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The retinoblastoma protein (pRb) is a central regulator of the cell cycle, controlling passage through G1 phase. Moreover, pRb has also been shown to play a direct role in the differentiation of multiple tissues, including nerve and muscle. Rb null mice display embryonic lethality, although recent data have indicated that at least some of these defects are due to placental insufficiency. To investigate this further, we have examined the role of pRb in early development of the frog Xenopus laevis, which develops without the need for a placenta. Surprisingly, we see that loss of pXRb has no effect on either cell cycling or differentiation of neural or muscletissue, while overexpression of pXRb similarly has no effects. We demonstrate that, in fact, pXRb is maintained in a hyperphosphorylated and therefore inactive state early in development. Therefore, Rb protein is not required for cell cycle control or differentiation in early embryos, indicating unusual control of these G1/G0 events at this developmental stage.
Fig. 1. Expression pattern of the Xenopus retinoblastoma gene. XRb expression was analysed by whole mount in situ hybridisation (stained in purple) in embryos at a range of stages. XRb is expressed in the anterior placodes (P) from stage 16 and increases by stage 19, especially in the optic vesicles (OV). Between stages 26 and 30, strong expression is seen in the optic vesicles, neural tube (NT) and branchial arches (BA). (A) Stage 16, anterior view; (B) stage 19, anterior view; (C) stage 26, lateral view; (D) stage 28, dorsal view; (E) stage 30, lateral view. Anteriors to the right (C, D, E).
Fig. 2. XRb morpholino specifically depletes XRb protein but does not affect the number of cells in mitosis or S phase in the neural tube, or the number of cells undergoing apoptosis or S phase in the epidermis. (A) Embryos were injected with 20 ng XRb morpholino (Mo) or control Mo in both blastomeres at the two-cell stage. Extracts made from these embryos at stages 10, 15 and 22 were Western blotted and probed with anti-Rb antibody (XZ160). The same blot was stripped and probed with an anti-β-actin antibody to show equal loading. XRb Mo depletes XRb protein at all three stages, while control Mo does not affect its level. (B) Embryos were injected with 20 ng XRb Mo and β-galactosidase as a tracer (visible as green autofluorescence) in one dorsal cell at the four-cell stage. At stage 21, embryos were sectioned and immunostained using an anti-phospho histone H3 antibody to visualise mitotic cells (red fluorescence) in the neural tube (NT). Hoechst staining of nuclei (blue). (C) Embryos were injected in one cell at the two-cell stage with 20 ng XRb Mo (Ca–Cc) or control Mo (Cd–Cf) and β-galactosidase as a tracer (blue) and then injected with BrdU at stage 20. After 2 h they were fixed and epidermal cells in S phase were visualised by whole mount BrdU immunostaining (purple). There is no difference between the density of cells in S phase in the Mo-injected sides of the embryos (Ca and Cd) and their density in the uninjected sides of the same representative embryos (Cc and Cf). Dorsal views with injected sides on the left and uninjected sides on the right (Cb and Ce). (D) Embryos were injected with 20 ng XRb Mo, with or without subsequent BrdU injection (as in C), sectioned and immunostained with anti-phospho histone H3 or anti-BrdU antibody. The numbers of cells in mitosis or S phase in both injected and uninjected sides of the neural tubes were counted in serial sections (n = 6 embryos for each marker, average of 41 sections per embryo). Average percentage differences between the sides are shown, with error bars showing the standard errors. pXRb depletion results in no significant change in the number of mitotic cells on the injected side of the neural tube compared to the uninjected side (average 8% increase, p = 0.2), or in the number of cells in S phase (average 28% increase, p = 0.43). (E) Embryos injected with 20 ng XRb Mo (Ea and Eb) or control Mo (Ec and Ed) with β-galactosidase as a tracer (blue) in one cell at the two-cell stage were fixed at stage 22. Apoptotic cells in the epidermis were visualised by whole mount TUNEL staining (purple). (F) The numbers of TUNEL-stained cells were counted in sample areas on both the Mo-injected and uninjected sides. Average percentage differences between the sides are shown, with error bars showing the standard errors. Neither XRb Mo (p = 0.44; n = 10) nor control Mo injection (p = 0.1; n = 11) affects the number of apoptotic cells in the epidermis (cf. injected sides, Ea and Ec, with uninjected sides, Eb and Ed).
Fig. 3. Depletion of XRb protein does not affect neural or muscle differentiation. Embryos were injected in one blastomere at the two-cell stage with 20 ng XRb Mo with β-galactosidase as a lineage tracer (blue). (A–H) Expression of a range of markers of neural differentiation was examined by in situ hybridisation (stained in purple) at stage 15. NCAM (A); neural β-tubulin (B); Engrailed (C); Otx2 (D); Pax6 (E); Xbf1 (F). Neurite outgrowth was examined by immunostaining for neural β-tubulin at stage 28: XRb Mo-injected side (G) and uninjected side (H) of the same embryo; magnified views of the neurites of the same embryo (G′ and H′). Arrow indicates normal structure of XRb Mo-injected somites (G′). pXRb depletion does not alter the expression patterns of any of these markers. (I, J) Expression of the markers of muscle differentiation muscle actin (I) and myosin heavy chain (J) was visualised by in situ hybridisation (purple) at stage 15: they are not affected by pXRb depletion. Dorsal views; anteriors to the right; injected sides uppermost (A, B, I, J). Anterior views; injected sides to the left (C, D, E and F). Lateral views; anterior to the right (G, G′, H and H′).
Fig. 4. Overexpression of XRb does not significantly affect the number of cells in mitosis or S phase in the neural tube or epidermis, or the number of apoptotic cells in the epidermis. (A) Embryos were injected in both blastomeres at the two-cell stage with increasing doses of XRb mRNA. Extracts made from stage 19 embryos were Western blotted and probed with an anti-human pRb antibody (XZ160) that cross-reacts with XRb. (B and C) Embryos were injected with 2 ng mRNA encoding XRb or GFP and β-galactosidase (blue) as a marker in one ventral cell at the four-cell stage. At stage 22 epidermal mitotic cells were stained by whole mount immunostaining (purple) with anti-phospho histone H3 antibody. Dorsal views; anteriors to the right; injected sides uppermost. (D) Embryos injected in one dorsal cell at the four-cell stage with 2 ng XRb mRNA and β-galactosidase (blue), with or without BrdU injection at stage 20, were sectioned and immunostained (purple) using anti-phospho histone H3 antibody to visualise mitotic cells or anti-BrdU antibody to visualise cells in S phase. The numbers of positive cells in both injected and uninjected sides of the neural tubes and the epidermis were counted in serial sections: graph shows the average percentage differences between the sides, with error bars showing standard errors (n = 6 embryos for each marker, average of 39 sections per embryo). XRb overexpression causes no significant difference in the number of mitotic cells on the injected side of the neural tube (average 8% decrease; p = 0.3) or in the epidermis (average 6% decrease; p = 0.2), or in the number of cells in S phase in the neural tube (average 22% increase; p = 0.5) or in the epidermis (average 15% decrease; p = 0.1). (E and F) Embryos injected with 2 ng XRb mRNA and β-galactosidase (blue) in one cell at the two-cell stage were fixed at stage 22 and apoptotic cells in the epidermis were visualised using whole mount TUNEL staining (purple). XRb overexpression has no effect on the density of apoptotic cells in the epidermis: XRb-injected side (E) and uninjected side (F) of the same embryo. (G) Apoptotic cells were counted in sample areas of the epidermis on both injected and uninjected sides of whole mount TUNEL-stained embryos. Graph shows the average percentage difference between the numbers of apoptotic cells on the two sides of embryos injected with XRb or GRP mRNA, with error bars showing standard errors. XRb overexpression does not make a significant difference to the number of apoptotic cells in the epidermis (average 72% increase; p = 0.09; n = 8); GFP injection also causes no difference (average 4% decrease; p = 0.28; n = 10).
Fig. 5. Overexpression of XRb does not affect neural or muscle differentiation or patterning. Embryos were injected in one blastomere at the two-cell stage with 2 ng XRb mRNA and β-galactosidase as a lineage tracer (blue). (A--H) Expression of a range of markers of neural and muscle differentiation was examined by in situ hybridisation (stained in purple) at stage 15. NCAM (A); neural β-tubulin (B); Engrailed (C); Otx2 (D); Pax6 (E); Xbf1 (F); muscle actin (G); myosin heavy chain (H). The expression patterns of none of these markers were affected by XRb overexpression. Lateral view of an XRb-injected embryo at stage 36 (I) and magnified view of its somites (I′), showing normal somite structure. Arrow indicates normal somite structure (I′). Dorsal views; anterior to the right; injected sides uppermost (A, B, C, G and H). Anterior views; injected sides to the left (D, E and F). Lateral view; anterior to the right (I and I′).
Fig. 6. XRb is predominantly hyperphosphorylated during Xenopus embryogenesis. (A) Extracts made from embryos at a range of stages between 1 and 30 were either untreated (â) or treated with lambda phosphatase (λ) and immunoblotted with anti-Rb antibody (XZ160). Endogenous Rb protein is detected at all stages between 8 and 30 in its hyperphosphorylated form (upper bands). (B) Extracts made from embryos at stage 22 (30 μg total protein) or stage 50 (50 μg total protein) were either untreated (â) or treated with lambda phosphatase (λ) and immunoblotted with anti-Rb antibody (XZ160). pXRb was predominantly hyperphosphorylated at stage 22 but exists in hyper- and hypophosphorylated forms at stage 50. (C) Extracts made from uninjected or XRb mRNA (2 ng)-injected embryos at stage 22 were either untreated (lanes 1 and 3) or treated with lambda phosphatase (lanes 2 and 4) and immunoblotted with anti-Rb antibody (XZ160). Both endogenous pXRb, seen in uninjected extracts (lanes 1 and 2), and exogenous pXRb, seen in extracts from XRb-injected embryos (lanes 3 and 4), are predominantly in the hyperphosphorylated form (upper bands) at stage 22. Hyperphosphorylated XRb (Hyperphos XRb); hypophosphorylated XRb (Hypophos XRb); stage (St).
