1 The Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard
Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA 02114,
USA
2 Developmental Biology Program, Sloan Kettering Institute, New York, NY 10021,
USA
Author for correspondence (e-mail:
tli{at}meei.harvard.edu)
Accepted 27 January 2004
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SUMMARY |
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Key words: FKBP8, FKBP38, Sonic hedgehog, Neural tube patterning, Signaling, Mouse
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Introduction |
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Consistent with the role of SHH defined in embryological experiments, null
mutations in the murine Shh gene or in genes required to transduce
the SHH signal abolish ventral fates in both the spinal cord and brain
(Chiang et al., 1996;
Wijgerde et al., 2002
). The
cells at the ventral pole of the neural tube instead acquire dorsal or lateral
identities that are normally repressed by SHH signaling. Excessive SHH
signaling results in an opposite phenotype: cells inappropriately adopt
ventral identities in favor of dorsal identities
(Echelard et al., 1993
;
Goodrich et al., 1997
). Thus,
increased or decreased levels of SHH signaling have pronounced effects on the
patterning of the central nervous system.
Hedgehog signal transduction occurs through a series of poorly understood
signaling events that modulate the activities of a family of transcriptional
factors, the GLI proteins. In Drosophila, the transcription factor
Cubitus interruptus (Ci, the ortholog of vertebrate GLI) mediates Hedgehog
(Hh)-induced transcriptional activation
(Methot and Basler, 2001). The
hedgehog ligand binds to the multipass transmembrane protein Patched,
preventing its normal inhibition of Smoothened
(Chen and Struhl, 1996
;
Marigo et al., 1996
). The Hh
signal is transduced through Smoothened to a complex containing Ci
(Robbins et al., 1997
).
Downstream of Smoothened, protein kinase A (Pka) phosphorylates Ci and
facilitates its proteolytic processing into a truncated transcriptional
repressor (Price and Kalderon,
1999
). Hh signaling reverses this effect and promotes the
accumulation of full-length Ci, which is a transcriptional activator of
Hh-response genes (Ohlmeyer and Kalderon,
1998
). Vertebrate orthologs of several Drosophila
hedgehog signaling components have been identified and appear to be generally
conserved in the vertebrate pathway (reviewed by
Marti and Bovolenta,
2002
).
The FK506-binding protein 8 (FKBP8, also known as FKBP38)
(Pedersen et al., 1999;
Shirane and Nakayama, 2003
) is
a member of the immunophilin family of proteins that bind immunosuppressant
drugs such as cyclosporin A or FK506
(Snyder et al., 1998
). FKBPs
comprise a large family, the members of which share a conserved
peptidyl-prolyl isomerase (PPI) domain. Proteins with PPI activity can alter
the conformation of other proteins by allowing free rotation around prolyl
peptide bonds. This activity may allow the immunophilins to function as
accessory folding proteins or as scaffold proteins that facilitate
protein-protein interactions. FKBP12, a relatively well-studied member of the
family, has several defined functions. Like other immunophilins, FKBP12
associates with and inhibits the Ca2+-calmodulin activated protein
phosphatase calcineurin upon binding FK506. In T-lymphocytes, FKBP12 activity
prevents the dephosphorylation of NF-AT and prevents this transcription factor
from entering the nucleus (Clipstone and
Crabtree, 1992
; Hemenway and
Heitman, 1999
). FKBP12 also binds to and regulates the activity of
the inositol (1,4,5) triphosphate and ryanodine receptors, which regulate
intracellular Ca2+ release
(Cameron et al., 1995
;
Jayaraman et al., 1992
). The
presence of tetratricopeptide repeats (TPR) in addition to the PPIase domain
in FKBP8 makes it more closely related to the larger members of the FKBP
family such as FKBP52, which functions as a specialized co-chaperone
(Young et al., 2003
).
The in vivo function of FKBP8 is unclear, although recent data from cell
culture experiments indicate that FKBP8 binds calcineurin in an
FK506-independent manner and targets BCL2 and BCL2L to mitochondria, thus
inhibiting apoptosis (Shirane and
Nakayama, 2003). To define the in vivo role of FKBP8, we disrupted
its function in mice by gene targeting. Our data show that FKBP8 has an
essential role during development. Loss of FKBP8 leads to inappropriate
activation of the SHH signaling pathway in the caudal neural tube, where
ventral fates were dramatically expanded at the expense of dorsal fates. The
direct role of FKBP8 in regulation of SHH signaling was confirmed genetically,
because SHH-dependent neural fates are restored in
Shh-/-;Fkbp8-/- double mutants. Our
data indicate that FKBP8 is primarily required for regulation of the hedgehog
signaling pathway in neural tissues.
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Materials and methods |
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To determine the distribution of FKBP8, tissues were extracted in RIPA buffer/1 mM DTT/protease inhibitors. Total protein from adult tissues was quantified by the modified Lowry assay. In the immunoblot shown in Fig. 1B, 5 µg total protein was loaded per lane. To determine membrane association of FKBP8, rat retinal proteins were extracted under the following conditions: 20 mM Tris-HCl, pH 7.4, 0.15 N NaCl (labeled as 0.15 N NaCl in Fig. 1C); 1 N KCl (1 N KCl); 0.1 M NaHCO3-Na2CO3 (pH 11.5); 20 mM Tris-HCl, pH 7.4, 0.15 N NaCl, 0.5% CHAPS (CHAPS); 6 M urea (6 M urea). All solutions also contained 1 mM DTT. Extracts were centrifuged at 200,000 g for 1 hour at 4°C. Both supernatants and pellets were analyzed by immunoblotting.
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Results |
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FKBP8 is required for neural tube development
Both RNA and protein analyses indicated that disruption of the
Fkbp8 gene generated a null allele
(Fig. 2A). We found that
homozygous embryos died around embryonic day (E)13.5. The
Fkbp8-/- mutant phenotype became morphologically apparent
by E10.5 (Fig. 2B). At this
stage, the mutant caudal neural tube appeared translucent and irregular. By
E12.5, the caudal neural tube of the mutant embryo was dilated and appeared as
a fluidfilled sac. In about 20% of the Fkbp8-/- mutant
embryos, a clump of pigmented cells was seen in place of the eye
(Fig. 2B), whereas the eyes
appeared smaller in the remaining mutant embryos. The limb buds, branchial
arches and somites of the mutants appeared normal. Histological examination
revealed a dilated caudal neural tube and dorsally displaced, smaller dorsal
root ganglia (Fig. 2B). By
contrast, the rostral mutant neural tube appeared morphologically normal. The
limbs developed normally in the mutant, and major organs such as the heart,
lung and gut also appeared normal in histological sections (not shown).
Experiments using cultured cells
(Shirane and Nakayama, 2003)
indicate that FKBP8 blocks apoptosis by recruiting BCL2 and BCL2L to
mitochondria. We considered the possibility that increased apoptosis could be
the underlying primary defect for the phenotype we observed in the
Fkbp8-/- mutant. We monitored apoptosis using an antibody
against activated caspase 3 but found no difference between wild-type and
mutant embryos in the affected lumbar neural tube at E10.5
(Fig. 2C). Similarly, no
differences were observed between genotypes in the rostral neural tube or in
non-neural tissues (data not shown). Thus, the phenotypic features observed in
Fkbp8-/- mutants do not stem from excessive apoptosis.
To determine the basis for the tissue-restricted developmental defects of the mutant, we examined the tissue expression pattern of Fkbp8 in E9.5 embryos by in situ hybridization and by immunostaining (Fig. 3). By whole-mount in situ hybridization (Fig. 3A), Fkbp8 mRNA was found uniformly distributed throughout the E9.5 embryo. On sections, the in situ hybridization signals (Fig. 3B) showed no discernable variation along either the anteroposterior axis or the dorsoventral axis. Similar observations were made by immunostaining for the FKBP8 protein, either in whole-mount embryos (not shown) or on cross-sections through the neural tube (Fig. 3C). These data suggest that the rostral-caudal differences in the mutant neural tube morphology do not stem from regional differences in Fkbp8 expression.
|
To address this possibility in more detail, we examined the expression
pattern of markers for dorsoventral neural cell fates in
Fkbp8-/- mutants from E9.5 to E12.5 by in situ
hybridization and immunohistochemistry
(Fig. 4, data not shown). The
expression domains of dorsoventral markers were clearly altered in mutant
embryos in sections through the caudal neural tube just anterior to the
hindlimbs. Foxa2 expression normally marks the most ventral neural
cell fate, the floor plate (Sasaki and
Hogan, 1993). Foxa2 expression was dramatically expanded
in the Fkbp8-/- mutant, such that roughly the ventral half
of the neural tube expressed this marker. Pax6 and Dbx1 are
normally expressed at high levels in the lateral regions of the neural tube;
Dbx1 expression is strictly limited to a small population of lateral
cells (including V0 interneuron progenitors), whereas Pax6 expression
is also observed at lower levels in ventrolateral and dorsal cells
(Pierani et al., 2001
;
Shoji et al., 1996
;
Walther and Gruss, 1991
). In
the Fkbp8-/- mutant, both Pax6 and Dbx1
were expressed ectopically in dorsal regions instead of their normal lateral
domains. Markers for the most dorsal fates, such as Wnt1, were not
expressed in the mutant. The expression domains of other markers for specific
dorsoventral fates (including Shh, Nkx2.2, Isl2, Pax3, Pax7 and
Gdf7, see below and data not shown) were all affected in a manner
consistent with ventralization of neural cell fates. In the rostral neural
tube, at the level of the forelimbs, all of the markers examined showed a
normal expression pattern (data not shown). Thus, defective neural patterning
in the Fkbp8-/- mutant is limited to the posterior of the
prospective spinal cord.
|
By E9.5, Shh is normally expressed in the floor of the
diencephalon located between the optic stalks
(Ishibashi and McMahon, 2002).
Pax2 expression marks proximal fate (optic nerve) and Pax6
expression marks distal fates (retina and lens) in the developing eye. Excess
SHH signaling, caused by ectopic expression of Shh or blocking of the
hedgehog pathway antagonist PKA, promotes Pax2 expression at the
expense of Pax6 expression (Ekker
et al., 1995
; Macdonald et
al., 1995
; Perron et al.,
2003
). In E11.5 Fkbp8-/- mutant embryos,
development of the retina and pigmented epithelium was severely reduced in the
ventral half of the eye (Fig.
5A). A similar phenotype was observed when PKA function was
blocked in Xenopus embryos
(Perron et al., 2003
). The eye
defect in the Fkbp8-/- mutant embryos is reminiscent of
the small eye phenotype caused by Pax6 haploinsufficiency
(Hill et al., 1991
). In the
E10.5 Fkbp8 mutant, PAX2 was expressed ectopically in the dorsal
optic vesicle (Fig. 5B). PAX6
expression was clearly reduced in the neural retina of the mutant at E11.5
(Fig. 5C). Similar defects have
been observed in mutants for the hedgehog pathway antagonist Rab23
(Gunther et al., 1994
)
(J.T.E., unpublished) and are consistent with inappropriate activation of the
SHH pathway in the optic vesicle.
|
To distinguish among these possibilities, we analyzed the phenotype of
Shh-/-;Fkbp8-/- double mutant. At
E11.5, the Shh-/- mutant exhibited a variety of defects
including severe growth retardation, cyclopia/holoprosencephaly, absence of
ventral cell types in the neural tube, abnormal somites and deformed limbs
(Fig. 6)
(Chiang et al., 1996). In
contrast to the Shh-/- mutant, the double mutant developed
two distinct eyes separated by ventral neural tissue in the forebrain
(Fig. 6). The double mutant
caudal neural tube showed the dilated morphology characteristic of
Fkbp8-/- single mutants. The somites and limbs were
indistinguishable from that of the Shh-/- single mutant;
the limbs of both Shh-/-;Fkbp8-/-
double mutants and Shh single mutants failed to grow out along the
proximodistal axis and the somites of both mutants were small and abnormally
shaped. These data suggest a partial rescue of SHH-dependent neural
development in the Shh-/-;Fkbp8-/-
double mutant.
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|
FKBP8 appears dispensable for SHH signaling in non-neural tissues
The overall morphology of Shh-/- single and
Shh-/-;Fkbp8-/- double mutants
suggested that non-neural tissues requiring SHH signaling, such as the limbs,
branchial arches and somites, do not activate the SHH signaling pathway in the
absence of FKBP8. In support of this, Ptch1 and Gli1 were
expressed in the medial portion of wild-type somites but not in
Shh-/- or
Shh-/-;Fkbp8-/- somites
(Fig. 8). Maintenance of
Pax1 expression in the sclerotome depends on SHH signaling
(Chiang et al., 1996). At
E11.5, Pax1 was not expressed in the Shh-/-
mutants nor was it expressed in the somitic mesoderm of the double mutant,
indicating that sclerotomal fate is not restored by the loss of FKBP8.
In addition to expressing Shh, the notochord requires SHH
signaling for its own maintenance (Chiang
et al., 1996). We confirmed the absence of the notochord in
Shh-/- mutants using a 5' RNA probe that could
detect Shh transcript from the mutant allele; there was clear
staining in the Shh-/- mutant gut
(Fig. 8R, inset) but no
staining in the position of the notochord
(Fig. 8R). Although loss of
FKBP8 clearly rescued neural tube Shh expression in the double mutant
(Fig. 8T; in an expanded
ventral domain), there was no morphological notochord and no Shh
expression was observed in cells ventral to the neural tube. These data
indicate that FKBP8 plays no detectable role in SHH signaling in the somites
and the notochord.
The Fkbp8 mutant phenotype is not caused by ectopic expression of Indian hedgehog or desert hedgehog
The other hedgehog family members, Indian hedgehog (Ihh) or desert
hedgehog (Dhh), are not normally expressed in the neural tube or
notochord although they are capable of inducing ventral neural fates
(Pathi et al., 2001). To rule
out ectopic activity of Ihh and Dhh as a cause for the
ligand-independent activation of the SHH pathway, we examined their expression
in the Shh-/-;Fkbp8-/- double mutant.
Although normal expression of Ihh and Dhh was observed in
the gut and gonad, there was no ectopic expression in the neural tube or
notochord by in situ hybridization (Fig.
9A). In addition, no hedgehog proteins were detected in the neural
tube of the double mutant by immunofluorescence
(Fig. 9B) using a monoclonal
antibody (clone 5E1) that crossreacts with all three hedgehog ligands
(Wang et al., 2000
). Thus,
activation of the SHH pathway in the double mutant is independent of all
hedgehog family members.
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Discussion |
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The TGFß (BMP/GDF) and WNT signaling pathways are also important in
the specification of dorsal cell fates in the spinal cord
(Lee et al., 1998;
Liem et al., 1995
;
Muroyama et al., 2002
). Thus,
it is formally possible that FKBP8 acts by promoting TGFß or WNT
signaling, which would account for the absence of dorsal neural fates in the
Fkbp8-/- mutant. A role for FKBP8 in the BMP signaling
pathway seems unlikely, given the normal expression of BMP signaling targets
MSX1 and MSX2 in the Fkbp8-/- mutant. Moreover, the
specification of ventral neural fates is not default; the lack of TGFß or
WNT signaling is not sufficient for the development of ventral fates in the
absence of hedgehog signaling (Ericson et
al., 1997
; Liem et al.,
2000
; Megason and McMahon,
2002
). Thus, the rescued development of ventral neural fates in
the Shh-/-;Fkbp8-/- double mutant is
most probably due to the loss of an essential antagonist in the hedgehog
signaling pathway.
Hedgehog signaling antagonists
Several antagonists of the mouse hedgehog pathway have been characterized
genetically, including PTCH1, RAB23, GLI3 and PKA. Mutations in any of these
components lead to expansion of ventral cell types in the neural tube.
Ptch1-null mutants show the strongest phenotype; cells at all
positions in the Ptch1-/- neural tube acquire the floor
plate fate, although cells also express markers of other ventral cell types,
including V3 interneuron progenitors and motoneurons
(Goodrich et al., 1997;
Motoyama et al., 2003
). No
lateral or dorsal cell types (marked by Pax6 and Pax3
expression, respectively) are specified in the Ptch1-/-
mutant. Mutants lacking Fkbp8, Rab23, or with partial
loss-of-function of Ptch1 or Pka (Ptch1-plof and
Pka-plof) show a less severe phenotype in which the neural tube is
partially ventralized (Eggenschwiler and
Anderson, 2000
; Huang et al.,
2002
; Milenkovic et al.,
1999
). The least severe phenotype is observed in the
Gli3-null mutant, in which the expression domains of some lateral
markers expand into slightly more dorsal regions
(Persson et al., 2002
).
There are interesting differences between the neural patterning phenotypes of Fkbp8-/-, Rab23, Pka-plof and Ptch1-plof mutants. For example, the dorsal cell fates marked by expression of Pax3 and Pax7 are completely deleted in Rab23-/-, Pka-plof and Ptch1-plof mutants but not in the Fkbp8-/- mutant. At the same time, ventral cell types, the floor plate and V3 progenitors are more strongly affected in the Fkbp8-/- mutant than in the other mutants of this class. Thus, Rab23 and PKA appear to be more important than FKBP8 in repressing hedgehog signaling in dorsal cells, and FKBP8 plays a more important role in ventral cells. These results suggest that the requirement for each of these factors in hedgehog signaling is not identical in all cells along the dorsoventral axis and may reflect differences in their mechanisms of action.
Although FKBP8 is required for neural tube patterning, this requirement
varies along the anteroposterior axis. An overt dorsoventral patterning defect
was seen only in the neural tube at lumbosacral levels, whereas patterning of
the thoracic neural tube was normal. Further anteriorly, in the cephalic
neural tube, a role for FKBP8 in SHH signaling is again apparent. Here, eye
development was either delayed or disrupted in the
Fkbp8-/- mutant, suggesting that excessive SHH signaling
occurs in the ventral domain of the brain and optic vesicles. Consistent with
this interpretation, the eye defects were accompanied by downregulation of
PAX6 expression and ectopic PAX2 expression. In addition, ventral forebrain
development in the Shh-/- mutant was rescued by the
concomitant loss of FKBP8. The variable phenotype with respect to the
anteroposterior neural axis does not appear to be related to regional
differences in Fkbp8 expression as the gene is expressed uniformly
along the anteroposterior and dorsoventral axes of the neural tube. Similar
anteroposterior differences have been observed in mutants lacking the hedgehog
signaling antagonists RAB23 or PKA
(Eggenschwiler and Anderson,
2000; Huang et al.,
2002
), and in mutants lacking both GLI3 and SHH
(Litingtung and Chiang, 2000
).
In each of these cases, the embryos showed pronounced ventralization of caudal
neural tube but minimal changes in the thoracic neural tube. Although the
reason for this is unclear, it has been shown that the balance of activating
and repressing functions of GLI3 vary along this axis
(Litingtung and Chiang, 2000
;
Motoyama et al., 2003
),
suggesting that different effects of the Rab23, Pka and
Fkbp8 mutations on GLI3 and GLI2 activity might account for this
phenomenon.
A tissue-specific role for FKBP8 in SHH signaling
We did not observe patterning defects in any of the non-neural tissues
examined in the Fkbp8 mutant. The limbs, somites or branchial arches
all appeared normal. These are SHH-responsive tissues affected in other HH
pathway mutants, such as Shh-/-,
Smo-/-, Ptch1-/-,
Rab23-/- and Gli3-/-
(Chiang et al., 1996;
Eggenschwiler et al., 2001
;
Goodrich et al., 1997
;
Hui et al., 1993
;
Zhang et al., 2001
). The
Shh mutant phenotype was rescued only in the caudal neural tube and
the brain of the Shh-/-;Fkbp8-/-
double mutant; there was no apparent rescue of the somite, limb or branchial
arch defects. Indeed, Ptch1, Gli1 and Pax1 expression was
not restored in the sclerotome of the double mutant somites, nor was
Shh expression rescued in the double mutant notochord. These data
suggest that the role of FKBP8 in the SHH signaling pathway is restricted to
neural tissues.
There are several precedents for tissue-specific action of
Drosophila HH pathway components. For example, in the
Drosophila embryonic ectoderm, Fused kinase is required for HH
responses in cells anterior to, but not posterior to, the hh
expression domain, even though both types of cells respond to and require HH
signaling (Therond et al.,
1999). In the mouse, reduction of PKA activity affects neural
tissues preferentially (Huang et al.,
2002
). Tissue-specific differences in the requirement for GLI
transcription factors has also been observed. The effects of the Gli3
mutation on limb development are much more dramatic than its effects on neural
patterning, whereas the opposite is true for the Gli2 mutation
(Matise et al., 1998
;
Persson et al., 2002
;
te Welscher et al., 2002
),
indicating that different GLI transcription factors mediate responses to SHH
signals depending on cell type. Thus, although some components of the hedgehog
signal transduction mechanism, such as Ptch1 and Smo, are required in nearly
all cell types (Goodrich et al.,
1997
; Zhang et al.,
2001
), signal transduction downstream of these components can vary
significantly depending on context.
The hedgehog signaling pathway in mammals
FKBP8 joins a group of recently uncovered components of the mammalian
hedghog signaling pathway including SIL, HIP, GAS1, megalin, RAB23, IFT172,
polaris/TG737 and KIF3A (Chuang and
McMahon, 1999; Eggenschwiler
et al., 2001
; Huangfu et al.,
2003
; Izraeli et al.,
2001
; Lee et al.,
2001
; McCarthy et al.,
2002
). Other components, such as PTCH1, dispatched, SMO, PKA,
suppressor of fused, and the hedghog and GLI families play analogous roles in
Drosophila and mammals (reviewed by
Ingham and McMahon, 2001
;
Nybakken and Perrimon, 2002
).
However, in several cases novel components that are present in the mammalian
genome have no clear orthologs in Drosophila, such as SIL, HIP and
GAS1. Drosophila homologs of other components, such as
polaris/TG737 (nompB) and Kif3a (Klp64D),
have been identified but these genes appear to have roles unrelated to
hedgehog signaling in the fly (Kernan et
al., 1994
; Ray et al.,
1999
). FKBP8 shares sequence similarity with an uncharacterized
Drosophila gene product, CG5482
(http://flybase.bio.indiana.edu).
The predicted CG5482 protein has the same overall domain structure, as well as
a membrane insertion site at the extreme C terminus. There are no known
mutations in the CG5482 gene, nor are there mutations in other
Drosophila FKBP genes that suggest their involvement in hedgehog
signaling. Similarly, genes such as Rab23 and Ift172 have
homologs in the Drosophila genome but the roles of these genes in
Drosophila are not yet clear.
Taken together, these data suggest that during the evolution of mammals, significant differences in the mechanism of hedgehog signaling regulation have developed. These differences underscore the importance of complementing genetic studies of hedgehog signaling in Drosophila with similar studies in mice to gain insight into how the pathway functions in humans.
Biochemical functions of FKBP8
We do not understand how FKBP8 acts in the hedgehog pathway at the
molecular level. We have observed that some endogenous FKBP8 protein is
present in a complex with the RII subunit of protein kinase A in vivo (O.V.B.
and T.L., unpublished), raising the possibility that FKBP8 has a role in
PKA-dependent phosphorylation of the GLI proteins. This possibility is
supported by the similarity in phenotype between mutants deficient in FKBP8
and PKA (Huang et al., 2002).
Total PKA activity in Fkbp8 mutant embryos, measured in vitro, is not
diminished, suggesting that FKBP8 is not required for general PKA activity
(O.V.B. and T.L., unpublished). Shirane and Nakayama
(Shirane and Nakayama, 2003
)
found that FKBP8 associates with and inhibits the phosphatase calcineurin.
This raises the possibility that activated calcineurin can promote hedgehog
signaling. Regardless of the biochemical functions of FKBP8 in hedgehog
signaling, it seems likely that its activities ultimately converge on the GLI
transcription factors. Given that Gli2-null mutants have a
considerably more severe neural patterning phenotype than Gli3 or
Gli1 null mutants, we favor GLI2 as the principal target of FKBP8
function. This hypothesis is currently being tested though genetic
epistasis.
FKBP8 is widely expressed in both adult and embryonic tissues. The function described in this study identifies the earliest requirement for FKBP8 during mouse development, leaving open the possibility that FKBP8 has other roles later in the embryo and in the adult. Failure to regulate hedgehog signaling properly in humans is associated with a variety of disorders ranging from birth defects such as holoprosencephaly to cancers of the brain and skin. Thus, identification of components of this signaling pathway and understanding how they function at the molecular level will have significant implications for the diagnosis and treatment of human diseases.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Princeton University, Department of Molecular Biology,
Princeton, NJ 08544, USA
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