IntroductionHyaluronan, or hyaluronic acid (HA) is a linear, highmolecular-weight (mega-Dalton) polymer comprised of repeating disaccharide units of (β1→3)D-glucuronate-(β1→4)N-acetyl-D-glucosamine. HA is synthesized by integral plasma membrane glycosyltransferases and is exported directly into the extracellular space (1, 2). Although HA is chemically homogeneous, there are three distinct mammalian HA synthases (designated Has1, Has2, and Has3), encoded by related but nonlinked genes (3-9). Each synthase has distinct catalytic properties, and the distribution and abundance of each varies during development of the mouse (6, 10). These observations suggest that the different Has enzymes play distinct roles.HA binds salt and water, expanding the extracellular space (11)(12)(13)(14). HA is especially prominent at sites where cell migration occurs, such as pathways of neural crest cell migration and in the developing cardiovascular system. In vivo, HA interacts with other extracellular matrix molecules, typically via an HA-binding domain called the link module (15). These interactions create a supramolecular architecture of the extracellular matrix, i.e., the composite matrix network of HA, link protein, and aggrecan that plays a critical role in load-bearing articular cartilage (16)(17)(18).In addition to its important physical properties, the overexpression of Has genes results in increased anchorage-independent growth and metastasis of transformed cells (19,20), suggesting a link between HA and transformation. HA is also implicated in receptor-mediated cell adhesion and intracellular signaling (21,22). Taken together, such observations suggest that HA plays a vital role in diverse cellular events, including cell migration, tissue remodeling, and metastasis. However, the near-ubiquitous distribution of HA in vivo, the biological activity of HA fragments released by degradative enzymes (23), and the inability to inhibit HA synthesis in vivo have hindered definitive analysis of the physiological roles of HA. Accordingly, we used a genetic approach to investigate the roles of HA in vivo and to identify the HA synthase that is critical during embryogenesis.Expression of Has2 appeared to correlate with expansion of cardiac cushion tissue and subsequent transformation of endocardial cells into mesenchyme. The tar- We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2 -/-embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2 -/-embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2 -/-explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, ...
