QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 2 December 2004
doi: 10.1242/dev.01563

This Article Summary Full Text Full Text (PDF) A corrigendum has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tian, H. Articles by McMahon, A. P. Search for Related Content PubMed PubMed Citation Articles by Tian, H. Articles by McMahon, A. P.

Mouse Disp1 is required in sonic hedgehog-expressing cells for paracrine activity of the cholesterol-modified ligand

Hua Tian^1,*, Juhee Jeong^2,*, Brian D. Harfe³, Clifford J. Tabin⁴ and Andrew P. McMahon^5,

¹ One DNA Way, Genentech Incorporated, South San Francisco, CA 94080, USA
² 1550 Fourth Street, Room 282, UCSF Mission Bay Campus, San Francisco, CA 94158, USA
³ University of Florida College of Medicine, Department of Molecular Genetics and Microbiology, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA
⁴ Department of Genetics, Harvard Medical School, NRB room 360, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
⁵ Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA

View larger version (57K): [in a new window]   Fig. 1. ShhCre was effective in removing Shh activity from all Shh-producing cells. (A,B) The distribution of Shh mRNA (A) and lacZ activated in compound heterozygous for the ShhCre and R26R reporter allele (B) are very similar. External morphology of E10.5 embryos (C-E). Shh-null embryo (D) is almost identical to ShhCre/C (E). In Shh-/- mutants (G,J,M,P), the floorplate (G) and distinct ventral progenitors [Olig2+ (J), Nkx6.1+ (M) and Nkx2.2+ (P) cells] are absent. Furthermore, Pax7 (M) and Pax6 (P), negative targets of Hh signaling, move ventrally to occupy the entire ventral neural tube. In the ShhCre/C mutant, one or two Nkx6.1+ cells remain (arrow in N) in the ventral neural tube, which is indicative of some low level signaling prior to Cre activity.

View larger version (97K): [in a new window]   Fig. 2. Attenuating Disp1 activity specifically in Shh-producing cell phenocopies Disp1 hypomorphic mutants. (A-E) External facial morphology of E18.5 embryos. Disp1 conditional mutants (C,E) display a spectrum of midline facial defects that result in a pointed face and nose that are similar but slightly milder than Disp12/2, Shh+/- and Disp1C829F/2, Shh+/- embryos (B,D). (F-J) Fgf8 in situ to demarcate the epithelium of the nasal pit. Two nasal pits, which are positioned well apart in wild type (F), are brought closer to the midline in Disp12/2, Shh+/- and Disp12/2C, ShhCre/+ (G,H), and are fused at the midline in Disp12/C829F, Shh+/- (I). The fusion of the nasal pits occurs at a more medial position in Disp1C829F/2C, ShhCre/+ embryos (J). (K-O) Alcian Blue (non-mineralized cartilage) and Alizarin Red (mineralized cartilage and bone) stained skeletons of E18.5 embryos. The premaxilla and upper incisor are missing from all mutants. The premaxilla, upper incisor and parietal bone are missing from Disp12/C829F, Shh+/- (N) but not in the conditional mutant Disp1C829F/2C, ShhCre/+ (O). Midline facial defects are due to attenuation of Shh induction and signaling in the ventral forebrain. (P-Y) Whole-mount in situ of Ptch1 (P-T) and Shh (U-Y) at E9.5 show an absence of Ptch1 upregulation in the frontal nasal process of the mutants and a failure of Shh induction in the ventral forebrain.

View larger version (137K): [in a new window]   Fig. 3. Attenuation of Disp1 activity in the Shh-producing notochord leads to a similar disruption of ventral neural tube patterning, as observed in Disp1 hypomorphic mutants. Sections through the neural tube of wild-type (A-E), Disp1 hypomorphic mutants (Disp12/2, Shh+/-) (F-J), Disp1C829F/2, Shh+/- (P-T), and Disp1 conditional mutants [Disp12/2C, ShhCre (K-O) and Disp1C829F/2C, ShhCre/+ (U-Y)]. In Disp12/2, Shh+/- mutant (F-J): the floorplate is absent (F); Nkx2.2+ and Olig2+ cells are greatly reduced in number (G); Nkx2.2+ cells occupy the ventral midline (G); Nkx6.1+-positive cells are also affected (H); and the dorsal marker Pax7 is restricted to the dorsal domain (H). The conditional mutant Disp12/2C, ShhCre/+ maintains the early floorplate marker Foxa2 but no Shh expression is observed in the floorplate (K). Nkx2.2+ and Olig2+ cells are reduced in number to about 50% of the wild-type control levels and Nkx2.2+ cells occupy the ventral midline (L). In Disp12/C829F, Shh+/- mutant (P-T), ventral progenitor cell numbers are further reduced compared with Disp12/2, Shh+/-. No Nkx2.2+ cells are present (S) and the Pax7 and Pax6 domains move ventrally (R,S). Nkx2.2+ cells are still present in Disp1C829F/2C, ShhCre/+ mutants (X).

View larger version (32K): [in a new window]   Fig. 4. N-Shh rescues Disp1C829F/C829F mutant at E10.5. Gross morphology of the (A) wild-type, (B) Disp1C829F/C829F, (C) N-ShhC/Shhn; Sox2Cre, (D) N-ShhC/Shhn; Sox2Cre; Disp1C829F/C829F embryos at E10.5.

View larger version (61K): [in a new window]   Fig. 5. Shh signaling is restored in the limb bud of N-Shh rescued Disp1-null mutants. Whole-mount in situ hybridizations with antisense riboprobes for Shh (A-C) and Ptch1 (D-F) in limb buds of wild-type (A,D), N-ShhC/Shhn; Sox2Cre (B,E) and N-ShhC/Shhn; Sox2Cre; Disp1C829F/C829F (C,F) embryos at 10.5 dpc as indicated. Shh gene expression is restricted to the posterior mesenchyme in the wild type (A). N-Shhp from this domain diffuses anteriorly, which then leads to a graded Ptch1 expression across the entire posterior half of the limb field (D). The expression of N-Shh is induced in the posterior mesenchyme in N-Shh mutant (B). Ptch1 is reduced and restricted to the posterior mesenchyme (E). High levels of Ptch1 expression are restricted to Shh-producing cells and to cells immediately anterior to this domain. No gradient of expression is apparent across the AP axis. Similar levels of Shh and Ptch1 expression are observed in limb buds of N-Shh rescued Disp1 mutant embryos (C,F).

View larger version (56K): [in a new window]   Fig. 6. Ventral spinal cord patterning defects in Disp mutants are rescued by N-Shh. Floorplate and ventral progenitors see in wild type (A,G) are not induced in Disp1C829F/C829F mutants (B,H). Similarly, floorplate and pV3 progenitors (Nkx2.2+) cells are not induced in Disp1C829F/2, Shh+/- mutant (E,K). N-Shh signaling induces floorplate and ventral neural progenitors in the absence of Disp1 activity in Disp1C829F/C829F; NShhC/Shhn; Sox2Cre mutant (D,J) and in Disp1C829F/2, N-ShhC/Shhn, Sox2Cre embryos (F,L), similar to the pattern observed in N-ShhC/Shhn, Sox2Cre (C,I) embryos. Shh signaling is restored in the ventral neural tube as shown by Ptch1 expression (M-R). Although N-Shh is not detected by immunofluorescence in the floorplate of N-ShhC/Shhn, Sox2Cre embryos (C,D,F, compare with A), Shh is expressed at the ventral midline, indicating that floorplate induction has occurred (S-X) in the floorplate of N-ShhC/Shhn, Sox2Cre embryos independent of Disp1 activity.