Fig. 1. Spry2-GOF, a conditional Spry2 gain-of-function allele, and its expression following recombination by En1^Cre. (A) Schematic diagram illustrating the Spry2-GOF transgene. The CAGG promoter drives expression of the β-Geo gene, which is followed by a triple polyadenylation sequence (3x pA). Cre-mediated recombination deletes β-Geo, and mouse Spry2 and human placental alkaline phosphatase (PLAP) cDNAs are expressed as a bicistronic mRNA containing an internal ribosome entry site (IRES) that directs translation of PLAP. (B) Assays in whole mount for β-Geo expression (β-GAL activity) in embryos hemizygous for Spry2-GOF. (C-K) Analysis of expression of the recombined Spry2-GOF transgene in En1^Cre/+;Spry2-GOF (mes/r1-S2GOF) mutants. (C-E) Assays for PLAP activity in embryos and postnatal brain at the stages indicated. Blue staining identifies cells in which recombination of the transgene occurred. The arrow in D indicates two ventrolateral stripes in the spinal cord. (F-K) RNA in situ hybridization assays in whole mount at the stages indicated, using a mouse Spry2 probe. The broken white lines outline the left side of mes/r1 in the control embryos, where the probe detects endogenous Spry2 expression. In mes/r1-S2GOF embryos, the Spry2 RNA detected represents the sum of expression from the endogenous Spry2 gene and the recombined transgene. Regions in which there is ectopic Spry2 expression in mes and in r1 are indicated by broken red lines (I,K) and by a red arrow (K), respectively. AER, apical ectodermal ridge of the limb bud; BA1, first branchial arch; CbV, cerebellar vermis; CbH, cerebellar hemispheres; Mb, midbrain; mes; mesencephalon; r1, rhombomere 1; so, somites.