Groucho Central Domains Instruct Development Figure 5. Central domains define patterns of embryonic Gro target gene expression. Fluorescence in situ hybridization analyzing the mRNA products of the Gro targets hkb, tll, and sna in cellular blastoderm embryos containing maternally overexpressed Gro variants. Embryos are oriented with the anterior to the left and ventral at the bottom. Embryo images were obtained using a 206objective. Scale bars = 50 mm. (A–D) Wild- type expression patterns. (E–H) GroWT overexpression leads to spatial patterning defects and reduced expression of all three target genes. Posterior hkb expression was absent and anterior hkb expression was significantly reduced (E). Posterior and anterior tll expression were reduced with a greater effect at the posterior (F). The ventral sna stripe was narrowed ventrally and retracted from both termini (G). (I–P) Overexpression of GroDGP (I–L) and GroDCcN (M–P) led to no changes in target gene expression patterns. (Q–T) GroDSP overexpression led to defects in spatial patterning and a reduction in the expression of all three target genes that is more severe than that resulting from GroWT overexpression. hkb (Q) and tll (R) expression were completely abolished at both termini. The sna stripe was substantially narrowed in the posterior region (as indicated by the arrow) and retracted from the termini, especially the anterior terminus (S). (U–X) GroDCR overexpression resulted in expansion of the posterior hkb domain (U), and an expansion of the anterior tll domain toward the ventral midline of the embryo (arrow) (V). The sna domain is expanded towards both termini (W). Arrowheads (panels A, C, G, S, U, and W) indicate borders of the wild-type expression domains. (Y–Z) Quantification of changes in expression pattern. (Y) 20 stage 5 embryos overexpressing each variant were scored according to whether the anterior and posterior expression domains of hkb and tll appeared wild-type, were reduced, were missing altogether, or were expanded. (Z) The same 20 embryos were scored according to whether the sna expression domain showed no retraction, mild retraction, strong retraction, or expansion at the anterior and posterior termini. doi:10.1371/journal.pone.0030610.g005 edges of the developing wing. As a result, misexpression of Gro in the wing disc results in ectopic repression of a lacZ reporter under the control of a vg cis-regulatory module termed the vg quadrant (vgQ) enhancer [1]. To assess the ability of the Gro variants to repress a Brk target, we generated clones of Gro variant overexpressing cells that overlap the vgQ-lacZ expression domain. PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30610