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First published online 19 July 2006
doi: 10.1242/dev.02493

Development 133, 3179-3190 (2006)
Published by The Company of Biologists 2006
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Proper patterning of the optic fissure requires the sequential activity of BMP7 and SHH

Julian Morcillo1, Juan Ramon Martínez-Morales1,*, Françoise Trousse1,{dagger}, Yasmin Fermin1, Jane C. Sowden2 and Paola Bovolenta1,{ddagger}

1 Departamento de Neurobiología del Desarrollo, Instituto Cajal, CSIC, Dr Arce 37, Madrid 28002, Spain.
2 Developmental Biology Unit, Institute of Child Health, 30 Guilford Street, University College London, London WC1N 1EH, UK.


Figure 1
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  		Fig. 1. Bmp7-null embryos with a microphthalmic phenotype lack a morphologically visible RF. (A-C) Lateral view of intact embryos and Cresyl Violet stained frontal (D-F) or parasagittal (G-I) sections from E12.5 (A-F) or E11.5 (G-I) wild-type (A,D,G) and Bmp7-/- mouse embryos affected by an anophthalmic (B,E,H) or microphthalmic (C,F,I) phenotype. The anophthalmic eye is rudimentary, consisting of a small heavily pigmented neuroepithelium with no evidence of the RF (arrowheads in E,H). Embryos with a microphthalmic phenotype show a well-developed optic cup but a complete absence of the RF (arrowheads in F,I) when compared with wild-type embryos (arrows in D,G). The optic stalk is composed of two juxtaposed neuroepithelial layers (arrow in F). Scale bar: 300 µm in A-C; 100 µm in D,F; 50 µm in E,G-I.

 

Figure 2
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  		Fig. 2. Development of PAX2-positive RF and IB4-positive hyaloid artery precursors is impaired in Bmp7-/- embryos. (A-P) Parasagittal sections at different distoproximal levels of E10.5 (A-F) or E12.5 (G-P) optic cup from wild-type or microphthamic Bmp7-/- embryos stained with antibodies against PAX2 or with the isolectin B4 (IB4) as indicated. At E10.5, PAX2-positive staining is progressively reduced along the distoproximal axis (arrows in D-F), and by E12.5 is completely absent from the optic cup (arrows, J-L) of Bmp7 null embryos. PAX2-positive RF precursors are absent from the prospective ventral neural retina in the proximal optic cup from Bmp7-null embryos (empty arrows in E,K) when compared with the wild type (arrow in B,H). The ventral invagination of PAX2-positive precursors is nearly absent in the proximal optic cup of the mutants (empty arrow in F,L) when compared with the wild type (arrow in C,I). By contrast, PAX2-positive precursors are normally aligned on the wall of the optic stalk in both wild-type and mutant embryos (insets in I and L), although the stalk is smaller in the latter. Staining with the IB4 highlights the presence of hyaloid artery precursors in wild-type (arrow in N) but not in mutant (arrow in P) embryos, while IB4-positive vessels are visible in the distal optic cup in both wild type (M) and mutants (arrow in O). (Q) Schematic drawing summarising the morphological alterations observed in the mutant optic cup. In the mutant, ventral RF precursors are diminished and do not invaginate into the ventral optic cup. pnr, prospective neural retina; prpe, prospective retina pigmented epithelium; rfp, RF precursors. Scale bar: 50 µm in A,B,D,E; 25 µm in C,F;100 µm in G-P.

 

Figure 3
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  		Fig. 3. OD precursors, characterised by the combined expression of Pax2, Vax1, Raldh3 and netrin 1 are absent in Bmp7-null embryos. Frontal sections of the optic cup from wild-type (A,C,E,G) and Bmp7-null (B,D,F,H) E12.5 embryos were immunostained with antibodies against PAX2 (A,B) or hybridised with specific probes for netrin 1 (C,D), Raldh3 (E,F) and Vax1 (G,H). Arrowheads in A,C,E,G indicate labelled OD precursors in wild-type optic cup. No stained precursors are observed in corresponding sections from mutant embryos. However, staining in the optic stalk (arrows in B,D,H) or ventral retina (arrow in F) precursors is observed in mutants as in wild type (arrows A,C,E,G). (I) The schematic drawing summarises the expression of the different markers shown above for E10.5 (left) and E12.5 (right) optic cups. The top drawings represent PAX2 expression in parasagittal sections at different proximodistal levels (as indicated) of the cup. The overlap between the different markers demarcates the OD. Scale bar: 50 µm

 

Figure 4
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  		Fig. 4. RGC axons fail to exit the eye in Bmp7 null embryos. (A,B) The posterior view of the eye globes from E18.5 embryos shows the complete absence of the optic disc (open arrow in B) and the presence of hypopigmented streaks (arrowheads) in the Bmp7-null microphthalmic embryos when compared with wild-type littermates (arrow in A). (A',B') The images show cryostat sections of the same eyes immunostained with anti-neurofilament antibodies (red). The arrowheads in B' indicate the abnormal accumulation of fibres in the subretinal space. (C,D) Frontal cryostat sections from E14.5 wild-type (C) and Bmp7-/- (D) optic cups were immunostained with antibodies against PAX2 (green) and Tuj1 (red). PAX2-postive cells are absent in the mutant and there is the aberrant fibre growth in the subretinal space or vitreal surface (arrowheads in D,D').

 

Figure 5
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  		Fig. 5. Dorsoventral polarity markers are normally expressed in Bmp7 null optic cups. Frontal vibratome sections from wild-type (A,C,E) and Bmp7-null (B,D,F) optic cups were hybridised with probes specific for Vax2 (A,B), Bmp4 (C,D) and Raldh3 (E,F). The pattern of expression of both ventral (Vax2) and dorsal (Bmp4) markers is unchanged in the mutant. Expression levels for Raldh3 are reduced in the mutant, particularly at the presumptive RF (arrows in E,F). Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. Cell proliferation and apoptotic cell death are reduced in Bmp7-/- optic cups. (A-C) Pregnant mice were injected with BrdU and sacrificed 40 minutes later. Embryos were fixed and frontal cryostat sections were immunostained with antibodies against BrdU. The number of BrdU-positive cells present in the dorsal and ventral halves of the optic cup from wild-type (A) or Bmp7-/- embryos (B) was calculated. The dorsal border (broken lines) of Vax2 expression (insets) was considered as the dividing landmark. (C) The graph represents the percentage of BrdU-positive cells in the ventral and dorsal wild-type (blue) or mutant (purple) optic cups. The decrease of BrdU-positive cells in the ventral optic cup of the mutants is statistically significant (***P<0.0001). (D-I) Parasagittal cryostat sections at different proximodistal levels of wild-type or mutant optic cups were processed for TUNEL staining. In wild type, apoptotic cells are concentrated along the prospective RF (arrowheads in E,F). Apoptotic cells are almost absent in the equivalent sections from the mutants. Abbreviation: lv, lens vesicle; ns; no significance. Scale bar: 60 µm (30 µm in insets).

 

Figure 7
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  		Fig. 7. BMP7 requirement for RF formation is independent of Chx10 function. Frontal (A-D,G-J) and parasagittal (E,F) vibratome (A-D,I,J) or cryostat (E-H) sections through the optic cup of wild-type, Bmp7-/- and orJ embryos (as indicated) were hybridised with probes specific for Chx10, Bmp7 and Math5 (as indicated) or immunostained with antibodies against PAX2 and TuJ1 (as indicated). Chx10 expression is unchanged in the Bmp7-null mice (A,B) and Bmp7 expression is normal in the orJ mutant (C,D). PAX2-postive RF precursors are initially present in the orJ mutants (arrow in F) but disappear at later stages of development (open arrow in H). (I,J) Changes in the OD are associated with a strong decrease in the number of Math5-positive RGC precursors in the orJ retinas (J) when compared with wild-type embryos (I). Scale bar: 50 µm in A-F; 100 µm in G,H; 200 µm in I,J.

 

Figure 8
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  		Fig. 8. BMP7 and SHH are sequentially required for Pax2 expression in the distoventral optic cup. Optic vesicle (A-E;A'-E') or optic cups (F-J) from wild-type (A-C,A'-C',F-H), Bmp7-/- (D,E,D',E') or orJ (I,J) embryos were dissected and cultured as described in the Materials and methods in the absence (A,A',D,D',F,I) or presence (B,B',E,E') of BMP7, follistatin (C,C',G), SHH (J), or the 5E1 anti SHH blocking antibody (H). After 2 days in culture (2DIV) optic cups were fixed and hybridised with a Pax2-specific probe. Images show whole-mount views (A'-E',F-J) or cryostat sections (A-E) of same cultured cups. Addition of BMP7 does not significantly modify the expression of Pax2 in wild-type vesicles (B,B') but it rescues that of Bmp7-null optic vesicles (arrows in E). By contrast, follistatin completely abrogates Pax2 signal in the optic vesicle (arrow in C) but not optic cups (arrow in G) from wild-type embryos. SHH rescues Pax2 expression in orJ cups (arrow in J), while the 5E1 antibodies prevents expression (arrow in H). Asterisks in H-J indicate RPE remnants; in these vesicles, the lens is visible through the tissue. Scale bar: 50 µm in A-F; 100 µm in G,H; 200 µm in I,J.

 

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© The Company of Biologists Ltd 2006