Fig. 10. Lvpmar1 does not activate the skeletogenic GRN network during NSM transfating. (A) The Lvpmar1 locus. (B) Developmental RT-PCR analysis of Lvpmar1 expression. Stages shown are unfertilized egg (UE), 4-, 8-, 16-, 32- and 64-cell stages, late cleavage (LC), early blastula (EB) and mesenchyme blastula (MB). (C) Overexpression of Lvpmar1 activates the PMC GRN in all cells of the embryo. Lvpmar1 mRNA (100 µg/ml) was injected into fertilized eggs and after 24 hours the embryos were fixed and immunostained with mAb 6a9. Lvpmar1 causes a transformation of all cells to a mesenchymal, 6a9-positive phenotype. (D) RT-PCR analysis of Lvpmar1, Lvp16 and Lvactin mRNA expression in PMC-deficient embryos. MB, control mesenchyme blastula stage embryos. Other lanes show PMC-deficient embryos, collected 0, 3, 6 and 9 hours after PMC depletion. Lvp16, a target of Lvpmar1 and Lvalx1, is expressed within 3 hours after PMC depletion and at higher levels at 9 hours. Lvpmar1 mRNA is not detectable at any of the stages examined (the amount of starting material at each stage was equivalent to 1 embryo). Bottom panels show control experiments using identical RT-PCR conditions but with cell lysates prepared from 16-cell stage embryos. Lvpmar1 can be detected when the amount of starting material is equivalent to just 1/100 embryo. R1-R3 are three independent replicates of the experiment, performed using three different batches of embryos.