O'Reilly MA et al. (1995), Patterning of the mesoderm in Xenopus: dose-dep...

XB-ART-19751

Development 1995 May 01;1215:1351-9. doi: 10.1242/dev.121.5.1351.

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Patterning of the mesoderm in Xenopus: dose-dependent and synergistic effects of Brachyury and Pintallavis.

O'Reilly MA , Smith JC , Cunliffe V .

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Widespread expression of the DNA-binding protein Brachyury in Xenopus animal caps causes ectopic mesoderm formation. In this paper, we first show that two types of mesoderm are induced by different concentrations of Brachyury. Animal pole explants from embryos injected with low doses of Xbra RNA differentiate into vesicles containing mesothelial smooth muscle and mesenchyme. At higher concentrations somitic muscle is formed. The transition from smooth muscle formation to that of somitic muscle occurs over a two-fold increase in Brachyury concentration. Brachyury is required for differentiation of notochord in mouse and fish embryos, but even the highest concentrations of Brachyury do not induce this tissue in Xenopus animal caps. Co-expression of Brachyury with the secreted glycoprotein noggin does cause notochord formation, but it is difficult to understand the molecular basis of this phenomenon without knowing more about the noggin signal transduction pathway. To overcome this difficulty, we have now tested mesoderm-specific transcription factors for the ability to synergize with Brachyury. We find that co-expression of Pintallavis, but not goosecoid, with Brachyury causes formation of dorsal mesoderm, including notochord. Furthermore, the effect of Pintallavis, like that of Brachyury, is dose-dependent: a two-fold increase in Pintallavis RNA causes a transition from ventral mesoderm formation to that of muscle, and a further two-fold increase induces notochord and neural tissue. These results suggest that Pintallavis cooperates with Brachyury to pattern the mesoderm in Xenopus.

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Species referenced: Xenopus
Genes referenced: acta2 acta4 actc1 actl6a foxa4 gsc mcidas nog tbx2 tbxt

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	Fig. 1. Injection of increasing amounts of Xbra RNA does not saturate the translation machinery. The indicated quantities of Xbra RNA, together with 1 mCi/ml [35S]methionine, were injected into Xenopus embryos at the 1- cell stage. Animal caps were dissected at the mid-blastula stage and cultured in 75% NAM to control stage 12, when cellular proteins were subjected to immunoprecipitation using an anti-Xbra antiserum. Immunoprecipitates were analyzed by polyacrylamide gel electrophoresis followed by fluorography.
	Fig. 2. Activation of muscle-specific cardiac actin genes requires a threshold quantity of injected Xbra RNA. Embryos at the 1-cell stage were injected with the indicated quantities of Xbra RNA. Animal caps were dissected from these embryos at the mid-blastula stage and cultured to stage 17/18. RNA was analyzed for expression of cardiac (muscle-specific) actin by RNAase protection.
	Fig. 3. Histological analysis of animal caps derived from embryos injected with increasing quantities of Xbra RNA. Embryos at the 1-cell stage were injected with increasing amounts of Xbra RNA. Animal caps were dissected from these embryos at the mid-blastula stage and cultured to stage 42 when they were fixed, sectioned at 7 mm and stained by the Feulgen technique and with Light Green and Orange G. (A) An uninjected animal cap forms atypical epidermis. (B) Animal caps from embryos injected with 0.5 ng Xbra RNA. This ventral tissue is typical of caps derived from embryos injected with 0.12 ng to 1 ng Xbra RNA. (C) Animal caps from embryos injected with 4 ng Xbra RNA. This muscle-like tissue is typical of caps from embryos injected with 1 ng to 4 ng Xbra RNA. Scale bar in C is 100 mm, and also applies to A and B.
	Fig. 4. Immunohistochemical analysis of animal caps injected with increasing quantities of Xbra RNA. a-smooth muscle actin and somitic muscle were defined with monoclonal antibodies a-SM actin (Saint-Jeannet et al., 1992) and 12/101 (Kintner and Brockes, 1984), respectively. Embryos at the 1-cell stage were injected with increasing amounts of Xbra RNA. Animal caps were dissected from these embryos at the midblastula stage and cultured to stage 47, when they were fixed and embedded in acrylamide prior to sectioning at 10 mm (see Cunliffe and Smith, 1994). Sections were immunostained with a-SM actin (A-C) and 12/101 (D-F), and a FITC-conjugated goat anti-mouse secondary antibody. (A,D) Uninjected animal caps. (B,E) Animal caps from embryos injected with 0.5 ng Xbra RNA. (C,F) Animal caps from embryos injected with 4 ng Xbra RNA. Results are summarised in Table 2. Scale bar in F is 50 mm, and also applies to A to E.
	Fig. 5. Co-expression of zebrafish goosecoid and ntl RNAs does not induce cardiac (muscle-specific) actin expression in Xenopus animal caps. Embryos at the 1-cell stage were injected with RNA encoding zebrafish goosecoid (1 ng) and/or ntl (0.4 ng) and/or Xenopus noggin (100 pg) as indicated. Animal caps were dissected at the mid-blastula stage and cultured to stage 28, when they were analyzed for expression of actin genes by RNAase protection. 0.4 ng ntl did not induce expression of muscle-specific actin genes, but did cause formation of ventral mesoderm as judged by morphological and histological criteria (not shown). Co-expression of noggin and ntl, but not goosecoid and ntl, induces expression of muscle-specific actin RNA.
	Fig. 6. Xbra and noggin act synergistically to induce expression of Pintallavis. Embryos at the 1-cell stage were injected with RNA encoding Xbra (1.0 ng) and/or noggin (200 pg) as indicated. Animal caps were dissected at the mid-blastula stage and cultured to stage 12, when they were analyzed for expression of Pintallavis by RNAase protection. noggin did not induce expression of Pintallavis and Xbra caused weak induction, but co-expression of the two genes elicited significant expression. This experiment was performed three times with similar results.
	Fig. 7. Co-expression of Pintallavis and Xbra RNAs induces expression of muscle-specific actin RNA in Xenopus animal caps. Embryos were injected at the 1- to 2-cell stage with the indicated combinations of Pintallavis and Xbra RNAs. Animal caps were excised at the mid-blastula stage and cultured to stage 31 when they were analyzed by RNAase protection. Maximal expression of muscle-specific actin RNA is seen in explants co-injected with 1.3 ng Xbra RNA and 0.8 ng Pintallavis RNA. This experiment was performed four times with similar results.
	Fig. 8. Histological analysis of animal caps derived from embryos co-injected with ntl RNA and increasing quantities of Pintallavis RNA. (A) Ventral mesoderm resulting from injection of 0.4 ng ntl RNA and 0.2 ng Pintallavis RNA. (B) Muscle masses resulting from injection of 0.4 ng ntl RNA and 0.4 ng Pintallavis RNA. (C) Notochord, muscle and neural tissue resulting from injection of 0.4 ng ntl RNA and 0.8 ng Pintallavis RNA. Notochord is marked with arrows. Scale bar in C is 100 mm and also applies to A and B.
	Fig. 9. Distribution of Pintallavis RNA in Xenopus embryos visualised by whole-mount in situ hybridisation. (A) Vegetal view of early gastrula stage showing expression of Pintallavis in prospective dorsal mesoderm. (B) Dorsal view of early neurula showing Pintallavis expression in midline structures including the notochord. (C) Horizontal section through the marginal zone of an early gastrula as in A, showing graded distribution of Pintallavis RNA in dorsal mesoderm. (D) Computer densitometric analysis of the section shown in C. The image was converted to greyscale and after background subtraction different degrees of grey were assigned false colours, with 1-7 units of grey as yellow, 8-15 units as green, and 16- 30 units as red. Scale bar in A is 0.5 mm and also applies to B-D.