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

Summary

When Transforming Growth Factor ß family precursor proteins are ectopically expressed in Xenopus laevis embryos, they dimerize, get cleaved and are secreted into the blastocoele, which begins at the late blastula to early gastrula stage. We describe a method for aspirating cleavage products from the blastocoele cavity for immunoblot analysis.

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.

Introduction

Members of the Transforming Growth Factor ß (Tgfß) superfamily are synthesized as inactive, dimerized precursor proteins. The precursors are then cleaved by members of the proprotein convertase (PC) family, either within the secretory pathway or outside of cells. This creates an active, disulfide-bonded ligand dimer and two prodomain fragments¹. Although it has been known for over 30 years that the prodomain of Tgfß family precursors is required to generate an active ligand², the understanding of how prodomains contribute to ligand function is incomplete.

Although the understand....

Protocol

All procedures described are approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah. Frogs are housed in an IACUC approved facility. Male frogs are euthanized by immersion in tricaine following by clipping the ventricle of the heart. Female frogs are housed in the laboratory for a maximum of 24 h following hormonal induction of spawning to allow for egg collection and then returned to the care facility.

1. Collection of X. laevis Testes

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Discussion

The main advantages to the protocol described here are that it can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions. Another advantage is that it allows one to analyze epitope-tagged proteins and thus circumvent the shortage of commercially available antibodies that recognize most Tgfß family prodomain. Although it is also possible to analyze the cleavage of epitope-tagged Tgfß precursor proteins i.......

Materials

Name	Company	Catalog Number	Comments
ß-mercaptoethanol	Fisher	AC125470100
Bromophenol Blue	Fisher	B392-5
Calcium Chloride	Fisher	C79-500
Calcium Nitrate	Fisher	C109-500
Disposable Pellet Pestle/Tissue Grinder	Fisher	12-141-364
Dumont #5 forceps	Fine Science tools	11251-10
Fetal Bovine Serum	Atlanta Biologicals	S11150H
Ficoll 400	Sigma Aldrich	F9378-500G
Glass capillary, 1 X 90 mm	Narshige	G-1
Glycerol	Fisher	G33-4
HEPES	Fisher	BP310-500
Human chorionic gonadotropin	Sigma Aldrich	CG10-10VL
Injection Syringe, 1 mL	Fisher	8881501368
L-Cysteine	Sigma Aldrich	C7352
Magnesium Sulfate	Fisher	M63-500
Needle, 26 G	Fisher	305111
Penicillin/Streptomycin	Gibco	15140148
Picoliter Microinjector	Warner Instruments	PLI-100A
Pipette Puller	Narashige	PC-100
Potassium Chloride	Fisher	P217-500
PVDF Membrane	Sigma Aldrich	IPVH00010
Sodium Bicarbonate	Fisher	S233-500
Sodium Chloride	Fisher	S271-10
Sodium Dodecyl Sulfate	Fisher	BP166-500
Sodium Hydroxide	Fisher	S318-500
Tricaine-S	Pentair	TRS5
Tris	Fisher	BP152-5