As we enter the era of vertebrate genomics, the complete definition of regulatory gene networks, which provides the basis for any dynamic biological system beyond the mere structural definition of genetic elements, seems to come into reach. In recent years, amphibians as a traditional system in experimental embryology with vertebrates has regained quite a lot of attraction. This is due to a number of advantages that this system has to offer in comparison to mouse, chicken or fish. In Xenopus laevis, a frog from South Africa, it is relatively easy to generate hundreds or thousands of staged embryos; early development is easily accessible for experiÐmental analysis because it occurs outside of the mother, and also because of the relatively large size of early embryos. Overexpression studies can be performed by simply injecting synthetic mRNA into fertilized eggs, and loss of function effects can be generated using the same strategy with dominant negative variants of a given regulatory protein. More recently, it has also become possible to generate large numbers of transgenic frogs by an efficient and relatively simple approach.

The initial goal of our research interest with Xenopus embryos was to identify zinc finger transcription factors (ZFPs) with a regulatory function in early vertebrate development. This has led to the characterization of several, evolutionarily highly conserved ZFPs, which play roles in either early mesoderm or neural differentiation. The activity or expression of these proteins is controlled by different secreted signals via the corresponding signal transduction cascades. For example, expression of the ZFP Xegr-1 in the Spemann organizer region, that serves as an organizing center for the anteroposterior body axis during gastrulation/neurulation, is regulated via the eFGF induced MAP-kinase cascade; expression of the ZFP XMyT1, which functions in primary neuronal differentiation, is regulated via the Notch/Delta signal transduction pathway, and the activity of the Gli family of ZFPs, which have multiple functions in mesoderm and neural development, is regulated via the Hedgehog induced cascade. Finally, Schnurri-type ZFPs appear to be directly involved in mediating TGFb signaling on the level of transcription regulation. We are currently trying to unravel the mechanisms of these different regulatory circuits in more detail.

In a different line of experiments, we have begun to perform a random screen for novel genes with a regulatory function in vertebrate embryogenesis. For this purpose, large numbers (hundreds) of randomly picked cDNA clones are characterized in respect to their embryonic expression characteristic. After sequencing of differentially expressed genes, it turns out that approximately 50% of these clones encode putative regulatory molecules. Thus, this approach identifies a plethora of putative developmental regulators which will be subjected to a more detailed functional analysis making use of the advantages that Xenopus embryos offer as an experimental system. As a first example for a novel regulatory gene in early patterning events, we have the differentially expressed retinoic acid metabolizing enzyme XCYP26 as serving as a key player in establishing morphogen signalling boundaries in the early embryo.

Abbildung:

Expression pattern analysis of a retinoic acid signals producing enzyme (RALDH2, red) and of a retinoic acid degrading enzyme (CYP26, purple) in early neurula stage Xenopus embryos.

Selected recent publications:

1. Rudt, F. and Pieler, T. (1996) Cytoplasmic retention and nuclear import of 5S ribosomal RNA containing RNPs. EMBO J. 15, 1383-1391.

2. Bellefroid, E., Bourguignon, C., Hollemann, T., Ma, Q., Anderson, D.J., Kintner, C. and Pieler, T. (1996) X-MyT1a Xenopus C2HC type zinc finger protein with a regulatory function in neuronal differentiation.Ê Cell 87, 1191-1202.

3. Panitz, F., Krain, B., Hollemann, T., Nordheim, A. and Pieler, T. (1998) The Spemann orgnizer-expressed zinc finger gene Xegr-1 responds to the MAP kinase/Ets-SRF signal traSelectnsduction pathway. EMBO J. 17, 4414-4425.

4. Hollemann, T., Chen, Y., Grunz, H. and Pieler, T. (1998) Regionalized metabolic activity establishes boundaries of retinoic acid signalling. EMBO J. 17, 7361-7372.

5. Hollonet, M., Hollemann, T., Pieler, T. and Gruss, P. (1999). Mutation of Vax1, a novel homeobox-containing gene, leads to defective development of the basal forebrain and visual system. Genes and Dev., in press.