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Figure 2. The C-terminus of FoxD4/FoxD4L1 from frog and mammals contains a novel specific conserved motif, which we term the Fox homology motif 2 (FH2).(A) The sequence logo of the 10 amino acid FH2 motif. (B) The FH2 motif is outlined in red on the FoxD4/FoxD4L1 sequence alignment.
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Figure 3. Conserved amino acids in the extreme C-terminus of FoxD4/FoxD4L1 proteins.(A) CLUSTALW alignment [64], viewed in ESPript [65], of the extreme C-terminal region of human FoxD4 (UniProtKB/Swiss Prot accession number Q12950), human FoxD4L1 (Q9NU39), mouse FoxD4 (Q60688), Danio FoxD4L1 (O73784) and Xenopus laevis FoxD4L1 (Q9PRJ8). The black boxes highlight identical amino acids, the light boxes highlight conserved amino acids and the bold letters indicate identical amino acids within a conserved region. The blue line denotes the amino acids in the Xenopus sequence predicted to form an α-helix, and the red line denotes Motif 6 (Fig. 1A). Arrows denote amino acid substitutions in the C-terminal mutants used in this study (L>A; Q>R; GARQ>GARG; GARQ>GARP). (B) Amino acid changes made in the C-terminal mutants used in this study.
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Figure 4. Mutant FoxD4L1 proteins are adequately expressed.Western blots of lysates from oocytes injected with mRNAs encoding C-terminus mutants (A) or Acidic Blob mutants (B) show expression of each mutant protein. Un, lysates from uninjected oocytes; Wt, lysates from wild-type FoxD4L1 injected oocytes.
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Figure 5. The ability to down-regulate zic3 and irx1 is lost in the GARP mutant.(A) The FoxD4L1 C-terminal mutant expressing clones, marked by nuclear β-Gal (pink dots), are located in the neural ectoderm. For L>A, Q>R and GARG mutants, the βGal labeled cells are less intensely stained (blue) than their neighboring cells (e) expressing endogenous levels of zic3 or irx1. For GARP, the βGal labeled cells are stained at the same intensity as the neighboring cells (e). Insets are higher magnifications of the clone, the position of which is indicated on the whole embryo by a bracket. For zic3, images are dorsal views with vegetal pole towards the bottom; for irx1, images are frontal views with dorsal towards the top. (B) The percentage of embryos in which the FoxD4L1 C-terminal mutants caused down-regulation of zic3 or irx1 in the dorsal neural ectoderm. Numbers on each bar indicates sample size; * indicates significant difference from wild type (WT) at the p<0.001 level. Data for WT, ÎRII-Cterm and A6 are from [39].
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Figure 6. FoxD4L1 mutant proteins have access to the nucleus in a pattern similar to wild-type FoxD4L1.(A) Left panel: epifluorescence image of wild-type, myc-tagged FoxD4L1 protein in neural ectoderm of stage 13 embryo. Tagged protein is in the cytoplasm and in the nucleus (arrows). Middle panel: confocal image of a similar sample shows that the protein (green) is localized in the periphery of the nucleus (blue) where chromatin is concentrated in non-mitotic cells. Right panel: example from a similar sample in which a 32-channel signature spectral curve analysis was performed. Red pixels around the periphery of the nucleus represent sites of DNA (blue) and protein (green) colocalization. (B) Left panel: DAPI nuclear staining of cells in the superficial neural ectoderm of stage 12 embryo. Middle panel: Myc-tagged GARP protein (green), like wild-type protein, is found in the cytoplasm and in the periphery of the nucleus. Right panel: a 32-channel signature spectral curve analysis was performed to demonstrate with confidence nuclear localization of the tagged protein. Magenta pixels represent sites of DNA (blue) and protein colocalization. (C) Left panel: DAPI nuclear staining of cells in the deep layer of the stage 14 neural plate. Middle panel: Myc-tagged AB4 protein (green) also is found in the cytoplasm and in the periphery of the nucleus. Right panel: a signature spectral curve analysis was performed: magenta pixels represent sites of DNA (blue) and protein colocalization. White bars indicate 7 µm.
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Figure 7. Grg4 binds to the FoxD4L1 C-term mutants.(AâD) Myc-tagged versions of wild-type (WT), as well as mutants harboring amino acid substitutions downstream of the Eh-1 domain (QR, GARG, LA, GARP) in FoxD4L1 were expressed in Xenopus oocytes along with HA-tagged wild-type Xenopus Grg4. Co-immunoprecipitation (IP) and Western blot (WB) analyses of oocyte lysates expressing HA- and Myc-tagged constructs are indicated. (A) All four constructs bind with Grg4. The control panels (BâD) show that the IPs contain similar levels of FoxD4L1 wild-type and mutant proteins (B), as do the direct lysates (C). Grg4 expressing lysates also show similar levels of this protein (D). Note: Although the co-expression of Grg4 along with the wild-type and mutant Fox constructs shows similar protein levels and binding in the IPs, it is worth noting that there is a marked reduction in expression of all Fox proteins in the presence of Grg4. This may be due to degradation, rather than competition for ribosomes that affects translation, since Grg4 levels are not affected.
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Figure 8. Conserved amino acids in the Acidic Blob region of FoxD4/FoxD4L1 proteins that were mutated for this study.(A) CLUSTALW alignment of the N-terminal region including the Acid Blob (AB, denoted by red line), as in Figure 3. The highly conserved IDIL sequence is predicted to form a short β-strand (green line). Six amino acids, denoted by the blue line, were deleted in the AB1 construct. The amino acid substitutions made in the AB2 and AB4 constructs are noted. (B) Predicted protein folding within the Acidic Blob of the wild-type (Wt) and AB mutated Xenopus FoxD4L1 proteins. Red lines denote the short β-strand, and the blue ribbon denotes a 1.7 turn α-helix predicted to form by the 6 alanine residues. Dashes over the aspartic (D) and glutamic (E) acid residues indicate negative charges.
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Figure 9. The ability to up-regulate gem and zic2 is lost in the AB4 mutant.(A) The FoxD4L1-AB mutant expressing clones, marked by nuclear βGal (pink dots), are located in the neural ectoderm. For AB1 and AB2, the βGal labeled cells are more intensely stained (darker blue) than their neighboring cells (e) expressing endogenous level of gem or zic2. For AB4, the βGal labeled cells are stained at the same intensity as the neighboring cells (e). Insets are higher magnifications of the clone, the position of which is indicated on the whole embryo by a bracket. For gem, images are dorsal views with vegetal pole to the top; for zic2, images are vegetal views with dorsal to the top. (B) The percentage of embryos in which the FoxD4L1-AB mutants caused up-regulation of gem or zic2 in the dorsal neural ectoderm. The data for the ÎAB mutant (14aa deletion in Fig. 8A) is shown for comparison. Numbers above each bar indicates sample size; * indicates significant difference from wild type (WT) at the p<0.001 level. Data for WT and ÎAB are from [39].
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Figure 10. The ability to ectopically induce gem and zic2 is lost in the AB4 mutant.(A) Ventral ectopic expression of gem and zic2 after injection of each FoxD4L1-AB mutant mRNAs into an epidermal precursor blastomere. Clones are indicated by βGal-positive pink dots. In AB1 and AB2 clones, most cells exhibit a high level of expression (dark blue stain), compared to neighboring cells showing endogenous expression levels (e). Cells in the AB4 clones do not express the genes at levels above endogenous (e). gem-AB1, zic2-AB1, and zic2-AB2 are ventral views with animal cap to the bottom; gem-AB2, gem-AB4, zic2-AB4 are animal cap views. (B) The percentage of embryos in which the FoxD4L1-AB mutants induced gem or zic2 expression in the ventral ectoderm. Labeling is as in 9B.
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