EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is requir ed for and sufficient to induce the formation of midbrain and cerebellar st ructures. To address through what genetic pathways FGF8 acts, we examined t he epistatic relationships of mid/hindbrain genes that respond to FGF8, usi ng a novel mouse brain explant culture system. We found that En2 and Gbx2 a re the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expres sion in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax 5 do not require the En genes for initiation of expression, but do for thei r maintenance, and Pax6 expression is expanded caudally into the midbrain i n the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/ hindbrain region, forebrain mutant explants were treated with FGF8 and, sig nificantly, the EN transcription factors were found to he required for indu ction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN p roteins, and a factor other than FGF8 could be involved in initiating norma l Pax5 expression in the mesencephalon/metencephalon. The En genes also pla y an important, but not absolute, role in repression of Pax6 in forebrain e xplants by FGF8, Previous Gbx2 gain-of-function studies have shown that mis expression of Gbx2 in the midbrain can lead to repression of Otx2, However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in m idbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mu tant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2, Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repres sion of Wnt1. This is the first such epistatic study performed in mouse tha t combines gain-of-function and loss-of-function approaches to reveal aspec ts of mouse gene regulation in the mesencephalon/metencephalon that have be en difficult to address using either approach alone.