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First published online 10 May 2006
doi: 10.1242/dev.02393
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Department of Developmental and Cell Biology, and Developmental Biology Center, University of California, Irvine, CA 92697-2300, USA.
Author for correspondence (e-mail:
kwcho{at}uci.edu)
Accepted 4 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Xenopus, Midline, Cholesterol, Floor plate, Smith-Lemli-Opitz syndrome (SLOS), Sonic hedgehog
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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45 kDa precursor to generate two mature proteins, a
N-terminal `signaling' ligand and a C-terminal cleavage product
(Lee et al., 1994
).
Autoproteolysis occurs concomitantly with the covalent attachment of
cholesterol to the C terminus of the Hh ligand
(Porter et al., 1996). Hh
ligands are also palmitoylated at a highly-conserved Cys residue
(Chamoun et al., 2001;
Pepinsky et al., 1998) and
these lipid modifications lead to production of a soluble multimeric protein
complex that is essential to ensure effective potency and range for Hh
signaling (Chen et al., 2004;
Lewis et al., 2001;
Zeng et al., 2001).
Hh proteins bind to their receptor Patched (Ptc), a 12-pass transmembrane
protein (Ingham and McMahon,
2001; Lum and Beachy,
2004). This binding relieves the inhibitory effect of Ptc on the
seven-pass transmembrane protein Smoothened (Smo), thereby transducing the Hh
signal to the interior of the cell. Once Smo activity is triggered,
Gli/Ci transcription factors are activated that induce downstream
target genes. At present, the details of the Hh signaling cascade between the
relief of Smo inhibition and the activation of Gli/Ci are not well
understood. However, in vertebrates, the activity of the Gli protein is
negatively regulated by protein kinase A (PKA)
(Hammerschmidt et al.,
1996).
Involvement of cholesterol in Hh signaling was further suggested by
sensitivity of the response to perturbation of cholesterol homeostasis. A
number of reagents known to inhibit cholesterol transport, such as
cyclopamine, jervine, progesterone and U1866A, block Hh signaling in chick
neural plate explants (Cooper et al.,
1998). Among these agents, cyclopamine has also been shown to
specifically bind to Smo (Chen et al.,
2002). Additionally, AY-9944, an inhibitor of cholesterol
biosynthesis, also blocks Shh signaling in vitro
(Cooper et al., 1998;
Incardona et al., 1998).
Together, these results point to the importance of cholesterol regulation
during embryogenesis, with the implication that disturbance of this process
could result in the types of developmental defects known to be associated with
abnormal Hh signaling.
Aberrant Hh signaling underlies a number of human diseases and congenital
defects, including basal cell carcinoma, polydactyly and holoprosencephaly
(HPE) (Ingham and McMahon,
2001). HPE is a patterning defect characterized by incomplete
separation of the two halves of the forebrain at the midline. Similarly, HPE
is also observed in the offspring of pregnant rats treated with AY-9944, which
inhibits cholesterol biosynthesis by inhibiting the enzymatic activity of
7-dehydrocholesterol reductase (DHCR7)
(Roux and Aubry, 1966). DHCR7
is a nine-pass membrane protein that catalyzes the final cholesterogenic step
in the endoplasmic reticulum by reducing 7-dehydrocholesterol (7-DHC) to
cholesterol (Fitzky et al.,
1998; Wassif et al.,
1998). Milder manifestations of HPE have been observed in 5%
of individuals with Smith-Lemli-Opitz Syndrome (SLOS), which is an autosomal
recessive human disorder that results from mutations in DHCR7
(Kelley et al., 1996). In
addition to midline defects, individuals with SLOS also suffer from
malformations in the limbs, heart, kidneys, pancreas and genitals
(Kelley and Hennekam, 2000).
Explanations of how blockade of the cholesterol biosynthetic pathway leads to
these malformations include (1) low levels of cholesterol and/or (2) the
accumulation of sterol precursors in tissues, either of which are proposed, in
turn, to affect the development of various tissues and organs during
embryogenesis.
Several groups have shown that cholesterol deficiency can interfere with
normal Hh signaling, and have proposed that this deficiency is the root cause
of some of the developmental abnormalities seen in individuals with SLOS
(Cooper et al., 2003;
Guy, 2000). However, direct
evidence demonstrating that impaired DHCR7 function leads to cholesterol
deficiency that, in turn, blocks Hh signaling is notably lacking. Because
cholesterol can be supplied to early embryos maternally, it is difficult to
assess the requirement of DHCR7 activity during early development.
Additionally, although some correlations exist between decreased levels of
plasma cholesterol and increased levels in the clinical severity of SLOS,
there are also inconsistencies between the acuteness of the SLOS phenotype and
the plasma levels of cholesterol and its precursor 7-DHC
(Witsch-Baumgartner et al.,
2000). Finally, neither treatment of cells with AY-9944 nor
deletion of DHCR7 in mouse embryonic fibroblasts has proven effective in
blocking sterol adduction of Shh (Cooper
et al., 1998; Cooper et al.,
2003).
In order to uncover the function of DHCR7 during mouse embryogenesis, two
DHCR7 mutants have been generated in this animal model. In the first, exon 8
was removed, generating the mutation DHCR7
EX8, which
resembles DHCR7IVS8-1G>C, a mutation frequently encountered in
individuals with SLOS (Fitzky et al.,
2001). Another mutant, DHCR73-5, was produced by
deleting exons 3, 4 and 5, thus eliminating most of the transmembrane-spanning
region (Wassif et al., 2001).
Both of these alterations inactivate the reductase activity of DHCR7,
resulting in impaired conversion of 7-DHC to cholesterol. Further
characterization of the mutant mice also revealed an overall accumulation of
sterol precursors and reduction in tissue cholesterol levels; importantly,
similar changes have been observed in human SLOS. However, neither mutant
displayed morphological defects in the developing central nervous system or
limbs, tissues where Shh is known to play crucial roles. Therefore, the
precise relationships between DHCR7 activity, Hh signaling and SLOS need to be
clarified.
We identified Xenopus DHCR7 in a microarray-based screen to uncover genes involved in chordamesodermal specification during gastrulation. To characterize the role of DHCR7 during early vertebrate development, we have performed both loss- and gain-of-function analyses in early Xenopus embryos. We report that DHCR7 functions contrary to previously held notions. DHCR7 acts as a negative regulator of the response to Shh signaling during early embryogenesis, and its inhibitory affects appear to impinge on the Hh signaling pathway at the level of smoothened (Smo).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos were microinjected and maintained with or without
5-100 µM AY-9944. Embryos were treated with cyclopamine continuously from
the blastula stage (Perron et al.,
2003). Capped mRNAs were prepared using an Ambion mMessage Machine
kit. Animal cap explants were dissected and cultured as described
(Cho et al., 1991). For
luciferase reporter assays, Gli-luciferase reporter (0.2 ng)
(Sasaki et al., 1997) was
microinjected into two-cell stage embryos alone or with combinations of
Chordin, Shh-N and DHCR7 mRNAs. Animal caps were dissected
(stage 8), cultured until control embryos reached stages 12-18 and subjected
to luciferase assays. All reporter gene experiments were repeated a minimum of
three times. Animal cap conjugation experiments were conducted by recombining
two animal caps expressing indicated mRNAs and a reporter gene. The conjugates
were cultured and assayed at indicated stages.
Whole-mount in situ hybridization
(Harland, 1991) was performed
using BM purple (Roche) as substrate. In situ probes for Shh, DHCR7, Pax6,
Pax2, HNF3ß/FoxA2, Ptc1 and Gli1 were prepared by
digesting the DNAs with EcoRI and transcribing with T7 RNA
polymerase.
Microarray analysis
Total RNA was isolated from microdissected notochordal and presomitic
mesodermal tissue fragments from early neurulae. Fluorescent labeled probes
were generated based on a T7-based RNA amplification method
(Wang et al., 2000). Four
micrograms of amplified RNA were reverse transcribed in the presence of
amine-modified random hexamers, and subsequently fluorescent labeled using a
Cy3- and Cy5 labeling kit (Amersham). Generation of microarray slides, slide
hybridization and scanning were as described previously
(Shin et al., 2005). A GenePix
Pro image acquisition program and Expressionist software (GeneData) were used
for array data analyses.
Plasmids
Xenopus DHCR7 was subcloned into pCS2+
(Turner and Weintraub, 1994)
to generate DHCR7 mRNA transcripts. A DHCR7 rescue construct
was created by inserting a PCR fragment between the EcoRI and
AscI sites of pCS2 AT+ (Tsuji and
Hashimoto, 2005). Primers used were DHCR7wt-F
(5'-GGAATTCCGCACCATGGGAGAGCGGAGAAGAG-3') and DHCR7wt-R
(5'-GGCGCGCCAAAGGTGAGGCGGTAAAACA-3'). DHCR7 mutants were generated
by PCR-based mutagenesis using the full-length DHCR7 cDNA as a template. The
combinations of the primers used to generate DHCR7 mutants were: DHCR7wt-F,
DHCR7 wt-R, DHCR7R350W-F
(5'-TACATCTTCTGGATGACCAATCAC-3') and DHCR7R350W-R
(5'-GCTGACACGTAAAAACATTCGATG-3') for DHCR7R350W;
DHCR7wt-F and DHCR7IVS8-1G>C-R
(5'-GGCGCGCCCTACTACTGTAGGGTATAGAGGTAGGG-3') for
DHCR7IVS8-1G>C; DHCR7wt-F and DHCR7W149X-R
(5'-GGCGCGCCGCAGCCCATT-3') for DHCR7W149X; and
DHCR7wt-F, DHCR7wt-R, DHCR7N1-F
(5'-CTGTGGTGCGCCAATCTC-3') and DHCR7N2-F
(5'-ATGGGAGAGCGGAGAAGAGCGAATCTGTGGTGCGCCAATCTC-3') for
DHCR7N. All DHCR7 templates were digested with NotI
and transcribed using SP6 polymerase. Full-length Shh and
Shh-N mRNA were synthesized with T7 polymerase using
EcoRI-linearized XshhTST7 or Xshh TST7-N templates
(Ekker et al., 1995a).
NotI-linearized pCS2+dnPKA (Ungar
and Moon, 1996) and pCS2+XSmoM2-FLAG
(Koebernick et al., 2003)
templates were transcribed with SP6 polymerase. chordin RNA was
synthesized as described (Blitz et al.,
2000).
Morpholinos and RT-PCR
Morpholino oligos (GeneTools) used to inhibit DHCR function were DHCR7a-MO
(5'-TCTGCTCTTTGTGTTCTGCTTATCT-3') and DHCR7b-MO
(5'-GTCCCCAGCAGCTCTCCCCATGTAG-3'). An equimolar mixture of these
MOs is referred as DHCR7-MO. Control MO
(5'-CCTCTTACCTCAGTTACAATTTATA-3') has no observable effect on
Xenopus development.
RT-PCR primers used to amplify histone H4 were as described
previously (Blitz and Cho,
1995). DHCR7 primers were
5'-AGACCTCTTCCGACGCACTA-3' and
5'-CCAGGGACCCCATTAAATCT-3'; Shh primers were
5'-CAGCGACTTCCTCATGTTCA-3' and
5'-GGCAGTTAGAGGCGCATAAG-3'; Gli1 primers were
5'-GAGCTAGTGACCCTGCAAG-3' and
5'-CATCGGGACCTGCTGTTTCC-3'; Ptc1 primers were
5'-TGCAAGGACTGCAAGATACG-3' and
5'-ATCCACCAACCGCTGTTTAG-3'; FoxA2 primers were
5'-CTTTCCCAGCACCATTCTGT-3' and
5'-ATGGGGGACCCGTAACTAGA-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8500 transcripts (Shin
et al., 2005). Hierarchical clustering analysis identified several
genes, including Pentraxin, Caveolin-1, FoxA1, Chordin, Xnot, FK506BP,
FoxA4A and DHCR7, which are differentially expressed in the
developing notochord (Fig. 1).
Among these, DHCR7 (7-dehydrocholesterol reductase, EC 1.3.1.21) was
particularly interesting as this enzyme has a presumed role in Hh signaling.
Whole-mount in situ hybridization confirmed the expression of DHCR7
in the midline of early neurula stage embryos
(Fig. 1).
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DHCR7 expression in Xenopus embryos
As DHCR7 has been implicated in Hh signaling, we looked for clues to its
relationship with Shh during Xenopus embryogenesis. Temporally,
DHCR7 transcripts are first detected at the blastula stage (stage 9).
As Shh is initially transcribed at the early gastrula stage (stage
10) (Ekker et al., 1995a),
DHCR7 expression precedes that of Shh. Both DHCR7
and Shh transcripts continue to be expressed during early
embryogenesis at least until tadpole stages
(Fig. 2A).
Whole-mount in situ hybridization revealed that shortly after the onset of gastrulation, DHCR7 is expressed in the organizer and extends broadly along the marginal zone (Fig. 2B, part a), whereas Shh is first detected weakly in the organizer with expression restricted to the dorsal midline of the gastrula (Fig. 2B, part e). As neurulation proceeds, expression of both DHCR7 and Shh mRNA extend along the embryonic dorsal midline (Fig. 2B, parts b, f). By late neural stages (stage 20), DHCR7 is expressed in the presumptive telencephalon and anterior ventral endomesoderm (ave) with weak expression in the neural plate, epidermis and notochord (Fig. 2B, parts c,d,j), while Shh is expressed in the floor plate, prechordal plate and notochord (Fig. 2B, parts g,h,n). At the tailbud stage, both DHCR7 and Shh are similarly expressed in the notochord, ventral central nervous system, floor plate, branchial arches, frontonasal region, and presumptive anterior gut endoderm, and in a very limited region of the ave (Fig. 2B, parts k,l,o,p). In conclusion, although there are differences in the details of their expression, the overall patterns are similar for Shh and DHCR7, allowing for the existence of a functional link between their activities.
We then used assays in animal cap (ectodermal) tissue explants to examine how the expression of DHCR7 is controlled. Overexpression of Shh, BMP or Wnt in animal caps did not induce DHCR7 (data not shown). However, treatment with activin protein, a member of the TGFß superfamily, induced DHCR7 expression, and this induction was direct as DHCR7 was upregulated even in the presence of the protein synthesis inhibitor cycloheximide (Fig. 2C). These results implicate a potential role for TGFß signaling in the regulation of the Shh signal cascade during early Xenopus embryogenesis.
Gain-of-function analysis suggests DHCR7 is an inhibitor of Shh signaling
Sterol modification of Shh appears to be obligatory for proper Shh
signaling in the processing of the precursor to produce the Hh ligand,
suggesting that DHCR7 is an important enzyme positively influencing the
activity of Shh. To explore the function of DHCR7 during early embryogenesis,
we examined the effects of DHCR7 overexpression. Embryos overexpressing
DHCR7 mRNA appeared to develop relatively normally until tailbud
stages (data not shown). At the swimming tadpole stage, however, embryos
overexpressing DHCR7 developed ectopic pigmentation in the optic stalk
(Fig. 3A; 63%, n=46).
This is a surprising observation as this phenotype resembles that reported in
mice deficient in Shh in retinal ganglion cells
(Dakubo et al., 2003) and
suggests that DHCR7 may function as a negative regulator of Hh signaling. We
therefore examined whether DHCR7 is capable of reversing phenotypes induced by
Shh overexpression. To accomplish this, we injected Shh mRNA alone or
together with DHCR7 mRNA, and examined the expression of genes
influenced by Shh. Previous work has shown that enhancement of Shh signaling
promotes the expression of Pax2 at the expense of Pax6
expression in optic vesicles
(Hammerschmidt et al., 1996;
Macdonald et al., 1995;
Ungar and Moon, 1996).
Pax6 expression, which is inhibited by Shh, is rescued by
co-injection of DHCR7 mRNA (Fig.
3B). Likewise, the expression of Pax2, Gli1 and
Ptc1, which is stimulated by Shh overexpression, can be rescued by
co-expression of DHCR7 mRNA.
Next, we examined the behavior of DHCR7 in a more defined system: animal
cap assays. When the BMP antagonist Chordin is expressed in animal cap
ectodermal explants, the caps are converted to a neural fate (Sasai et al.,
1994). Chordin expression in the presence of Shh effectively induces Shh
targets such as Gli1 (Tsuda et
al., 2002) (Fig.
3C, lane 2). However, expression of DHCR7 significantly reduced
the expression of Gli1, Ptc1 and FoxA2
(Fig. 3C, lane 3) consistent
with an inhibitory role for DHCR7 in Shh signaling. To examine whether this
inhibitory behavior of DHCR7 is independent of the cholesterol adduction of
Shh, we examined the effect of DHCR7 on a Shh-N-mediated Hh response. Shh-N, a
N-terminal Shh ligand produced from recombinant Shh mRNA lacking the
C-terminal domain, does not require cholesterol adduction for its activity,
but is as active as full-length Shh construct in overexpression studies
(Ekker et al., 1995a;
Lai et al., 1995). DHCR7
efficiently blocked Shh-N-induced Gli1, Ptc1 and FoxA2
expression in both animal caps (Fig.
3C, lanes 4 and 5) and embryos (data not shown), suggesting that
the role of DHCR7 in antagonizing Shh signaling does not occur at the level of
cholesterol modification of Shh ligand.
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), which was shown to respond to Hh signaling directly.
The Gli reporter is activated in animal caps expressing Chordin and Shh-N, and
this activation is blocked in the presence of DHCR7
(Fig. 3D). This Gli reporter
activation is due to Hh signaling, as mGli-BS (a reporter gene with mutations
in Gli-binding sites) was not induced by Shh-N. We observed the effect of
Shh-N on Gli transcription as early as stage 12 embryos. We also wished to determine whether DHCR7 inhibits Hh signaling by blocking responding cells from reacting to the signal. To address this issue, we used an animal cap conjugation assay (Fig. 3E). Animal caps expressing Chordin with or without Shh-N mRNA were used to provide the Shh protein to conjugated animal caps injected with Gli-reporter in the presence or absence of DHCR7. The recombined caps were then incubated and subjected to a luciferase assay. We found that induction of Gli-BS reporter was effectively blocked by DHCR7 (Fig. 3F), indicating that DHCR7 inhibits signaling in Hh responding cells and not at the level of production of the ligand.
Loss-of-function analysis: DHCR7 is an inhibitor of Shh signaling
Antisense morpholino oligonucleotides (MOs) designed to recognize both
DHCR7a and DHCR7b were injected into developing
Xenopus embryos. DHCR7-MO-injected embryos had developed
small eyes with reduced retinas and pigmented epithelia in the ventral half of
the eye (Fig. 4B,E; 90%,
n=48). This phenotype was efficiently rescued by co-expression of
DHCR7 mRNA lacking the MO-binding sequence
(Fig. 4C,F; 78%,
n=40). Combined injection of MOs designed against both
DHCR7a and DHCR7b gave stronger phenotypes than injection of
the individual MOs alone (data not shown). Interestingly, this DHCR7 phenotype
is similar to loss-of-function phenotypes for the known Hh antagonists Rab23
and FKBP8, which result from an inappropriate activation of Shh signaling
within the optic vesicle (Bulgakov et al.,
2004; Eggenschwiler et al.,
2001). The similarities between these phenotypes are consistent
with the notion that DHCR7 functions as a negative, rather than a positive,
regulator of Shh signaling. To address whether the defects in optic vesicle
patterning are due to the effect of DHCR7 on Shh signaling, rather than by
other indirect effects, we injected DHCR7-MO into Xenopus embryos and
examined the expression patterns of marker genes known to be regulated by Shh
signaling. Expression of Pax6 was reduced in DHCR7 MO-injected
embryos (compare Fig. 4G with
H; 70%, n=30), accompanied by slightly expanded
expression of Pax2 in the ventral eye
(Fig. 4K,L; 60%,
n=28). Gli1 was also broadly upregulated in MO-injected
embryos (Fig. 4O,P; 58%,
n=22). Thus, the DHCR7 loss-of-function effects resemble the
phenotypes observed after Shh overexpression or PKA inhibition in
Xenopus and zebrafish (Ekker et
al., 1995b; Macdonald et al.,
1995; Perron et al.,
2003).
As loss-of-function suggests that DHCR7 is an inhibitor of Shh signaling,
we examined whether loss of DHCR7 enhances responsiveness towards Hh
signaling. Therefore, we co-injected DHCR7-MO with low doses of Shh-N
mRNA that, alone, only weakly alter the expression of Pax2, Pax6 and
Gli1 expression. Stimulation of Hh signaling by injecting 50 pg of
Shh-N mRNA alone resulted in a reduction of Pax6
(Fig. 4I; 80%, n=16)
with concomitant expansion of Pax2
(Fig. 4M; 80%, n=22)
in the ventral half of the eye. Importantly, embryos co-injected with DHCR7-MO
and Shh-N mRNA showed complete suppression of Pax6
expression (Fig. 4J; 80%,
n=16) and expansion of Pax2 in the entire optic vesicle
(Fig. 4N; 80%, n=16).
These effects were similar to those of embryos receiving high Hh signaling
(Lupo et al., 2005),
suggesting that the loss of DHCR7 enhances the cellular response towards Hh
signaling during optic vesicle pattering.
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; Briscoe et al.,
2000). All these phenotypes were effectively rescued by
overexpression of DHCR7 mRNA (Fig.
5C,F,I,L,O; Fig.
6D,G,J). These phenotypes are consistent with the notion that
DHCR7 loss-of-function causes partial ventralization of the neural tube owing
to upregulation of Shh signaling.
De novo cholesterol synthesis is not essential for Shh signaling during Xenopus early embryogenesis
We next examined how DHCR7 might function together with an inhibitor of
cholesterol biosynthesis, in particular AY-9944, a pharmacological inhibitor
of the enzymatic activity of DHCR7 (Roux
and Aubry, 1966). When Xenopus embryos were continuously
treated with AY-9944 (up to 100 µM) from the early gastrula stage onwards,
no obvious morphological defects were noted (compare
Fig. 7C with 7D). Similarly,
when animal cap explants were treated with 10 µM AY-9944, a fivefold higher
concentration than that necessary to inhibit completely FoxA2
induction by Shh-N in chick neural plate explants
(Cooper et al., 1998),
induction of the Gli-reporter by Shh-N was largely unaffected
(Fig. 7B). These results
suggest that inactivation of the enzymatic activity of DHCR7 by AY-9944 alone
may not be sufficient to inhibit Shh signaling in Xenopus embryos.
However, as the knockdown of DHCR7 clearly shows its requirement in Shh
signaling, we considered the possibility that a non-enzymatic activity of
DHCR7 is responsible for its inhibition of Shh activity.
Therefore, we treated DHCR7 mRNA-injected embryos with AY-9944. The embryos developed noticeable microcephaly and a shortened body axis when compared with DHCR7 alone or with AY-9944 alone (compare Fig. 7C-F; 76%, n=58). We also assessed the affects of AY-9944 on Gli reporter activity. Induction of Gli reporter was suppressed more strongly in DHCR7-expressing embryos treated with AY-9944 than that of either AY-9944 or DHCR7 alone (Fig. 7B). These data support the idea that AY9944 augments the antagonistic activity of DHCR7 towards Shh by influencing an activity other than its reductase activity.
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;
Witsch-Baumgartner et al.,
2000). Another mutation, DHCR7IVS8-1G>C, is the most
frequently encountered DHCR7 mutation in individuals with SLOS. This defect
causes aberrant splicing of exon 8, generates a truncated protein that is
missing part of the sterol sensing domain (SSD; amino acid residues 318-471),
and is also significantly reduced in its reductase activity
(Fig. 7A). Several missense
mutations mapped within the DHCR7 SSD have also been identified among some
individuals with SLOS. Overexpression of either DHCR7R350W or
DHCR7IVS8-1G>C resulted in blockade of induction of Gli reporter
in the animal cap conjugation assay (Fig.
7L), despite the fact that the mutant proteins have presumably
lost essentially all of their reductase activity. Additionally, embryos
expressing either DHCR7IVS8-1G>C or DHCR7R350W
displayed severe microcephaly and a shortened body axis
(Fig. 7G,H; 90%, n=44
and 78%, n=44, respectively). When such severely affected embryos
were stained for the eye marker, Pax6, a single small `cyclopic'
eyespot was detected (Fig. 7K).
It is noteworthy that cyclopia is frequently observed in mouse embryos
deficient in Shh and in individuals with holoprosencephaly (HPE)
(Chiang et al., 1996;
Cohen, Jr and Shiota, 2002).
The experiments presented in the current study suggest that mutant DHCR7
proteins associated with SLOS are still capable of inhibiting Shh signaling
despite their lack of reductase activity.
Next, in order to determine the contribution of the SSD on Hh signaling, we
generated a Xenopus counterpart (DHCR7W149X) of human
DHCR7W151X, which completely lacks the SSD. Additionally,
DHCR7N lacking most of the N terminus of DHCR7 was
generated. When DHCR7W149X was expressed, it caused microcephaly in
Xenopus embryos (Fig.
7I; 70%, n=20), and effectively blocked the induction of
the Gli-reporter in the animal cap conjugation assay
(Fig. 7L), despite the fact
that the entire SSD is missing in the mutant protein. Importantly, expression
of DHCR7N did not affect cephalic development
(Fig. 7J; 100%, n=30)
but instead moderately upregulated the expression of the Gli-reporter gene
(Fig. 7L). Altogether, these
results show that the inhibition of Hh signaling by DHCR7 is mediated via the
N-terminal domain of DHCR7.
Epistasis analysis: DHCR7 acts at the level of Smoothened
At what level does DHCR7 inhibit Hh signaling? The inhibition by DHCR7 of
Hh signaling does not appear to be at the level of cholesterol adduction of
the ligand, as loss of reductase activity does not appear to be essential and
as Shh-N, which is not cholesterol modified, is also inhibited by DHCR7. Using
an animal cap conjugation assay, we also showed that DHCR7 inhibits signaling
in Hh responding cells and not at the level of production of the Shh ligand.
To determine where in the signal transduction pathway DHCR7 acts, we performed
epistasis experiments. First, we examined whether the cyclopic phenotype
caused by the expression of DHCR7R350W was due to a defect in Shh
signaling. The expression patterns of FoxA2, Shh and Ptc1 in
these embryos were examined. Injected embryos
(Fig. 8) exhibited significant
reductions of FoxA2 (100%, n=20), Shh (100%,
n=34) and Ptc1 (92%, n=38) transcripts. We also
examined whether dominant-negative Protein Kinase A (dnPKA) could block the
midline defects induced by DHCR7R350W. dnPKA reversed the
microcephalic/cyclopic phenotype caused by DHCR7R350W
(Fig. 8G-J; 96%,
n=46), demonstrating that DHCR7 acts as an inhibits Shh signaling by
acting upstream of PKA.
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). In
order to determine whether DHCR7 functions upstream or downstream of Smo,
embryos injected with DHCR7 MO were treated with cyclopamine and its effects
on eye development were examined. Cyclopamine blocked both the inhibition of
Pax6 and induction of Pax2 and Gli1 by DHCR7-MO or
Shh-N overexpression (Fig.
4G'-J',K'-N',O'-R'). One
interpretation of these observations is that DHCR7 functions to inhibit Hh
signaling by acting either at the level of, or upstream of, Smo.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Requirement for DHCR7 activity during early development
The fact that DHCR7 is a key enzyme in cholesterol biosynthesis,
responsible for the final step in the process, would suggest that DHCR7
stimulates Hh signaling through increased production of cholesterol. However,
results of our current loss- and gain-of-function analyses do not support this
view. Instead, our data indicate that DHCR7 serves to inhibit Hh signaling
during early Xenopus development. Overexpression of DHCR7 inhibits
the effects of ectopic expression of Shh, as well as endogenous Shh activity
(Figs 3,
7 and
8), whereas knockdown of DHCR7
by morpholino antisense oligonucleotides leads to enhancement of Shh activity
in both the eye and neural tube (Figs
4,
5,
6). Importantly, inhibition of
the enzymatic activity of DHCR7 either by treatment of embryos with the
pharmacological inhibitor AY-9944 or by overexpression of mutant DHCR7
proteins deficient in reductase activity, both still lead to inhibition of Shh
signaling (Fig. 7), suggesting
that some of the observed effects of DHCR7 on Shh signaling are independent of
the enzymatic properties of DHCR7.
Previous work using DHCR7-deficient mouse fibroblasts has suggested that
DHCR7 is required for cellular response to Shh
(Cooper et al., 2003). This is
in contrast to our finding that DHCR7 knockdown promotes Shh signaling (Figs
3,
4), and the reason for this
discrepancy is unclear at present. However, it is important to note that the
Shh response of fibroblast cells lacking DHCR7 was impaired only when residual
endogenous cholesterol was depleted. As embryos from many non-placental
vertebrates, including Xenopus, stockpile cholesterol maternally [in
contrast to mammals, where cholesterol is supplied by yolk sac/placental
transfer (Fitzky et al., 2001;
Wassif et al., 2001)],
differences in cellular cholesterol content may contribute to the difference
in DHCR7 loss-of-function phenotypes.
DHCR7 and Shh signaling in midline development
Shh secreted from the notochord and floor plate forms a concentration
gradient along the dorsoventral (DV) axis of the neural tube, thereby
controlling the expression of specific homeodomain transcription factors
(Ericson et al., 1997). Shh
signaling is also negatively regulated by the Hh antagonists Patched1, Rab23,
PKA, Hip1 and FKBP8 (Bulgakov et al.,
2004; Eggenschwiler et al.,
2001; Goodrich et al.,
1997; Hammerschmidt et al.,
1996; Jeong and McMahon,
2005). Defects in these components lead to ventralization of the
neural tube. In the present study, a similarly ventralized neural tube
phenotype was also observed in Xenopus embryos when DHCR7 activity
was inhibited (Figs 5 and
6). We note that mice deficient
in DHCR7 do not display obvious defects in the developing central nervous
system (Fitzky et al., 2001;
Wassif et al., 2001), but our
findings are consistent with the recent observation that the formation of
serotonin (5-HT) neurons, which is dependent on high Shh signaling, showed a
threefold increase in the number of neurons in the ventral CNS of
DHCR73-5 mutant mice
(Waage-Baudet et al., 2003).
In addition to its role in the CNS, Shh is also required in the formation of
the notochord and for maintenance of Shh signaling in that tissue once formed
(Chiang et al., 1996). That
DHCR7 is expressed both in the notochord and ventral CNS, and that the loss of
DHCR7 enhances the transcription of Shh and FoxA2 in these
tissues (Fig. 5), lead us to
surmise that DHCR7 functions to negatively regulate levels of Shh signaling in
both these structures.
|
DHCR7 functions intracellularly at the level or downstream of Smo and
possesses a conserved sterol-sensing domain (SSD) that is found predominantly
among proteins involved in vesicular trafficking
(Kuwabara and Labouesse,
2002). Ptch1 and Disp1 are implicated in the vesicular trafficking
of Hh, whereas SCAP, NPC1 and NPC1L are involved in sterol trafficking.
Accordingly, fibroblasts isolated from many individuals with SLOS show
abnormal storage of sterols, presumably resulting from defects in sterol
trafficking (Wassif et al.,
2002). Given that the Ptc protein is largely detected within
intracellular vesicles (Incardona et al.,
2000), and that both Ptc and Smo appear to shuttle between the
plasma membrane and cytoplasmic vesicles in response to the presence/absence
of Hh ligand (Denef et al.,
2000), it is tempting to speculate that DHCR7 participates in the
regulation of vesicular trafficking of Hh signaling components. However, our
current DHCR7 mutational analyses confound this simple interpretation, as a
DHCR7 mutant containing an intact N terminus (but lacking the SSD) is
sufficient to inhibit the action of Hh signaling. It is currently unclear what
the role of this N-terminal region might be, and further work will be required
to fully understand the function of each domain of DHCR7.
The role of DHCR7 in the pathogenesis of SLOS
One curious aspect of SLOS is the wide range of symptoms seen in this
disease. Individuals appear to display opposing phenotypes for the same
underlying genetic condition. For example, some individuals are polydactyl
(extra digits), while others are monodactyl (a single digit); some show
holoprosencephaly, while others are macrocephalic
(Kelley and Hennekam, 2000).
How can mutations in a single gene cause such seemingly divergent phenotypes?
As DHCR7 appears to have two distinct activities, a Hh antagonistic activity
and a reductase enzymatic activity, perhaps, depending on the precise nature
of the specific mutations, the activity of DHCR7 is oppositely modulated to
bring phenotypes of opposing directionality. Additionally, mutations in
DHCR7 can have the effect of altering intracellular sterol
concentrations, which may affect cellular responses to Hh signaling
(Cooper et al., 2003).
An important topic to address in the future is the biochemical nature of the DHCR7 mutants, especially how point mutations in the SSD domain or deletions affect the antagonistic activity of Shh relative to its reductase activity. Additionally, it is important to address how DHCR7 expression is regulated and its relationship to the occurrence of SLOS as proper expression of DHCR7 is essential for normal midline development.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/12/2395/DC1
* Present address: Laboratory of Neurobiology of Synapses, RIKEN Brain
Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 
Present address: Department of Molecular Pharmacology, Medical Research
Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
|
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