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 (Chx+dex, 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-/dex+), 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 (Sia-hGR, 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 (Chx+/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 (Chx+/dex+) 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 (Chx+/dex+) 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.