Genetic analysis of ligand-receptor interactions in the TGF-beta superfamily during early embryonic development

Abstract: During early development, cells differentiate and acquire specific fates according to their position in concentration gradients of morphogens. These substances often spread from a local region, termed an organizer, and exert different effects at distinct thresholds. The transforming growth factor beta (TGF-β) superfamily comprises more than 30 factors that have previously been shown to induce specific cell-types and to act as morphogens. The work presented here identified critical roles for ligand-receptor interactions within the TGF-β superfamily during formation and functioning of organizing centers in the mouse embryo. TGF-β ligands bind and signal through a heteromeric receptor complex consisting of type I and type II receptors. Although most of the TGF-β ligands were identified more than ten years ago, interactions with their cognate receptors remain largely unknown. The number of ligands exceeds the number of receptors, indicating the existence of redundancy among the ligands. Indeed, in vitro experiments performed during the course of these studies revealed that three ligands, Nodal, GDFI, and GDF3 could all signal through the type 1 receptors ALK4 and ALK7 in a complex with the type II receptors Acvr2 and Acvr2b. The most critical time during embryogenesis is prior to- and during gastrulation, which is a phase of development that is characterized by extensive cell migration and morphological change. Data presented here show that, in the pre-gastrulation embryo, GDF1 and GDF3 cooperate during formation of anterior visceral endoderm, a tissue that is responsible for initiation of the anterior-posterior axis and forebrain induction. At gastrulation, GDF1 and GDF3 was shown to synergize with Nodal during the specific allocation of mesendoderm precursors into prechordal plate and foregut endoderm, tissues that are necessary for maintenance of the anterior axis and forebrain identity. Despite the ability of these ligands to signal through both ALK4 and ALK7 in vitro, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for anterior axis development. Since these three ligands have overlapping expression patterns and signal through a common pathway, it was not surprising that functional redundancy occurred at multiple sites, as revealed by genetic interactions in mutant mice. Thus, Nodal, GDF1 and GDF3 form a robust signaling network of major importance during early embryonic development. Given the high degree of convergence on a limited set of receptors, this mode of action may be widespread among other members of the TGF-β superfamily. Subsequent to gastrulation, during which the basic body plan is set up, elongation and regionalization of the embryo is coordinated by structures in the tail bud. GDF11 is a ligand that is expressed in the tail bud, and has been shown to affect these processes in a dosedependent manner by signaling through Acvr2b, although the identity of the corresponding type I receptor was unknown. Therefore, several distinct biochemical assays were undertaken to examine ligand binding and signaling properties, and complemented with in vivo experiments to establish physiological relevance. Despite in vitro evidence showing that GDF11 can signal promiscuously, signaling specificity was revealed in vivo by a genetic interaction between Alk5 and Acvr2b, which resulted in an enhanced homeotic transformation. Thus, functional specificity was only observed when taking a genetic approach to a mechanistic question. Taken together, this thesis provides insight into the mechanisms by which TGF-β ligands cooperate and are functionally integrated during embryonic development.