(A) Schematic diagram of laser capture microscopy to isolate the periocular mesenchyme for RNA-seq analysis. (B) Dot plot of genes downregulated at least two folds in the Shp2 mutants. The Alx1 and Alx4 genes are marked by arrows. (C) qRT-PCR confirmed the deletion of Shp2 and down regulation of Fgf10, Alx1 and Alx4 in the laser captured periocular mesenchyme from Shp2 mutants. Student’s t test: *P<0.001, n = 3. (D) Shp2 deletion reduced Alx4 expression in the cranial mesenchyme, especially at the periocular region next to the future lacrimal gland at E13.5, which was ameliorated in Wnt1-Cre;Shp2f/f;LSL-KrasG12D embryos. Arrow: Alx4 expression in the cranial mesenchyme at E10.5 and E11.5. Arrowhead: Alx4 expression in the periocular mesenchyme at E12.5 and E13.5. (E) In Shp2 mutants, Alx4 was progressively reduced in the periocular mesenchyme adjacent to the conjunctival epithelium from E12.5 to E13.5. By E14.5, both Alx1 and Alx4 were lost. Arrow: Alx4 immunostaining in the periocular mesenchyme. Arrowhead: Alx1 expression surrounding the lacrimal gland bud. Lacrimal gland primordia are outlined in dotted lines.