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Figure 3. Opposite Roles of TCF3 and HIPK2 in Dorsoanterior Development(A) CoMO (60 ng), HK2MO (60 ng), or TCF3MO (40 ng) were injected four times into the marginal zone of four-cell embryos as indicated. Anterior views are shown; dorsal is up. HK2MO enhances, whereas TCF3MO inhibits, the dorsoanterior fate, assessed by head and cement gland development at stage 28 (top). Effects of HIPK2 and TCF3 MOs on Vent2 (middle) and Otx2 (bottom) were analyzed by whole-mount in situ hybridization at stages 14 and 17, respectively. CoMO does not affect Vent2 and Otx2 gene expression (95%, n = 56, and 96%, n = 43, respectively). HK2MO suppresses (68%, n = 56), whereas TCF3MO expands (56%, n = 45), Vent2 expression in the anterior and ventral region. HK2MO expands (56%, n = 50), whereas TCF3MO suppresses (72%, n = 50), the Otx2 expression domain. Arrowheads demarcate cement gland (CG) boundaries (top), and the boundaries of the Vent2 (middle) and the Otx2 (bottom) expression domains.(B) Inhibition of anterior development by TCF3MO is rescued by TCF3 RNA. Embryos shown represent phenotype classification based on CG size at stage 28. Class I, CG is between one-third and one-half of normal size; class II, CG is less than one-third of normal size.(C) HK2MO enhances anterior development; this defect is rescued by RNA encoding HIPK1, a related protein kinase, and partially reversed by TCF3MO. Phenotype classes are based on cement gland size at stage 26. Class I, CG size is increased less than twice; class II, CG size is increased more than twice. RNAs or MOs were injected into the animal pole (B) or the marginal zone (C) of four-cell embryos at indicated doses.(D) HK2MO inhibits Vent2-Luc reporter activation via the TCF-binding site. MOs and reporter DNA were injected six times into the animal pole and the marginal zone for luciferase activity determination analysis at late gastrula stages. Data are shown as means ± SD. |
