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doi: 10.1242/10.1242/dev.00178

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Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling

Takatoshi Kawakami^*, T'Nay Kawcak^*, Ya-Jun Li, Wanhui Zhang, Yongmei Hu and Pao-Tien Chuang

Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA

View larger version (29K): [in a new window]   Fig. 3. Targeted disruption of the Disp gene. (A) Schematic diagram showing the Disp genomic locus, the targeting vector and the mutant allele. The top line shows a partial restriction map of the Disp genomic locus. The Disp genomic locus consists of eight exons (E1-E8). The second exon (E2) contains the translation start ATG and is followed by a 50 kb intron. A large Disp genomic locus suggests that Disp may be subject to intricate transcriptional regulation. The regions between the broken lines represent the 5' and 3' regions of homology and X indicates events of homologous recombination. The location of the fragments used as the 5' or 3' external probes in Southern blotting are shown, as well as the sizes of the fragments detected for wild-type and targeted alleles. (B) Southern blot analysis of targeted Disp E8 allele. Southern blot analysis of genomic DNA from 9.5 dpc embryos generated from matings between Disp E8+/- heterozygous animals. DNA was digested with EcoRI and hybridized with the 3' probe. The resulting 4.5 kb and 5.2 kb bands correspond to the wild-type and targeted allele, respectively.

View larger version (92K): [in a new window]   Fig. 1. Mouse dispatched (Disp) belongs to an emerging family of proteins containing a sterol-sensing domain (SSD). (A) Predicted 1521 amino acids translation product of the Disp gene. The SSD (blue) and the 12 putative transmembrane domains (red) are colored. Transmembrane domain prediction was performed using the TopPred2 program (http://www.sbc.su.se/~erikw/toppred2). (B) Amino acid alignment between SSD-containing proteins. In addition to dispatched, several other major classes of SSD-containing proteins are incorporated in the alignment, including patched 1 (PTC1 in figure), the Hh receptor (Goodrich et al., 1996); the sterol regulatory element-binding protein [SREBP]-cleavage activation protein (SCAP) (Brown and Goldstein, 1999; Goldstein and Brown, 1990); NPC1, a protein affected in the lipid storage disorder Niemann-Pick disease type C1 (Carstea et al., 1997; Loftus et al., 1997); and HMG CoA reductase (HMGCR), a cholesterol biosynthetic enzyme (Gil et al., 1985). Che-14 encodes a C. elegans orthologue of disp and is likely to be involved in apical secretions of proteins (Michaux et al., 2000). KIAA 1742 encodes the Disp-related protein and its function is unknown. Only the SSD domains are shown and conserved amino acid residues are shown in green. Numbers to the right of the genes represent the amino acid positions in the corresponding protein used in the sequence alignment. Sequence alignment was performed using the CLUSTAL W algorithm (Thompson et al., 1994) in the DNASTAR program.

View larger version (79K): [in a new window]   Fig. 2. Expression of Disp overlaps with Hh expression in the mouse embryo. (A-H,J-L) Whole-mount in situ hybridization, using digoxigenin-labeled Shh and Disp riboprobes on wild-type mouse embryos at different stages of development from 7.75 to 10.5 dpc. (I,M-P) Section in situ hybridization using 33P-UTP-labelled Disp and Ihh riboprobes on paraffin wax sections of wild-type mouse embryos from 9.5 to 16.5 dpc. (A,B) Lateral view of late streak, head process stage egg cylinder (7.75 dpc). Arrow in A indicates Shh expression in the node. (C,D) Ventral anterior view of head fold stage embryos just prior to somite formation (8 dpc). Arrowheads in D indicate Disp expression in cells immediately adjacent to the midline mesoderm and arrows indicate Disp expression at junctions between neural and surface ectoderm. (E,F) Stage showing 13-20 somites (9 dpc). Lateral view. (G,H) Stage showing 20-25 somites (9.5 dpc). Lateral view. (I) Cross-section of a wild-type 9.5 dpc mouse embryo at the forelimb level. Arrowhead indicates the notochord. (J) Dorsal view of H at the forelimb level. White arrows indicates Disp expression in the forelimb buds. (K,L) Stage showing 31-35 somite (10.5 dpc). Lateral view. (M) Cross-section of a wild-type 10.5 dpc mouse embryo at the forelimb level. (N) Cross-section of a wild-type 13.5 dpc mouse embryo through the thoracic cavity. (O,P) Longitudinal section through the metatarsal bones of the hindlimb of a wild-type 16.5 dpc mouse embryo. Phalanges (not shown) are to the right of the pictures. nt, neural tube; fp, floor plate; nc, notochord.

View larger version (46K): [in a new window]   Fig. 4. Disp null mutants phenocopy Smo mutants. (A-D) External morphology of wild-type (A), Disp-/- (B), Smo-/- (C) and Shh-/- (D) embryos at 9.5 dpc. All views are lateral except B,C, which represent lateral ventral views. Note that embryos in B,C have initiated but failed to complete turning. By contrast, Shh-/- embryo (D) collected at a similar stage has completed embryonic turning. (E-H) Cross-sections of 9.5 dpc wild-type (E), Disp-/- (F), Smo-/- (G) and Shh-/- (H) embryos at the level of the heart tube stained with Hematoxylin and Eosin. Arrows in F,G indicate the linear heart tube in Disp (F) and Smo (G) mutants, when compared with the multichambered heart in the wild-type (E) and Shh-/- (H) embryos. All major cell types are present in a grossly normal organization in Disp mutants (F).

View larger version (90K): [in a new window]   Fig. 5. Disp null mutants are defective in Hh signaling. (A-H,O-P) Whole-mount in situ hybridization using digoxigenin-labeled riboprobes on wild-type (A,C,E,G,O) and Disp-/- (B,D,F,H,P) embryos at 9.5 dpc. All views are lateral. (A,B) Shh expression; (C,D) Ptch expression; (E,F) Hip1 expression; (G,H) Gli1 expression; (O,P) Disp expression. (I-N) Isotopic section in situ using 33P-UTP-labeled riboprobes on wild-type (I,K,M) and Disp-/- (J,L,N) embryos at 9.5 dpc. (J) Crosssection at the heart level. (I,K,L,M,N) Cross-sections at the forelimb level. (I,J) Shh expression; (K,L) Ptch expression; (M,N) Disp expression. nt, neural tube; fp, floor plate; nc, notochord.

View larger version (70K): [in a new window]   Fig. 6. Disp mutants exhibit multiple defects because of loss of Hh signaling. (A-D,Q-Z,AA-FF) Whole-mount in situ hybridization using digoxigenin-labeled riboprobes on wild-type (A,C,Q,S,U,W,Y,AA,CC,EE) and Disp-/- (B,D,R,T,V,X,Z,BB,DD,FF) embryos at 9.5 dpc. All views are lateral except (Y,Z,AA,BB), which represent dorsal views at the forelimb level. (A,B) Brachury (T) expression; (C,D) Hnf3b expression; (Q,R) Pax1 expression; (S,T) Pax3 expression; (U,V) Myf5 expression; (W,X) myogenin expression; (Y,Z) Hand2 (dHand) expression; (AA,BB) Hoxd13 expression; (CC,DD) Fgf4 expression; (EE,FF) Fgf8 expression. Bracket in Y and arrow in Z indicate Hand2 expression in the limb, whereas the line next to the bracket indicates the extent of the limb bud viewed at this angle. Arrow in CC indicates Fgf4 expression in the posterior AER of the forelimb of a wild-type embryo. (E-P) Isotopic section in situ hybridization using 33P-UTP-labeled riboprobes on paraffin sections of wild-type (E,G,I,K,M,O) and Disp-/- (F,H,J,L,N,P) embryos at 9.5 dpc. (E,F,I) Cross-section at the hindbrain level; (G,H,J-P) cross-section at the forelimb level. (E,F) Hnf3b expression; (G,H) Pax3 expression; (I,J) Pax6 expression; (K,L) Dbx1 expression; (M,N) islet 1 expression; (O,P) Wnt3a expression. nt, neural tube; fp, floor plate; bp, branchial pouch.

View larger version (55K): [in a new window]   Fig. 7. Shh protein is processed in Disp mutant embryos. Western blot of lysate from wild-type, Disp E8+/-, Disp E8-/-, Shh-/- embryos collected at 9.5 dpc and COS7 cells transfected with expression constructs that encode either the full-length Shh protein (Shh) or the unmodified N-terminal fragment (Shh-N) probed with anti-Shh antibodies. Approximately equal amounts of proteins were loaded onto each lane. Both unprocessed (Shh,.upper arrow) and processed (Shh-Np, lower arrow) forms of Shh are detected from COS7 cells expressing the full-length Shh and are absent in lysate from Shh mutant embryos. A major band running at the same position as processed Shh was detected in lysate from wild-type, Disp E8+/- and Disp E8-/- embryos. The doublet observed in COS7 cells transfected with Shh-N could represent Shh-N proteins with different lipid modifications at its N terminus. A nonspecific band (or immunoreactivity with another Hh protein) was detected in lysates from embryos only and conveniently serves as a loading control. A very faint band representing the unprocessed Shh can be detected in lysates from wild-type, Disp E8+/- and Disp E8-/- embryos upon longer exposure (data not shown).

View larger version (72K): [in a new window]   Fig. 8. Shh protein is restricted to its site of synthesis in Disp mutants. Cross-sections of wild-type (A) and Disp-/- (B) embryos at 9.5 dpc at the heart level. In the wild-type (A) sections, Shh immunoreactivity (brown) is strong in the notochord and floor plate and it extends out bi-directionally in a graded fashion (arrows and arrowheads). In sections of Disp-/- embryos (B), Shh immunoreactivity is only detected in the notochord (arrows) and no extended staining is present. nt, neural tube; fp, floor plate; nc, notochord.