CELL BIOLOGY OF DEVELOPMENT
Research Focus
Australian Collaborators
- Dr Klaus Matthaei, Gene Targeting Laboratory, Division of Molecular Bioscience,
John Curtin School of Medical Research, ANU.
- Professor Ian Young, Cytokine Molecular Biology and Signalling Group, Division
of Molecular Bioscience, John Curtin School of Medical Research, ANU.
- Dr Carolyn Behm, School of Biochemistry and Molecular Biology, ANU.
- Dr Barry Powell, Department of Paediatrics, University of Adelaide, and
Laboratory of Epithelial Biology, Child Health Research Institute, North Adelaide,
SA.
- Dr Allison Cowin, Department of Surgery, University of Adelaide, and Laboratory
of Epithelial Biology, Child Health Research Institute, North Adelaide, SA.
- Dr Michael Crouch, TGR BioSciences, Adelaide, SA.
- ARC/NHMRC Research Network in
Genes and Environment in Development (NGED)
International Collaborators
- Professor Michael Stallcup, Departments of Biochemistry & Molecular
Biology and Pathology, University of Southern California, Los Angeles CA,
USA.
- Dr Heidi Scrable, Department of Neuroscience, University of Virginia, Charlottesville
VA, USA.
- Professor Dennis Roop, Departments of Molecular & Cellular Biology and
Dermatology, Baylor College of Medicine, Houston TX, USA.
Mammalian homologues of Drosophila melanogaster
genes involved in development and neurobiology
The laboratory is studying the mammalian homologues of novel Drosophila melanogaster
(fruit fly) genes involved in developmental and neurobiological processes. Genes
with novel encoded proteins and possessing interesting mutant phenotypes were
chosen for study from a group of previously uncharacterised genes mapping to
the Drosophila X chromosome. The approach then involves identifying the mammalian
and other homologues of these genes using PCR, or, increasingly, bioinformatic
methods. We then use gene targeting and transgenics in the mouse in our initial
approaches to the biology of the genes in mammals. We also look at the cell
biology of the mammalian proteins using antipeptide antibodies and reporter
gene fusions. We are also studying the genes from an evolutionary point of view
and in collaborative studies, use RNAi in C. elegans to gain insight into the
function of the genes in this simple model organism.
The main gene under study is the Drosophila flightless I gene. In the fruit
fly, null mutations in this gene result in embryonic lethality with defects
first being observed in cellularization of the syncytial blastoderm, a process
with parallels to cytokinesis. Certain point mutations in the gene result in
adult flies that are unable to fly due to defective indirect flight muscles.
We have identified the human and mouse orthologues of flightless I. The protein
contains a domain involved in interaction with Ras, and possibly related GTPases,
as well as additional ligands such as FLAP1 and FLAP2. It also contains a gelsolin-like
domain involved in interaction with actin. We have used gene targeting to inactivate
this gene in mice. When the gene is absent, mouse embryos degenerate at around
the time of uterine implantation, so the gene is required for early mammalian
development, just as it is required for early fly development. Currently our
investigations are aimed at finding out more about the exact role of the gene
during development and in normal cellular function.
We have previously made transgenic mice carrying the human gene on a 40 kb fragment
and showed that the human gene is capable of fully replacing the mouse gene.
In order to facilitate further manipulation of flightless transgenes and to
eliminate other genes present on the original cosmid, we have now subcloned
a 17.8 kb fragment consisting of the human FLII gene and its 5´-flanking
region, and have made transgenic mice carrying this fragment. We have crossed
this onto the mouse flightless knockout background and shown that this transgene
is also capable of fully rescuing the embryonic lethality. The available heterozygous
knockout and transgenic strains have enabled us to show an important role for
FLII in mammalian biology. We have also used site-directed mutagenesis to create
a mutated form of the 17.8 kb FLII gene to begin dissecting the exact role of
the gene, and have prepared transgenic ES cells. Initial attempts to make mice
using one of these lines have been unsuccessful for technical reasons, but we
are pursuing construction of these mice using additional lines of transgenic
ES cells carrying the mutated fragment. We have also prepared a form of the
17.8 kb FLII gene where the GFP coding region is fused to the C-terminal region
of FLII and have prepared transgenic ES cells carrying this fusion to enable
us to visualise FLII in real time in transgenic mice and in cells and tissues
derived from them. In collaborative studies, we have found that the flightless
protein is involved as a transcriptional coactivator for certain nuclear hormone
receptors including the estrogen and thyroid hormone receptors, indicating an
important new role for members of the gelsolin family. We are analysing the
protein-protein interactions of the C. elegans flightless protein, including
those relating to the nuclear hormone receptors and associated factors, using
the yeast two hybrid system and have uncovered novel interactions. We have used
RNAi in C. elegans to reveal a novel knockdown phenotype for flightless and
have shown that FLAP exhibits a similar knockdown phenotype, supporting the
hypothesis that the interaction of flightless and FLAP is of functional significance.
Genome-wide microarray analysis of flightless knockdown worms has been carried
out in collaboration with the Kim lab (Stanford). Several genes significantly
up- or down-regulated in three replicate experiments have been uncovered and
are currently under study. These findings with C. elegans may have important
implications for the mammalian flightless work.
Publications (since 2000)
Archer, S.K., Claudianos, C. and Campbell, H.D. (2005). Evolution of the gelsolin
family of actin-binding proteins as novel transcriptional coactivators. BioEssays,
27, 388-396.
Archer, S.K., Behm, C.A., Claudianos, C., Campbell, H.D. (2004) The flightless
I protein and the gelsolin family in nuclear hormone receptor-mediated signalling.
Biochemical Society Transactions 32: 940-2
Lee, Y.H., Campbell, H.D., Stallcup, M.R. (2004) Developmentally essential
protein flightless I is a nuclear receptor coactivator with actin binding activity.
Molecular and Cellular Biology 24: 2103-17
Campbell, H.D., Fountain, S., McLennan, I.S., Berven, L.A., Crouch, M.F., Davy,
D.A., Hooper, J.A., Waterford, K., Chen, K.S., Lupski, J.R., Ledermann, B.,
Young, I.G., Matthaei, K.I. (2002) Fliih, a gelsolin-related cytoskeletal regulator
essential for early mammaliar embryonic development. Molecular and Cellular
Biology 22, 3518-26
Kamei, M., Ades, L.C., Eyre, H.J., Callen D.F., Campbell, H.D. (2002). SOLH,
a human homologue of the Drosophila melanogaster small optic lobes gene is deleted
in ATR-16 syndrome. Applied Genomics and Proteomics 1, 65-71.
Davy, D.A., Campbell, H.D., Fountain, S., de Jong, D., Crouch, M.F. (2001).
The flightless I protein colocalizes with actin- and microtubule-based structures
in motile Swiss 3T3 fibroblasts: Evidence for the involvement of PI 3-kinase
and Ras-related small GTPases. J. Cell Sci. 114, 549-562.
Campbell, H.D., Fountain, S., Young, I.G., Weitz, S., Lichter, P., Hoheisel,
J.D. (2000). Fliih, the murine homologue of the Drosophila melanogaster flightless
I gene: nucleotide sequence, chromosomal mapping and overlap with Llglh. DNA
Sequence 11, 29-40.
Campbell, H.D., Kamei, M., Claudianos, C., Woollatt, E., Sutherland, G.R.,
Suzuki, Y., Hida, M., Sugano, S., Young, I.G. (2000). Human and mouse homologues
of the Drosophila melanogaster tweety (tty) gene: A novel gene family encoding
predicted transmembrane proteins. Genomics 68, 89-92.
Campbell, H.D., Young, I.G., Matthaei, K.I. (2000). Mammalian homologues of
the Drosophila melanogaster flightless I gene involved in early development.
Current Genomics 1, 59-70.
Davy, D A., Ball, E.B., Matthaei, K.I., Campbell, H.D., Crouch, M.F. (2000).
The flightless I protein localizes to actin-based structures during embryonic
development. Immunol. Cell Biol. 78, 423-429.
Kamei, M., Webb, G.C., Heydon, K., Hendry, I.A., Young, I.G., Campbell, H.D.
(2000). Solh, the mouse homologue of the Drosophila melanogaster small optic
lobes gene: organization, chromosomal mapping, and localization of gene product
to the olfactory bulb. Genomics 64, 82-89.
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