Genetic analysis of tissue interactions required for otic placode induction in the zebrafish

Development of the vertebrate inner ear begins during gastrulation with ind uction of the otic placode. Several embryonic tissues, including cephalic m esendoderm, notochord, and hindbrain, have been implicated as potential sou rces of otic-inducing signals. However, the relative contributions of these tissues have not been determined, nor have any genes affecting placode ind uction been identified. To address these issues, we analyzed otic placode i nduction in zebrafish mutants that are deficient in prospective otic-induci ng tissues. Otic development was monitored by examining mutant embryos for morphological changes and, in some cases, by visualizing expression pattern s of dlx-3 or pax-2.1 in preotic cells several hours before otic placode fo rmation. In cyclops (cyc(-)) mutants, which develop with a partial deficien cy of prechordal mesendoderm, otic induction is delayed by up to 1 h. In on e-eyed pinhead (oep(-)) mutants, which are more completely deficient in pre chordal mesendoderm, otic induction is delayed by 1.5 h, and morphology of the otic vesicles is abnormal. Expression of marker genes in other regions of the neural plate is normal, suggesting that ablation of prechordal mesen doderm selectively inhibits otic induction. In contrast, the timing and mor phology of otic development is not affected by mutations in no tail (ntl) o r floating head (flh), which prevent notochord differentiation. Similarly, a mutation in valentine (val), which blocks early differentiation of rhombo meres 5 and 6 in the hindbrain, does not delay otic induction, although sub sequent patterning of the otic vesicle is impaired. To test whether inducti ve signals from one tissue can compensate for loss of another, we generated double or triple mutants with various combinations of the above mutations. In none of the multiple mutants do the flh or vol mutations exacerbate del ays in placode induction, although val does contribute additively to defect s in subsequent patterning of the otic vesicle. In contrast, mutants homozy gous for both oep and ntl, which interact synergistically to disrupt differ entiation of cephalic and axial mesendoderm, show a delay in otic developme nt of about 3 h. These data suggest that cephalic mesendoderm, including pr echordal mesendoderm and anterior paraxial mesendoderm, provides the first otic-inducing signals during gastrulation, whereas chordamesoderm plays no discernible role in this process. Because val(-) mutants are deficient for only a portion of the hindbrain, we cannot rule out a role for that tissue in otic placode induction. However, if the hindbrain does provide otic-indu cing signals, they apparently differ quantitatively or qualitatively from t he signals required for vesicle patterning, as vol disrupts only the latter . (C) 1999 Academic Press.