Key processes in embryonic development are recapitulated in a pathologic manner during oncogenesis and metastasis, suggesting an aberrant regulation or re-activation of such processes. My lab has identified several new developmental control genes (transcription factors) that appear to be involved in regulating the formation of, and the pattern of structures in the primary embryonic axis and the limb axis. We are analyzing their normal function and potential role in oncogenesis. The limb arises from a bud by progressive branching of skeletal precursors from proximal to distal (ie. shoulder to hand). The pattern of skeletal elements that form from anterior to posterior (thumb to little finger) is regulated by secreted sonic hedgehog (shh) signals from the posterior edge of the limb bud. We found that misexpression of the posteriorly expressed homeobox gene Hoxd-12 in limb buds of transgenic mice alters skeletal growth causing posterior transformations of anterior digit morphologies, and sometimes produces mirror-image digit duplications due to secondary induction of shh in the anterior limb bud. Since shh induces Hoxd-12, this indicates that a positive feedback loop between shh and Hoxd-12 may act to reinforce signals critical for the ongoing process of limb patterning. In a mouse model, we have also found a genetic interaction between Hoxd-12 and a transcriptional repressor of shh, Gli3. Thus, Gli3 may repress shh through down-regulating Hoxd-12 expression. Hoxd-12 is a trans-activator and strategies to identify other target genes are being developed. We have identified the c-Fos proto- oncogene as a potential target. Fos plays a role in cartilage proliferation and formation of normal growth plates in bone. Activation of Fos by Hoxd-12 illustrates the link between patterning and growth during development and suggests Hoxd-12 could play a pathogenic role in bone tumors. A conditional transgenic expression system is being developed to selectively analyze late functions of Hoxd-12 in growth plates of bone and oncogenic potential. During gastrulation, a single cell layer embryo (epiblast) is converted to three cell layers and forms an elongated axis, via cell movements and through the actions of an organizer that can induce a new embryonic axis when grafted to a host embryo. Epiblast cells migrate into and through a furrow, the primitive streak, to form mesoderm and endoderm. An organizing center (the node) forms at the edge of this streak and produces a specialized mesodermal cord, the notochord, which in turn regulates patterning of the CNS and of the somites. We have isolated two homeobox genes, Gnot1 and Gnot2, specifically expressed in organizer tissues; node and notochord. During gastrulation, very rapid and dynamic changes in gene expression are critical, likely involving post- transcriptional mechanisms. Cycloheximide dramatically increases Gnot RNA levels (by 40X), implying the presence of a labile inhibitory factor. We are analyzing this regulation, which involves specific sequences in the 3UTR, using defective Adenoviral vectors and electroporation into chick embryos to study transcript processing in the natural host cell. Gnot1 is also expressed in the limb and may regulate pattern along the proximodistal limb axis. Early and late Gnot functions are being analyzed using several genetic and embryologic approaches in chick embryos. The mouse T gene (brachyury) is critical for normal mesoderm migration and patterning of the embryonic axis (notochord function). This transcription factor belongs to a new multigene (T-box) family that binds DNA as a dimer. We have isolated three chick genes, Ch-TbxT, -Tbx6L and Ch-T. Ch-TbxT and Ch-Tbx6L are expressed in two different subdomains of Ch-T expression (node/notochord and primitive streak respectively), and may modify Ch-T function. Murine genes have been isolated for TbxT and for a novel Tbx6 family member that is expressed exclusively in primitive streak and anterior neural folds. Functional analyses of these genes using targeted gene disruption in mice are underway in collaboration with Dr. Chuxia Deng. Genetic analyses of these genes are also being utilized in the chick embryo system (see below). T-box family members also appear to be involved in mesoderm formation and regulating the relative migratory and adhesive behavior of mesoderm. We are evaluating possible roles for these genes in genesis of embryonal mesodermal tumors. T is expressed in chordoma, confirming the presumed notochordal origin and providing the first molecular marker for this tumor. We have also analyzed tail development in the chick embryo as a potential model for evaluating function of Not-homeobox and T-box genes during gastrulation. Tail development is both experimentally highly accessible and is more readily amenable to infection with retroviral expression vectors expressing genes of interest than is early gastrulation (which proceeds too rapidly for viral infection studies). We have found that tail development occurs mechanistically as a continuation of gastrulation, with respect to both neural and mesoderm induction, and have identified a tail organizer that functions as a signalling center analogous to the node during gastrulation. Not-homeobox and certain T-box genes continue to be expressed in the developing tail bud and retroviral experiments are underway to evaluate the effects of altering this expression. Retinoids and FGFs have both been implicated in initiating limb development at particular positions along the embryonic axis. Retinoids regulate the expression of Hox genes in multiple tissues along the anterior-posterior axis of the embryo and patterns of Hox gene expression in lateral plate have been linked to positioning of limbs. FGFs can induce ectopic limbs in flank with accompanying changes in Hox expression, and a relay of local FGF signals from midline to peripheral tissues has been proposed to regulate the positon of limb initiation. How these signals are relayed, localized, and interrelate to each other is a matter of active inquiry. We have found that the prototype T-box transcription factor, T, is expressed at some possible relay sites and plan to determine if T plays a role in limb intitiation or outgrowth. Since T is involved in a positive feedback loop with eFGF in the early Xenopus embryo, a similar positive feedback loop could also propagate signals through different tissues by a relay mechanism. Our lab is also engaged in a collaborative project with Dr. Glenn Merlino to evaluate the roles of fibroblast growth factor (FGF) signals during embryonic development, particularly limb development and mesoderm formation during gastrulation and tail development. We are using a novel approach involving soluble extracellular FGF receptors that are stable and efficiently bind specific types of FGFs and consequently have a dominant negative effect by sequestering FGF ligands. These will serve as useful tools to document FGF relay sites in the embryo and as an adjunct to evaluate the possible role of T as an intracellular component of the relay. Retinoids have been implicated in positioning both sites of limb initiation and of limb polarizing regions, as well as having potent effects during gastrulation and axis formation. It remains uncertain whether these processes require localized regions of higher RA or differential tissue sensitivities. We are developing methods to sensitively evaluate RA sources in situ during early stages of development where the embryo is rapidly changing. In a collaborative effort with Dr. Gordon Hager, we are developing a rapid in situ assay using a chimeric glucocorticoid/retinoic acid receptor fusion protein which we have generated. This chimeric protein demonstrates RA- dependent nuclear translocation, providing for a rapid, real time read-out assay by culturing embryos on reporter cells that express the chimera.
