Pecorino L T, Brockes J P, Entwistle A
Ludwig Institute for Cancer Research, London, United Kingdom.
J Histochem Cytochem. 1996 Jun;44(6):559-69. doi: 10.1177/44.6.8666741.
Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic acid (RA) such that proximal structures are formed. To establish an assay for positional identity, cells of distal and RA-treated distal blastemas are labeled by transfection with an alkaline phosphatase marker gene using particle bombardment (biolistics). After grafting the distal blastema to a proximal stump, a context known as intercalary regeneration, the proximodistal distribution of labeled cells in the resulting regenerate is an index of positional identity. We use enzyme-labeled fluorescence (ELF) in conjunction with laser scanning microscopy to detect transfected cells within a section of the entire regenerate. A semi-automated analysis of the positional distribution of marked cells along the proximal-distal axis demonstrates that cells from both distal and RA-treated blastemas contribute to the regenerate. This procedure provides an efficient and accurate tool for positional analysis of transfected cells, and should be applicable for studying genes that play a role in specifying cell position during morphogenesis.
蝾螈等有尾两栖动物的肢体再生是研究细胞位置身份的关键系统。再生组织局部起源于芽基细胞，即间充质祖细胞，它们通常形成截肢平面远端的结构，但通过视黄酸（RA）处理可重新指定（近端化），从而形成近端结构。为了建立位置身份检测方法，使用粒子轰击（生物弹道学）通过转染碱性磷酸酶标记基因来标记远端和经RA处理的远端芽基细胞。将远端芽基移植到近端残端后，即所谓的间插再生环境，在所得再生组织中标记细胞的近端-远端分布是位置身份的指标。我们将酶标记荧光（ELF）与激光扫描显微镜结合使用，以检测整个再生组织切片内的转染细胞。对标记细胞沿近端-远端轴的位置分布进行半自动分析表明，来自远端和经RA处理的芽基的细胞都对再生组织有贡献。该方法为转染细胞的位置分析提供了一种高效且准确的工具，并且应该适用于研究在形态发生过程中对指定细胞位置起作用的基因。
