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First published online 23 June 2005
doi: 10.1242/dev.01901

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Depletion of Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive brain formation in Xenopus embryos

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA

View larger version (76K): [in a new window]   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).

View larger version (118K): [in a new window]   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).

View larger version (114K): [in a new window]   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.

View larger version (90K): [in a new window]   Fig. 4. BMP depletion causes ventral mesoderm to acquire dorsal fates, animal caps to become neural, and primordial germ cell losses. (A) RT-PCR analysis of Bmp4/Bmp7-depleted VMZ explants. Microinjections of Bmp4/7 MOs cause VMZ to adopt a DMZ fate with upregulation of neural markers, such as Ncam, N-Tubulin, Otx2, and dorsal midline markers, like Shh and Xnot. (B,C) Histological sections of control VMZ compared with Bmp4/Bmp7-depleted VMZ. Absence of Bmp4/Bmp7 signaling in VMZ induces massive cell proliferation accompanied by muscle differentiation (mu), and neural tissue (ne) and cement gland (cg) formation. Insets show external views of the explants. en, endoderm; epi, epidermis; m, mesothelium; c, coelomic cavity; lpm, lateral plate mesoderm. (D-G) Animal caps depleted of Bmp4, Bmp7, or both, contain neural tissue as shown by the expression of N-Tubulin, a marker of differentiated neurons (n=12 per experimental set). (H) RT-PCR analysis of animal cap (AC) explants. Inhibition of Bmp4/Bmp7 results in neural differentiation in animal caps as shown by the expression of Ncam and Otx2 in the absence of mesodermal marker -Actin. ODC provides a loading control and Xag is a cement gland marker. (I-L) Germ cells, marked by the probe Xpat (small dots in the endodermal region), fail to develop in embryos injected with Bmp2/Bmp4/Bmp7 MOs. Primordial germ cells still form, albeit in reduced numbers, in Bmp4 or Bmp4/Bmp7 MO-injected embryos (n=8 per experimental set).

View larger version (102K): [in a new window]   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.

View larger version (85K): [in a new window]   Fig. 6. Inhibition of Bmp2/Bmp4/Bmp7 signaling in UV-treated embryos or ß-Catenin-depleted embryos results in radial brain formation. Bmp2/Bmp4/Bmp7 MOs were injected four times radially (12 ng each) into two-cell-stage UV-irradiated embryos, or were co-injected with ß-Catenin MO (7 ng total) (n=45 or more per experimental set). White arrowheads point to the blastopore/anus. (A,B) Depletion of Bmp4/Bmp7 in wild-type embryos leads to tail defects, but leaves the head and trunk regions mostly unaffected [Dorso Anterior Index, DAI=6.6 (Kao and Elinson, 1988)]. (C) UV-treated embryos are radially ventralized and develop into ventral `belly pieces' lacking all CNS and head structures (DAI=0.7). They are devoid of neural tissue, as shown by the lack of Sox2 expression at stage 20 (inset). (D) UV-treated embryos injected with Bmp4/Bmp7 MOs are radially dorsalized, and lack trunk and tail structures (DAI=8.1). At stage 20, half of the ectoderm of UV-treated Bmp2/Bmp4/Bmp7 morphants expresses the pan-neural marker Sox2 (inset). (E) ß-Catenin MO-injected embryos, like UV embryos, develop into radially ventralized belly pieces (DAI=0.3). They are also devoid of neural tissue, as shown by the lack of Sox2 expression at stage 20 (inset). (F) Depletion of Bmp4/Bmp7 activity in ß-Catenin MO-injected embryos leads to dramatically hyperdorsalized embryos (DAI=9.5) with a radial cement gland (compare with B). At stage 20, half of the ectoderm of ß-Catenin/Bmp2/Bmp4/Bmp7 morphants expresses the pan-neural marker Sox2 (inset). (G) Embryos dorsalized by LiCl treatment (DAI=8.6). (H) Western blot analysis of endogenous phosphorylation of Smad1 at gastrula stage 11. Embryos ventralized by ß-Catenin MOs have high levels of Smad1 phosphorylation, which require Bmp4/Bmp7 signals.

View larger version (96K): [in a new window]   Fig. 7. Depletion of Bmp2/Bmp4/Bmp7 causes the formation of head-like structures with a radially patterned CNS in embryos ventralized by ß-Catenin MOs. Wild-type, ß-Catenin or triple ß-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 ß-Catenin MO-injected embryos, and greatly expanded in embryos depleted of ß-Catenin and Bmp4/Bmp7. (D-F) Expression of N-Tubulin in differentiated neurons in the posterior CNS is abolished in ß-Catenin MO-injected embryos, but can be seen radially around the blastopore in embryos lacking ß-Catenin and Bmp4/Bmp7. (G-I) Gbx2 expression, which marks rhombomere 1 and otic vesicles (Von Bubnoff et al., 1996), is undetectable in ß-Catenin MO-injected embryos but can be visualized in two concentric rings in embryos depleted of ß-Catenin and Bmp4/Bmp7. (J-L) Engrailed-2 expression, which marks the midbrain-hindbrain boundary, is not present in ß-Catenin MO embryos but is expressed as a ring in embryos depleted for ß-Catenin and Bmp4/Bmp7. (M-O) Expression of the forebrain/midbrain marker Otx2 is absent in embryos injected with ß-Catenin MOs but is expressed circumferentially in embryos depleted of ß-Catenin and Bmp4/Bmp7. (P) Histological section through a ß-Catenin MO-ventralized embryo. ae, atypical ectoderm; en, endodermal tissue; m, mesothelium. (Q) Co-injection of ß-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 ß-Catenin and Bmp4/Bmp7-deficient embryos deduced from in situ hybridization studies.

View larger version (86K): [in a new window]   Fig. 8. Expression of Nieuwkoop, BCNE or Spemann organizer genes is not restored in ß-Catenin/Bmp4/Bmp7-depleted embryos. (A-D) Expression of Xnr6 in the vegetal dorsal side at blastula stage 9 in the Nieuwkoop center requires ß-Catenin, is unaffected by knockdown of Bmp4/Bmp7, and is not re-expressed in ß-Catenin/Bmp4/Bmp7 triple knockdowns. (E-L) Expression of the BCNE center genes Pintallavis/Hnf3ß and Chordin at blastula stage 9 requires ß-Catenin, is not affected by the depletion of Bmp4/Bmp7 and fails to be rescued in ß-Catenin/Bmp4/Bmp7 triple knockdowns. (M-P) Chordin expression in the Spemann organizer at gastrula stage 10 is eliminated by ß-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 ß-Catenin or ß-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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