrula, and neurula. By performing erbA alpha-specific PCR on reverse-transcribed RNAs from high-density sucrose gradient fractions prepared from early-stage embryos, we have demonstrated that these erbA transcripts are recruited to polysomes. Therefore, erbA is expressed in Xenopus development prior to the appearance of the thyroid gland anlage in tailbud-stage embryos. This implies that erbA alpha/thyroid hormone receptors may play ligand-independent roles during the early development of X. laevis. Quantitative PCR revealed a greater than 25-fold range in the steady-state levels of polyadenylated erbA alpha RNA across early stages of development, as expressed relative to equimolar amounts of total embryonic RNA. Substantial increases in the levels of erbA alpha RNA were noted at stages well after the onset of zygotic transcription at the mid-blastula transition, with accumulation of erbA alpha transcripts reaching a relative maximum in advance of metamorphosis. We also show that erbA alpha RNAs are expressed unequally across Xenopus neural tube embryos. This differential expression continues through later stages of development, including metamorphosis. This finding suggests that erbA alpha/thyroid hormone receptors may play roles in tissue-specific processes across all of Xenopus development.
原癌基因c-erbA编码甲状腺激素受体,它是一种依赖配体的转录因子,在脊椎动物的生长和发育中起重要作用。为了确定甲状腺激素受体在发育过程中的作用,我们开始研究非洲爪蟾个体发育过程中c-erbA基因的表达情况,在这种生物中,甲状腺激素对形态发生有充分记录的影响。利用聚合酶链反应(PCR)作为特定基因表达的灵敏检测方法,我们发现多聚腺苷酸化的erbAαRNA在非洲爪蟾细胞的早期发育阶段就已存在,包括受精卵、囊胚、原肠胚和神经胚。通过对从早期胚胎制备的高密度蔗糖梯度级分的逆转录RNA进行erbAα特异性PCR,我们证明这些erbA转录本被募集到多核糖体上。因此,在尾芽期胚胎的甲状腺原基出现之前,erbA就在非洲爪蟾的发育过程中表达。这意味着erbAα/甲状腺激素受体可能在非洲爪蟾的早期发育过程中发挥不依赖配体的作用。定量PCR显示,相对于等摩尔量的总胚胎RNA,在发育早期多聚腺苷酸化的erbAαRNA的稳态水平有超过25倍的变化范围。在中囊胚转换期合子转录开始后的阶段,erbAαRNA水平显著增加,erbAα转录本的积累在变态前达到相对最大值。我们还表明,erbAαRNA在非洲爪蟾神经管胚胎中的表达是不均一的。这种差异表达在发育后期包括变态过程中持续存在。这一发现表明,erbAα/甲状腺激素受体可能在非洲爪蟾整个发育过程中的组织特异性过程中发挥作用。