CELLULAR SIGNALING IN THE BLADDER

Laurence S. Baskin^1,3, Simon W. Hayward^1,2, Ronald A. Sutherland¹, Michael S. DiSandro¹, Axel A.Thomson² and Gerald R. Cunha^1,2

Department of Urology1, Anatomy2 and Pediatrics3 University of California, San Francisco San Francisco, California, 94143-0738

Received 11/13/97 Accepted 11/25/97

2.MESENCHYMAL-EPITHELIAL INTERACTIONS

Embryologically, the urinary bladder is derived from undifferentiated mesenchyme and endoderm of the urogenital sinus and allantois. During development, the undifferentiated mesenchyme differentiates into bladder smooth muscle (1). What are the signaling mechanisms that allow this process to occur? Previously, we have shown that epithelium is necessary for bladder smooth muscle differentiation (figure 1)(2). Using the rat as an experimental model, we first defined the ontogeny of both epithelial and smooth muscle differentiation using a panel of smooth muscle and epithelial proteins (1). The smooth muscle proteins, alpha-actin, myosin, vinculin, desmin, laminin and vimentin, are sequentially expressed as a function of developmental stage. In the embryonic rat bladder at 14 days gestation, none of these markers were detected by immunostaining, whereas at 16 days gestation, when smooth muscle is first noted by histologic criteria, alpha-actin stained cells are detected in the periphery of the bladder. Subsequently, these alpha-actin positive cells sequentially acquire the remaining smooth muscle markers. We hypothesized that epithelial mesenchymal signaling is required for the smooth muscle differentiation to occur (2). To test this hypothesis, 14 day embryonic rat bladders (prior to the differentiation of smooth muscle) were isolated and the epithelium was separated from the mesenchyme following tryptic digestion which degrades the epithelial basement membrane (3). The isolated bladder mesenchyme, bladder mesenchyme recombined with urothelium and intact embryonic bladders were then grown underneath the kidney capsule of syngeneic hosts. The intact 14 day embryonic bladders underwent normal differentiation, defined by the expression of smooth muscle proteins. In contrast, when isolated bladder mesenchyme was grown alone underneath the kidney capsule, the tissue survived, but the mesenchyme did not differentiate into smooth muscle. However, when bladder mesenchyme was recombined with bladder epithelium, smooth muscle differentiation occurred, although this was less organized than if the epithelium was left intact in its natural state.

Figure 1. Epithelium is required for bladder mesenchyme to differentiate into bladder smooth muscle. Signaling occurs via an unknown mechanism possibly diffusable growth factors (12) (Used with Permission) . The same experiments were repeated in vitro, where intact embryonic bladders prior to smooth muscle differentiation were grown on top of filter paper nourished by standard tissue culture medium. Compared to bladder mesenchyme cultured alone, there was impressive growth of the intact organs. When epithelium was recombined with bladder mesenchyme, again the growth was greatly improved compared to the mesenchyme alone. Labeling of the organ culture with 3H-thymidine showed extensive cellular proliferation in the tissue recombinants composed of bladder mesenchyme plus bladder epithelium and in intact bladders, whereas in the mesenchyme alone, little growth occurred. Histologically, tissue recombinants of bladder mesenchyme plus epithelium structures revealed healthy cellular histology where as the bladder mesenchyme alone showed evidence of cellular degeneration. A final experiment determined whether epithelium from a different species could induce bladder smooth muscle differentiation. This hypothesis was tested by performing a unilateral nephrectomy in nude mice (2). Undifferentiated, 14 day rat bladder mesenchyme with the epithelium removed was then surgically attached to the severed mouse ureter. The bladder mesenchyme and mouse ureter were allowed to grow for one month in situ, with the idea that the ureteral epithelium would induce smooth muscle differentiation of the rat bladder mesenchyme. As expected, the mouse urothelium induced smooth muscle differentiation in the rat bladder mesenchyme resulting in the expression of smooth muscle differentiation markers. Species specific probes confirmed that the epithelium was of mouse origin and the smooth muscle was from the rat. These experiments strongly support the concept that cellular signaling between the bladder epithelium and undifferentiated bladder mesenchyme, is required for the induction of bladder smooth muscle. A number of questions remain unanswered. Why does bladder smooth muscle initially form in the periphery of the bladder at the greatest distance from the signaling epithelium? Does signaling occur by diffusable factors, (a hypothesis that we have subsequently been testing) or does it occur by cellular contact of mesenchymal cells with the epithelial basement membrane followed by signaling peripherally through the undifferentiated mesenchyme to the sub-serosal zone? These questions are the basis for further research.