Traverso EE et al. (2007), Disruption of the dynamic sub-cellular localiza...

XB-ART-35783

Differentiation 2007 Dec 01;7510:947-56. doi: 10.1111/j.1432-0436.2007.00179.x.

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Disruption of the dynamic sub-cellular localization of the Xenopus tumorhead protein causes embryonic lethality at the early gastrula transition.

Traverso EE , Cho MS , Wu CF , Sater AK , Larabell CA , Kloc M , Etkin LD .

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The Xenopus laevis tumorhead (TH) protein, a positive regulator of cell proliferation during embryogenesis, shuttles from the cell periphery into the nucleus during embryogenesis. In these studies, we performed a detailed analysis of TH's subcellular localization pattern to characterize its dynamic behavior. We found that TH exhibits distinct patterns of localization in different germ layers. At the blastula stage, TH is present in the apical cell periphery of prospective mesodermal and ectodermal cells. At the gastrula stage, TH is distributed throughout the entire cytoplasm of prospective mesodermal and ectodermal cells, whereas it shows nuclear localization in presumptive endodermal cells. TH moves into the nucleus of mesodermal and ectodermal cells during the neurula and early tailbud stages. To understand if TH is regulated by changes in its subcellular localization, we used a TH mutant containing signals for farnesylation and palmitoylation to tether the protein to the plasma membrane. Ubiquitous overexpression of this mutant causes embryonic lethality at the early gastrula transition. Further examination using TUNEL assays indicated that wild-type TH overexpression induces apoptosis during gastrulation, and that this effect is exacerbated by the overexpression of the membrane-bound TH mutant. Taken together, our results suggest that changes in the sub-cellular localization of the TH protein are important for its function because blocking the nuclear translocation of overexpressed TH increases apoptosis and causes embryos to die. Our data also suggest that TH plays a role outside the nucleus when it is present at the cell periphery.

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Species referenced: Xenopus laevis
Genes referenced: myc trhd
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	Fig. 1 Tumorhead (TH) constructs used in these studies and their expression. (A) We used a construct for the expression of wild-type (WT)-TH and an additional construct for the expression of a TH mutant containing the sequence TCQCCVIM at the C-terminus, which confers signals for the covalent attachment of the palmitoyl and farnesyl (P/F) membrane anchoring elements. WT-TH and membrane-bound (MB)-TH were expressed as Myc epitope-tagged (MT) proteins to permit detection using anti-Myc antibodies. (B) Embryos were injected with 0.5 ng of mRNA encoding WT-TH or MB-TH at the two-cell stage into both blastomeres and grown to the gastrula stage (stage 12) or the neurula stage (stage 17). Cell extracts were prepared from these embryos as well as from uninjected (Un) embryos. The equivalent of a half embryo for each preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting using an antibody against the Myc epitope. Arrows point to the characteristic bands that correspond to the TH protein.
	Fig. 2 Subcellular localization pattern of wild-type tumorhead (WT-TH). mRNA (0.1 ng) encoding WT-TH was injected into embryos at the two-cell stage into both blastomeres. The embryos were grown until the midblastula (stage 8), early gastrula (stage 10), neurula (stage 17), or early tailbud (stage 22), and then fixed and sectioned. Immunostaining was performed with anti-Myc antibody and detection was done with fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Nuclei were stained with the Hoechst dye. (A) At the midblastula stage (stage 8), WT-TH is detectable in the apical surface of cells from the animal cap and the marginal zone, with a minor apical/basal gradient of cytoplasmic staining. (B) At the early gastrula stage (stage 10), WT-TH dissociates from the cell membrane and spreads through the cytoplasm in presumptive ectodermal and mesodermal cells, whereas it shows nuclear localization in endodermal cells. Ectoderm images show the animal cap. Mesoderm images show a section of the involuting marginal zone. Endoderm images show the vegetal yolk mass. (C) Examples of the nuclear translocation that occurs in the epidermal ectoderm during neurulation, and the presence of WT-TH in the nucleus of some endodermal cells at the neurula stage (stage 17). Ectoderm images show the epithelial layer of epidermal ectoderm. Endoderm images show the archenteron roof. Arrows point to the fibrous and punctuated patterns of subnuclear localization in ectodermal cells and the perinuclear localization pattern in endodermal cells. (D) Example of the predominant nuclear localization of WT-TH in somitic mesodermal cells at the early tailbud stage (stage 22).
	Fig. 3 Summary of the subcellular localization of wild-type tumorhead (WT-TH) during embryogenesis. WT-TH exhibits a dynamic and germ-layer-specific pattern of subcellular localization. The protein shows localization to the apical surface of ectodermal and mesodermal cells at the midblastula stage, with a minor apical/basal gradient of cytoplasmic localization. At the gastrula stages, a portion of WT-TH distributes throughout the cytoplasm of ectodermal and mesodermal cells, whereas it is nuclear in endodermal cells. WT-TH starts to migrate into the nucleus of ectodermal and endodermal cells at the neurula stages, localizing predominantly in the nucleus of cells in all germ layers by the early tailbud stage.
	Fig. 4 Subcellular localization pattern of membrane-bound tumorhead (MB-TH). mRNA (0.1 ng) encoding MB-TH was injected into embryos at the two-cell stage into both blastomeres. The embryos were grown until the cleavage (stage 6), midblastula (stage 8.5), or gastrula (stages 11–12) stages, and processed for immunostaining with anti-Myc antibody and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Nuclei were stained with the Hoechst dye. (A) MB-TH localizes predominantly to the cell periphery at the 32-cell stage (stage 6) although it shows a minor punctate cytoplasmic staining. (B) MB-TH localizes mainly to the cell periphery throughout the embryo at the midblastula stage (stage 8.5), showing no preference to the apical surface of the cells. (C) During gastrulation, MBTH exhibits predominant localization to the cell periphery in all three germ layers. Ectoderm images show the epithelial layer of the epidermal ectoderm of a stage 12 embryo. Mesoderm images show the involuting dorsal mesoderm of a stage 12 embryo. Endoderm images show the endodermal yolk mass of a stage 11 embryo.
	Fig. 5 Ubiquitous overexpression of wildtype tumorhead (WT-TH) causes shortening of the A/P axis, whereas membrane-bound tumorhead (MB-TH) overexpression results in lethality at the early gastrula transition. (A–C) 0.5 ng of in vitro synthesized mRNA encoding WT-TH or MB-TH was injected into embryos at the two-cell stage into both blastomeres. (A) Embryos at the tailbud stage (stage 26) were visualized under bright field illumination with a stereoscope. Overexpression of WT-TH causes shortening of the A/P axis. (B) Embryos at the late gastrula stage (stages 12–12.5) were visualized under bright field illumination. The blastopore (arrow) is misshaped and fails to close properly in embryos expressing the MB-TH mutant. (C) Embryos expressing MB-TH die throughout gastrulation. Only 9% of the embryos expressing MB-TH survive to the late gastrula/early neurula stage (stage 13). (D) One nanogram of transcripts encoding WT-TH or MB-TH was injected into embryos at the two-cell stage into both blastomeres. Embryos were grown until the early gastrula stage (stage 10) and were processed for TUNEL assays. MB-TH induces cell death in the lateral region (arrowhead) and the blastopore (arrows) in most of the embryos. WT-TH overexpression causes cell death in the lateral regions of the embryo (arrowhead) in a fraction of the embryos, but no cell death can be seen in the blastopore region at this stage. The vast majority of the mock-injected embryos (control) show no TUNEL staining.
	Fig. 6 Overexpression of wild-type tumorhead (WT-TH)-HA causes gastrulation morphological defects. One to two nanograms of in vitro synthesized mRNA encoding WT-TH-HA was injected into embryos at the two-cell stage into one blastomere with or without 10 ng of TH morpholino. (A) Embryos were grown until the late gastrula/early neurula stage (stage 13) and visualized under bright field illumination with a stereoscope. Overexpression of WT-THHA causes gastrulation defects as shown by incomplete blastopore closure. This effect was rescued with the coinjection of TH morpholino (WT-TH-HA1MO). (B) Cell extracts were prepared from embryos collected at the neurula stage (stage 17) and subjected to Western blot analysis. Note the decrease in the expression of WTTH- HA (TH) in the presence of the morpholino (TH1MO). Actin was used as a loading control. Un, uninjected. (C) The percentage of embryos showing the defective gastrulation phenotype (% P) is smaller in embryos injected with WT-TH-HA and TH morpholino (TH1MO) than in embryos injected with WT-TH-HA alone (TH). WT-TH-HA was more potent in stimulating gastrulation defects than WT-TH containing an N-terminal Myc tag. n, total embryos injected.