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First published online 12 November 2008
doi: 10.1242/dev.029835

Development 135, 4049-4058 (2008)
Published by The Company of Biologists 2008
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C2cd3 is required for cilia formation and Hedgehog signaling in mouse

Amber N. Hoover1, Aaron Wynkoop1, Huiqing Zeng1, Jinping Jia1, Lee A. Niswander2 and Aimin Liu1,3,*

1 Department of Biology, Eberly College of Science, The Pennsylvania State University, 201 Life Science Building, University Park, PA 16802, USA.
2 Howard Hughes Medical Institute and Department of Pediatrics, University of Colorado Health Sciences Center, 12800 E. 19th Avenue, Aurora, CO 80045, USA.
3 The Programs of Cell and Developmental Biology, Genetics and Neuroscience, Huck Institutes of the Life Sciences, The Pennsylvania State University, 201 Life Science Building, University Park, PA 16802, USA.


Figure 1
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  		Fig. 1. Mouse Hty mutants exhibit multiple defects in embryonic development. (A,B) E10.5 wild-type (wt) (A) and Hty mutant (B) embryos showing exencephaly and pericardial edema (outlined by dashed line). Inset in B shows front view of the mutant head; note the flat basal plate of the brain (arrowhead). (C,D) E9.5 wild-type and Hty mutant embryos showing tight mesencephalic flexure in mutant embryos (outlined). Insets show heart looping in the two embryos (direction of heart looping indicated by arrows). (E,F) At E12.5, Hty mutant embryos exhibit severe polydactyly. Forelimbs are shown. (G,H) Nodal is normally expressed asymmetrically in left lateral plate mesoderm (G, arrowhead), but is expressed on both sides in Hty mutants (H). (I,J) Lefty2 is normally expressed asymmetrically in left lateral plate mesoderm (I, arrowhead), but is expressed on both sides in Hty mutants (J). (G-J) In situ hybridization, ventral views. R, right; L, left.

 

Figure 2
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  		Fig. 2. Hty encodes a novel C2 domain-containing protein. (A) Genetic mapping of Hty to a 700 kb region on mouse chromosome 7. (B) A single nucleotide substitution (G to A; arrowheads) is present at the beginning of intron 4 of C2cd3. (C) RT-PCR using primers in exons 3 and 5 (arrowheads in D) of C2cd3 shows multiple abnormal transcripts (a-e) in an E9.5 Hty mutant embryo. A 100-bp DNA ladder (100bp) is included for reference. (D) C2cd3 contains five C2 domains. Abnormal splicing in Hty mutants (in red) results in either a severe truncation or in-frame deletions. The gene-trap allele (C2cd3GT, clone AG0177) is predicted to truncate the C2cd3 protein and fuses a β-galactosidase moiety (lacZ) to the remaining N-terminal fragment of C2cd3. The green region in the wild-type protein denotes the part of the C2cd3 protein that is not encoded by mutant transcripts d and e. (E) RNA in situ hybridization on E10.5 embryos using a sense (left) or an antisense (right) probe against C2cd3. (F) Hty/C2cd3GT transheterozygous embryos exhibit a similar phenotype to the original Hty mutants, confirming that C2cd3 is indeed the Hty gene. Shown are an E10.5 transheterozygous embryo and its wild-type littermate. Arrowhead in F points to pericardial edema.

 

Figure 3
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  		Fig. 3. Ventral spinal cord patterning is disrupted in both Hty mutant alleles. (A-C) The notochord, but not the floor plate, expresses Shh in Hty and C2cd3GT mutants. (D-F) Nkx2.2-expressing V3 interneurons are mislocalized to the ventral midline in Hty mutants, and are absent in C2cd3GT mutants. (G-I) Isl1-expressing motoneurons expand ventrally in Hty mutants, and are absent in C2cd3GT mutants. (J-L) Pax6 expression expands ventrally in Hty and C2cd3GT mutants. Shown are immunofluorescent images of transverse sections at the forelimb level of E10.5 mouse embryos. Spinal cords are outlined by dashed lines. Arrowheads point to the ventral midline of the spinal cord, which corresponds to the floor plate in the wild-type embryos.

 

Figure 4
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  		Fig. 4. Hh signaling is disrupted in Hty mutants. (A,B) Ptch1-lacZ is strongly expressed in Hh target cells in an E10.5 wild-type mouse embryo (A), but is greatly downregulated in an Hty embryo (B). (C,D) Transverse sections at the forelimb level to show Ptch1-lacZ expression in wild-type (C) and Hty mutant (D) embryos. (E,F) Dorsal views of E10.5 forelimb buds showing strong Ptch1-lacZ expression in the posterior region of the wild type (E), but no detectable expression in Hty (F; anterior is to the left). (G-L) Gli1 is normally expressed in hindbrain, otocysts (G), spinal cord (I, hindlimb level) and limb buds (K). In Hty mutants, Gli1 expression in all these tissues is greatly downregulated (H,J,L). Arrows point to expression of Ptch1-lacZ or Gli1 in wild-type embryos and the corresponding locations in Hty mutant embryos where expression of both genes is greatly reduced. Asterisks in G and H indicate the otocysts.

 

Figure 5
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  		Fig. 5. Hty is downstream of Rab23. (A,B) Foxa2 is expressed in the floor plate of the wild-type and Opb2 (Rab23) mutant spinal cord. (C,D) Foxa2 expression is absent in both Hty and Hty/Opb2 mutant spinal cords. (E) Nkx2.2 is expressed in V3 interneurons located adjacent to the floor plate in wild-type spinal cords. (F) In Opb2 mutants, Nkx2.2 expression is expanded dorsally to occupy most of the spinal cord. (G,H) In both Hty and Hty/Opb2 mutants, a few Nkx2.2-expressing cells are located at the ventral midline. (I) Pax6 is expressed in the dorsal two-thirds of the wild-type spinal cord. (J) In Opb2 mutants, Pax6 expression is restricted to a few cells in the dorsal-most region. (K,L) Pax6-expressing cells occupy the entire spinal cord in both Hty and Hty/Opb2 mutants. Shown are immunofluorescent images of transverse sections at the forelimb level of E10.5 mouse embryos. Spinal cords are outlined with dashed lines.

 

Figure 6
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  		Fig. 6. Spinal cord cells do not respond to Shh in the absence of C2cd3. (A,B) Pax7 is expressed in dorsal progenitor cells in wild-type spinal cord (A), but is expressed in all cells of Shh mutant spinal cord (B). (C,D) In both Hty and Hty/Shh double mutants, Pax7 expression remains dorsally restricted. (E) Lhx3 is expressed in V2 interneurons and motoneurons, which occupy the ventral-lateral region of wild-type spinal cord. (F) Lhx3 expression is absent in the Shh mutant. (G,H) In both Hty and Hty/Shh mutant spinal cords, the Lhx3 expression domain is expanded ventrally to include the ventral midline. (I) En1 is expressed in V1 interneurons located in small lateral domains of wild-type spinal cord. (J) En1 expression is absent in Shh mutants. (K,L) En1-expressing cells are scattered in the ventral half of Hty and Hty/Shh mutant spinal cords. Shown are immunofluorescent images of transverse sections at the forelimb level of E10.5 mouse embryos. Spinal cords are outlined with dashed lines.

 

Figure 7
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  		Fig. 7. Cilia formation is disrupted in Hty mutants. (A) SEM image of the ventral embryonic node of an E8.0 wild-type mouse embryo showing primary cilia on every cell. (B) In Hty mutants, most node cells do not have cilia. (C) In C2cd3GT mutants, cilia are absent from nearly all cells. (D) Wild-type cells kept at G0 for 48 hours develop cilia efficiently. (E) Most Hty mutant cells kept at G0 for 48 hours fail to develop cilia. In D and E, cilia are stained green and nuclei are stained blue. (F) The ratio of ciliated cells versus total cells after 48 hours in G0. Approximately 78% of wild-type cells are ciliated, whereas only ~20% of Hty mutant cells are ciliated.

 

Figure 8
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  		Fig. 8. C2cd3 regulates proteolytic processing of Gli3. (A) Western blot showing the levels of the full-length (Gli3-190) and processed (Gli3-83) forms of Gli3 in E10.5 wild-type, Hty and C2cd3GT mutant mouse embryos. (B) The ratio between Gli3-190 and Gli3-83. The results were obtained from four independent experiments. The number above each bar indicates the Gli3-190/Gli3-83 value.

 

Figure 9
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  		Fig. 9. C2cd3 is localized at the basal body. (A) C2cd3-GFP localized at one end of cilia labeled with antibody to acetylated tubulin (Ace-tub). (B) C2cd3-GFP is present at the basal body, around the centrioles labeled with antibody to {gamma}-tubulin (Gamma-tub). Arrows point to the locations of C2cd3-GFP signals.

 

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© The Company of Biologists Ltd 2008