Courtesy of Dirk Meyer, University of Freiburg, Germany. Sur mutant reprinted from Pogoda, H. M. et al. with permission from Elsevier Science © (2000).

Key morphogenetic events during vertebrate embryogenesis require the signalling molecule Nodal. These events include establishing and maintaining the embryonic organizer, inducing mesodermal formation, and specifying the left–right axis. As a result, vertebrate nodal genes, and their downstream targets, have been well studied, and it has been found that nodal expression is controlled by both positive and negative autoregulatory feedback loops. The recent identification of the zebrafish FoxH1 gene has added a new component to these regulatory pathways.

Foxh1 (or Fast1) is a forkhead (Fkh) transcription factor that has been shown to transmit inductive Nodal signals in frogs. However, in the absence of a foxH1 mutant, the relevance of this data remained unconfirmed. This problem was solved when two research groups recently tested foxH1 as a candidate gene for the zebrafish mutant, schmalspur ( sur), which shares certain phenotypic features with other nodal-pathway mutants. Both groups identified foxH1 mutations in sur zebrafish — Pogoda et al. also identified foxH1 mutations in another allele, called uncle freddy — and subsequently showed them to be loss-of-function mutations in the conserved Fkh domain.

FoxH1 partly functions through its Fkh domain, which probably mediates its DNA-binding activity and nuclear localization. With the sur mutations to hand, Pogoda and colleagues investigated the biological activity of FoxH1 by making FoxH1 chimeric proteins that could repress or activate FoxH1 target genes — when introduced into zebrafish embryos, these proteins antagonized Nodal signalling or rescued Nodal-pathway mutant phenotypes, respectively. When Fkh-domain mutations from sur mutants were introduced into these chimeric proteins, they produced no phenotypic effect in zebrafish embryos, showing that these mutations abolish the function of the Fkh domain and the chimeric proteins' effect on Nodal signalling.

Pogoda and colleagues next investigated where in the nodal pathway FoxH1 acts by examining the expression of two zebrafish nodal -related genes, squint (sqt) and cyclops (cyc) in sur embryos. In early sur embryos, sqt and cyc were initially expressed — although at reduced levels — and their expression was rapidly lost with development. Compared to embryos that lack Nodal signals, sur mutants have only mildly affected mesodermal and endodermal development, suggesting that mesoderm formation and differentiation is initiated, as supported by these findings. These results also indicate that foxH1 is unlikely to transmit inductive nodal signals but rather maintains and regulates nodal's expression. The expression of the Nodal antagonist antivin is also rapidly lost in sur mutants, suggesting that FoxH1 functions in both positive and negative nodal regulatory pathways.

Future work should reveal how FoxH1, and its related proteins, function to modulate and amplify nodal gene expression to determine its intensity, range and duration.