Development 109,449-459(1990) Printed in Great Britain © The Company of Biologists Limited J990 449 Cytoskeletal protein and mRNA accumulation during brush border formation in adult chicken enterocytes KARL R. FATHt, STEVEN D. OBENAUFf, and DAVID R. BURGESS* Department of Cell Biology and Anatomy, University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, Florida 33101, USA • Autho r for all communications. t These authors contributed equally to the contents of this paper and should be considered joint first authors Summary We have explored the development of the brush border in adult chicken enterocytes by analyzing the cytoskel- etal protein and mRNA levels as enterocytes arise from crypt stem cells and differentiate as they move toward the villus. At the base of the crypt, a small population of cells contain a rudimentary terminal web and a few short microvilli with long rootlets. These microvilli appear to arise from bundles of actin filaments which nucleate on the plasma membrane. The microvilli apparently elongate via the addition of membrane supplied by vesicles that fuse with the microvillus and extend the membrane around the actin core. Actin, villin, myosin, tropomyosin and spectrin, but not myosin I (previously called 110 kD; see Mooseker and Coleman, J. Cell Biol. 108, 2395-2400, 1989) are already concentrated in the luminal cytoplasm of crypt cells, as seen by immunoflu- orescence. Using quantitative densitometry of cDNA- hybridized RNA blots from cells isolated from crypts, villus middle (mid), or villus tip (tip), we found a 2- to 3-fold increase in villin, calmodulin and tropomyosin steady-state mRNA levels; an increase parallel to mor- phological brush border development. Actin, spectrin and myosin mRNA levels did not change significantly. ELISA of total crypt, mid and tip cell lysates show that there are no significant changes in actin, myosin, spec- trin, tropomyosin, myosin I, villin or a-actinin protein levels as the brush border develops. The G-/F-actin ratio also did not change with brush border assembly. We conclude that, although the brush border is not fully assembled in immature enterocytes, the major cytoskel- etal proteins are present in their full concentration and already localized within the apical cytoplasm. Therefore brush border formation may involve reorganization of a pool of existing cytoskeletal proteins mediated by the expression or regulation of an unidentified key pro- tein(s). Key words: crypt, differentiation, microvilli, rootlets, terminal web, villus. Introduction Two model systems that have been useful for under- standing the organization and assembly of actin cyto- skeletons are skeletal muscle, with its striking sarco- meric pattern, and the red blood cell, with its relatively simple cortical cytoskeleton. We have learned much about cytoskeletal protein gene expression in these cells, and how the proteins are arranged to generate contractile forces and how they stabilize the membrane cytoskeleton. The analysis of the developmental as- sembly of the red blood cell and muscle cytoskeleton has led to an understanding of the regulation of cytoskeletal formation at the gene transcriptional and post-translational levels. Studies with myoblasts during skeletal muscle differentiation show that the levels of mRNAs encoding sarcomeric-specific proteins (eg. tro- ponin, myosin heavy chains) increase many-fold and the levels of mRNAs encoding the non-muscle cytoskel- etal proteins decrease as myoblasts fuse (Shani et al. 1982; Wade and Kedes, 1989). In the erythrocyte, cortical cytoskeleton differentiation is controlled by a similar co-ordinate activation of specific genes; how- ever, the assembly of the spectrin heterodimer is regulated by post-translational modifications and differ- ential levels of a'- and /3-spectrin synthesis (Blikstad et al. 1983; Moon and McMahon, 1987). The simplicity and stereotypic arrangement of the brush border cytoskeleton have made the enterocyte another excellent model for analyzing the organization and development of an actin-based cytoskeleton (see reviews by Mooseker, 1985; Burgess, 1987). The ma- ture brush border is structurally divided into two parts; the microvilli and the terminal web. A microvillus contains a core bundle of actin filaments, which extends into the terminal web as the rootlet. The actin-binding proteins villin, myosin I and fimbrin are associated with the core filaments, and, in addition to tropomyosin, are