The long-term goal of this project is to understand the formation of the caudalmost part of the vertebrate embryo. This region gives rise to the trunk as well as to associated internal organs and external appendages. Secreted molecules (e.g. Retinoic acid [RA] and Fibroblast growth factor [Fgf]) are essential regulators of caudal development, as their inactivation leads to loss of caudal structures, but much remains to be learned about how these factors function. In particular, there is a paucity of identified genes regulated by RA and Fgf and the spatial control of Fgf and RA activity in vivo is poorly understood. The PI and his colleagues have isolated two Zn finger-containing genes (dpz and lmo4) expressed caudally in the zebrafish embryo which will address these issues. First, dpz and lmo4 will help in the study of two questions concerning the spatial regulation of Fgf and RA activity. 1) dpz and lmo4 expression covers both RA and Fgf dependent domains in the embryo. By analyzing the regulation of these genes the PI will learn how RA and Fgf signals are integrated spatially in vivo. 2) It has been suggested that Fgf affects mesoderm directly, but ectoderm indirectly via a secondary signal. Since dpz, and possibly lmo4, is expressed in both ectoderm and mesoderm the PI will address whether Fgf acts differently in these germ layers. Second, these genes are novel in that they, in contrast to most RA and Fgf regulated genes characterized to date, do not encode classical DNA-binding transcription factors. They may therefore function at different levels of the RA and Fgf signaling pathways than known RA and Fgf regulated genes. Two different approaches will be taken to determine how dpz and lmo4 act in caudal development. 1) Zebrafish mutant lines will be used and transient expression of dominant negative constructs in vivo to define where in the RA/Fgf signaling pathways dpz and lmo4 function. 2). The roles of dpz and lmo4 will be defined by performing rescue experiments. To this end dpz and lmo4 will be expressed in zebrafish embryos that lack RA or Fgf signaling. By determining which aspects of caudal development can be rescued by either gene, it will be possible to infer their normal function.

This work will have implications for human health in several ways: Defective caudal development caused by genetic defects or exposure to environmental agents results in birth defects such as spina bifida. This work will define steps in caudal development that may be affected by such external insults and suggest ways they can be reversed. Also, lmo4 is implicated in various cancers (e.g. breast cancer) and its oncogenic potential is likely related to its normal role in controlling basic processes such as differentiation. Thus, studying normal lmo4 function will help us understand aspects of tumorigenesis.

R03HD039156

2000-09-01

2002-06-30