Pbx and Meis form dimeric and trimeric complexes with Hox proteins in vitro and these complexes are thought to modulate Hox activity, primarily by conferring high-specificity

Vertebrate hox genes, like their Drosophila counterparts the HOM-C genes, play essential roles during embryogenesis. For instance, their expression in overlapping domains along the anteroposterior (AP) axis provides a ‘hox code’ that specifies AP positional identity, and changes in hox gene expression lead to homeotic transformations in the AP axis, wherein anterior structures acquire the character of more posterior structures (reviewed by Krumlauf, 1994). Genetic analyses in Drosophila revealed that many HOM-C genes require the extradenticle (exd) and homothorax (hth) genes for proper function (Rauskolb et al., 1993; Rieckhof et al., 1997). Homologs of exd and hth are encoded by the vertebrate pbx (Kamps et al., 1990; Monica et al., 1991; Nourse et al., 1990; Vlachakis et al., 2000) and meis/prep (Berthelsen et al., 1998; Moskow et al., 1995; Nakamura et al., 1996) gene families, respectively. Recently, the zebrafish lazarus mutation, which disturbs segmental patterning in hindbrain and trunk, was cloned (Popperl et al., 2000) and found to encode the previously reported pbx4 gene (Vlachakis et al., 2000), suggesting a role for pbx genes also in vertebrate development. Vertebrate meis genes also likely play a role, as misexpression of Xenopus Meis3 leads to abnormalities of the AP axis (Salzberg et al., 1999). Pbx and Meis form dimeric and trimeric complexes with Hox proteins in vitro and these complexes are thought to modulate Hox activity, primarily by conferring high-specificity DNA-binding (reviewed by Mann and Affolter, 1998). An in vivo role for such complexes is suggested by the effect of dominant negative forms of Hth in Drosophila (Jaw et al., 2000; Ryoo et al., 1999), by the finding that dimers (e.g. Chang et al., 1997; Knoepfler et al., 1997) and trimers (Ferretti et al., 2000; Shen et al., 1999) can be reconstituted in cell extracts and by the observation that Meis, Pbx and Hox binding sites are present in several Hox-dependent promoters (Ferretti et al., 2000; Jacobs et al., 1999; Pöpperl et al., 1995; Ryoo et al., 1999). pbx4, meis3 and hoxb1b are co-expressed in the caudal hindbrain primordium of the zebrafish embryo and Pbx4, Meis3 and Hoxb1b form complexes in vitro (Vlachakis et al., 2000). Here we explore the role of these proteins during zebrafish development and test whether they need to interact to function in vivo. We find that Hoxb1b and Pbx4 act together to induce ectopic hoxb1a expression in rhombomere (r) 2. In marked contrast, Hoxb1b and Pbx4 together with Meis3 induce massive rostral expression of several hindbrain genes (hoxb1a, hoxb2, krox20 and valentino) and cause anterior truncations, apparently due to the transformation of rostral (forebrain and midbrain) fates to caudal (hindbrain) fates. This transformation is extensive enough that we observe excess Mauthner neurons (normally found in r4) anteriorly. These effects are dependent on Meis3 and Hoxb1b having intact Pbx interaction domains, suggesting that they interact with Pbx4 in vivo. Our results also indicate that Meis3 must interact with Pbx4 to access the nucleus. Since meis3, pbx4 and hoxb1b are co-expressed in the 1299 Development 128, 1299-1312 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV2653