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Fig. S8. rfng overexpression can rescue the effect of Lfng depletion in the pronephros. We previously showed rfng translation is not inhibited by either lfng MO1 or lfng MO2 (see Fig. S1E in the supplementary material). Given the previously characterised functional homology between vertebrate Fringe proteins, we co-injected rfng mRNA with lfng MO1 and lfng MO2 and attempted to rescue expression of the proximal tubule marker, slc5a2. X. laevis embryos were injected at the eight-cell stage into a ventro-vegetal blastomere to target the presumptive pronephric region, with beta-galactosidase mRNA to act as a lineage tracer (red staining on injected side). (A-G) Embryos were cultured to stage 34 when whole-mount in situ hybridisation for expression of slc5a2 was detected. Injection of a control MO had no effect on proximal tubulogenesis (A). rfng overexpression, as reported in Fig. 6C, caused ectopic expression of slc5a2 in the distal pronephric region (white arrow, B). Injection of either lfng MO1 (C) or lfng MO2 (D) completely inhibits slc5a2 expression; these embryos have gross developmental defects, highlighted by curling and perturbed growth of the tail region (black arrows). Upon co-injection of rfng mRNA with lfng MO1 (E) or lfng MO2 (F), slc5a2 expression is rescued, and in some cases over-rescue is achieved with ectopic slc5a2 expression being observed (white arrows). Overall, gross developmental defects detected after single injections of lfng MO1 or lfng MO2 were also rescued, the embryos look healthy and morphologically similar to controls. A histogram illustrates rescue of Lfng depletion by rfng overexpression in the pronephros, with much fewer embryos having reduced slc5a2 expression after co-injection of lfng MO1 and lfng MO2 with rfng mRNA than after single injections of lfng MO1 or lfng MO2 (G). We propose that Rfng is able to rescue the effects of Lfng depletion. This conclusion suggests functional homology between Rfng and Lfng exists. Asterisk denotes injected side. In addition, we have also rescued the inhibitory effects lfng MO1 and lfng MO2 had on nephrin expression with rfng mRNA and also the effect lfng MO2 had on slc5a2 and nephrin expression with lfng mRNA. Image published in: Naylor RW and Jones EA (2009) Copyright © 2009. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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