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First published online 13 February 2008
doi: 10.1242/dev.012989

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BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions

Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Distribution of type I BMPRs in the lumbar region of E11.5 mouse spinal cord. (A) BmprIa is expressed throughout the VZ and is absent from the mantle layer, outlined in A and B. (B,C) BmprIb is expressed specifically in the dorsal and intermediate VZ, and in the dorsal-most neurons in the mantle layer (open arrowhead, C). The yellow box in B is shown at higher magnification in C. (D) BmprIb is expressed in an overlapping population of commissural progenitor cells and neurons with that labeled by antibodies against Math1 (open arrowheads, C,D). Scale bars: in A, 50 µm for A,B; in C, 25 µm for C,D.

View larger version (102K): [in this window] [in a new window]   Fig. 2. Dorsal neural cell identity is not affected if either BMPRIA or BMPRIB are constitutively activated in the chick spinal cord after HH stage 14/15. (A-H) Following electroporation of either CMV::caBMPRIA (A,B,E,F,I,J) or CMV::caBMPRIB (C,D,G,H,K,L) constructs in combination with a CMV::GFP vector, changes in cellular identity were examined in stage 22/23 embryos using antibodies against (A-D) pLh2 and (E-H) pIsl. There was no significant difference in the number of dI1 and dI3 neurons on the electroporated and non-electroporated sides of the spinal cord (electroporated side marked with +). (I-L) The activation status of the BMP-specific Smad (Smad1/Smad5/Smad8) second messenger intermediates was assessed using antibodies against the phosphorylated (phos) forms of Smad1/Smad5/Smad8, which endogenously labels the progenitor domain of dI1 neurons (open arrowheads, I,K). Both constructs can activate Smad1/Smad5/Smad8 to equally high levels. (M) Quantification of the activity levels of the CMV::caBMPRIA-HA or CMV::caBMPRIB-HA constructs, by assessing the percentage of HA+ cells that were simultaneously positive for phosSmad1/Smad5/Smad8. There was no significant difference (P>0.17) between the percentage of cells activated by BMPRIA (97.0%±0.6 s.e.m., n=97 sections from 8 embryos) and that activated by BMPRIB (96.0%±0.8 s.e.m., n=101 sections, 11 embryos). (N) Summary of spinal cell types labeled. (O) Quantification of the number of dI1 and dI3 neurons on the electroporated side verses the non-electroporated side following electroporation of either CMV::caBMPRIA or CMV::caBMPRIB at stage 11/12 or 14/15. For both receptors, there was a significant increase in the number of dI1 neurons (BMPRIA: P<0.0004, n=32 sections from 3 embryos; BMPRIB: P<3.8x10-5, n=32 sections, 2 embryos) and dI3 neurons (BMPRIA: P<0.0032, n=25 sections from 2 embryos; BMPRIB: P<0.019, n=24 sections, 2 embryos) on the electroporated side, following electroporation at stage 11/12. However, there was no increase in dI1 (BMPRIA: P>0.29, n=56 sections, 5 embryos; BMPRIB: P>0.48, n=36 sections, 4 embryos) or dI3 (BMPRIA: P>0.36, n=53 sections, 5 embryos; BMPRIB: P>0.22, n=35 sections, 6 embryos) cell numbers, following electroporation at stage 14/15. Scale bar in L: 100 µm for A-L.

View larger version (80K): [in this window] [in a new window]   Fig. 3. BMPRIB specifically mediates commissural axon outgrowth and guidance. (A-D) After the ubiquitous expression of caBMPRIA and GFP from the CMV enhancer, the (B) GFP+ (blue) and (C) Axonin1+ axons (green) cross the spinal cord normally at the FP (arrowhead, B). (D) The electroporated Axonin1+ axons also project around the circumference of the spinal cord similar to control axons. (E-H) By contrast, after misexpression of caBMPRIB and GFP from the CMV enhancer, (F) no GFP+ axons cross the FP (arrowhead), and (G,H) Axonin1+ axons are both mispolarized medially towards the lumen of the spinal cord (arrows, G,H) and stalled (open arrowhead) above the ventral midline. + indicates the electroporated side. Scale bar in A: 100 µm for A-H.

View larger version (100K): [in this window] [in a new window]   Fig. 4. Commissural axons are misguided following misexpression of Math1::caBMPRIB-IRES-fGFP in stage 15 chick spinal cords. (A-D) Electroporation of either Math1::fGFP (A,B) or Math1::caBMPRIA-IRES-fGFP (C,D) constructs results in GFP (blue) in all commissural neural processes, including the trailing processes (open arrowhead, B,D) and Axonin1+ axons (green) that cross the FP (arrowhead, B,D). Antibodies against Axonin1 also transiently label MNs (m). (E,F) By contrast, electroporation with a Math1::caBMPRIB-IRES-fGFP construct results in commissural axons being mispolarized towards the lumen (arrow, F) and stalling (arrowhead, F). (G,H) The GFP+ axons in control fillet preparations (G; dotted yellow lines in G and H indicate position of the RP and FP) project robustly to the FP, whereas GFP+ axons in the Math1::caBMPRIB-IRES-fGFP fillets (H) do not enter the ventral spinal cord (arrowhead). (I,J) The extent of the commissural axon outgrowth was quantified in stage 22/23 embryos by determining the number of GFP+ commissural axons that crossed lines drawn (J) in the mid-dorsal (MD), intermediate (INT) and mid-ventral (MV) spinal cord, and the FP. Of the control commissural neurons extending axons to the MD line, over 55% of these axons subsequently project to the MV line (n=158 sections from 10 embryos). By contrast, less than 23% of the Math1::caBMPRIB-IRES-fGFP commissural axons that extend to the MD line subsequently reach the MV line (n=145 sections, 15 embryos), a figure significantly different from control (P<2.4x10-31). Scale bar in F: 100 µm for A-F.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Commissural axons are mispolarized in BmprIb loss-of-function mutant mice. (A-C) In transverse sections of the spinal cord taken from hind-limb levels from E11.5 wild-type (A) and Math1::cre; BmprIaflox/flox (B) embryos, Tag1+ neurons are highly polarized, with the overwhelming majority (99.9%±0.1 s.e.m., n=27 sections from 4 embryos) extending axons to the FP. Many commissural axons also extend towards the FP in BmprIb-/- embryos (C); however, a small number of axons (1.8%±0.4 s.e.m., n=50 sections, 5 embryos) project aberrantly towards the lumen (open arrowhead, C). (D) A significantly higher (P<0.001) percentage of commissural axons are mispolarized medially in BmprIb-/- embryos compared with their wild-type littermates. (E,F) The Math1::cre line drives expression of Cre recombinase (green) specifically in the pLh2+ (red) population of commissural neurons. Scale bar in A: 75 µm for A-C, E,F.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Commissural axons are dorsally mispolarized in vivo in BmprIb-/- single mutants and Math1::cre;BmprIaflox/flox; BmprIb-/- double mutants. (A,B) In either wild-type (A) or BMPRIA-deficient (B) fillets of the spinal cord, very few Tag1+ axons extend towards the RP (A, open arrowhead) and no axons cross the RP. (C-F') By contrast, many Tag1+ axons extend into the RP in either BmprIb-/- single mutants or Math1::cre;BmprIaflox/flox; BmprIb-/- double mutants (open arrowheads, C,F), with commissural axons (closed arrowheads, D',F') now observed to cross the RP (outlined in D,F). (G) There is no significant difference (P>0.27) between the percentage of mispolarized axons in BmprIaflox/flox control fillets (0.75%±0.19 s.e.m., n=8213 pLh2+ neurons from 9 embryos) and the BMPRIA-deficient (Math1::cre;BmprIaflox/flox) fillets (0.95%±0.20 s.e.m., n=9262 pLh2+ neurons, 9 embryos). By contrast, a significant increase (P<0.002) is observed in BmprIb-/- mutants (1.32%±0.14 s.e.m., n=7940 pLh2+ neurons from 10 embryos) compared with wild-type litter-mates (0.73%±0.10 s.e.m., n=10379 pLh2+ neurons, 12 embryos). The percentage of mispolarized commissural axons seen in fillets from the Math1::cre;BmprIaflox/flox; BmprIb-/- double mutant embryos (3.5%±0.35 s.e.m., n=2114 pLh2+ neurons, 2 embryos) is statistically identical (P>0.4) to that seen in fillets from Gdf7-/- embryos (Butler and Dodd, 2003). Scale bar in B: 10 µm for A-F'.

View larger version (66K): [in this window] [in a new window]   Fig. 7. In the absence of BMPRIB, mouse commissural axons are significantly less responsive to a rat RP explant. (A,A') E11.5 rat RP explants deflect Tag1+ commissural axons (green) growing within E10.5 wild-type mouse dorsal spinal cord (d-sc) explants by an average angle of reorientation of 21.8°±2.0 s.e.m. (n=28). (B,B') Commissural axons deficient in BMPRIA (Math1::cre;BmprIaflox/flox) are reoriented by a rat RP explant, to a similar extent (P>0.4) as BmprIaflox/flox control littermates, with an average reorientation angle of 20.4°±2.0 s.e.m. (n=18). (C,C') By contrast, BmprIb-/- commissural axons are significantly less reoriented by a rat RP explant than wild-type littermates (P<1.3x10-7), with an average angle of reorientation of 9.6°±1.9 s.e.m. (n=19). Note that in the tracings in A', B' and C', only the axons deriving from the mouse d-sc explant are illustrated. (D) Schematic to illustrate the orientation of the E11 rat RP explant and the E10.5 mouse d-sc explant. (E) Histogram of the average angles of reorientation. Scale bar in C: 75 µm for A-C.

View larger version (29K): [in this window] [in a new window]   Fig. 8. BMPs have diverse functions for progenitor and postmitotic neurons in the dorsal spinal cord during development. (A) Graded signalling from the RP-derived BMPs is sufficient to induce the dI1, dI2 and dI3 cell fates. BMPRIA and BMPRIB have a shared redundant activity mediating dorsal neural cell fate specification, presumably acting through the Smad transcriptional regulator. (B) Subsequently, BMP heterodimers act as a diffusible chemorepellent to direct (dI1) commissural axons away from the RP. This activity is predominantly mediated by BMPRIB, which acts through an as yet unknown second messenger intermediate to locally reorganize the cytoskeleton.

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