Members of the Meis/Prep Family Synergize: with Pbx4 and Hoxb1b in Prompting Hindbrain fates in the zebrafish

Publication Date

June 2001

Document Type

Doctoral Dissertation


Graduate School of Biomedical Sciences, Department of Biochemistry and Molecular Pharmacology


Zebrafish; Homeodomain Proteins; Rhombencephalon; Academic Dissertations


Hox as well as Meis proteins are known to bind DNA as heterodimers with members of the Pbx family, and it is believed that such complexes mediate the in vivo functions of Hox and Meis. To begin exploring the role of hoxb1b and meis3 in vertebrate development, we isolated and characterized a zebrafish pbx cDNA which encodes a novel member of the pbx family, which we called pbx4. In situ analysis revealed that pbx4 RNA is maternally deposited and is detected throughout the zebrafish embryo during blastula stages. It becomes excluded from ventroanterior structures at late gastrula stages and is detected within the developing central nervous system during segmentation stages. pbx4 expression overlaps with that of hoxb1b and meis3, in the region of the presumptive caudal hindbrain during gastrula stages. In vitro binding experiments revealed that Pbx4/Meis3 and Pbx4/Hoxb1b, as well as a novel trimeric complex containing Pbx4, Meis3 and Hoxb1b form in vitro.

Thus, protein complexes of different combinations of Pbx4, Meis3 and Hoxb1b form in vitro and importantly pbx4, meis3 and hoxb1b are coexpressed in a domain at the level of the presumptive caudal hindbrain during zebrafish gastrula stages. These findings raised the possibility that similar complexes may exist in vivo and may be involved in the specification of distinct developmental fates.

To address this possibility we overexpressed meis3,pbx4 and hoxb1b in zebrafish embryos and we tested for the effect on endogenous gene expression, morphology and neuronal specification. Our results demonstrate that Hoxb1b/Pbx4/Meis3-containing complexes induce extensive expression of several hindbrain genes (hoxb1a, hoxb2, krox20 and valentino) anterior to their normal expression domains, and mediate the transformation of anterior (forebrain and midbrain) fates to posterior (hindbrain) ones, including the formation of excess ectopic Mauthner neurons. Ectopic expression of Hoxb1b/Pbx4/Meis3-containing complexes also leads to truncation of the embryonic axis anteriorly. In contrast, Hoxb1b/Pbx4 expression induces ectopic expression of only hoxb1a (primarily in r2), but does not mediate axial truncations, and Hoxb1b (or its mouse homolog, HoxA1) has been reported to induce an ectopic pair of Mauthner neurons in r2 (Alexandre et al., 1996). Thus, binding of Meis3 to Hoxb1b/Pbx4 generates Hoxb1b/Pbx4/Meis3-containing complexes that have qualitatively (e.g. induction of hoxb2 expression) and quantitatively (e.g. larger number of ectopic Mauthner neurons) different effects than Hoxb1b/Pbx4-containing complexes.

These results suggest that Meis3/Pbx4/Hoxb1b-containing complexes may be responsible for specification of hindbrain fates in vivo. In addition to meis3, three other members of the meis/prep gene family are expressed during early embryogenesis in zebrafish. Analysis of gene expression patterns revealed both common as well as unique spatial and temporal expression patterns for each of these genes. This finding raises the question of whether all family members are functionally similar to meis3 or meis3 performs unique functions. To address this question we overexpressed meis1.1, meis2.2 and prep1 in zebrafish embryos and we asked whether they are able to induce hindbrain fates like meis3 does. Overexpression of any Meis protein, or Prep, along with Pbx4 and Hoxb1b resulted in embryos that were truncated anteriorly and exhibited massive ectopic hoxb1a and hoxb2 expression anterior to their normal expression domains. Furthermore, in vitro analysis demonstrated that they are all able to form dimers with Pbx4 in vitro. In addition, analysis of their subcellular localization defined Pbx4 interaction as a prerequisite for nuclear localization of all Meis and Prep proteins. Thus, at least in the overexpression assay there are no functional differences among meis/prep genes.

These results raise the question of what is exactly the function of Meis/Prep proteins. Is binding to Pbx proteins and to DNA their only function, or do they have additional roles? To address this question we performed a deletional analysis of Meis3 protein and we tested the requirement of each domain in the overexpression assay. Our experiments revealed that the domain N-terminal to the Pbx-Interaction-Domain (PID) as well as the domain C-terminal to the Homeodomain are not required for the function of Meis3, at least in the overexpression assay. Furthermore, the homeodomain and the domain between the PID and the homeodomain are not required. From our previous analysis (Vlachakis et al., 2001) we know that the PID is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates. Our deletion analysis extended this fmding showing that the PID is also sufficient to provide the Meis3 function in vivo, at least in our overexpression assay. Furthermore, a mutant PID that does not bind Pbx, when fused to the Pbx4 homeodomain induced hindbrain fates upon overexpression along with Hoxb1b. This finding suggests that the PID (motifs M1, M2 and the domain in between them, ID) besides binding to Pbx may also bind another protein that is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates.

Taking all our results together, we propose the following roles for Meis proteins in the transcriptional activation complexes. First, they are involved in the nuclear localization of Pbx4. Second, they bind to DNA as heterodimers with Pbx4 facilitating binding of Hoxb1b to Pbx4, which occurs only on DNA. In doing so, they provide the specificity for DNA binding since the Meis3/Pbx4 dimer first recognizes the "hox response element" and then the Hox protein is recruited. Third, they stabilize the binding of Hoxb1/Pbx4 complex on DNA. Fourth, they are responsible for recruiting additional factors to DNA, necessary for activation of target genes. The complicate and dynamic spatial and temporal expression patterns of the meis/prep genes, suggest that they are involved in many different processes during embryogenesis, as well as in the adult organism. We believe that one or more members of the Meis/Prep family execute some of the functions listed above at different times and places during development, although all member are probably capable of executing all these functions.


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