Molecular studies towards understanding the role of Prep1 (Pbx regulation protein 1) in segmental expression of Hox proteins in the vertebrate hindbrain.

The homeotic genes of the Hox cluster specify segmental diversity along the body axis of vertebrates. This segmental modulation of target genes produces morphologically distinct segments termed rhombomers which define the fate of cells on the antero-posterior body axis of embryos (McGinnins and Krumlauf, 1992). Members of the Hox gene family are sequence-specific DNA binding transcription factors which show high conservation within the DNA binding homeodomain. Consequently, the DNA binding properties of Hox proteins are very similar, raising the question of how proteins with equivalent biochemical properties can achieve in vivo distinct regulatory effects and define precise developmental programs (Ghering et al, 1994; Lu et al, 1996). The mechanisms by which the different Hox protein and their cofactors generate those diverse fates remain unclear (Mann, 1995). Both of evidence supports the model where the DNA binding sites that directly interact with Hox/PBC heterodimers determine which member of the Hox protein family will bind and thereby regulate a given target element (Rauskolb et al, 1993; Popperl et al, 1995; Di Rocco et al, 1997). Furthermore, the Meinox protein family (Prep, Meis and Hht) form specific heterodimers with the PBC proteins acting as cofactors. Thus, the identification of Meinox proteins that can regulate the PBC proteins introduces an additional level of complexity in the regulation of Hox protein function. We have isolated two members of the Meinox family: Prepl and Prep2. We also showed that Prepl and Prep2 represent a new sub class of Meionox subfamily, distinct from Meis. We analyzed the expression of Prepl both in adult and in embryonic murine tissues showing that Prepl is ubiquitously expressed in developing embryos as well as in adult mice. We also showed that Prepl is expressed early in the mouse embryo and is already present in the cytoplasm of murine oocytes, suggesting it involvement in the initial stages of embryonic development. Prepl is always found in a DNA-binding complex with members of the Pbx protein family, whose identity varies among different tissues and during embryogenesis. Thus, the Prepl-Pbx heterodimers show a tissue and development-dependent composition, suggesting specific regulation properties for the different complexes. We found that Prepl, Pbxl and Pbx2 are present in the hindbrain at the time when Pbx is controlling Hox expression. This colocalization is compatible with a role for Prepl as a regulator of Pbx-Hox interaction and function in vivo. Furthermore, we have demonstrated that r4-specific Hoxb1 and Hoxb2 and r5/ r6 specific Hoxa3 enhancers are complex elements containing separated Pbx/Hox (PH) and Pbx/Meinox (PM) binding sites. We have also shown that the PM-PH motifs are elements present in several Hox enhancers and conserved in different species from fly to mammals. Moreover we have shown that both the PM and PH sites are required for the in vitro formation of a Prepl-Pbx-Hox ternary complex. We also showed that, in vivo, the PM site of Hoxbl enhancer is essential for Hoxb2 expression in r4. On the contrary, mutations in the PM site of Hoxb1 and Hoxa3 enhancers do not alter Hoxbl r-4restricted and Hoxa3 r5/r5 restricted expression (Ferretti et al, 2000; Manzanares et al, 2001). We propose that the additional PH sites in Hoxb1 and Hoxa3 enhancers modulate the activity of those enhancers and may explain the different in vivo behaviors. We demonstrated, that in vitro, one of the additional PH site (R2) in the Hoxb1 enhancer exerts an inhibitory effect on the ternary complex formation. We have further demonstrated that the PM-PH elements from Hoxb1 and Hoxb2 enhancers are sufficient to direct the r4-specific expression of the reporter gene in r4 of electroporated chicken embryos. Moreover, in vivo like in vitro, the presence of R2 exerts an inhibitory effect by blocking the r4-restricted expression of the reporter gene in the chicken embryos. Finally we have inactivated Prepl both in mouse and in zebrafish showing that Prepl has an essential role in embryonic development. Prepl -/- mice show an embryonic lethal phenotype, dieing in uterus at 16.5 d.p.c. Gross morphologic analysis of Prepl-/- embryos revealed massive subcutaneous edema, generalized pallor diminished vascularization, smaller livers and abnormally orientated forelimbs. All these abnormalities observed in Prepl-/- embryos and those presented by Pbxl-/-embryos are similar, suggesting a genetic interaction between these two factors. Inactivation of prep1.1 in zebrafish results in a lethal phenotype, prep1.1 morphants show severe alteration in cranial-facial chondrogenesis resulting from of the incapacity of neural crest to differentiate into chondroblast.