Regulation of Gap Junction Differentiation

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably R N A as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells. Recent observations on the construction of gap junctions in vitro (18) as well as in vivo (1, 3, 6, 35, 36, 48) portray junctional precursors assembling in an orderly fashion. Common features among developing gap junctions include the: (a) appearance of formation plaques (18); (b) clustering and/or aggregation of large precursor particles accompanying a reduction of the extracellular space; (c) polygonal packing of particles into recognizable junctions; and (d) enlargement of junctions through the recruitment of nearby granules and/or fusion of smaller aggregates (I, 3, 6, 18, 36). Although minor variations on this theme may exist (3), the similarities are truly remarkable. Moreover, the developing gap junction provides an ideal model system to assess some of the macromolecular requirements for regional plasma membrane differentiation. While a morphological picture of gap junction assembly is slowly emerging, macromolecular events concerned with the control of junctional differentiation remain to be elucidated. In one instance, the coordinate appearance of gap junctions and electrotonic coupling between reaggregating Novikoff hepatoma cells (18) appears insensitive to drugs like cycloheximide (9) which inhibit protein synthesis. In another, cycloheximide prevents cell-cell interaction between cultured myoblasts (47) and, therefore, may preclude the development of gap junctions and low-resistance coupling (33) between these cells. Events accompanying myoblast fusion (47) imply that the production of new membrane proteins is essential in the development of multinucleate myotubes. The synthesis and insertion of gap proteins into the myoblast plasma membrane are thought to be crucial in the coordination of myogenic cell fusion (33). The present experiments, employing hypophysectomized Rana pipiens larvae, reveal that the synchronous differentiation of ependymoglial cells requires thyroid hormones. One feature of glial THE JOURNAL OF CELL BIOLOGY " VOLUME 69, 1976 pages 669-685 669 on July 4, 2017 jcb.rress.org D ow nladed fom different iat ion encompasses the format ion of gap junct ions between adjacent cells. The mode of junct ional assembly resembles tha t depicted in reaggregat ion of Novikoff hepa toma cells (18); however, new protein synthesis is an absolute requirement for junc t ional format ion during thyroxine-mediated glial differentiat ion. M A T E R I A L S A N D M E T H O D S