Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos

Abstract

The two arms of the Transforming Growth Factor ß (Tgfß) superfamily, represented by Tgfß/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgfß family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgfß receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgfß precursor proteins. RNA encoding Tgfß precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions.

Figure 1.. Injection and aspiration needles.

Photographs of representative needles that have been pulled but not clipped (A) pulled and clipped for use as an injection needle (B) or pulled and clipped for use as an aspiration needle (C) are shown. The arrow and arrowhead in (A) indicate the point at which the glass was clipped to generate a needle for injection and aspiration, respectively. Please click here to view a larger version of this figure.

Figure 2.. Analysis of cleaved Bmp ligands extracted from X. laevis blastocele fluid.

(A) Cleavage products generated from Bmp4 and Bmp7 homodimeric or heterodimeric precursor proteins and position of myc epitope tag (silver bar) is shown schematically (B) Schematic diagram showing the timing of injection and blastocoele fluid extraction. (C-D) Xenopus embryos were injected with 200 pg of Bmp7 or Bmp4 RNA, or with Bmp4 and Bmp7 RNA mixed together (100 pg each). At the gastrula stage, fluid was extracted from the blastocoele of the same number of embryos in each experimental group. Proteins present in the blastocoele fluid were deglycosylated and separated by SDS-PAGE. Antibodies recognizing the myc-epitope tag were used to probe immunoblots. The relative position of immunoreactive ligand monomers and dimers is shown schematically to the right of each gel. Black line in (C) indicates removal of an intervening lane on the blot. Data shown are extracted from a previously published study. Please click here to view a larger version of this figure.