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Fig. 4.
Separating the function of NEDD4L in mesoderm induction and convergent extension. (A) Whole mount in situ hybridization for mesodermal genes Xbra in embryos with indicated treatments. xNEDD4L MO (6 ng) was injected into two dorsal animal blastomeres at the 4-cell stage. The rescuing mRNA (120 pg) was injected at 2-cell stage. The embryos were collected and processed for in situ hybridization of at st10.5. All embryos are viewed from the vegetal pole with the dorsal side upwards in (A). (B) Effects of NEDD4L depletion in dorsal region on the expression of mesodermal genes. Under the same experimental settings stated in (A), the dorsal marginal zones of control, MO-injected and MO/mRNA coinjected embryos at stage 10.5 were dissected and subject to RT-PCR analysis. Asterisks indicate p<0.05 in student t-test. (C) Activin treated animal explants. MO (10 ng) or xNEDD4L mRNA (500 pg) or both together were injected into the animal pole at 2-cell stage. Animal caps were then dissected at st8.5–9 and cultured with (left panel) or without (right panel) Activin (25 ng/mL). Numbers in ratio format indicate the proportion of explants looking like the ones shown in picture. Quantitative RT-PCR analysis of the expression of Xbra, Gsc and Chrd in the animal caps (ACs) in response to Activin induction. The animal cap explants were collected at st11.5. Dorsal mesodermal markers goosecoid (Gsc) and chordin (chrd), and pan-mesodermal marker Xbra were examined. Error bars represented s.d. from three independent experiments. (E) Quantitative RT-PCR analysis of the expression of Gsc, Mespo, Xbra and MyoD in the animal caps (ACs) in response to the conjugated vegetal bases from normal blastula. Animal caps from control (ctrl) or MO-injected (xN4L MO, 25 ng) were dissected at st8.5 and conjugated with vegetal bases dissected from control embryos. The animal caps were then stripped off after 4 h culture and subject to RT-PCR analysis. (F) Nieuwkoop recombinants recorded at the sibling stage 18. All vegetal bases were dissected from control embryos. Animal caps were dissected from control (upper left panel), MO (25 ng, lower left panel), NEDD4L mRNA (100 pg, upper right panel), or both MO and mRNA (lower right panel) injected embryos. Scale bars in A, C and F: 0.5 mm. |
