Malcolm Whitman - US grants

Extracellular signalling molecules responsible for the establishment of early embryonic pattern in vertebrates have recently begun to be identified and characterized. Little is known, however, about how these signals act on target cells to specify developmental fate. The proposed research will investigate the early, pre-transcriptional events at the plasma membrane and in the cytosol involved in mediating the action of these extracellular signals. The primary focus of this work will be on the signals responsible for the induction of mesoderm during early frog embryogenesis. This study will provide important insights into the molecular mechanisms by which intercellular interactions establish the body plan of a vertebrate embryo. Two agents (fibroblast growth factor, polyoma middle T) known to activate cellular tyrosine kinases during mitogenic stimulation of fibroblasts have previously been found to respecify prospective embryonic ectoderm to form mesoderm. To determine the role of tyrosine phosphorylation in mesoderm induction, anti-phosphotyrosine antibodies will be used to identify substrates for tyrosine phosphorylation in response to mesoderm inducing factors. Preliminary experiments have identified MAP kinase as such a substrate. The phosphorylation and activation of MAP kinase in response to inducing factors will be further characterized. Several approaches to inhibition and activation of MAP kinase will be used to test the importance of its role in inductive signal transduction. The c-ras proto-oncogene product, p2lras, has also been implicated in the action of mitogenic signals in fibroblasts. Overexpression of dominant inhibitory and constitutively active mutants of P21ras by RNA microinjection will be used to investigate the role of this putative signal transducer during mesoderm induction. Immunoprecipitation of endogenous p2l ras from embryos will be used to assess P21ras activation during mesoderm induction. To study endogenous signalling events during early development, both anti-phosphotyrosine antibodies and SH2 domains from several receptor tyrosine kinase associated signalling enzymes will be used as probes for whole mount affinity cytochemistry of embryos. This will provide a novel tool for visualization of spatial and temporal patterns of receptor tyrosine kinase activated signal transduction. This technique will be used to study patterns of signalling during normal and experimentally manipulated early development.

DESCRIPTION (appended verbatim from investigator's abstract): Members of the TGFB superfamily of growth factors play a central role in the establishment of axial pattern in the early vertebrate embryo. We have used the Xenopus embryo as a model system to identify a novel maternal transcription factor, FAST-1, that is a key regulator of the specification of the axial mesodermal gene program by TGFB ligands during early embryogenesis. We have also established that FAST-1 is regulated by direct interaction with Smads, a recently identified class of TGFB signal transducers. We will now examine: 1) how FAST-1 contributes to patterning of early embryonic gene expression 2) how FAST-1 is regulated, and 3) how the endogenous spatial and temporal patterns of Smad activation are regulated in the embryo. FAST-1 is necessary for the expression of a wide variety of early mesodermal genes, but we do not understand the contribution of transcriptional regulation by FAST-1 to specific patterns of endogenous gene expression. We will use transgenic analysis of FAST-1 regulated genomic reporters to investigate the role of FAST-1 in the regulation of complex patterns of mesodermal gene expression in response to endogenous inducing signals. Correct developmental patterning depends as much on the competence of embryonic cells to respond to specific signals as it does on the correct localization of the signals themselves. We have identified several ways in which the competence of embryonic cells to respond to inducing signals is regulated. One involves the regulation of FAST-Smad complexes at the start of gastrulation, a second involves promoter-specific regulation of responsiveness to activated FAST-1 after the end of gastrulation, a third involves interaction between FGF and TGFB signals in the pre-gastrula embryo. We plan to determine the mechanistic basis for these various mechanisms for competence regulation to clarify the fundamental problem of how single intercellular signals can regulate different developmental events as embryogenesis progresses. Although we know that Smads are important for early embryonic patterning, we do not know where and when these regulators are active in early development. We have developed a new approach to the study of the endogenous state of activation of Smads that is applicable to early embryos. Investigation of the regulation of endogenous Smad activity will allow us to critically test a variety of hypotheses concerning the role of TGFBs in early development.

[unreadable] DESCRIPTION (provided by applicant): Our long term goal is to understand how members of the TGF[unreadable] superfamily act to exert a wide range of cell- type specific actions during development. Our current focus is on the role of TGF[unreadable] ligands and their primary signal transducers, the Smads, in two sets of developmental events: 1) the regulation of migration of cell populations that establish the craniofacial skeleton and the body wall musculature; 2) the normal growth of the tail and the regeneration of this structure following surgical extirpation. Migration of cell populations over extended distances in the embryo prior to their terminal differentiation is a critical component of the establishment of embryonic pattern. These migrations involve cell behaviors and regulatory programs which may be recapitulated during tumor metastasis, making an understanding of their regulation important for tumor biology as well as embryology. The craniofacial skeleton is made up primarily of neural crest cells that migrate from the edge of the anterior neural plate into the craniofacial region, where they differentiate into cartilage and bone. The muscle of the body wall is made up of muscle precursor cells that migrate from the somites to the ventro-lateral body wall, where they differentiate into muscle. In each case, preliminary work implicates BMP signals as regulators of the cell migration and/or the subsequent differentiation of the migratory cell. We will use a novel conditional inhibitor developed in our lab to understand how BMPs regulate these processes. The Xenopus tail has been shown to be a powerful system for studying the molecular basis of complex regenerative events. We have identified a TGF[unreadable] superfamily ligand, GDF11, that controls outgrowth of the normal tail through the activation of Smad2. We plan to explore how GDF11 and Smad2 activation during tail regeneration interacts with other signaling pathways to establish the regenerative program. Understanding how extracellular factors control cell and tissue migration during normal development, during regenerative healing following extensive tissue damage, and during pathological processes such as tumor metastasis, provides a basis for new paths to therapeutic regulation of these events. The study of TGF[unreadable] superfamily ligands provides a common approach, and a common set of molecular tools, with which to understand the regulation of these important physiological processes. [unreadable] [unreadable]