RESEARCH ARTICLES CURRENT SCIENCE, VOL. 96, NO. 9, 10 MAY 2009 1217 *For correspondence. (e-mail: ghaskadbi@gmail.com) Fibroblast growth factor regulates early mesoderm and neural development in chick embryo through its action on brachyury, goosecoid, ERNI and noggin Seema Borgave and Surendra Ghaskadbi* Division of Animal Sciences, Agharkar Research Institute, Pune 411 004, India We have analysed the molecular mechanism of action of fibroblast growth factor (FGF) in the development of mesodermal and neural structures in chick embryo. Experimentally altered levels of FGF signalling were found to differentially modulate expression of brachyury, goosecoid, ERNI and noggin, genes implicated in meso- dermal and neural development. These effects became evident within 2 h and persisted for 6 h post-treatment, with either exogenous FGF or FGF inhibitor. Signifi- cantly, changes in gene expression correlate well with the abnormal mesodermal and neural phenotypes observed after 22 h. Thus FGF seems to regulate the development of mesodermal and neural structures in early chick embryo through its action on specific genes. Keywords: Chick embryo, developmental gene expres- sion, fibroblast growth factor signalling, mesoderm, nerv- ous system. OUR earlier work has demonstrated that appropriate levels of fibroblast growth factor (FGF) signalling are essential for mesodermal and neural development in chick 1 . Expression of brachyury, essential for induction and dif- ferentiation of mesoderm 2 and noggin, involved in the pattering of the nervous system 3 and somites 4 , was differ- entially modulated within 2 h in embryos with altered levels of FGF signalling. This suggested involvement of brachyury and noggin in the early molecular events eli- cited by FGF 1 . Changes in the expression of brachyury and noggin, however, account only partially for the array of abnormalities seen in FGF- and suramin-treated embryos 1 . Some of the spectacular developmental outcomes of altered FGF signalling were irregular notochord forma- tion and shortening of body axis 1 . Goosecoid, an orga- nizer-specific homeobox gene, is important in regulating gastrulation movements, specification of dorsal meso- derm and formation of body axis 5,6 . ERNI (Early Response to Neural Induction), a pre-neural marker, represents cells that receive the initial signals for neural induction 7 . FGF signalling is necessary for the acquisition of neural fate in cultured explants 8 and in whole chick embryos 7 . In the light of these observations, we have studied the expression of goosecoid and ERNI in chick-embryo explants with altered FGF signalling. Further, along with goosecoid and ERNI, we have extended studies on brachy- ury and noggin expression to examine if the effects of altered FGF signalling persist for longer duration. The data reveal that action of FGF is mediated through four developmentally crucial genes; brachyury, goose- coid, ERNI and noggin. This conclusion supports our contention that FGF signalling is required for the devel- opment of neural and mesodermal structures in chick embryo. Materials and methods Freshly laid White Leghorn chicken eggs were obtained from a local hatchery. Suramin and human recombinant bFGF were procured from Sigma (USA). In vitro culture and treatment of chick embryos Eggs were incubated for 18 h at 37.5°C to obtain Ham- burger Hamilton (HH) stage 4 embryos 9 , cultured in vitro by New’s single ring technique 10 and treated with either PC saline, BSA (100 ng/culture), 6 nM bFGF or 2 mM suramin, as described 1 . Embryos treated with either PC saline 11 or PC saline containing BSA served as control. At the end of 2, 4 or 6 h of incubation at 37.5°C, the em- bryos were either fixed in 4% paraformaldehyde for whole mount in situ hybridization or homogenized in TRIZOL reagent (Gibco BRL) for RNA extraction 12 . Study of gene expression DIG-labelled UTPs (Roche Molecular Biologicals, Ger- many) were used to generate the anti-sense riboprobes. cGsc plasmid (kind gift from Dr J. C. Izpisúa-Belmonte,