Klein SL and Moody SA (2015), Early neural ectodermal genes are activated by ...

XB-ART-50588

Genesis May 1, 2015; 53 (5): 308-20.

Early neural ectodermal genes are activated by Siamois and Twin during blastula stages.

Klein SL , Moody SA .

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: [+]

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

	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.
	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.
	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.
	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.
	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).