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BMC Dev Biol
2015 Feb 03;15:9. doi: 10.1186/s12861-015-0057-x.
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The medaka dhc2 mutant reveals conserved and distinct mechanisms of Hedgehog signaling in teleosts.
Yamamoto T
,
Tsukahara T
,
Ishiguro T
,
Hagiwara H
,
Taira M
,
Takeda H
.
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BACKGROUND: Primary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction.
RESULTS: To precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling.
CONCLUSIONS: Ciliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.
Figure 1.
Morphological phenotypes of
dhc2
mutants. (A-D) Frontal views of the heart at 6 days post fertilization (dpf). (E-L) Lateral views of the ventricle (E-H) and the somite (I-L) at 3 dpf. (M-T) Transverse section of nephric duct (M-P) and tail morphology at 7 dpf (Q-T). v, ventricle; a, atrium; Scale bars: 100 μm in D, H, L, P; 200 μm in T.
Figure 2.
Generation of Maternal-Zygotic
dhc2
mutant. Germ-line replacement strategy using the rhodamine-dextran labeling technique. (A) Overview of transplantation strategy showing the transfer of cells from the margin of rhodamine-dextran-labeled mutant donor embryos into the animal pole of dead end-MO injected WT (Tg[olvas-GFP], germ cells are labeled with GFP [29]) hosts. A morpholino antisense oligonucleotide (Genetools) to dead end was complementary to a region covering the splicing site for exon 2 and intron 2, 5′-TGTTCAGAACTGGCCTCTCACCATC-3′. (B) Chimeric host embryos were screened at 2 dpf for the presence of rhodamine-labeled donor PGCs that had migrated successfully into the gonadal mesoderm (arrowhead). Host embryos also showed somatic contribution of rhodamine-dextran-labeled donor cells to anterior neuroectoderm lineages (*). (C) Chimeric host embryos were screened again at 4–6 dpf for the lost of GFP-labeled host PGCs at the dorsal region of the gut (arrowhead). Scale bars: 500 μm in B-C.
Figure 3.
Cilia and Neural patterning in MZ
dhc2
mutants. (A) SEM analysis of the ventricular surface of the neural tube at 16-somite stage. (B) Schematic view of opening of the apical surface of neural tube with forceps. (C) Expression of neural tube markers in a cross-sectional view at 16-somite stage (Dashed line in Additional file 3: Figure S3 indicates section plane). (D) Representation of the size of each progenitor domain along the DV axis. Scale bars: 5 μm in A; 20 μm in C.
Figure 4.
A schematic drawing explaining the similarities and differences in ciliary and neural tube phenotypes between fish and mouse
dhc2
/
dnchc2
mutants. Ciliary phenotypes and dorsal expansion of olig2 domain in MZdhc2 mutants are nearly identical to those in mouse mutant but nkx2.2 expression was reported to be lost in mouse mutant [14,15]. D, dorsal; V, ventral.
Figure 5.
Hh signaling activity is partially defective in MZ
dhc2
mutants but Ptch1 is localized to the cilia. (A) The percentage of nkx2.2-positive embryos with the graded series of cyclopamine treatment (Sample numbers are indicated in Additional file 6: Table S3). (B) Dorsal view of nkx2.2 expression in 0.5 μM cyclopamine treated and control (DMSO-treated) embryos. (C) Localization of Ptch1 on cilia stained with the anti-acetylated α-tubulin antibody in the neuroepithelium at 16-somite stage. (D) Transplantation of biotin-injected WT cells into MZdhc2 cells, with its schematic view, resulted in ectopic olig2 expression of WT cells in the dorsal region of MZdhc2 neural tube (arrowhead). Scale bars: 100 μm in B, 5 μm in C; 20 μm in D.
Figure 6.
fused
is a Hh target gene in medaka fish. (A-B)
nkx2.2 expression in 600 μM fused-MO injected WT embryos (A), and shh, nkx2.2 and olig2 expression in 300 μM fused-MO injected embryos (B). (C)
fu expression in a cross-sectional view and a lateral view (dashed line indicates section plane). (D)
fu expression in 5 μM cyclopamine-treated embryos. (E)
fu overexpression induced ectopic nkx6.1 and olig2 expression (arrowheads). (F) The loss of nkx2.2 expression in 2.5 μM cyclopamine-treated embryos was rescued by overexpression of fused. Scale bars: 500 μm in A, B, C (lower panel), D, F; 20 μm in C (upper panel), E.
Figure 7.
Proposed model of the distinct features of Hh signal transduction in insect, fish and mammal.
fu is expressed in a Hedgehog-dependent fashion and is also one of the components of the Hedgehog pathway in fish. Fused negatively regulates Suppressor of Fused (SuFu), which is a negative regulator of Gli/Ci in Hh signaling. The transcription of fused in fish could lead to Hh activation. This positive-feedback loop amplifies Hedgehog pathway in fish downstream of cilia.
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