Subtilisin-like proprotein convertase activity is necessary for left-right axis determination in Xenopus neurula embryos.

Signaling by members of TGF-beta superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left-right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left-right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left-right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left-right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left-right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left-right axis determination of the heart and gut in Xenopus embryos.