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First published online 21 July 2004
doi: 10.1242/dev.01257

Development 131, 4021-4033 (2004)
Published by The Company of Biologists 2004
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Dose dependency of Disp1 and genetic interaction between Disp1 and other hedgehog signaling components in the mouse

Hua Tian, Toyoaki Tenzen and Andrew P. McMahon*

Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA



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  		Fig. 1. The Disp1 gene genetically interacts with Hh-signaling pathway components, including Ptch1 and Shh gene. External facial morphology of E18.5 embryos (A-E). The midline facial defects, clearly visible in Disp1{Delta}2/{Delta}2 (B), are rescued in Disp1{Delta}2/{Delta}2, Ptch1+/– (D), but exacerbated in Disp1{Delta}2/C829F (C), and Disp1{Delta}2/C829F, Shh+/– (E). Alcian Blue- and Alizarin Red-stained skeletons of E18.5 embryos (F-J). The premaxilla and upper incisor are missing from Disp1{Delta}2/{Delta}2 (G), but restored in Disp1{Delta}2/{Delta}2, Ptch1+/– (I). The premaxilla, upper incisor and parietal bones are missing from Disp1{Delta}2/C829F and Disp1{Delta}2/C829F, Shh+/– (H,J). Fgf8 in situ to demarcate the epithelium of the nasal pit (K-O). Two nasal pits, which are positioned well apart in wild type (K), are brought closer to the midline in Disp1{Delta}2/{Delta}2 (L), fused in the midline in Disp1{Delta}2/C829F (M), and Disp1{Delta}2/C829F, Shh+/– (O), and are separated to normal distance in Disp1{Delta}2/{Delta}2, Ptch1+/– (N).

 


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  		Fig. 2. The induction of Shh and its targets at the ventral forebrain is delayed or attenuated at E8.5. Whole-mount in-situ hybridization of Shh (A-D); Nkx2.1(E-H); Nkx2.2 (I-L); Ptch1 (M-P) of E8.5 dpc wild-type and mutant embryos as indicated. Arrow in (A) indicates the Shh expression in the axial mesoderm notochord. Arrowhead in (B) indicates expression in the ventral forebrain.

 


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  		Fig. 3. Dorsoventral patterning of telencephalon is affected in Disp1 mutants. Coronal sections of E12.5 embryo of wild type, Disp1{Delta}2/{Delta}2, Disp1{Delta}2/C829F, and Disp1{Delta}2/C829F, Shh+/– were assayed for expression of region-specific homeobox gene Nkx2.1 (A-D), Mash1 (E-H), Dlx2 (I-L) and Ngn2 (M-P). Progressive attenuation of Shh-signaling strength through genetic manipulation of Disp1 and Shh genes leads to gradual restriction of Pan-ventral gene markers, including Nkx2.1, Mash1 and Dlx2, to the midline; and corresponding expansion of Pan-dorsal gene Ngn2.

 


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  		Fig. 4. Absence of floor plate, reduction of ventral neural progenitor population in response to attenuated Shh signaling in Disp1 hypomorphic mutants. Sections through the neural tube of control (A-D), Disp1 hypomorphic mutants (as indicated). In Disp1{Delta}2/{Delta}2 mutant (E-H): the floor plate is absent (E); Nkx2.2 and Olig2 positive cells are reduced by over 50% (F); Nkx2.2 cells occupy the ventral midline (F); and Nkx6.1-positive cells are also affected (G). The dorsal marker Pax7 is restricted to the dorsal domain (G). In Disp1{Delta}2/C829F mutant (I-L), both Nkx2.2 and Olig2 are further reduced (J). The Pax7 and Pax6 domains move ventrally (K,L). In Disp1{Delta}2/C829F, Shh+/– mutant (M-P), Nkx2.2-positive cell is absent (N). Reducing the sequestration by Ptch1 rescues the Disp1{Delta}2/{Delta}2 spinal cord phenotype in Disp1{Delta}2/{Delta}2, Ptch1+/– mutant (Q-T).

 


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  		Fig. 5. Ventral neural precursor patterning defects in Disp1 hypomorphic mutants. Motoneuron precursors (MNR+ cell) occupy the ventral midline in Disp1{Delta}2/C829F (I) and Disp1{Delta}2/C829F, Shh+/– mutants (M), instead of its normal lateral position in wild type (A). V2 (Chox10+) and V1 (En1+) interneurons are present in comparable numbers in Disp1 mutants as the wild type (B,F,J,N). V0 (Evx1/2+) precursors expand ventrally in Disp1 mutants (C,G,K,O). Lim1/2-positive cells, which demarcate the p5, V0 and V1 precursor populations also expand ventrally in Disp1 mutants (D,H,L,P).

 


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  		Fig. 6. Cephalic mesoderm patterning is affected in Disp1{Delta}2/C829F mutants. (A-B) Alcian Blue- and Alizarin Red-stained skull from E18.5 dpc embryos. (C) Model of skeletal elements of the vertebrate skull. Elements formed by neural crest are labeled green and those by mesoderm are labeled purple. (D) Skull elements altered in Disp1{Delta}2/C829F mutants are highlighted in red, which overlap with most of the skeletal elements with mesoderm origin. (E,F) Ventral view of the base of the brain; note that the basioccipital bone (bo) and basisphenoid bones (bs) are misshapen and greatly reduced in Disp1{Delta}2/C829F mutant. Whole-mount in-situ hybridization of paraxial mesoderm markers FoxD1 (G,H), FoxD2 (I,J) show clear reduction in the cephalic mesoderm area in Disp1{Delta}2/C829F mutant.

 


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  		Fig. 7. Disp1 genetically interacts with Ihh signaling at the early stage of embryonic development from E7.5 to 9.5. External morphology of E9.5 dpc embryos: (A) wild type; (B) Shh–/–; (C) Disp1{Delta}2/C829F, Shh–/–. Heart looping is clearly visible in wild-type (A) and Shh–/– (B) mutant. No heart looping occurs in Disp1{Delta}2/C829F, Shh–/– (C). Whole-mount in-situ of Dbx1 expression in wild type (D), Shh–/– (E) and Disp1{Delta}2/C829F, Shh–/– (F).

 


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  		Fig. 8. Analysis of Disp1 topology, localization and role in Shh secretion. (A) The predicted sequence of Disp1, with transmembrane helix highlighted in green and red. The blue-shaded sequence indicates what has been deleted in Disp1{Delta}2 mutation. (B) No signal was detected by immunostaining on unpermeabilized Cos7 cell using DspN and DspC antibodies. After permeabilization with Triton, Disp1 immunostaining were co-localized with ER marker Bip, and Golgi marker GM130. The protein also displayed a typical cell-surface distribution. (C) Proposed topology of Disp1 with sterol-sensing domain highlighted in red. (D) Immortalized Disp1C829F/C829F mutant fibroblast was transfected with expression constructs as indicated (a). After 2 days, the media were collected and Shh protein was immuno-pulled down with anti-Shh 5E1 monoclonal antibody, separated by SDS-PAGE, and immuno-detected with anti-Shh AB80 polyclonal antibodies. (b) Cell-surface proteins were labeled by biotin and pulled down by streptavidin, then separated by SDS-PAGE and immuno-detected with AB80. L, cell lysate input before biotinylation; S, cell-surface fraction pulled down with streptavidin.

 

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© The Company of Biologists Ltd 2004