AbstractThe functional specialization or redundancy of the ubiquitous 14-3-3 proteins constitutes a fundamental question in their biology and stems from their highly conserved structure and multiplicity of coexpressed isotypes. We address this question in vivo using mutations in the two Drosophila 14-3-3 genes, leonardo (14-3-3ζ) and D14-3-3ϵ. We demonstrate that D14-3-3ϵ is essential for embryonic hatching. Nevertheless, D14-3-3ϵ null homozygotes survive because they upregulate transcripts encoding the LEOII isoform at the time of hatching, compensating D14-3-3ϵ loss. This novel homeostatic response explains the reported functional redundancy of the Drosophila 14-3-3 isotypes and survival of D14-3-3ϵ mutants. The response appears unidirectional, as D14-3-3ϵ elevation upon LEO loss was not observed and elevation of leo transcripts was stage and tissue specific. In contrast, LEO levels are not changed in the wing disks, resulting in the aberrant wing veins characterizing D14-3-3ϵ mutants. Nevertheless, conditional overexpression of LEOI, but not of LEOII, in the wing disk can partially rescue the venation deficits. Thus, excess of a particular LEO isoform can functionally compensate for D14-3-3ϵ loss in a cellular-context-specific manner. These results demonstrate functional differences both among Drosophila 14-3-3 proteins and between the two LEO isoforms in vivo, which likely underlie differential dimer affinities toward 14-3-3 targets.