XB-ART-50588
Genesis
May 1, 2015;
53
(5):
308-20.
Early neural ectodermal genes are activated by Siamois and Twin during blastula stages.
Abstract
BMP signaling distinguishes between neural and non-neural fates by activating
epidermis-specific transcription and repressing neural-specific transcription. The
neural ectoderm forms after the
Organizer secrets antagonists that prevent these BMP-mediated activities. However, it is not known whether neural genes also are transcriptionally activated. Therefore, we tested the ability of nine
Organizer transcription factors to ectopically induce the expression of four neural ectodermal genes in epidermal precursors. We found evidence for two pathways:
Foxd4 and
Sox11 were only induced by
Sia and Twn, whereas
Gmnn and
Zic2 were induced by
Sia, Twn, as well as seven other
Organizer transcription factors. The induction of
Foxd4,
Gmnn and
Zic2 by
Sia/Twn was both non-cell autonomous (requiring an intermediate protein) and cell autonomous (direct), whereas the induction of
Sox11 required
Foxd4 activity. Because direct induction by
Sia/Twn could occur endogenously in the dorsal-equatorial
blastula cells that give rise to both the
Organizer mesoderm and the
neural ectoderm, we knocked down
Sia/Twn in those cells. This prevented the
blastula expression of
Foxd4 and
Sox11, demonstrating that
Sia/Twn directly activate some neural genes before the separation of the
Organizer mesoderm and
neural ectoderm lineages.
PubMed ID:
25892704
Article link:
Genesis
Grant support:
[+]
R01 DE022065 NIDCR NIH HHS
Species referenced:
Xenopus laevis
Genes referenced:
ctnnb1
foxd4l1.1
foxd4l1.2
gmnn
hesx1
lhx1
pou3f4
sia1
sia2
sox11
zic2
Morpholinos:
foxd4l1.1 MO1
foxd4l1.2 MO1
sia1 MO2
sia2 MO2
Phenotypes:
Xla Wt + sia1 MO + sia2 MO
(fig.4)
Article Images:
[+] show captions
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FIG. 1. NE genes are ectopically induced in the ventral epidermis by different sets of Organizer transcription factors. (a) The percentage
of embryos in which an ectopic ventral patch of gene expression (Foxd4, Sox11, Gmnn, Zic2) was observed after mRNA encoding an Organizer
transcription factor, indicated by colored bars (key is across the top), was injected into a ventral epidermis progenitor (V1.1; Moody,
1987). The number of embryos analyzed is shown over each bar. (b) Examples of ectopic ventral induction of the four NE genes in response
to injection of Organizer transcription factor mRNAs. Induced clones are outlined in black. Foxd4 and Sox11 are ectopically induced by Sia,
but not by Gsc, Otx2, Foxa4 or Xnot2, whereas Gmnn and Zic2 are ectopically induced by each of these genes. Images are of the ventral
sides of gastrula stage embryos, animal pole to the left. The cells expressing the Organizer transcription factor (noted to the left of each
embryo) are identified by pink nuclei (nbgal staining). The NE genes, labeled at the top of each column, were detected by ISH (purple reaction
product). Higher magnification insets in the top row show examples of nbgal-positive cells that are also NE gene-positive (red arrows);
for these cells the induction is likely cell autonomous. Blue arrows indicate cells that are nbgal-negative and NE gene-positive; for these
cells the induction is non-cell autonomous. For Sox11, the blue bar indicates a broad region of ectopic induction in which there are no
nbgal-positive cells, demonstrating that most of its induction is indirect. Black arrows indicate the endogenous expression domain of the
NE gene on the dorsal side of the embryo.
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FIG. 2. NE genes are directly induced in the ventral epidermis by
Sia or Twn. (a) The percentage of embryos in which an ectopic ventral
patch of gene expression (Foxd4, Sox11, Gmnn, Zic2) was
observed after injection of hGR-Sia mRNA. Embryos were either
not treated with hormone (no dex, blue bars), treated with hormone
at the 64-cell cleavage stage (dex-CL, red bars), treated with hormone
at the stage 8 blastula (dex-BL, green bars), or pretreated at
stage 8 with a protein synthesis inhibitor 40 minutes before hormone
treatment (Chx1dex, purple bars). The high frequency of
induction of ectopic gene expression with dex treatment alone at
either cleavage or blastula stages indicates that the construct is
hormone-inducible. Foxd4, Gmnn and Zic2 are likely direct targets
of Sia because in the presence of Chx they are induced at the
same frequency as dex treatment alone, and at a significantly
greater frequency compared to no dex embryos (*,p<0.001);
Sox11 is not a direct target because it is not induced when protein
synthesis is blocked by Chx. The number of embryos analyzed is
shown over each bar. (b) The percentage of embryos in which an
ectopic ventral patch of gene expression (Foxd4, Sox11, Gmnn,
Zic2) was observed after injection of hGR-Twn mRNA. The data are
presented as described in A. Gmnn and Zic2 are likely direct targets
of Twn (*,p<0.001), whereas Foxd4 and Sox11 are not. The
number of embryos analyzed is shown over each bar. (c) When an
embryo is injected with Sia-hGR mRNA and not treated with Chx
or dex (Chx-/dex-) (left image), NE genes (in this case Foxd4) are
not induced; the Sia-hGR expressing cells (outlined, and in the
inset) have pink nuclei but no purple reaction product. When an
embryo is injected with Sia-hGR or Twn-hGR mRNA and treated
with only dex (Chx-/dex1), NE genes are induced (outlined). For
Sox11, there is considerable non-cell autonomous induction (in
area indicated by blue arrow), whereas for Gmnn nearly all the
labeled cells have pink nuclei (inset). Black arrows indicate endogenous
expression domain. (d) Examples of direct induction of NE
genes by hGR-Sia or hGR-Twn mRNAs. The injected mRNAs (SiahGR,
Twn-hGR) are indicated to the left of each image, and the
assayed NE gene is indicated at the upper left of each row.
Embryos treated only with Chx do not show ectopic induction
(Chx1/dex-); only pink nuclei are visible. Two examples of
responses to Sia-hGR and one example of responses to Twn-hGR
direct induction at blastula stages (Chx1/dex1) are shown for
Foxd4, Sox11, and Gmnn; two examples of responses to Twn-hGR
and one example of response to Sia-hGR direct induction at blastula
stages (Chx1/dex1) are shown for Zic2. The region of ectopic
induction (purple) is outlined by red dashes in each case. Sia
directly induces Foxd4, Gmnn and Zic2; Twn directly induces
Gmnn and Zic2. Sox11 is not directly induced by either.
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FIG. 3. Foxd4 mediates some of the ectopic induction of Sox11,
Gmnn and Zic2. The percentage of embryos in which an ectopic
patch of gene expression (Sox11, Gmnn, Zic2) was observed after
injection of Sia (blue bars) or Twn (green bars) mRNA in a ventral
epidermal lineage in which Foxd4 translation was knocked-down
(FoxMO). Sox11 induction by Sia and Twn was dramatically
reduced in the absence of Foxd4. Gmnn and Zic2 induction by Sia
was more strongly affected by the absence of Foxd4 than was their
induction by Twn (***,p<0.001; **, p<0.01; *, p<0.05). The number
of embryos analyzed is shown over each bar. Examples of
ectopic ventral expression of Sox11, Gmnn and Zic2 in response
to injection of Twn mRNA in the presence of Foxd4 MOs. Black
arrows point to the endogenous expression domains of the genes
on the dorsal side. In most cases, there was no detectable Sox11
expression at the site of Twn expression (pink nuclei without purple
reaction product; inset). For Gmnn and Zic2, some embryos
showed no induction in the absence of Foxd4 (Gmnn left embryo;
pink nuclei without purple reaction product; inset), whereas in
others there was robust induction despite the knock-down of
Foxd4 (Gmnn, right embryo and Zic2; pink nuclei surrounded by
purple reaction product; insets). All embryos are oriented with animal
pole to left and vegetal pole to right.
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FIG. 4. Sia/Twn are required for blastula expression of Foxd4 and Sox11. Paired bright field (top row) and fluorescence (bottom row)
images of blastula stage embryos in which Sia1 Twn MOs were injected into a 16-cell blastomere that gives rise to the dorsal-equatorial
blastula precursors of the Organizer mesoderm and neural ectoderm (marked by a bracket in the top row). The location of the MOs is visualized by red (lissamine) fluorescence on the left side of the embryo (above the white arrows in the bottom row). Normal Foxd4 and Sox11
expression is more restricted to these dorsal-equatorial cells (brackets), whereas Zic2 and Gmnn normally are expressed throughout the
animal hemisphere including the dorsal-equatorial cells (shown in right side of each embryo; below the black arrows). Foxd4 and Sox11
expression is greatly reduced in the region containing Sia/Twn MOs (above the black arrows in the top row). In contrast, Gmnn and Zic2
expression appears unaffected. The numbers refer to the number of embryos in which gene expression was repressed by Sia/Twn knock
down. Dorsal views of embryos are oriented with animal pole to the right.
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FIG. 5. Summary of Sia/Twn time and location of induction of NE
genes. At the blastula stage, cells that are fated to give rise to both
Organizer mesoderm and neural ectoderm express Sia and Twn,
which in turn directly activate Foxd4, Gmnn and Zic2; Sox11 activation
requires the presence of Foxd4. At the gastrula stage, when
the descendants of these cells have segregated into the Organizer
mesoderm and the neural ectoderm, NE genes are up-regulated
indirectly by Organizer transcription factors that regulate the
expression of secreted factors that inhibit the BMP and Wnt pathways.
Previous studies show that Sox11, Gmnn and Zic2 are
expressed by inhibiting BMP, whereas Foxd4 is best expressed
when both BMP and Wnt signaling pathways are blocked (refs in
text). In the neural ectoderm, Foxd4 directly activates Sox11,
Gmnn and Zic2 (Yan et al., 2009).
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