Figure 6. Quadruple Knockdown of BMP2/4/7 and ADMP Results in Ubiquitous CNS Formation In Vivo.

(A) CNS marked by Sox2 in control embryos; inset shows frontal view (DAI = 5.2).

(B) Knockdown of ADMP moderately increases Sox2 expression in anterior CNS (inset) (DAI = 6.2).

(C) Triple knockdown of BMP2/4/7 results in embryos with a neural plate approximately 5 times the width of that of control siblings (transversal section shown in inset).

(D) Quadruple inactivation of BMP2/4/7 and ADMP (9 ng total each) results in radially dorsalized embryos and ubiquitous neural differentiation in the ectoderm (DAI = 9.7; n = 59).

(E) Cytokeratin expression marks epidermal cells on the surface of control embryos.

(F) Expression of epidermal Cytokeratin is abolished in Bmp2/4/7/Admp morphants at stage 16.

(G and H) BMP2/4/7/ADMP-deficient embryos express radial Otx2, Rx2a, and Krox20.

(I) Quadruple Chordin/Noggin/Follistatin/Cerberus MO-injected embryos are strongly ventralized (DAI = 2.3) but retain posterior CNS (compare to [K]).

(J) ADMP is held inactive by forming a stable inhibitory ternary complex with Chordin and Tsg. DN-Xlr inhibits Xolloid-related (Xlr), preventing the proteolytic cleavage of Chordin at two specific sites.

(K) Sox2 in wild-type embryos.

(L) Injection of DN-Xlr mRNA (400 pg) broadens Sox2 expression.

(M) Simultaneous knockdown of BMP2/4/7/ADMP and of Chordin/Noggin/Follistatin/Cerberus display ubiquitous neural induction, indicating that BMP ligands are epistatic to their secreted antagonists.

(N) Blastocoele injection (40 nl) of Noggin-Fc (0.5 μM) or Chordin (1 μM) protein causes complete neuralization of ectoderm.

(O) BMP2/4/7-depleted embryos retain nonneural ectoderm.

(P) Ubiquitous neural differentiation in BMP2/4/7-depleted embryos injected with DN-Xlr mRNA; this experiment suggests that Chordin cleavage by Xlr is required for ADMP-mediated epidermal differentiation.