(A) Gene expression analysis of markers of differentiation 2 days after knockdown of Sfrp1 on Day 2 of Stg3. Sfrp1 knockdown produced similar changes in its downstream genes, Pax3, Wnt1, Lmx1a, SHH and Foxa2 as DM/SB treatment at Stg2. (B) Treatment with EMD Millipore 344300, a chemical inhibitor of Sfrp1, at Stg2 increased mRNA expression levels of mDA markers SIP1, Sfrp1, Wnt1 and Lmx1a as DM/SB treatment at Stg2. (C) Sfrp1 inhibitor, like DM/SB, increased Wnt signaling as indicated by the rise in active β-catenin on Western blot analysis. As Sfrp1 inhibition occurs downstream of Sip1, no change in Sip 1 was noted as with DM/SB treatment. (D) Quantification of Western blot results shown in panel C.