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Meis family proteins are required for hindbrain development in the zebrafish

Seong-Kyu Choe*, Nikolaos Vlachakis* and Charles G. Sagerström{dagger}

Department of Biochemistry and Molecular Pharmacology, and Program in Neuroscience, University of Massachusetts Medical School, Worcester, MA 01655, USA
* These authors contributed equally to this work



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  		Fig. 1. Prep1 retains functions similar to Meis3. (A) Prep1 protein. Letters indicate the name of individual domains; the Meinox domain includes the M1, I and M2 domains. Numbers on top represent amino acid positions in Prep1 and numbers on the bottom indicate percent identity of each domain between Prep1 and Meis3. (B-I) Expression pattern of prep1 during zebrafish embryogenesis. An antisense (B,D,E,F,H) or sense (C,G,I) probe for prep1 was hybridized to zebrafish embryos at the two-cell stage (1 hpf; B,C), early gastrula (6 hpf; D), late gastrula (9 hpf; E), early segmentation (13 hpf; F,G) and late segmentation (25 hpf; H,I). (B,C) Lateral views with animal pole towards the top. (D) An animal pole view. (E) A lateral view with dorsal towards the right and anterior towards the top. (F-I) Dorsal views with anterior towards the left. (J) Prep1 binds to Pbx4/Lzr in vitro. Pbx4/Lzr was in vitro transcribed in the presence of 35S-methionine together with MycMeis3 (lanes 1, 2), MycPrep1 (lanes 3, 4) or by itself (lanes 5, 6), immunoprecipitated with anti-Myc antibody, resolved on a 10% SDS-PAGE gel and exposed to film. (K,L) Prep1 is brought to the nucleus by Pbx4/Lzr. One- to two-cell stage embryos were injected with 300 pg MycPrep1 mRNA by itself (K) or together with 300 pg pbx4/lzr mRNA (L), raised to 5 hpf and stained with anti-Myc antibody. (M-O) Prep1 induces hindbrain fates in the same way as Meis3. One- to two-cell stage embryos were injected with 500 pg lacZ RNA (M), meis3+pbx4+hoxb1b mRNA (N; 165 pg each), or prep1+pbx4+hoxb1b mRNA (O; 165 pg each), raised to 25 hpf and analyzed for hoxb2 expression by in situ hybridization. All three embryos are dorsal views with anterior to the left. (P) MycMeis3 and MycPrep1 are expressed at similar levels. One- to two-cell stage embryos were injected with 300 pg MycMeis3 mRNA or MycPrep1 mRNA, raised to 5 hpf, lysed, resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Myc antibody.

 


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  		Fig. 2. Meis constructs. Meis3 deletion constructs (A) and fusions with Pbx4/Lzr (B) are shown schematically on the left. Columns on the right indicate whether each protein binds Pbx4/Lzr and displays activity in vivo. Asterisks indicate two constructs that have drastically reduced Pbx binding and in vivo activity, but retain some function (see text for details). na, not applicable [because the fusion constructs were designed not to require Pbx binding (see text for details)]. Meis3 is blue, except for the homeodomain (HD; white) and M1 and M2 (red). Yellow indicates sequences from the Prep1 C terminus that were inserted in place of the I domain in the C->IMeis3 construct. The M1 and M2 domains in several fusion constructs (B) were mutated to abolish Pbx binding (purple). These domains are referred to as BM1 and BM2 in the text. Pbx4/Lzr is green.

 


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  		Fig. 3. The M1 domain is sufficient to confer Meis activity. (A) All constructs used are expressed at comparable levels in embryos. One- to two-cell stage embryos were injected with 300 pg of each mRNA encoding Myc-tagged constructs as indicated at the top of each lane. Embryos were raised to 5 hpf, lysed, resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Myc antibody. (B-M) Analysis of Pbx4/Lzr-mediated nuclear localization of Meis constructs. One- to two-cell stage embryos were injected with 300 pg of each mRNA as indicated at the bottom right of each panel, raised to 5 hpf and stained with anti-Myc antibody. All Meis constructs were Myc-tagged, whereas Pbx4/Lzr was untagged. (N-U) Analysis of in vivo activity of Meis constructs. One- to two-cell stage embryos were injected with 500 pg lacZ RNA (control) or 165 pg of each mRNA as indicated in the lower right corner of each panel, raised to 25 hpf and analyzed for expression of hoxb1a (N-Q) or hoxb2 (R-U) by in situ hybridization. All embryos are dorsal views with anterior to the left. (V) Meis3-Pbx4 fusion constructs do not bind endogenous Pbx. One- to two-cell stage embryos were injected with 300 pg MycMeis3 (lane 1) or MycBMNPbx4 (lane 2) and raised to 10 hpf. Embryos were lysed, immunoprecipitated with anti-Myc, resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Pbx4 antiserum (left panel) or anti-Myc antiserum (right panel). Note that the BMNPbx4 fusion protein in lane 2 of the left-hand panel is detected by the anti-Pbx4 antiserum. MycMeis3 and BMNPbx4 are the same size. IgH, antibody heavy chain; IgL, antibody light chain. (W) Meis3-Pbx4 fusion proteins remain stable at 12 hpf. One- to two-cell stage embryos were injected with 300 pg MycBMNPbx4 or MycIPbx4 mRNA and harvested at 5 hpf or 12 hpf. Embryos were lysed, and three embryo equivalents were resolved on a 10% SDS-PAGE gel, western blotted and probed with anti-Myc antiserum.

 


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  		Fig. 4. Loss of Meis function disrupts hindbrain development. (A) {Delta}CPbx4 construct with amino acid positions indicated at the bottom. The red boxes indicate the PBC-A and PBC-B domains. The blue domain represents a biotin tag introduced at the C terminus. (B-D) {Delta}CPbx4 sequesters Meis3 in the cytoplasm. One- to two-cell stage embryos were injected with 300 pg of Myc{Delta}CPbx4 (B), MycMeis3 (C) or {Delta}CPbx4 +MycMeis3 (D), raised to 12 hpf and stained with anti-Myc antibody. (E-R) {Delta}CPbx4 affects gene expression in the hindbrain. One- to two-cell stage embryos were injected with 300 pg of {Delta}CPbx4 mRNA (F,H,J,L,N,P,R) or lacZ mRNA (E,G,I,K,M,O,Q), raised to 14 hpf (M,N) or 24 hpf (E-L,O-R) and analyzed by in situ hybridization for the genes indicated at the bottom of each panel. Black asterisks indicate the level of the otic vesicle on the right side of each embryo. Black asterisks on left side in Q, and R indicate rhombomere boundaries. Black triangle in R indicates region of strong pax6 expression. (S-W) {Delta}CPbx4 affects neuronal differentiation. One- to two-cell stage embryos were injected with 300 pg of {Delta}CPbx4 mRNA (S,U,V) or lacZ mRNA (T,W), raised to 48 hpf (S,T) or 28 hpf (U-W) and stained with anti-islet (S,T) or 3A10 (U-W) antibody. Black asterisks indicate the otic vesicle and rhombomeres are numbered on the left.

 

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© The Company of Biologists Ltd 2002