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Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.
Fig. 1. Wee2 expression correlates with the lack of mitotic cells within the dorsal and paraxial mesoderm. (A) Panels 1 and 3 are sagittal sections of representative stage 12.5 and 18 embryos that were subjected to in situ hybridization for Wee2. Panels 2 and 4 are composite images of 10 serial sagittal sections of representative stage 12.5 and 18 embryos that were subjected to whole-mount immunocytochemistry against phospho-histone H3 (αPH3). Note the absence of mitotic cells (black dots) in the dorsal and paraxial mesoderm. Dorsal towards the right, anterior towards the top. pne, presumptive neural ectoderm; dm, dorsal mesoderm; pm, paraxial mesoderm; ey, eye anlage; sm, somite. (B) Wee2 protein is expressed from mid-gastrula stages onwards. Indicated developmental stages were subject to αWee2 andα Cdk1/2 western analysis.
Fig. 3. Wee2 is required for anterior-posteriorembryo 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.
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.
Fig. 5. Rescue of Wee2-depleted embryos. Both blastomeres of two-cell embryos were microinjected with a combination of 40 ng W2MO.1 and either injection buffer (panels 1-4), 20 pg Wee2 mRNA (panels 5-8) or 40 pg Wee2 (panels 9-12). These were allowed to develop until non-injected siblings (panels 13-16) reached stage 19. Subsequently, the embryos were subjected to MyoD or Sox3 in situ analysis as indicated. Lateral limits of paraxial mesoderm (MyoD expression) are indicated by broken red lines. Lateral limits of presumptive neural tissue (Sox3 expression) are indicated by red arrows and vertical lines.
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).
Fig. 6. Expression of constitutively active Cdk2 phenocopies the convergent extension defects observed in Wee2-depleted embryos. (A) Both blastomeres of two-cell embryos were microinjected with 230 pg of Cdk2WT or Cdk2AF mRNA as indicated. These embryos were allowed to develop until stage 18 before being processed for MyoD or XNot in situ analysis. Lateral limits of paraxial mesoderm (MyoD expression) are noted by broken lines. Arrow indicates lack of anterior neural fold. (B) The paraxial mesoderm of Cdk2 AF-treated embryos fails to converge towards the midline. Representative Cdk2WT or Cdk2AF treated, MyoD stained, stage 18 embryos from 6A were serially sectioned transversely. The anteroposterior positions of the shown sections are indicated by letters in right lower corner of panels as per Fig. 4E. Dorsal towards the top. Black arrow denotes the forming somitic ridge. (C) Expression of Cdk2AF causes cell proliferation within the paraxial mesoderm but not the axial mesoderm. The total number of nuclei within the paraxial and axial mesoderm of representative embryos from 6A was determined as in Fig. 2E. (D) Cdk2 AF treatment compromises convergent extension driven elongation of dorsal explants. Embryos were injected with Cdk2 WT or Cdk2 AF mRNA as in A and then processed for dorsal explants. Explants were photographed when controls reached stage 24. Scale bar: 1 mm. (E) Mesoderm specific gene expression is unchanged in Cdk2 AF-treated embryos. Quantitative RT-PCR for MyoD, XNot, Vent1, MA, MHC and ODC from whole, stage 18 embryos injected as in A with Cdk2 AF mRNA, Cdk2 WT mRNA, or nothing (Sibling).
Fig. 7. Paraxial mesoderm targeted expression of wild-type Cdc25 disrupts convergent extension. (A) Dorsal and posterior views of embryos unilaterally injected with 100 pg of the indicated Cdc25 plasmid DNA. Embryos were allowed to develop until stage 19 before being processed for GFP in situ analysis. Cells expressing GFP-Cdc25 stain blue. WT [wild type (active)] and PD [phosphatase-dead (inactive)] forms of Cdc25A or Cdc25C. Lateral limits of GFP-Cdc25 positive cells are indicated by broken lines. Arrows indicate the lack of a somatic ridge. (B) Dorsal, anterior and sectioned views of embryos unilaterally injected and treated as in A, except 300 pg of Cdc25A plasmid DNA was used. Arrow indicates somitic ridge.
