Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis

Abstract

Although the study of rostral-caudal segmentation of the insect body has been a rich source of information about embryonic pattern formation, relatively little is known of the process of proximal-distal segmentation of insect appendages. Here we demonstrate that during the period of limb segmentation, five segmentally iterated, sharply demarcated bands of cell surface alkaline phosphatase activity are expressed in embryonic grasshopper limbs. These bands span each intersegmental boundary in the limb as well as one boundary within the tarsus. Within appendages, expression is restricted to epithelial cells, where activity is present on both apical and basolateral surfaces. This epithelial alkaline phosphatase remains active at neutral pH, is insensitive to levamisole inhibition, and is strongly inhibited by nucleoside monophosphates. Treatment of embryos with phosphatidylinositol-specific phospholipase C releases almost all visible chromogenic activity, indicating that the epithelial alkaline phosphatase is anchored to the plasma membrane by glycosyl-phosphatidylinositol. When material released by phosphatidylinositol-specific phospholipase C is separated on native polyacrylamide gels, a single broad band of enzymatic activity is detected following incubation with substrate. A polyclonal antiserum raised against a 55 × 10(3) M(r) alkaline phosphatase from shrimp recognizes a single band of 56 × 10(3) M(r) on immunoblots of grasshopper membrane proteins. The spatially restricted expression of epithelial alkaline phosphatase suggests that it may be involved in epithelial cell rearrangements or shape changes associated with limb segmentation and morphogenesis. It also may contribute to definition of axon routes in the limb, since pioneer afferent growth cones turn at, and migrate along, the edge of one alkaline phosphatase-expressing epithelial domain.