Reversade B , Kuroda H , Lee H , Mays A , De Robertis EM .

R37 HD021502-18 NICHD NIH HHS , R37 HD21502-19 NICHD NIH HHS , R37 HD021502 NICHD NIH HHS

	Fig. 1. Antisense MOs against Bmp2, Bmp4 and Bmp7 inhibit endogenous Smad1 phosphorylation and cause dorsalization and posterior truncations of Xenopus embryos. (A) Design of Bmp4, Bmp7 and Bmp2 antisense MOs that target both pseudoalleles expressed in the subtetraploid species X. laevis. (B) In vitro transcription/translation of Bmp4, Bmp7 and Bmp2 is specifically inhibited by the respective MOs. (C) MOs for Bmp2, Bmp4 and Bmp7 (at 12 ng each) were injected either alone or in combinations at the four-cell stage radially in each blastomere. (D) Endogenous carboxy-terminal Smad1 phosphorylation in stage 11 embryos is decreased by co-injection of multiple Bmp MOs. (E-H) Bmp4 MO-injected embryos (F) are dorsalized with enlarged heads (compare with control embryos, E). Red arrowheads delineate the spinal cord marker Hoxb9 (>85%, n=60). (G) Bmp4 MO-dorsalized phenotype is rescued by microinjection of 100 pg of mouse Bmp4 mRNA. (H) Microinjection of mouse Bmp4 mRNA alone (100 pg) results in ventralized embryos with small heads, no eyes and reduced spinal cord structures (red arrowheads). (I-L) Bmp4-depleted embryos (J) develop into swimming tadpoles with no ventral fins and slightly larger heads than control embryos (I). Note the position of the anus, which is displaced (posteriorized) to the tip of the tail (white arrowhead; >72%, n=61). (K) Bmp7-depleted tadpoles develop with a partial loss of ventral fin and a posteriorized anus (white arrowhead;> 79%, n=51). (L) Double knockdown of Bmp4 and Bmp7 results in tadpoles lacking tail structures (>90%, n=39).
	Fig. 2. Triple depletion of Bmp2, Bmp4 and Bmp7 results in greater dorsalization of the embryo than any single or double BMP knockdown. Embryos were fixed at tailbud stage and expression domains of four markers (Otx2, Krox20, Myod and Sizzled) were compared with control siblings (n=15 per experimental set). (A-H) Lateral view, anterior to the left. (A′-H′) Ventral view, anterior to the top. (H′) Dorsal view, anterior to the top. (A-D′) Uninjected controls and single Bmp2, Bmp4 and Bmp7 knockdowns. (E-G′) Double BMP knockdowns (2/4, 2/7 and 4/7). Note the dorsalized phenotype with increased Otx2 expression, radial expression of Myod in the posterior, and ventral loss of Sizzled. (H-H′) Triple knockdown of Bmp2, Bmp4 and Bmp7 produces further dorsalization. Expression of Krox20 in rhombomere 5 was seen over the entire circumference of the embryo down to the tip of the tail (white arrowheads).
	Fig. 3. BMP knockdown induces neuronal differentiation in posterior epidermis and cooperates with Fgf8 signaling. (A) The differentiated neuronal marker N-Tubulin is expressed in the CNS of uninjected control embryos at tailbud stage (inset: dorsal view, anterior to the top). (B-D) Inhibition of endogenous Bmp4, Bmp2/Bmp4 and Bmp4/Bmp7 activities results in ectopic neuronal differentiation in the posterior ectoderm (inset: dorsal view, anterior to the top). Comparable results were obtained with Bmp2/Bmp7 and triple Bmp2/Bmp4/Bmp7 MO injections (data not shown). (E,F) Control embryos or Bmp4 MO embryos were injected at the blastula stage with 1.5 ng of recombinant mouse Fgf8 protein into the blastocoele cavity and analyzed at tadpole stage for N-Tubulin expression. Fgf8 protein injection in wild-type embryos resulted in minor ectopic neural differentiation, which greatly synergized with Bmp4 depletion.
	Fig. 5. Bmp4 antagonizes Chordin activity in the embryo. Four-cell stage embryos were injected, into each blastomere, with Bmp4 MOs (12 ng) or Chordin MOs (7 ng), or co-injected with a mixture of the two (n=15 per experimental set). (A-C) Sizzled is expressed in the ventral center at stage 12 (A) and on the ventral side (B,C) at tailbud stage. (D-F) Depletion of Bmp4 results in the lack of Sizzled expression at late gastrula stage (D) and in significant reductions at tadpole stage (E,F). Expression of Six3, an eye marker, is expanded. (G-I) Chordin depletion gives rise to a phenotype opposite to that seen in Bmp4 MO embryos, with increased Sizzled expression and reduced Six3. (J-L) Bmp4 is epistatic to Chordin in co-injection experiments.
	Fig. 7. Depletion of Bmp2/Bmp4/Bmp7 causes the formation of head-like structures with a radially patterned CNS in embryos ventralized by β-Catenin MOs. Wild-type, β-Catenin or triple β-Catenin/Bmp4/Bmp7 MO-injected embryos were tested for the expression of CNS markers (n=9 or more per experimental set). (A-C) Expression of Sox2, demarcating the CNS at stage 15 in a control embryo, is abrogated in β-Catenin MO-injected embryos, and greatly expanded in embryos depleted of β-Catenin and Bmp4/Bmp7. (D-F) Expression of N-Tubulin in differentiated neurons in the posterior CNS is abolished in β-Catenin MO-injected embryos, but can be seen radially around the blastopore in embryos lacking β-Catenin and Bmp4/Bmp7. (G-I) Gbx2 expression, which marks rhombomere 1 and otic vesicles (Von Bubnoff et al., 1996), is undetectable in β-Catenin MO-injected embryos but can be visualized in two concentric rings in embryos depleted ofβ -Catenin and Bmp4/Bmp7. (J-L) Engrailed-2 expression, which marks the midbrain-hindbrain boundary, is not present in β-Catenin MO embryos but is expressed as a ring in embryos depleted for β-Catenin and Bmp4/Bmp7. (M-O) Expression of the forebrain/midbrain marker Otx2 is absent in embryos injected with β-Catenin MOs but is expressed circumferentially in embryos depleted of β-Catenin and Bmp4/Bmp7. (P) Histological section through a β-Catenin MO-ventralized embryo. ae, atypical ectoderm; en, endodermal tissue; m, mesothelium. (Q) Co-injection ofβ -Catenin and Bmp4/Bmp7 MOs revealed enhanced cell proliferation in ectoderm with neural tissue (ne) differentiation and radial cement gland formation (cg). (R) Schematic depiction of the anteroposterior polarity of the radial CNS formed in β-Catenin and Bmp4/Bmp7-deficient embryos deduced from in situ hybridization studies.
	Fig. 8. Expression of Nieuwkoop, BCNE or Spemann organizer genes is not restored inβ -Catenin/Bmp4/Bmp7-depleted embryos. (A-D) Expression of Xnr6 in the vegetal dorsal side at blastula stage 9 in the Nieuwkoop center requires β-Catenin, is unaffected by knockdown of Bmp4/Bmp7, and is not re-expressed in β-Catenin/Bmp4/Bmp7 triple knockdowns. (E-L) Expression of the BCNE center genes Pintallavis/Hnf3β and Chordin at blastula stage 9 requires β-Catenin, is not affected by the depletion of Bmp4/Bmp7 and fails to be rescued in β-Catenin/Bmp4/Bmp7 triple knockdowns. (M-P) Chordin expression in the Spemann organizer at gastrula stage 10 is eliminated by β-Catenin depletion but is unchanged in Bmp4/Bmp7-depleted embryos. Note that embryos shown in panels D,H,L and P do not express any of the dorsal genes tested, yet still give rise to hyperdorsalized embryos with large head structures. (Q) RT-PCR analysis at stage 9. Dorsal markers are not expressed in embryos lacking β-Catenin orβ -Catenin/Bmp4/Bmp7. (R-V) A simplified model highlighting the interactions taking place at blastula stage between BMP signals and the dorsal BCNE and Nieuwkoop centers.

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