Machingo Quentin J, Fritz Andreas, Shur Barry D
Department of Cell Biology, Emory University, Atlanta, GA 30329, USA.
Dev Biol. 2006 Sep 15;297(2):471-82. doi: 10.1016/j.ydbio.2006.05.024. Epub 2006 May 27.
Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.
我们对复杂碳水化合物在胚胎发育过程中如何发挥作用的理解仍然非常有限，这主要是由于目前已知大量糖基转移酶参与其合成。为了克服这些限制，我们利用斑马鱼系统，通过下调特定糖基转移酶的表达来分析复杂碳水化合物在发育过程中的功能。在此，我们报告了哺乳动物β1,4-半乳糖基转移酶I（β4GalT1）的斑马鱼直系同源物的鉴定，以及其在原肠胚形成过程中对正常会聚延伸运动的需求。β4GalT1在卵母细胞中表达，并在发育的最初24小时内贯穿整个胚胎。通过两种独立的吗啉代寡核苷酸敲低斑马鱼β4GalT1会导致胚胎出现前后轴截断，以及体节拉长和头部间充质模式的中度缺陷。共注射斑马鱼β4GalT1 mRNA可使半乳糖基转移酶活性恢复到对照水平，并挽救吗啉代寡核苷酸产生的缺陷。各种分子标记的原位杂交显示，在β4GalT1 morphant胚胎中，外包期胚胎的轴向中胚层异常变宽，这表明会聚延伸异常。与此一致，相对于注射对照的胚胎，前后轴伸长率降低，这与已知的会聚延伸突变体中观察到的情况相似。β4GalT1的许多潜在底物之一是层粘连蛋白，它是细胞外基质的主要成分，支持细胞运动，如在会聚延伸过程中发生的运动。先前的体外研究表明，层粘连蛋白的半乳糖基化状态直接影响其支持细胞铺展和迁移的能力。在这方面，从β4GalT1 morphant胚胎中分离的层粘连蛋白半乳糖基化程度很低，这可能导致会聚延伸运动期间细胞迁移缺陷。这项工作表明，斑马鱼可用于鉴定特定糖基转移酶的关键发育作用，否则这些作用不会很明显，例如在会聚延伸运动期间对β4GalT1的绝对需求。
