Identification of ephrin-A3 and novel genes specific to the midbrain-MHB in embryonic zebrafish by ordered differential display

Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). We set out to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIMIs1-3) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. We therefore searched for genes that may be either up- or down-regulated by overexpression of LIMIs1-3 by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function was repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encoded Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of our strategy. We identified four other partial cDNA clones that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum. and three cDNAs expressed in neural crest cells including those derived from the midbrain level. The ephrin-A3 gene was specifically expressed in posterior tectum in a gradient that decreased anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos, the superior/inferior colliculi, mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.