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Figure 2. CV2 and Chordin Compensate for Each Other in Xenopus D-V Patterning(A) Uninjected embryo showing CV2 expression, which is used here as a BMP4 signaling readout (n = 17). Inset shows mid-gastrula embryo stained for Chordin (Chd) and Sizzled (Szl) (n = 24).(B) CV2 depletion upregulates its own expression (n = 15), as well as increasing Szl and decreasing Chd (n = 20).(C) Depletion of the BMP antagonist Chordin also increases the ventral CV2 and Szl expression domains (n = 13 and n = 18, respectively).(D) When coinjected, CV2 MO and Chd MO show a marked expansion of the CV2 and Szl expression domains (n = 15 and n = 25, respectively).(E–H) qRT-PCR analyses of single and double CV2 and Chd morphants for the D-V markers Szl, CV2, Gsc, and Chd at late gastrula stage 12.5.(I) Endogenous Smad1 phosphorylation is increased by coinjection of Chd MO and CV2 MO in stage 11 embryos.(J) The CV2 cleavage sequence contains the conserved low-pH GDPH autocatalytic site present in mucins. hMuc2, human mucin-2.(K) Chordin, but not full-length CV2, is cleaved by the extracellular zinc-metalloproteinases Xolloid-related (Xlr) and BMP1 (lanes 1–6). However, cleavage of full-length CV2 (80 kD band) is triggered by low pH (lanes 9 and 10).(L–N) Ventral injections of mRNAs encoding full-length CV2 (CV2-FL, n = 56, of which 95% had partial secondary axes, three independent experiments), N-terminal CV2 fragment terminating at the GDPH cleavage site (CV2 N-Ter, n = 42, no secondary axes observed), or a secreted C-terminal fragment encoding most of the vWFd domain (CV2 C-Ter, n = 45, no secondary axes observed). The insets show injected embryos at late neurula stage hybridized with the panneural marker Sox2. |
