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 co-injected, 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 co-injection 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 pan-neural marker Sox2.