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First published online 15 August 2007
doi: 10.1242/dev.005967

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BMP signalling inhibits premature neural differentiation in the mouse embryo

¹ Molecular Embryology Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK.
² Transgenic Facility, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK.
³ Molecular Developmental Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Bmpr1a is essential for BMP signalling in the early mouse embryo. (A) At 5.5 dpc, pSmad1/5/8 expression is observed in the majority of visceral endoderm cells and in a mosaic fashion in the epiblast of control embryos, but (B) is completely lost in Bmpr1a mutant embryos (n=4). (C) At 6.5 dpc, pSmad1/5/8 is expressed in the proximal epiblast and visceral endoderm of control embryos. (D) In Bmpr1a-/- embryos, expression is either present in the visceral endoderm and lost from the epiblast (n=2) or (E) strongly downregulated in the epiblast (n=6). (F) At 7.5 dpc, pSmad1/5/8 expression can still be observed in the proximal epiblast and visceral endoderm of control embryos, but (G) is completely lost in Bmpr1a mutant embryos (n=4). (A'-G') Nuclear DAPI-stained image; (A''-G'') overlay of pSmad1/5/8 and nuclear staining.

View larger version (124K): [in this window] [in a new window]   Fig. 2. Premature neural differentiation of the epiblast occurs in Bmpr1a-/- embryos. (A,B) At 5.5 dpc, Oct4 expression is observed throughout the epiblast of control and Bmpr1a mutant mouse embryos (n=5). (C,D) At 6.5 dpc, Oct4 is expressed in the epiblast of controls but its expression is downregulated in 50% of Bmpr1a-/- embryos (n=4/8). (E,F) Nanog expression is observed in the proximal posterior epiblast at 6.5 dpc in controls, but not in Bmpr1a mutant embryos (n=6). (G,H) At 6.5 dpc, Fgf5 is expressed throughout the epiblast of controls but is strongly downregulated or absent in Bmpr1a-/- embryos (n=5). (I-L) At 7.5 dpc, Six3 and Hesx1 are expressed in the anterior neural ectoderm of controls, but in Bmpr1a mutant embryos expression of both these genes is observed throughout the epiblast (n=7/8 and n=4/6, respectively). (M,N) Sox1 expression is not observed at 7.5 dpc in control embryos, but in Bmpr1a-/- embryos expression is observed in the entire epiblast (n=7/8). (O,P) No Foxg1 expression is observed in control embryos at 7.5 dpc, but in Bmpr1a-/- embryos Foxg1 is expressed in a discrete patch of cells at the boundary between the embryonic and extra-embryonic region (n=4/6). (Q,R) At 6.5 dpc, Six3 is not expressed in control embryos but is present throughout the epiblast of two-thirds of Bmpr1a-/- embryos (n=3/5). (S,T) Hesx1 is expressed in the anterior visceral endoderm at 6.5 dpc, but in a third of Bmpr1a mutant embryos expression is observed throughout the epiblast (n=3/10).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Suppression of mesoderm in Bmpr1a mutant mouse embryos. (A,B) At 6.5 dpc, Fgf8 is expressed in the posterior epiblast and visceral endoderm of controls, but is lost from the posterior epiblast of Bmpr1a mutant embryos (n=5). (C-F) eomesodermin and brachyury (T) are expressed at 6.5 dpc in the posterior epiblast and extra-embryonic ectoderm of controls, but in Bmpr1a-/- embryos their expression is lost from the posterior epiblast and is downregulated in the extra-embryonic ectoderm (n=9 and n=4). (G-J) Nodal and Cripto are expressed in the posterior epiblast at 6.5 dpc in controls, but are lost in Bmpr1a mutant embryos (n=5 and n=4, respectively). (K,L) At 6.5 dpc, Wnt3 is expressed in the posterior epiblast and posterior visceral endoderm of the control but is strongly downregulated in Bmpr1a-/- embryos (n=4). All are lateral views, anterior to the left.

View larger version (103K): [in this window] [in a new window]   Fig. 4. BMP signalling is required in the epiblast for mesoderm specification and to inhibit neural differentiation. (A,B) At 5.5 dpc, pSmad1/5/8 expression is slightly decreased in the epiblast of Bmpr1a-/fx; Sox2Cre+/- mouse embryos (n=4/7) as compared with controls. (C,D) At 6.5 dpc, in Bmpr1a-/fx; Sox2Cre+/- embryos pSmad1/5/8 expression is severely reduced in the epiblast (n=4/7) and (E,F) by 7.5 dpc is almost lost (n=4/4), although some expression in the visceral endoderm remains at both these stages. (A'-F') Overlay of pSmad1/5/8 and nuclear staining. (G,H) At 6.5 dpc, Fgf8 is expressed in the posterior epiblast of controls but its expression is reduced or lost in the majority of Bmpr1a-/fx; Sox2Cre+/- embryos (n=4/6). (I,J) Cripto is expressed in the posterior epiblast of controls at 6.5 dpc but is lost in a proportion of Bmpr1a-/fx; Sox2Cre+/- embryos (n=3/9). (K,L) At 6.5 dpc, Nodal is expressed in the posterior epiblast of controls, but when Bmpr1a is deleted from the epiblast Nodal expression is reduced or lost (n=3/4). (M,N) At 7.25 dpc, Hesx1 is expressed in the anterior definitive endoderm and anterior neural ectoderm of controls. A large expansion in the domain of Hesx1 expression is observed in the epiblast of Bmpr1a-/fx; Sox2Cre+/- embryos (n=4/6). (O,P) Six3 is a marker of the anterior neural ectoderm at 7.5 dpc. In Bmpr1a-/fx; Sox2Cre+/- embryos, Six3 expression is greatly expanded (n=7/8). (Q,R) At 7.5 dpc, Sox1 is not expressed in control embryos but can be found throughout the epiblast of Bmpr1a-/fx; Sox2Cre+/- embryos. All are lateral views, anterior to the left.

View larger version (106K): [in this window] [in a new window]   Fig. 5. Bmp7 signalling via Acvr1 cooperates with Bmp2/4 signalling to maintain pluripotency. (A) Western blot showing pSmad1/5/8 expression (upper) and loading control (lower) after Bmp4 and Bmp7 stimulation of control and Bmpr1a-/- ES cells. (B) Upregulation of Acvr1 is observed in Bmpr1a-/- cells by RT-PCR. (C) Ventral view of a medium-contribution chimera generated with Bmpr1a-/- lacZ ES cells. Red arrow, allantois. (D-F) Transverse sections of a medium-contribution chimera. Black arrows mark (D) the surface ectoderm, (E) the endoderm-derived gut and (F) the mesodermally derived notochord. (G,H) Bmpr1a-/- embryos display self-renewal defects when cultured in serum-free conditions and Bmp4. (I,J) The self-renewal defects are rescued when Bmpr1a-/- embryos are cultured in serum-free conditions plus Bmp7.

View larger version (57K): [in this window] [in a new window]   Fig. 6. Nodal signalling can resue the pluripotency defect of Bmpr1a mutant mouse embryos. (A,B) At 4.5 dpc, Nodal is expressed in the epiblast and primitive endoderm in both control and Bmpr1a-/- mouse embryos (n=5/5). (C,D) At 5.5 dpc, Nodal is expressed in the epiblast and visceral endoderm of controls but is severely downregulated in a proportion of Bmpr1a mutant embryos (n=2/3). (E,F) Cripto is expressed in the epiblast at 5.5 dpc of controls, but is lost in a proportion of Bmpr1a-/- embryos (n=4/7). (G,H) At 5.5 dpc, Gdf3 expression is observed throughout the epiblast of control and Bmpr1a-/- embryos (n=5/5). (I,J) Bmpr1a-/- embryos display self-renewal defects when cultured in serum-free conditions and Bmp4. (K,L) 20 ng/ml activin can rescue the self-renewal defects of Bmpr1a-/- embryos (n=5/7).

View larger version (49K): [in this window] [in a new window]   Fig. 7. Inhibition of FGF signalling does not block neural specification in Bmpr1a-/- mouse embryos. (A,B) No Hesx1 expression is observed in 6.5 dpc control embryos treated with either DMSO or 80 µM SU5402 and cultured for 24 hours. (C,D) Hesx1 is expressed in 6.5 dpc Bmpr1a-/fx; Sox2Cre+/- embryos after 24 hours culture in DMSO or SU5402. (E-H) Hesx1 expression is observed in 5.5 dpc control and Bmpr1a-/fx; Sox2Cre+/- embryos after 48 hours culture with SU5402, but not when cultured with DMSO. (I,J) 5.5 dpc Bmpr1a-/- embryos show similar levels of Hesx1 expression after 48 hours culture in DMSO or SU5402.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Model for how Bmp2/4 and BMPs of the 60A subgroup cooperate to maintain epiblast pluripotency in mouse. Prior to gastrulation, Bmp2 and Bmp4 maintain pluripotency and inhibit neural differentiation of the epiblast. As gastrulation commences, BMPs of the 60A subgroup cooperate with Bmp2 and Bmp4 to reinforce the inhibition of neural fate. Neural differentiation occurs in the anterior epiblast when BMPs are antagonised, first by factors secreted by the AVE and later by the axial mesendoderm. AVE, anterior visceral endoderm; ExE, extra-embryonic ectoderm.