Using the frog Xenopus laevis and the zebrafish Danio rerio as experimental systems, the laboratory is engaged in studies of molecular-genetic mechanisms of early vertebrate development.

The Small GTPases Rho and Rac Are Components of the Noncanonical Wnt Pathway in Controlling Gastrulation Movements
Habas, Dawid; in collaboration with He
The work of many laboratories has demonstrated a role for Wnt signalling in gastrulation movements. Wnt signals are transduced by three distinct pathways in different biological contexts. Gastrulation movements require the so-called noncanonical pathway that is mediated by frizzled receptors and the Dishevelled (Dsh) protein but does not result in activation of bcatenin. In studying the mechanism of signal transduction downstream of Dsh, we have shown previously that the small GTPase Rho is activated as a response to Wnt signalling and that the novel formin-family protein, Daam1, is required in this process (Habas et al., 2001). Rho-family GTPases are known regulators of cell physiology with important functions in cytoskeletal dynamics and effects on gene activation. More recent studies have demonstrated that activation of Rho family GTPase Rac is also required for gastrulation movements. Rac activation is mediated by Wnt signalling through the function of Dsh, but the structural requirements in Dsh for Rho and Rac activation differ. Inhibition of either Rho or Rac activation alone leads to failure of gastrulation, indicating that the independent activation of both GTPases downstream of Wnt signalling is required for early development.

The Role of Lim1 in Gastrulation
Hukriede, Habas, Dawid, in collaboration with Weeks, Tam
The Lim1 gene encodes a LIM-homeodomain protein that is expressed in the organizer region of all vertebrate embryos. A knock-out in the mouse, generated by Shawlot and Behringer, has shown that the Lim1 gene is required for head formation. Further mechanistic studies carried out in Xenopus used antisense oligonucleotide depletion techniques developed by Weeks and Dagle, University of Iowa. Lim1-depleted frog embryos failed to form head structures, providing a phenocopy of Lim1-deficient mice. Most organizer-specific gene expression was retained in the affected embryos, but cell movements characteristic for gastrulation were impeded. The defect was traced to impaired expression of para-axial protocadherin (PAPC), an adhesion molecule shown by De Robertis and colleagues, UCLA, to be involved in gastrulation. This defect in PAPC expression correlated with a failure of Rho activation on the dorsal side of the embryo. Rho activation has previously been shown to be required for gastrulation movements, as discussed above. We conclude that a loss of PAPC expression, directly and indirectly through Rho activation, affects gastrulation movements in Lim1-depleted embryos. Complementary studies in the mouse showed that Lim1-/- cells fail to participate in gastrulation movements when transplanted into wild-type embryos. Together, these studies indicate that control of gastrulation movements is a major function of the Lim1 gene in different vertebrate embryos.

Sef, a Novel Feedback Inhibitor of Fgf Signalling
Tsang, Kudoh, Dawid; in collaboration with Friesel
Fibroblast growth factors constitute a family of signalling factors that regulate many different developmental and physiological processes, and mutations in Fgf receptors are associated with several disorders in humans. The Fgf signal transduction pathway has been studied extensively in many systems, yet it was possible to isolate a novel feedback inhibitor of the pathway by observing a gene with Similar Expression to Fgfs (Sef) in zebrafish. The group of C. and B. Thisse, University of Strasbourg, independently identified Sef, which is a transmembrane protein whose expression is controlled by Fgf signalling. Its role, as studied by gain-of-function and loss-of-function approaches, is to attenuate the Fgf signal in the embryo. Thus, Sef joins the ranks of many factors identified in various pathways that have a role in limiting the extent or range of their cognate signalling pathway within the organism.

Interplay of Different Signalling Pathways in Patterning the Neural Ectoderm in Zebrafish
Kudoh, Dawid; in collaboration with Wilson
In a model proposed some time ago by the great embryologist Pieter Nieuwkoop, the nervous system is first induced by action of the organizer with anterior characteristics and is subsequently patterned by influences from the mesoderm that give a portion of the developing neural ectoderm more posterior characteristics. Many studies have implicated three classes of signalling factors, Fgf, Wnt, and retinoic acid (RA), in posteriorization of the neural ectoderm. We have studied the regulatory interrelationships between these signalling factors in the zebrafish. By a combination of gain-of-function and loss-of-function experiments, we determined the effects of each of these signals on the neural ectoderm as well as the relationships between them in controlling neural patterning. From these data, we conclude that each of the three signalling pathways alone is capable of inhibiting anterior development in the neural ectoderm, but induction of posterior development has an obligatory requirement for RA action. Thus, RA function is epistatic to both Wnt and Fgf signalling in posteriorization of the neural ectoderm, but all three pathways must interact in precise ways for the generation of the normal anteroposterior pattern of the zebrafish nervous system.