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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.
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.
