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First published online 1 June 2005
doi: 10.1242/dev.01894

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Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors

Aimin Liu¹, Baolin Wang² and Lee A. Niswander^1,*,

¹ Department of Pediatrics, University of Colorado at Denver and Health Sciences Center, Mailstop 8322, Box 6511, Aurora, CO 80045, USA
² Department of Genetic Medicine, Cornell University Weill College of Medicine, New York, NY 10021,USA

View larger version (59K): [in a new window]   Fig. 1. Ift88hypo limbs exhibit polydactyly and anterior expansion of posterior genes. (A,C) Normal forelimb (A) and hindlimb (C) bud at E12.5 with five digits; (B,D) Ift88hypo forelimb (B) and hindlimb (D) bud at E12.5 show polydactyly. Arrowheads indicate digit condensations. (E) Fgf4 is normally expressed in the posterior AER; (F) Fgf4 is expressed in the entire AER of Ift88hypo mutant limb bud at E10.5. (G,I) Hoxd11 (G) and Hoxd13 (I) are normally expressed in the posterodistal mesenchyme of E10.5 limb; (H,J) in Ift88hypo mutants, Hoxd11 (H) and Hoxd13 (J) expression is expanded anteriorly and proximally. Green and red arrowheads in E-J indicate the anterior boundary of the AER in wild-type and Ift88hypo limbs, respectively.

View larger version (75K): [in a new window]   Fig. 2. Hh signaling and limb prepattern is defective in Ift88hypo mutant limbs. (A,B) E10.5 forelimbs. (A) Shh is expressed in the posterior mesenchyme in wild-type limbs at E10.5. (B) Shh expression is normal in Ift88hypo limbs. (C) Ptch1-lacZ is strongly expressed in cells adjacent to the Shh expression domain in wild-type E11.5 limbs. (D) Ptch1-lacZ (Goodrich et al., 1997) is expressed at very low levels in Ift88hypo E11.5 limbs. (E,F) E10.5 hind limbs. (E) Gli1 is expressed in the posterior region of the wild-type limb bud at E10.5. (F) Gli1 expression is downregulated in Ift88hypo limbs at E10.5; (G,I) Hand2 (dHand in figure) is expressed in the posterior region of the wild-type limb bud at E10.25 and E10.5, respectively; (H,J) Hand2 expression is upregulated and anteriorly expanded in Ift88hypo limbs at both E10.25 and E10.5. (K,M) Gli3 RNA is expressed throughout the wild-type limb bud, except in the most posterior domain at E10.25 and E10.5. (L,N) In Ift88hypo limbs at E10.25, Gli3 is expressed as in wild type, but later expands into the posterior mesenchyme. Green arrows indicate gene expression in normal limbs; red arrows indicate gene expression in Ift88hypo limbs; brackets indicate gene expression in Ift88hypo limbs.

View larger version (69K): [in a new window]   Fig. 3. Loss of IFT52 function leads to multiple defects in embryogenesis. (A) Wild type IFT52 encodes for a protein of 356 amino acids. In the first mutant line (XL826), the N-terminal 257 amino acids of IFT52 are fused to ß-galactosidase protein. In the second mutant line (RRJ295), the N-terminal 309 amino acids of IFT52 are fused to ß-galactosidase protein. (B) A wild-type E10.5 embryo has round smooth mesencephalic flexure (outlined). (C) An Ift52hypo E10.5 embryo has tight mesencephalic flexure (outlined). (D) The heart tube in wild-type embryo loops to the left. (E) The heart tube in Ift52hypo loops to the right. (F) The left and right olfactory pits (red arrowheads) and maxillary arches (blue arrowheads) are separated at E10.5 in wild-type embryos but are fused in the ventral midline in Ift52hypo E10.5 embryos (G). Broken lines in F,G outline the ventral boundaries of the mandibular processes. (H) A limb bud of wild-type E12.5 embryo with five digits (white arrowheads). (I) A limb bud of Ift52hypo mutant with six digits (white arrowheads). (J) Ptch1 expression in an E10.5 wild-type limb bud. (K) Ptch1 expression is not detectable in an E10.5 Ift52hypo limb bud.

View larger version (115K): [in a new window]   Fig. 4. Both Ift88hypo and Ift52hypo mutant embryos have ventral spinal cord defects. (A-D) In the wild-type spinal cord, (A) Shh is expressed in both the notochord and floorplate, (B) Nkx2.2-expressing cells are adjacent to the floor plate, (C) Isl1/2-expressing motoneurons are dorsal to Nkx2.2 cells, and (D) Pax6 is expressed weakly in dorsal neural progenitors and strongly in the intermediate regions in the spinal cord. (E-L) In both IFT mutants (E,I), the Shh expression in the floorplate is absent, (F,J) a reduced number of Nkx2.2 expressing cells are present in the ventral midline of the spinal cord, (G,K) Isl1/2-expressing cells are found scattered in the ventral midline, and (H,L) cells strongly expressing Pax6 are found scattered ectopically in the ventral most region of the spinal cord. Red arrows indicate the ventral most part of the spinal cord.

View larger version (129K): [in a new window]   Fig. 5. IFT88 is essential for Hh signaling in the spinal cord. (A) Pax7 is expressed in the dorsal neural progenitors in the spinal cord in normal embryos. (B) Pax7 is expressed in the entire spinal cord, except for the most dorsal part in Shh mutants. (C) Pax7 expression remains dorsally restricted in Ift88null mutant (C) and in Shh/Ift88null double mutant (D) spinal cord. (E) Pax6 is weakly expressed in the dorsal spinal cord and strongly expressed in the intermediate region in wild-type embryos. (F) Strong Pax6 expression is shifted to the ventral most region of the spinal cord in Shh mutants. (G) Strong Pax6 expression is expanded to the ventral region of the spinal cord in Ift88null. It also appears to be expanded more dorsally. (H) Strong Pax6 expression appears to be expanded both dorsally and ventrally in Shh/Ift88null double mutants. (I) Lhx3 is expressed in differentiated motoneurons and V2 interneurons in normal embryos. (J) No or few Lhx3-expressing cells are present in ventral midline of Shh mutant spinal cord. (K,L) Lhx3 expression is expanded to the ventral midline of the spinal cord in Ift88null (K) and Shh/Ift88null double mutants (L). (M) En1-expressing V1 interneurons are dorsally adjacent to V2 interneurons in normal embryos. (N) There are no or very few En1-expressing cells present in the ventral midline of the Shh mutant spinal cord. (O,P) En1 expression is expanded to the ventral midline of the spinal cord in both Ift88null (O) and Shh/Ift88null mutant (P) embryos. Broken yellow lines outline the spinal cord. Arrowheads in A,C,D indicate the ventral border of the Pax7 expression domains. Arrowheads in E-H indicate the boundary between the dorsal weak Pax6 domain and the strong Pax6 domains. Arrowhead in I,K,L and M,O,P indicate the ventral border of the expression domains of Lhx3 and En1, respectively.

View larger version (70K): [in a new window]   Fig. 6. Mutation in IFT88 rescues limb formation in Shh mutants. (A) At E12.5, Shh mutant embryos are smaller and have a cyclopic eye, proboscis-like facial structure and curly tail. (B) Shh/Ift88hypo double mutants are of normal size, have separated eyes, partially rescued facial structure and normal looking tail. (C) Ift88hypo embryos have very mild ventral midline defects and sometimes exencephaly. (D) Shh mutant embryos have only one digit in all four limbs. (E) Shh/Ift88hypo double mutant limbs and (F) Ift88hypo mutant limbs display multiple digits.

View larger version (34K): [in a new window]   Fig. 7. IFT88 mutations affect Gli3 processing. (A) Immunoblots show the levels of Gli3-190 and Gli3-83 in wild type, Ift88null and Ift88hypo mouse embryos at E10.5. In wild-type embryos, the majority of Gli3 protein is proteolytically processed to form Gli3-83, whereas in both Ift88null and Ift88hypo mutants, the Gli3 processing is impaired. The bands marked with asterisks in lanes 1 and 4 are nonspecific and not consistently detectable. (B) Graphical representation of the ratio of Gli3-190 versus Gli3-83 in wild type, Ift88null and Ift88hypo mutant embryos. The results were obtained from three independent experiments. Numbers above the bars show an average of the ratio.