DEVELOPMENTAL BIOLOGY 67, 137-151 (1978) Differential Distribution of Poly(A)-Containing RNA in the Embryonic Cells of Oncopeltus fasciatus Analysis by in Situ Hybridization with a [3H]poly(U) Probe DAVID G. CAPCO AND WILLIAM R. JEFFERY Department of Zoology, University of Texas at Austin, Austin, Texas 78712 Received March 16, 1978; accepted in revised form June 23, 1978 The regional distribution of poly(A)+ RNA was examined in the embryonic cells of the milkweed bug, Oncopeltus fasciatus, by in situ hybridization of histological sections with a [:‘H]poly(U) probe. As shown by a number of control experiments, this probe interacts specifically with poly(A) sequences preserved in the sections. Using this method, it was shown that labeling of periplasmic and vitellophage nuclei increases markedly early during syncytial blastoderm formation. At this time, label also increases in the vitellophage cytoplasm but not in the cytoplasm surrounding the blastodermal nuclei. Labeling continues to increase in the blastodermal nuclei during cellularization and germ band differentiation without a concomitant accumulation in the blastodermal cell cytoplasm. At the time of germ band invagination, the region of the most intense subcellular labeling shifts from the nucleus to the cytoplasm of the invaginated cells. This shift is not evident in the blastodermal cells which remain at the surface of the egg to become the serosa. In the serosa and the vitellophage energids, labeling then decreases as histogenesis proceeds. Significant labeling of the nuclei and cytoplasm of the invaginated germ band cells continues through germ layer formation. It is concluded that poly(A)’ RNA, probably synthesized de novo following oviposition, is subject to differential intracellular distribution in three types of Oncopeltus embryonic cells which may reflect cell-specific patterns of mRNA or poly(A) metabolism. INTRODUCTION Although considerable information is available concerning the temporal aspects of RNA metabolism in whole embryos (Davidson, 1976), little is currently known about the activities of these macromole- cules within individual embryonic cells. Moreover, total embryonic RNA, rather than RNA of a particular class, has been examined in the few previous studies that have addressed this question (Czihak et al., 1967; Karp, 1973; Zalokar, 1976). The anal- ysis of particular RNA classes, such as mes- senger RNA (mRNA), is often limited by the lack of methodology for distinguishing these molecules from the usually more abundant nontemplate RNA species. The present investigation was undertaken with the aim of developing a method for the analysis of mRNA distribution in sections of fixed embryonic material and utilizing this technique to examine the regional lo- calization of mRNA during embryogenesis of the milkweek bug, Oncopeltus fasciatus. The presence of a polyadenylic acid [poly (A)] sequence in many eukaryotic mRNA molecules (Brawerman, 1974) provides a potentially valuable means for the detec- tion of poly(A)-containing RNA [poly(A)+ RNA], which we assume is mRNA, in cy- tological preparations by in situ hybridiza- tion (Hennig, 1973; Pardue and Gall, 1975) with a radioactive homopolymer probe. Ac- cordingly, Lamb and Laird (1976) have been able to observe the appearance of poly(A)+ RNA in preparations of nuclei obtained from ruptured Drosophila mela- nogaster embryos by in situ hybridization with [3H]poly(U). In the present investiga- 137 0012-16os/78/0671-0137$02.00/0 Copyright 0 1978 by Academic Press,Inc. All rights of reproduction in any form reserved.