XB-ART-366Development June 1, 2006; 133 (12): 2395-405.
Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase.
Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.
PubMed ID: 16687448
Article link: Development
Species referenced: Xenopus laevis
Genes referenced: chrd.1 dbx1 dhcr7 foxa2 gli1 h4c4 nkx2-2 ntn1 pax2 pax6 ptch1 ptx shh smo thbs3
GO keywords: cholesterol biosynthetic process
Morpholinos: dhcr7 MO1 dhcr7 MO2
Disease Ontology terms: Smith-Lemli-Opitz syndrome
OMIMs: SMITH-LEMLI-OPITZ SYNDROME; SLOS
Article Images: [+] show captions
|Fig. 1. Expression of DHCR7 in developing notochord. (Top) Hierarchical clustering analysis of microarray data. Each column represents a single hybridization and each row a single clone. The colors ranging from red to green correspond to increased and decreased gene expression. LPM, left presomitic mesoderm; NTC, notochord; RPM, right presomitic mesoderm. (Bottom) Whole-mount in situ hybridization revealing the expression of DHCR7 in the midline of a Xenopus neurula stage embryo.|
|Fig. 2. Developmental expression profiles of DHCR7 and Shh. (A) RT-PCR analysis of DHCR7 and Shh expression during Xenopus development. H4, histone H4. (B) Whole-mount in situ hybridization of DHCR7 and Shh at gastrula (a,e), neurula (b-d,f-h,i,j,m,n), tadpole (k,l,o,p) stages. (a,b,d,e,f,h) Dorsal views. (c,g) Anterior views. (i-p) Lateral views with anterior towards the left and dorsal upwards. dl, dorsal lip; fp, floor plate; arc, archenteron; tel, telencephalon; ave, anterior ventral endoderm; ba, branchial arches; fnr, frontonasal region; age, presumptive anterior gut endoderm; nd, notochord; ov, otic vesicle. (C) Induction of DHCR7 by activin in animal cap explants. CHX, cycloheximide; WE, whole embryos.|
|Fig. 3. Inhibition of Shh signaling by DHCR7. (A) DHCR7 mRNA (2 ng) was microinjected unilaterally into one blastomere of the two-cell stage embryo. (B) Two-cell stage embryos were injected with 250 pg of Xenopus Shh mRNA (middle row), or together with 500 pg of DHCR7 mRNA (bottom row; Shh + DHCR7). Embryos were subjected to whole mount in situ hybridization at stage 35 (tailbud) using indicated probes. When Shh mRNA was co-injected with DHCR7 mRNA, marker gene expression was restored to the wild-type level (top row). The rescue efficiency was: Pax6, 87% (n=46); Pax2, 90% (n=48); Gli1, 91% (n=34); Patched1, 93% (n=28). (C) RT-PCR analysis of RNA isolated from animals caps injected with Chordin (50 pg) plus Shh (100 pg) (lane 2), Chordin plus Shh with 1 ng of DHCR7 (lane 3), Chordin plus 100 pg of Shh-N (lane 4) and Chordin plus Shh-N with 1 ng of DHCR7 (lane 7) mRNA. Uninjected stage 28 equivalent animal cap control (lane 1). (D) Gli-reporter assays using animal cap assay. (E) Schema of a conjugation experiment. (F) Gli-reporter gene assay using animal cap conjugates.|
|Fig. 4. Loss-of-DHCR7 affects cellular response toward Hh signaling in optic vesicle. (A-F) DHCR7 knockdown by MOs leads to eye defects. Four-cell stage Xenopus embryos were injected marginally in all four blastomeres with 5 ng of control MO (A,D), DHCR7 MOs (B,E) or DHCR7 MOs together with 125 pg of full-length DHCR7 rescue mRNA (C,F). Additional defects detected were in the heart and gut of the swimming tadpole stages (data not shown). (G-R) Loss of DHCR7 affects cellular response towards Hh signaling. Four-cell stage Xenopus embryos were injected with 20 ng of control MO (G,G′,K,K′,O,O′), 20ng of DHCR7 MOs alone (H,H′,L,L′,P,P′), 50 pg of Shh-N mRNA alone (I,I′,M,M′,Q,Q′) or 20 ng of DHCR7 MOs with 50 pg of Shh-N mRNA (J,J′,N,N′,R,R′). Knockdown of DHCR7 reduces Pax6 expression (G-J), expands the expression of Pax2 expression (K-N) and upregulates Gli1 expression (O-R). (G′-R′) Continuously treatment of embryos with cyclopamine (100μ M) from the blastula stage blocks Hh signaling (G-J,K-N,O-R). Circled areas mark optic vesicles.|
|Fig. 5. Expansion of Shh and FoxA2 expressing domains in MO-injected embryos. Four-cell stage Xenopus embryos were injected with 20 ng of control MO (A,D,G,J,M), with 20 ng of DHCR7 MOs alone (B,E,H,K,N) or with 500 pg of DHCR7 rescue mRNA (C,F,I,L,O). Whole-mount in situ hybridization of the tailbud stage embryos microinjected with DHCR7-MOs shows expansion of floor plate as marked by FoxA2 (A-C) and Shh (D-O) expression. (B,E) DHCR7 MOs injected embryos expand FoxA2 and Shh expression domains in frontal nasal region, branchial arches and anterior gut endoderm. (M-O) Transverse sections of embryos indicated in D,E. (K) Ectopic expression of Shh was detected in the notochord region of DHCR7 MO injected embryos (40%, n=28). ave, anterior ventral endoderm; ba, branchial arches; fl, floor plate (arrowheads); fnr, frontonasal region; age, presumptive anterior gut endoderm. White arrowheads indicate ectopic Shh expression.|
|Fig. 6. Neural patterning modulated by DHCR7. (A) Schema of unilateral injection experiment. Eight-cell stage Xenopus embryos were unilaterally (right side) microinjected with 20 ng of control MO (B,E,H) or 20ng of DHCR7 MOs alone (C,F,I) or with 500 pg of DHCR7 rescue mRNA (D,G,J). Transverse section of whole-mount in situ hybridization of the tailbud stage embryos microinjected with DHCR7-MOs shows ventralization of neural tube as marked by Shh (B-D), Nkx2.2 (E-G), FoxA2 and Dbx1 (H-J) expression. Arrowheads indicate the dorsal expression limit of the indicated markers. (K) Summary of ventralization of neural tube by loss of DHCR7.|
|Fig. 7. Shh signaling and cholesterol synthesis. (A) Various DHCR7 mutant constructs. (B) Inhibition Hh signaling is enhanced by treatment with AY-9944. Embryos were injected with the reporter construct 8X3′Gli-BS Luc with or without Shh-N, DHCR7 mRNA and treatment with 10 μM AY-9944. (C-J) Microinjection of DHCR7 mutants that are defective in reductase activity causes cyclopic phenotypes. The overexpression of DHCR7 mRNA (E) or treatment with AY-9944 (D) caused minor defects, but a combination of both causes severe defects (F). Embryonic phenotypes caused by microinjection of 1 ng of DHCR7R350W (H), DHCR7IVS8-1G>C (G), DHCR7δN (J) and DHCR7W149X (I) mutants. (K) Pax6 staining of tailbud stage embryos injected with DHCR7R350W. Arrow marks `cyclopic' eye. (L) Modulation of Gli reporter by DHCR7 mutants using animal cap conjugation. Embryos were injected with Chordin (with or without Shh-N mRNA) or with Gli-BS Luc (with or without DHCR7 mRNA).|
|Fig. 8. The effects of DHCR7 impinges on Smo. (A-F) Microinjection of DHCR7R350W mutant inhibits endogenous Shh signaling. Embryos microinjected with 1 ng of DHCR7R350W mRNA inhibits the expression of FoxA2, Shh and Ptc-1. (G-I) Microinjection of mRNA (500 pg) encoding dnPKA inhibits the cyclopic phenotype caused by DHCR7R350W. (J) Control uninjected embryo. (K) Smo-M2 rescues DHCR7-mediated Hh signaling inhibition. Embryos were co-injected with 8×3′Gli-BS Luc together with Chordin, Shh-N, DHCR7 or Smo-M2 mRNA (0.5 ng), or in combination, and the reporter gene activity was measured. (L) DHCR7IVS8-1G>C blocks Gli reporter activation mediated by Smo-M2. Embryos were co-injected with Gli reporter together with Chordin, DHCR7IVS8-1G>C or Smo-M2 mRNA, or in combination, and reporter gene activity was measured. (M) Model for the role of DHCR7 in Shh signaling. Shh inactivates Ptc, thus permitting the activation of Smo. Activated Smo transmits a Shh signal to activate Gli protein. PKA inhibits Gli activation. DHCR7 inhibits Shh signaling either at the level of or downstream of Smo.|
Bijlsma, A dual role for 7-dehydrocholesterol reductase in regulating Hedgehog signalling? 2006, Pubmed