Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain

Experiments involving tissue recombinations have implicated both early vert ical and later planar signals in the specification and polarisation of the midbrain. Here we investigate the role of fibroblast growth factors in regu lating these processes in the avian embryo. We show that Fgf4 is expressed in the notochord anterior to Hensen's node before transcripts for the earli est molecular marker of midbrain tissue in the avian embryo, En1, are detec ted. The presence of notochord is required for the expression of En1 in neu ral plate explants in vitro and FGF4 mimics this effect of notochord tissue , Subsequently, a second member of the fibroblast growth factor family, Fgf 8, is expressed in the isthmus in a manner consistent with it providing a p olarising signal for the developing midbrain. Using a retroviral vector to express En1 ectopically, we show that En1 can induce Fgf8 expression in mid brain and posterior diencephalon, Results of the introduction of FGF8 prote in into the anterior midbrain or posterior diencephalon are consistent with it being at least part of the isthmic activity which can repolarise the fo rmer tissue and respecify the latter to a midbrain fate. However the abilit y of FGF8 to induce expression of genes which have earlier onsets of expres sion than Fgf8 itself, namely En1 and Pax2, strongly suggests that the norm al function of FGF8 is in maintaining patterns of gene expression in poster ior midbrain. Finally, we provide evidence that FGF8 also provides mitogeni c stimulation during avian midbrain development.