The goal of this proposal is to characterize 2 embryonic liver specific genes, ELF (embryonic liver fodrin) and ELM (embryonic liver MAGUK- Membrane Associated Guanylate Kinase), which by homology studies appear to be important in the establishment of cell polarity, and to establish a basis for their role in hepatic differentiation. Subtractive clones ELF and ELM potentially represent critically new genes expressed at a crucial point in liver development. These were selected from 2 group of clones form subtractive hybridization using embryonic mouse liver cDNA libraries at days 10.5,11.5,12.5,14.5 (constructed and characterized by the PI). Further preliminary data have revealed that by expression studies, ELF and ELM are stage specific and liver restricted; comparison of the complete nucleotide and amino acid sequence analysis utilizing the intelligenetics database has revealed these to be novel cell polarity genes. Northern analysis and in situ localization indicate ELF to represent the crucial mesodermal component of liver development. Zoo blot analysis has revealed that both ELF and ELM are conserved throughout evolution. Drosophila homologues of ELF and ELM (spectrin and d(l)gl respectively) have shown these to be of pivotal importance in epithelial cell polarization and Drosophila larval development it is therefore conceivable that the ELF and ELM may play a similar role to the homologues in determining liver cell fate. The first part of this project involves the completion of full length sequence determination and specific localization of ELF a ELM mRNA by in situ hybridization. The next step will involve identification of genomic clones and chromosomal localization of ELF and ELM. The third part of this project involves obtaining and studying polyclonal antibodies which will be used for localization in embryonic and adult mouse tissue sections as the first approach to the functional and physiological role of ELF and ELM in liver development problem for expression in various cell lines, in particular, human, mouse and rat HCC cell lines e.g., HepG2, HA22T, Hep3B, FOCUS, NGP, will also yield further information and provide essential tools for further functional vitro assays utilizing these cell lines. The last part of this proposal will focus on loss of function studies utilizing antisense oligonucleotides in cultured embryonic liver explants (the latter is a well established functional assay in this laboratory). These preliminary studies will determine the biological importance of ELM and ELF in hepatocyte formation. The long term objective of this research is to elucidate the pathways involved when a group of foregut endodermal cells differentiate into hepatocytes. The identification and characterization of such early liver specific growth and differentiating factors promises to establish a foundation for their future role in the medical treatment of diseases in which the formation of healthy new hepatocytes is of crucial importance, such as end stage chronic liver disease.
| Mishra, L; Cai, T; Yu, P et al. (1999) Elf3 encodes a novel 200-kD beta-spectrin: role in liver development. Oncogene 18:353-64 |
| Mishra, L; Cai, T; Levine, A et al. (1998) Identification of elf1, a beta-spectrin, in early mouse liver development. Int J Dev Biol 42:221-4 |
