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First published online 20 April 2005
doi: 10.1242/dev.01822

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Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm

¹ Howard Hughes Medical Institute, and Department of Biological Chemistry, University of California, Los Angeles, CA 90095-1662, USA
² Department of Orthopaedic Surgery, University of California, Los Angeles, CA 90095, USA

View larger version (72K): [in a new window]   Fig. 1. Tsg and Bmp7 interact biochemically and are expressed in overlapping domains. (A) Bmp7 co-immunoprecipitates with Tsg. Top panel was probed with an anti-Bmp7 antibody. Bmp7 was pulled down only in the presence of Tsg (compare lanes 1 and 2). This Tsg/Bmp7 interaction was not competed by a sixfold excess of TGFß2 (lane 3) but was partially competed by equimolar amounts of Bmp4 (lane 4). Middle panel provides a control for the amount of Tsg immunoprecipitated; the bottom panel serves as a loading control for antibodies on the beads. (A') Crosslinking of Tsg and Bmp7 proteins by DSS. A bimolecular complex of Tsg and Bmp7 is formed in lane 3. Tsg protein is HA tagged. Top panel (crosslinked) and middle panel (no crosslinker) were probed with an anti-HA antibody; bottom panel (no crosslinker) was probed with anti-Bmp7 antibody. Tsg protein forms a homodimer when crosslinked (lane 1, top panel). In the presence of Bmp7 (lane 3, top panel) a bimolecular complex (consistent with a homodimer of Tsg binding a homodimer of Bmp7) runs around 110 kDa. (B-D') Expression of Tsg and Bmp7 analyzed by whole-mount in situ hybridization and ß-gal staining. (B,B') Bmp7 and Tsg are co-expressed in allantois (al), axial and lateral mesoderm (am) and gut endoderm (en) at 8.25 dpc. Tsg expression is visualized via an in-frame fusion to lacZ. (C,C') At 9.0 dpc, Tsg and Bmp7 are found in gut endoderm, branchial arch one (a1), eye vesicle (ev) and otic vesicle (ov). (D,D') At 10.5 dpc, Tsg and Bmp7 are co-expressed in posterior ventral mesoderm (vm), limb bud (lb), first pharyngeal arch and basal telencephalon (bt). (E,F) Sections of 8.25 and 9.0 dpc embryos showing Tsg mRNA in endoderm, neurectoderm, head mesenchyme (hm) and ventral mesoderm (vm). (G) Section of a 9.0 dpc embryo showing expression of Bmp7 in ventral mesoderm and endoderm of the hindgut. Plane of sections are indicated in B',C,C'. h, heart; hg, hindgut; me, mesoderm; md, mandibular component of first pharyngeal arch; mx, maxillary component of first pharyngeal arch; ne, neuroectoderm; no, node.

View larger version (104K): [in a new window]   Fig. 2. Sirenomelia in Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- compound mutants. (A,B) Comparison of a wild-type neonate with a Tsg-/-;Bmp7-/- mutant displaying sirenomelia. The mutant appears normal up to the level of the hindlimbs which are fused. (C-F) Alcian Blue (cartilage) and Alizarin Red (bone) preparations of wild-type and Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- mutants. (C,D) Lateral view of the posterior region of a wild-type and Tsg-/-;Bmp7-/- skeleton. In the mutant, the tail is shorter and has fewer vertebrae. The single hindlimb contains two femurs (fe), two tibias (ti), a single foot and lacks both fibulae (fi). (E,F) Ventral views of the hindlimbs and pelvic region of a wild-type and Tsg+/–;Bmp7-/- neonate. The mutant hindlimb is formed by the fusion of two hindlimbs at the level of the ischium (is); two feet are present, fused at the level of the heel. Insets in D and F show higher magnification views of the feet of Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- neonates, respectively. Digits are indicated by numbers and digit duplications by letters. il, ilium; is, ischium; p, pubic bone; ps, pubic symphysis.

View larger version (57K): [in a new window]   Fig. 3. Tsg and Bmp7 cooperate in ventral development in Xenopus embryos. (A,F,K,P) Control and morpholino-injected embryos at stage 12 hybridized with sizzled probe. Expression of sizzled is reduced in Bmp7-MO- or Tsg-MO-injected embryos and almost completely lost in the Bmp7-MO/Tsg-MO-injected embryos. (B,G,L,Q) Expression of Otx2 in injected embryos at stage 13; there is an increase in Bmp7-MO/Tsg-MO-injected embryos. (C,H,M,R) Embryos at stage 18 hybridized with Otx2 and Myod1 probes. The expansion of Otx2 regulates and is no longer seen at this stage. (D,E,I,J,N,O,S,T) Phenotypes of morpholino-injected embryos at (D,I,N,S) stage 39 (2 day) and (E,J,O,T) stage 42 (3 day). Bmp7-MO-injected embryos have a reduced ventral fin (arrowhead); Tsg-MO-injected embryos have a bent tail and reduction of ventral fin. Co-injection of Bmp7-MO and Tsg-MO synergize, causing a severe loss of ventral fin (arrow in S) and at later stages truncation of the entire tail region (T).

View larger version (59K): [in a new window]   Fig. 4. VMZ explants co-injected with Tsg-MO and Bmp7-MO are strongly dorsalized and adopt the identity of DMZ explants. (A) Experimental design of the DMZ, LMZ and VMZ explants. (B) VMZs from wild-type embryos cultured until tailbud stage 20. (C) VMZs from Bmp7-MO-injected embryos are dorsalized and resemble LMZs (inset). (D) VMZs from Tsg-MO-injected embryos showing dorsalization (unpigmented regions). (E) VMZs from Tsg-MO- and Bmp7-MO-injected embryos elongate similarly to DMZ (Spemann organizer) explants (shown in the inset). (F) RT-PCR assays of stage 26 whole embryos (WE) (lane 1), DMZs (lane 2), LMZs (lane 3) and VMZs (lane 4) from uninjected embryos or from embryos injected with Tsg-MO (lane 5), Bmp7-MO (lane 9), with Tsg-MO or several doses of Bmp7-MO (lanes 6-8). VMZs from embryos injected with both MOs express the same markers as wild-type DMZ explants (compare lanes 2 and 8), indicating strong dorsalization and reduced Bmp signaling. VP, vegetal pole; WE, whole embryo.

View larger version (76K): [in a new window]   Fig. 5. Phenotypic abnormalities in Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- compound mutants in the mouse. (A) Wild-type embryo 10.5 dpc. (B) Side view of a Bmp7-/-,Tsg-/- mutant with sirenomelia; the hindlimb buds are fused into a single bud (arrow). (C) Ventral view of a severely affected Tsg+/–;Bmp7-/- mutant embryo with heart edema and underdeveloped anterior and posterior structures. (D-G') Histological analyses of the posterior region of 9.5 dpc wild-type and Tsg-/-;Bmp7-/- embryos. (D,D') Side view of the embryos indicating the level of sections in E-G'. The posterior arterial system is drawn schematically and superimposed; the dorsal aorta (da) curves ventrally in the tailbud, forming the recurved distal aorta (rda), from which the umbilical artery (ua) originates. (E-G') Transverse serial sections of embryos shown in D and D' stained with Hematoxylin and Eosin. (E,E') Section at the level of the right and left hindlimb buds (rhb, lhb) showing the absence of the rda, fusion of the hindlimb buds (hb) and a single coelomic cavity (cc) in the mutant. (F,F') More posterior sections showing a decreased diameter of the tail at the level of the aortic curvature. (G,G') Section at the tip of the tail showing fewer tailbud mesoderm cells and a normal neural plate in the mutant. hg, hindgut; lhb, left hindlimb bud; n, node; nt, neural tube; rhb, right hindlimb bud; s, somite; tbm, tail bud mesoderm; ua, umbilical artery; va, vitelline artery.

View larger version (100K): [in a new window]   Fig. 7. Analysis of the sirenomelia phenotype. (A,B) Side view of 9.5 dpc wild-type and mutant embryos hybridized with Bmp4 probe. (A',B') Ventral views of hindlimb bud region of embryos shown in A and B, respectively; the tail has been excised. In the mutant, expression of Bmp4 in the future hindlimb bud mesoderm (hb) forms a transverse horizontal domain instead of two lateral ones on the right (rhb) and left (lhb) hindlimb buds seen in the wild type. (C,D) Side view of 10.5 dpc embryos hybridized with Fgf8 probe, marking the apical ectodermal ridge (aer). (C',D') Ventral views of hindlimb bud region of embryos shown in C and D, respectively. In the mutant, the aer expression domain of Fgf8 adopts a horseshoe-like shape, with the two aer regions meeting posteriorly instead of forming two lateral arches. (E,F) Side view of 10.5 dpc embryos hybridized with sonic hedgehog (Shh) probe. (E',F') Ventral views of hindlimb bud region at higher magnification. Expression of Shh in the zone of polarizing activity (zpa) is not yet detected, and hindgut (hg) staining is reduced in the mutant. (G,H) Side view of 9.5 dpc embryos hybridized with brachyury probe. (G',H') Higher magnification of tail region of embryos stained for brachyury. Staining of tail bud mesoderm (tbm) is decreased ventrally in the mutant; note the abnormal indentation (black arrow) and narrowing of the tail in the mutant. no, node; vm, ventral mesoderm.

View larger version (97K): [in a new window]   Fig. 6. Onset of ventral and posterior mesoderm defects in Tsg-/-;Bmp7-/- and Tsg+/–;Bmp7-/- compound mutants. (A,A') Ventral view of posterior primitive streak (ps) region of wild-type and mutant embryos stained with brachyury. The expression of brachyury is unchanged at 8.25 dpc in the mutant. (B,B') Side view of 8.25 dpc embryos before turning hybridized with Fgf8. Staining of the primitive streak and posterior mesoderm (pm) is fainter in the mutant. (C,C') Dorsal view of the posterior region of embryos shown in B and B', respectively. Fgf8 expression is less intense in the mutant. (D,D') High magnification view of the tailbud region of embryos stained with brachyury; in the mutant staining of tailbud mesoderm at the tip appears fainter, but notochord (n) expression is unaffected. (E,E') Bmp4 expression in side view of 9.0 dpc embryos (after turning). The allantois (al) and lateral plate mesoderm (lpm) are stained. (F,F') Higher magnification view of the tailbud region of embryos hybridized with Bmp4. The tailbud and of the allantois are abnormal in shape. Upon dissection, the allantois was found not to be fused to the chorion in the mutant (chorio-allantoic fusion has normally completed by this stage).