Identification and characterisation of three novel eyes absent genes in the cranial sensory placodes, the developing inner ear and lateral line in the zebrafish Danio rerio

Sequence analyses of the zebrafish eya2, eya3 and eya4 proteins have revealed two highly conserved domains; the eya homology region (eyaHR) and eya domain 2 (ED2). Moreover all three eya proteins contain sequence motifs equivalent to the family of phosphohydrolases, recently described for mouse Eya proteins by LI (2003) and RAYAPUREDDI (2003). Therefore zebrafish eya 2-4 proteins function as transcriptional co-factors which possess protein phosphatase activity. As a first step towards a functional analysis of the eya gene during sensory organ development, the expression patterns of the three zebrafish eya genes were analysed and compared. Expression pattern analysis was carried out by RNA in situ hybridisation. Both eya2 and eya4 are expressed in distinct patches within the cranial placodes and their derivates, otic vesicle, and in the neuromasts of the lateral line system. Eya2 expression is observed in the several cranial placodes and their cranial ganglia. Eya4 transcripts are found in some of the corresponding cranial ganglia and in developing somites. The significance and biological relevance of this observation is tested by overexpression experiments and the analysis of gene knock-down experiments by using in vitro synthesised eya1, eya2, and eya4 mRNA as well as antisense eya2 and eya4 Morpholino oligonucleotides respectively. Ectopically upregulated function of eya1, eya2 and eya4 revealed a reduced level of apoptosis-mediated cell death, while the loss-of-function of eya2 and eya4 triggers programmed cell death. These results maintain the idea of apoptosis affective activity, which was described for the loss-of-function for eyes absent in Drosophila, zebrafish (eya1/dor-eared) and mouse(Eya1-/-), as a general function of eya gene relatives.