Wessel G M, Goldberg L, Lennarz W J, Klein W H
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Dev Biol. 1989 Dec;136(2):526-36. doi: 10.1016/0012-1606(89)90278-9.
Spatial diversification of the endoderm during gastrulation in the sea urchin Lytechinus variegatus was examined with an endoderm-specific cDNA clone. This cDNA clone, LvN1.2, was identified by a differential cDNA screen between the ectoderm and endoderm/mesoderm fractions from prism stage embryos. The LvN 1.2-kb mRNA was first detectable by Northern blots at the mesenchyme blastula stage just prior to gastrulation and then accumulated approximately 15-fold from gastrulation to the pluteus stage. In situ hybridization analysis demonstrated that the mRNA accumulated specifically in endoderm and was restricted to the hindgut-midgut regions. This restricted localization was apparent during gastrulation and predicted the morphological distinction between foregut and midgut eventually seen at prism and pluteus stages. Sequence analysis showed that the 189-amino acid open reading frame represented a novel protein. In vitro translation of synthetically produced LvN1.2 mRNA and Western blot analysis with antibodies to the protein sequence yielded the same 25-kDa polypeptide on SDS-PAGE. The LvN1.2 protein resided within discrete granules of the hindgut and midgut cells. These particles were concentrated to the luminal aspect of the cells, suggesting the LvN1.2 protein participates in the digestive function of this region of the gut.
利用一个内胚层特异性的cDNA克隆,研究了多色粗海胆原肠胚形成过程中内胚层的空间分化。这个cDNA克隆LvN1.2是通过对棱柱期胚胎的外胚层与内胚层/中胚层组分进行差异cDNA筛选而鉴定出来的。Northern印迹法显示,LvN 1.2-kb mRNA在原肠胚形成前的间充质囊胚期首次可检测到,然后从原肠胚形成期到长腕幼虫期积累了约15倍。原位杂交分析表明,该mRNA在内胚层中特异性积累,并局限于后肠-中肠区域。这种局限的定位在原肠胚形成过程中很明显,并预示了最终在棱柱期和长腕幼虫期出现的前肠和中肠之间的形态学差异。序列分析表明,189个氨基酸的开放阅读框代表一种新蛋白。对人工合成的LvN1.2 mRNA进行体外翻译,并使用针对该蛋白序列的抗体进行Western印迹分析,在SDS-PAGE上得到了相同的25-kDa多肽。LvN1.2蛋白存在于后肠和中肠细胞的离散颗粒中。这些颗粒集中在细胞的腔面,表明LvN1.2蛋白参与了肠道该区域的消化功能。