1 Department of Pediatrics, University of Colorado at Denver and Health Sciences
Center, Mailstop 8322, Box 6511, Aurora, CO 80045, USA
2 Department of Genetic Medicine, Cornell University Weill College of Medicine,
New York, NY 10021,USA
Author for correspondence (e-mail:
lee.niswander{at}uchsc.edu)
Accepted 27 April 2005
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SUMMARY |
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The developing limb provides an excellent system to study Hh signaling, in particular as it allows a biological and molecular readout of both Gli activator and repressor function. Here we report that homozygous mutants for flexo (Fxo), a hypomorphic allele of mouse IFT88 generated in our ENU mutagenesis screen, exhibit polydactyly in all four limbs. Molecular analysis indicates that expression domains of multiple posteriorly restricted genes are expanded anteriorly in the mutant limbs, similar to loss of Gli3 transcriptional repressor function. Sonic hedgehog (Shh) expression is normal, yet Ptch1 and Gli1, two known targets of Hh signaling, are greatly reduced, consistent with loss of Shh signaling. Expression of Gli3 and Hand2 in the mutant limb indicates that the limb prepattern is abnormal. In addition, we show that partial loss-of-function mutations in another mouse IFT gene, Ift52 (Ngd5), result in similar phenotypes and abnormal Hh signaling as Fxo, indicating a general requirement for IFT proteins in Hh signaling and patterning of multiple organs. Analysis of Ift88 and Shh double mutants indicates that, in mouse, IFT proteins are required for both Gli activator and repressor functions, and Gli proteins are insensitive to Hh ligand in the absence of IFT proteins. Finally, our biochemical studies demonstrate that IFT proteins are required for proteolytic processing of Gli3 in mouse embryos. In summary, our results indicate that IFT function is crucial in the control of both the positive and negative transcriptional activities of Gli proteins, and essential for Hh ligand-induced signaling cascade.
Key words: IFT88 (TTC10), IFT52, Gli, Hh signaling
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Introduction |
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Despite extensive studies of the Hh pathway, the transduction and
regulation of Hh signaling in vertebrate has yet to be fully understood
(Lum and Beachy, 2004). In
Drosophila, Hh protein binds and inactivates its receptor Patched
(Ptc), allowing the activation of Smoothened (Smo), another transmembrane
protein. The principal target of Hh signaling is the transcription factor
Cubitus interruptus (Ci). In the absence of Hh signaling, Ci is
proteolytically processed into a transcriptional repressor, whereas Hh
signaling inhibits such processing and allows the unprocessed Ci to act as a
transcriptional activator. In the mouse, homologues for some major components
of Hh signaling have been isolated and exhibit conserved functions in Hh
signaling. However, important divergence exists between the Hh signaling
pathway in insects and in vertebrates. First, owing to gene duplication, there
are three Hh proteins (Shh, Ihh and Dhh) and three Ci homologues (Gli1, Gli2
and Gli3) in the mouse. Among the three Gli proteins, Gli1 does not contain a
repressor domain and cannot be proteolytically processed, and thus appears to
be an obligate activator (Dai et al.,
1999
). Gli2 and Gli3 contain both repressor and activator domains
(Dai et al., 1999
;
Sasaki et al., 1999
). Gli3 can
be processed into transcriptional repressors in vivo
(Wang et al., 2000
), although
it remains to be determined whether Gli2 is processed in vivo. However, both
biochemical and genetic evidence indicates that Gli3 predominantly acts as a
repressor and Gli2 predominantly as an activator
(Ding et al., 1998
;
Hui and Joyner, 1993
;
Matise et al., 1998
).
Furthermore, novel regulators of Hh signaling that appear to be specific to
vertebrates have been isolated, suggesting that vertebrates have evolved
different mechanisms to regulate Hh activity
(Chuang and McMahon, 1999
;
Huangfu et al., 2003
;
Izraeli et al., 1999
;
Lee et al., 2001
;
McCarthy et al., 2002
). This
paper focuses on two novel regulators, both IFT proteins, to elucidate the
molecular mechanisms underlying their regulation of Hh signal
transduction.
The pattern of vertebrate limbs along the anteroposterior axis and
formation of digits are regulated by the interaction between sonic hedgehog
(Shh) and Gli3 (Niswander,
2003). Shh is expressed in a small group of mesenchymal
cells in the posterior distal region of the early limb bud (the zone of
polarizing activity, ZPA). Application of either ZPA cells or Shh protein to
the anterior region of the limb bud leads to the formation of extra digits
(polydactyly) in chick and mouse (Liu et
al., 1998
; Riddle et al.,
1993
). By contrast, all digits but one are lost in the absence of
Shh protein function, reflecting the requirement for Shh function in digit
formation (Chiang et al.,
1996
). Conversely, loss of Gli3 function as found in the mouse
mutant extra-toes results in the formation of multiple digits and
there is ectopic Shh expression in the anterior region of the limb
bud (Buscher et al., 1997
;
Hui and Joyner, 1993
). Ectopic
Hh activity, or loss of Gli3 repressor function, has also been observed in
several other mouse and chicken mutants with polydactyly
(Wang et al., 2000
;
Yang et al., 1998
).
Interestingly, in the absence of Shh and Gli3 function, skeletal formation is
rescued and multiple digits are formed although they are unpatterned
(Litingtung et al., 2002
;
te Welscher et al., 2002b
).
From this, it has been concluded that Shh-Gli3 interactions serve to control
the number and pattern of the digits.
Intraflagellar transport (IFT) proteins are required for the biogenesis of
flagella and cilia in multiple organisms, including the green alga
Chlamydomonas reinhardtii, C. elegans, insects and mouse
(Rosenbaum and Witman, 2002).
We have previously shown that in the mouse spinal cord, mutations in two IFT
proteins, IFT88 (TTC10 - Mouse Genome Informatics) and IFT172, lead to loss of
several ventral cell types and reduced expression of the Hh downstream gene
Ptch1 (Huangfu et al.,
2003
). However, the ventral defects in these mutants are not as
severe as those in Shh or Smo mutants, raising the issue of
whether these IFT proteins are essential components of the Hh pathway. In
addition, these IFT mutants die around E10.5, preventing more thorough
analysis of Hh signaling in other organs, especially in the limb where
requirement for the repressor function of Gli proteins can be better
addressed. Orpk, an existing hypomorphic allele of mouse
Ift88, survives to young adulthood with multiple defects, including
an extra thumb and polycystic kidneys
(Moyer et al., 1994
). However,
it has been reported that Hh signaling and anteroposterior patterning gene
expression is normal in Orpk limbs except for an anterior expansion
of Fgf4 expression in the apical ectodermal ridge (AER)
(Zhang et al., 2003
).
In this report, we show that flexo, a novel hypomorphic mutant of mouse IFT88, forms multiple digits in all four limbs despite the lack of ectopic Hh signaling and the downregulation of Hh activity in its normal domain. Hypomorphic mutants for another mouse IFT gene, Ift52, exhibit similar phenotypes to flexo mice in multiple organs. We also show, by double mutant analysis, that IFT proteins are required for tissues to respond to Hh ligand. Finally, we show that IFT proteins regulate Gli activity in part through the proteolytic processing of the Gli3 protein. Thus, IFT protein function is required for both the activator and repressor activities of the Gli proteins in the Hh pathway.
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Materials and methods |
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Tissue processing and histochemistry
Whole-mount RNA in situ hybridization on embryos was performed as described
(Liu et al., 1998).
Immunohistochemical study on frozen cryosectioned tissue was performed as
described (Timmer et al.,
2001
).
Western blot
Protein lysate was prepared from E10.5 wild type, Gli3-/-,
Ift88null Ift88hypo or
Ift52hypo mouse whole embryo, dissected spinal cord or
limb buds. Equal amounts of protein were loaded onto 7% SDS-PAGE gels and
western blotting was performed as described
(Wang et al., 2000). Anti
ß-tubulin antibody (Sigma, #T4026) was used as loading control. The
result of western blot was quantitated using NIH Image 1.60.
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Results |
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Ift88hypo mutants survive until E12.5, allowing us to
examine anteroposterior patterning in the Ift88hypo limb
bud. Normally, Fgf4 is expressed in the posterior aspect of the AER
at E10.5 (Fig. 1E) and begins
to be downregulated at E11.5 (Niswander
and Martin, 1992). We found that Fgf4 expression in
Ift88hypo embryos is expanded to the entire length of AER
at E10.5 (Fig. 1F) and its
expression is maintained at E11.5 in the anterior AER (data not shown).
Homeobox genes Hoxd11 and Hoxd13 are both normally expressed
in the posterior mesenchyme of the limb buds at E10.5 and E11.5
(Fig. 1G,I; data not shown),
and ectopic expression of Hoxd genes in the anterior parts of the limb has
been shown to cause polydactyly and homeotic transformation
(Knezevic et al., 1997
;
Morgan et al., 1992
). We found
that both genes are expressed in an expanded region at both E10.5 and E11.5
(Fig. 1H,J; data not shown).
Therefore, the formation of extra digits in the limb coincides with an
anterior-to-posterior transformation.
|
It has been suggested that a mutual repression between Gli3 and
Hand2 in the early limb buds sets up a limb prepattern that
determines the anteroposterior polarity of the limb
(te Welscher et al., 2002a).
This prepattern exists before Shh expression initiates in the limb
and hence is not affected by a mutation in the Shh gene
(Chiang et al., 2001
). We
therefore examined the expression of Hand2 and Gli3 at
E10.25 and E10.5 to determine whether the limb prepattern is altered in
Ift88hypo limbs. Normally, Hand2 is expressed in
the posterior mesenchyme of the early and later limb buds
(Fig. 2G,I). In
Ift88hypo limbs, Hand2 expression is both
upregulated and anteriorly expanded (Fig.
2H,J). This suggests that the limb prepatterning defect results
from a failure of Gli3 to repress Hand2 expression; however,
Gli3 RNA is present in Ift88hypo limbs
(Fig. 2K-N). Later,
Gli3 expression expands posteriorly
(Fig. 2N), which is probably
due, in part, to the loss of Shh signaling. Taken together with the data
above, the analysis of Ift88hypo limbs indicates loss of
both Gli activator function (downregulation of Ptch1 and
Gli1 expression) and Gli repressor function (Hand2
misexpression and polydactyly).
Ift52 mutants display similar embryonic defects as Ift88hypo mutants
Our results that both Hh signaling and limb patterning along the
anteroposterior axis are altered in Ift88hypo mutants are
in contrast to a previous report in which there was no change detected in the
expression patterns of Shh, Ptch1 or Hoxd genes in limbs of
another hypomorphic allele of mouse Ift88, Orpk
(Zhang et al., 2003). In
contrast to Ift88hypo, the Orpk mutant mice can
survive to postnatal stage and the polydactyly phenotype is much milder than
Ift88hypo (Moyer et
al., 1994
). Therefore, it is possible that an alteration in gene
expression would be too mild to detect in the Orpk mutant
embryos.
Ift88hypo is so far the only mouse IFT mutant to survive long enough to reveal the role for IFT88 in the regulation of both activator and repressor functions of Gli proteins. In order to determine whether the phenotype seen in Ift88hypo reflects a general requirement for IFT proteins in Gli-positive and -negative regulation, we sought additional IFT mutant alleles that allow a characterization of both CNS and limb development.
|
|
The tight mesencephalic flexure, left-right and ventral midline defects as
well as polydactyly seen in Ift52hypo embryos are very
similar to what is seen in Ift88hypo embryos. Loss of
Ptch1 expression in the Ift52hypo limb buds
further indicates that, similar to IFT88, IFT52 is also required for normal Hh
signaling. We therefore examined ventral patterning of the spinal cord in both
Ift52hypo and Ift88hypo mutant
embryos. Our published studies of Ift88 null mutants
(Ift88null) show that the floorplate, V3 interneurons and
motoneurons are all absent (Huangfu et
al., 2003). In Ift88hypo and
Ift52hypo mutant spinal cord, Shh-expression in
the floor plate is absent (Fig.
4A,E,I). Nkx2.2-expressing cells that give rise to V3
interneurons and that are normally dorsal and lateral to floorplate
(Fig. 4B) are still present in
the mutants, but they occupy the ventral most region of the spinal cord and
the number is greatly reduced (Fig.
4F,J). Motoneurons that express Isl1/2 are normally
located dorsally to V3 interneurons (Fig.
4C); in the two hypomorphic IFT mutants, these cells expand into
more ventral regions (Fig.
4G,K). Interestingly, scattered motoneurons are found in the
ventral midline of the spinal cord, presumably among V3 interneurons.
Pax6 is normally strongly expressed in motoneuron precursors and
weakly expressed in more dorsal regions of the spinal cord
(Fig. 4D). In the two mutants,
Pax6 is ectopically expressed in scattered cells in more ventral
regions of the spinal cord (Fig.
4H,L). Therefore, both Ift88hypo and
Ift52hypo mutant embryos exhibit similar defects in
ventral patterning of the spinal cord, and this phenotype is consistent with
compromised Hh signaling.
IFT88 is essential for Hh signaling
In order to further address the relationship between IFT proteins and Hh
signaling, we characterized the double mutant between Ift88 and
Shh. Ift88null mutants have defects in ventral spinal cord
patterning that is similar to Shh mutants. However, interesting
differences exist between the two mutants. In Shh mutants, the spinal
cord is severely dorsalized. Pax7, which labels the dorsal progenitor
cells, is expanded throughout the entire spinal cord
(Fig. 5A,B). Strong
Pax6 expression, which labels neural progenitor cells in the
ventral-intermediate region of the spinal cord, is shifted to the ventral most
part of the spinal cord (Fig.
5E,F). Lhx3 is normally expressed in both motoneurons and
V2 interneurons, and En1 is expressed in differentiated V1
interneurons that are dorsally adjacent to V2 interneurons
(Fig. 5I,M). In Shh
mutants, very few En1- and Lhx3-expressing cells are present
in the ventral midline of the spinal cord
(Fig. 5J,N). By contrast, in
the Ift88null spinal cord, Pax7 expression
remains dorsally restricted (Fig.
5C). Strong Pax6 expression is detected in a broader
domain expanded both ventrally and dorsally
(Fig. 5G). En1- and
Lhx3-expressing cells are both present but expanded to the ventral
midline (Fig. 5K,O). One
interpretation for the apparent milder ventral defects in
Ift88null is that Hh can partially signal through Gli in
the absence of IFT88, which would predict that loss of Hh ligand in addition
to IFT88 leads to a severe dorsalizing defect similar to that seen in
Shh mutants. We hence generated double Ift88null;
Shh-/- mutant embryos to determine whether this is indeed
the case.
|
|
It has been shown that anteroposterior patterning and formation of digits
in the mammalian limbs depends on the interaction between Shh and Gli
transcription factors, mainly Gli3
(Niswander, 2003), and that
the major function of Shh in the limb is likely to antagonize the repressive
function of Gli3 (Litingtung et al.,
2002
; te Welscher et al.,
2002b
). In E12.5 Shh mutant limbs, the posterior tissue
degenerates, leaving a narrow pointy bud with a single digit condensation
(Fig. 6D)
(Chiang et al., 2001
;
Kraus et al., 2001
). By
contrast, the Shh/Ift88hypo double mutants display
polydactyly (Fig. 6E), similar
to Ift88hypo mutants
(Fig. 6F). Moreover, the
polydactyly is similar to that observed following loss of Gli3. In the absence
of Gli3, multiple digits are formed, with the anterior expansion of
otherwise posteriorly located genes such as Fgf4, Hoxd11 and
Hoxd13 (Buscher et al.,
1997
; Hui and Joyner,
1993
). Ift88hypo mutant limb buds also display
an anterior expansion of Fgf4, Hoxd11 and Hoxd13, along with
the formation of extra digits, suggesting a loss of Gli3 repressor
function. The fact that the polydactyly of Ift88hypo
mutants is not altered by loss of Shh function also suggests that there is a
loss of Gli3 repressor function in Ift88hypo mutants.
However, the loss of the Shh targets Ptch1 and Gli1 in both
the limb and other places in Ift88hypo embryos, but not
Gli3 mutant embryos, also indicate a loss of Gli activator functions.
The simplest explanation for such paradoxical phenotype is that IFT proteins
are required, independently of Hh input, for both the activator and repressor
function of Gli transcription factors.
|
|
![]() |
Discussion |
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In Drosophila, Costal 2 (Cos2) and Fused
(Fu) appear to play similar bipartite roles in Hh signaling such that
both gain and loss of Hh signaling phenotypes are observed in such mutants
(Lefers et al., 2001;
Wang and Holmgren, 2000
).
Cos2, a Kinesin II-related protein without a functional motor, seems
to play multiple roles in Hh signaling, including recruiting Ci to the cell
surface through Smo and cytoplasmic retention of Ci
(Jia et al., 2003
;
Lum et al., 2003
;
Ogden et al., 2003
;
Ruel et al., 2003
;
Wang and Holmgren, 2000
). Fu
is a Kinase whose only function appears to be antagonizing the function of
Sufu, a negative regulator of Ci. Interestingly, when Fu is mutated, Ci
cleavage is blocked and full-length Ci accumulates in the cells
(Lefers et al., 2001
).
Hh signaling in vertebrates appears to be more complex than that in
insects. First, three Gli family members play different roles in mediating Hh
signals, whereas in Drosophila there is only a single Ci gene that
encodes both an activator and a repressor. Second, the existence and function
of Cos2 and Fu in the vertebrate has yet to be confirmed. Third, IFT proteins
that are crucial for mouse Hh signaling do not appear to mediate Hh signaling
in Drosophila (Han et al.,
2003; Sarpal et al.,
2003
). Therefore, vertebrates may use different sets of proteins
to regulate Gli3 processing and subcellular localization of Gli proteins.
Finally, as the loss of IFT functions leads to simultaneous loss of cilia and
abnormal Hh signaling, it is difficult to distinguish a more direct function
of IFTs as a component of the Hh pathway in the regulation of Gli protein
modification, or a more indirect function, through signals associated with
cilia.
Despite the divergence on the molecules involved, the vertebrate and
insects may share similar strategy in regulating Gli/Ci activities. Kif3a, a
subunit of mouse kinesin 2, plays important roles in Hh signaling
(Huangfu et al., 2003).
Although Kif3a does not seem to be the mouse ortholog of Cos2 according to
sequence homology, it may be functionally equivalent. IFT proteins may also be
a novel addition to the Gli-Kinesin (Ci-Cos2) complex that serves to bring Gli
proteins to Smo upon activation of the pathway similar to what has been shown
in Drosophila (Jia et al.,
2003
; Lum et al.,
2003
; Ogden et al.,
2003
; Ruel et al.,
2003
; Wang and Holmgren,
2000
). It will be interesting in the future to elucidate whether a
functional relationship exists between these proteins, and to determine the
subcellular site(s) of IFT action and their interacting protein partners.
In summary, we have shown that IFT proteins are required for the proteolytic processing of Gli3 protein. Although there is an increase in the ratio of full-length activator to repressor Gli3 protein forms, this is not reflected genetically as the IFT mutants lack the hallmarks of in vivo Gli activator functions. Our analysis of two novel IFT mutants, as well as double mutants for Ift88 and Shh, demonstrate that Gli activator as well as Gli repressor functions are disrupted and that IFT is an essential component of the Hh signaling pathway. Thus, IFT proteins are required for both the activator and repressor functions of Gli transcription factors.
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ACKNOWLEDGMENTS |
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Footnotes |
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