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Fig. 2. The low mitotic index of the paraxial mesoderm requires Wee2. (A) Wee2
targeted morpholinos (W2MO.1 and W2MO.2), but not a control morpholino (CMO)
or water (No MO), blocked the in vitro translation of a mRNA containing the
5'UTR of Wee2 fused to GFP. (B) W2MO.1 and W2MO.2, but not CMO, reduce
the endogenous levels of the Wee2 protein. 40 ng of CMO, W2MO.2 or W2MO.1 were
microinjected into each cell of two-cell embryos. Sibling is a non-injected
control. At stage 18, these embryos were analyzed for Wee2 and Myt1 protein
levels by western analysis. (C) W2MO.1 reduces the level of endogenous Wee2
protein in a dose-dependent manner. 40 ng CMO; or 10, 20 or 40 ng W2MO.1 were
microinjected into each cell of two-cell embryos and then processed as in B.
The total amount of morpholino injected is indicated. (D) Embryos were
microinjected as in B with CMO (odd panels) or W2MO.1 (even panels), and
allowed to develop to stage 18. Panels 1 and 2 are composite images of eight
serial sagittal sections from representative embryos that were processed for
PH3 staining. Note the increase in mitotic cells (black dots) within the
paraxial mesoderm of Wee2-depleted embryos (panel 2). Panels 3 and 4 are
sagittal sections of embryos processed for MyoD in situ analysis. These same
MyoD sections were subsequently stained with SYTOX Green to visualize the
nuclei (panels 5 and 6). Nuclei in the paraxial mesoderm appear dimmer owing
to the MyoD staining. Note the absence of somites in Wee2-depleted embryos.
Labels and orientation as in Fig.
1A. (E) Depletion of Wee2 protein causes cell proliferation within
the paraxial mesoderm but not the axial mesoderm. The total number of nuclei
within the paraxial and axial mesoderm was determined by counting all nuclei
within the MyoD-(paraxial mesoderm, PM) or XNot-(axial mesoderm, AM) positive
regions of representative embryos that were microinjected with either CMO or
W2MO.1. Every section of both transversely (MyoD and XNot) and sagittally
(MyoD) cut embryos was counted and totaled. Data are -fold change from the
control (CMO=1). (F) The total volume of MyoD-expressing tissue does not
change with depletion of Wee2 protein. The same transverse and sagittal MyoD
stained sections used in E were used to determine the area of MyoD expression.
Data are -fold change from the control (CMO=1).
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