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Fig. 4. Depletion of Wee2 protein disrupts convergent extension of the paraxial mesoderm during neurulation. (A-C,F) Temporal analysis of Wee2-depleted embryos during neurulation. Embryos were microinjected as in Fig. 2D with CMO (odd panels) or W2MO.1 (even panels), and were allowed to develop until controls reached the indicated stages before being processed for MyoD (A), XNot (B), MA and MHC (C), or Sox3 (F) in situ analysis as indicated. Green arrows indicate a closed blastopore. Lateral limits of paraxial mesoderm (MyoD expression) are indicated by broken red lines. White and yellow arrows indicate lack of anterior neural fold and somitic ridge, respectively. Lateral limits of presumptive neural tissue (Sox3 expression) are indicated by red arrows and vertical lines. (D) The paraxial mesoderm of Wee2-depleted embryos fails to converge towards the midline. Representative CMO and W2MO.1 treated, MyoD stained, stage 18 embryos from 4A were serially sectioned transversely. The anteroposterior positions of the shown sections are indicated by letters in right lower corner of panels as per E. Dorsal is towards the top. Black arrow indicates the forming somitic ridge. (E) Drawing reproduced, with permission, from Nieuwkoop and Faber (Nieuwkoop and Faber, 1994) showing position of cuts in D. |
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Fig. 3. Wee2 is required for anterior-posterior embryo elongation, somite formation, and convergent extension. (A) Wee2-depleted embryos fail to extend along the anteroposterior axis and fail to form somites. Embryos were treated as in Fig. 2D, but allowed to develop until the controls reached stage 25 before being processed for MyoD in situ analysis. Anterior towards the right, dorsal towards the top. Labels as in Fig. 1A. Scale bar: 300μ m. (B) Unilateral depletion of Wee2. One blastomere (asterisk) of a two-cell embryos was microinjected with 40 ng CMO or W2MO.1. These were allowed to develop until controls reached stage 19 and photographed. Dorsal view, anterior towards the top. Note curvature and reduced somitic ridge (arrow) on the Wee2-depleted side. (C) Mesoderm specific gene expression is unchanged in Wee2-depleted embryos. Quantitative RT-PCR for MyoD, XNot, Vent1, MA, MHC and ornithine decarboxylase (ODC) from whole, stage 18 embryos treated with W2MO.1, CMO or nothing (Sibling). (D) Depletion of Wee2 compromises convergent extension driven elongation of dorsal explants. Embryos were treated with W2MO.1 or CMO as in Fig. 2D and then processed for dorsal explants. Explants were photographed when controls reached stage 26. Scale bar: 1 mm. |
