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First published online 6 June 2007
doi: 10.1242/dev.003715
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1 Institut für Entwicklungs- und Molekularbiologie der Tiere (EMT),
Heinrich-Heine-Universität, 40225 Düsseldorf, Germany.
2 Department of Genetic Medicine, Weill Medical College of Cornell University,
New York, NY 10021, USA.
Author for correspondence (e-mail:
ruether{at}uni-duesseldorf.de)
Accepted 14 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Basal body, Left-right asymmetry, Limb development, Mouse mutant, Neural tube
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In invertebrates, such as Drosophila, Hh
binds its receptor Patched (Ptc) and thereby activates Smoothened (Smo),
another transmembrane protein. As a consequence, processing of Cubitus
interruptus (Ci) is inhibited and Ci enters the nucleus to act as a
transcriptional activator. In the absence of Hh, Ci is processed and acts as a
transcriptional repressor. Hh signalling in vertebrates is more complex,
involving three Hh (Shh, Ihh and Dhh) and three Ci homologues (Gli1, Gli2 and
Gli3). While Gli3 is known to be processed similarly to Ci, Gli2 processing in
vivo is much less efficient (Dai et al.,
1999; Wang et al.,
2000; Pan et al.,
2006). Gli3 seems to act predominantly as transcriptional
repressor, while Gli2 mainly acts as activator
(Ding et al., 1998;
Matise et al., 1998). Gli1
appears to be an obligate activator (Dai
et al., 1999). The ratio between Gli activators and repressors
realizes Hh-mediated signalling (Wang et
al., 2000; Briscoe and Ericson,
2001; Jacob and Briscoe,
2003).
During vertebrate development, Shh plays a crucial role in patterning of
the limb buds and the neural tube. In the early limb bud, Shh regulates
processing of Gli3 to lay the foundation for anteroposterior identities and
the number of digits to be formed
(Niswander, 2003). Shh
expression in the posterior mesenchyme, within the zone of polarizing activity
(ZPA), causes a Gli3 repressor gradient to form from anterior to posterior.
Loss of Shh leads to the loss of all digits except one, reflecting
the requirement of Shh function in digit formation
(Chiang et al., 1996). By
contrast, loss of Gli3 leads to polydactyly and an ectopic Shh
expression domain in the anterior limb bud
(Masuya et al., 1995;
Büscher et al., 1997).
Additionally, ectopic Shh expression or a reduction of Gli3 repressor
function has also been correlated with polydactyly
(Yang et al., 1998;
Wang et al., 2000).
During neural tube development, Shh is initially produced by axial
mesodermal cells from the notochord. The Shh signal from the notochord induces
the floor plate, the ventral signalling centre of the neural tube. The floor
plate in turn establishes a Shh gradient from ventral to dorsal. In response
to different Shh concentrations, subsets of transcription factors are
regulated, which define the five ventral neuronal subtypes: motoneurons (MN),
V3, V2, V1 and V0 interneurons (Briscoe and
Ericson, 2001). Based on their mode of regulation these
transcription factors can be subdivided into two classes: Class I genes such
as Pax6 and Irx3 are repressed by Shh, whereas class II
genes such as Nkx2.2 (also known as Nkx2-2 - Mouse Genome
Informatics) and Olig2 require Shh for their expression.
Additionally, the distinct progenitor domains of neuronal subtypes are refined
and maintained by cross-repressive interactions between class I and class II
proteins (Briscoe et al.,
2000).
Cilia are specialized structures with several functions during embryonic
development. They are involved in symmetry breaking within the node by
producing a leftward flow and thereby causing an asymmetric distribution of
signalling molecules. Analyses of different mouse mutations, such as
inversus viscerum (iv) and inversion of embryonic
turning (inv), revealed that both the motility of cilia and the
direction of the nodal flow are necessary for the establishment of left-right
asymmetry (Hirokawa et al.,
2006). The role of cilia in embryonic development is eminent.
Dysfunction of cilia is associated with several human disorders, such as
Bardet-Biedl syndrome (BBS). This disorder is characterized by retinitis
pigmentosa, renal malformations, situs inversus, cardiomyopathy, diabetes and
polydactyly. Mutations in a number of genes are known to cause BBS, and there
is evidence that all BBS proteins participate in a common cellular process,
given that mutations in any BBS gene result in clinically related phenotypes
(Katsanis, 2004;
Nishimura et al., 2005;
Stoetzel et al., 2006). BBS
proteins are located at the basal body of cilia and centrosomes (during cell
cycle), and it is proposed that these proteins assist microtubule-related
transport and cellular organization processes relating to ciliary and
centrosomal activities (Kim et al.,
2005).
Recently, the crucial function of cilia as a specialized compartment for
signal transduction has been shown. Several proteins involved in Hh-signal
transduction are localized within the cilia structure. suppressor of fused
(Sufu), a negative regulator of Hh signalling, and Gli proteins are localized
at the distal tip of the cilia (Haycraft
et al., 2005). Furthermore, Smo is enriched in cilia in the
presence of Hh ligand (Corbit et al.,
2005; May et al.,
2005). Genetic screens have identified additional genes required
for Hh signalling and the generation of ventral neural cell types. Several of
these genes are required for proper cilia formation and function [e.g.
intraflagellar transport (IFT) motors kinesin-2 and Dnchc2 (Dync2h1 - Mouse
Genome Informatics) and IFT particle subunits IFT88 and IFT172]. Based on
phenotypic and biochemical analysis, IFT proteins seem to act downstream of
the membrane-bound protein Smoothened and upstream of Gli transcription
factors. They are required to generate active Gli2 in response to Hh
signalling, and are required for processing of Gli3
(Scholey and Anderson, 2006).
Additionally, IFT proteins are necessary for anterograde and retrograde
transport, which is essential for cilia maintenance. In the absence of IFT
proteins, the cilia structure fails to form
(Pazour et al., 2000;
Cole, 2003). Despite the
finding that several proteins are essential for cilia function, the connection
between cilia and Hh signalling is still only beginning to be understood.
The Ftm (fantom; Rpgrip1l - Mouse Genome Informatics)
gene was originally identified in the mouse mutation Ft (fused toes;
also known as Fts - Mouse Genome Informatics). This mutation is
caused by a deletion of 1.6 Mb on mouse chromosome 8, affecting three members
of the Iroquois family of homeobox genes, Irx3, Irx5 and
Irx6, and three other genes, named Fts, Fto
(AJ237917) and Ftm
(Peters et al., 2002).
Development of embryos homozygous for the Ft mutation is delayed and
embryos die between embryonic day (E) 10.5 and 14.5. The embryos show severe
malformations of craniofacial and forebrain structures
(van der Hoeven et al., 1994).
Furthermore, they reveal polydactyly in fore limbs and syndactyly in fore- and
hind limbs (Grotewold and Rüther,
2002). In addition, establishment of left-right asymmetry is
disturbed, and ventral neural tube patterning and floor plate maintenance is
affected (Heymer et al., 1997;
Götz et al., 2005).
Interestingly, the majority of these defects point to a disturbed Hh
signalling, leading to the assumption that one of the genes deleted in the
Ft mutation might be involved in Hh signalling. To clarify which of
the six genes might be a component of Hh signalling, we started to generate
single knockout experiments in mice. Our data reveal that the absence of Ftm
seems to be the cause of the majority of defects observed in Ft/Ft
embryos. However, some phenotypes, such as fusion of phalanges or early
lethality in homozygous embryos, have not been observed, suggesting
involvement of other or additional genes in these Ft phenotypes. The
present study shows that Ftm is localized at the basal body of cilia and that
it is essential for Gli protein function. We conclude that Ftm is a novel
component of Hh signalling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Total RNA was isolated using Trizol (GibcoBRL) followed by DNaseI digestion.
First strand cDNA was synthesized with the Expand RT system (Roche) according
to the manufacturer's instructions. Detailed information and primer sequences
are available upon request.
Western blotting
Western blot analysis was done essentially as described using anti-Gli3
antibody (Wang et al., 2000)
or anti-Ftm antibody. The membrane was then incubated with anti-actin or
anti-tubulin antibody (Santa Cruz) as control for loading. NIH Image was use
to digitize and normalize the band intensities on scanned images.
Tissue processing and histochemistry
After dissection in ice-cold PBS, embryos were fixed in 4% paraformaldehyde
for 2 to 4 hours and washed in PBS. Afterwards they were incubated in 70, 80,
90 and 100% ethanol, respectively, for 2 hours, and then 1-butanol overnight
and transferred into paraffin for embedding. In situ hybridizations on
paraffin sections were carried out as previously described (Moormann et al.,
2001). Whole-mount RNA in situ hybridization on embryos and
immunohistochemistry study on frozen tissue was performed as described
(Timmer et al., 2001).
Morphological stainings on sections were performed with Hematoxylin and Eosin
and embryos were stained for cartilage and bones using standard Alcian Blue
and Alizarin Red staining.
Cell culture
Mouse embryonic fibroblasts (MEFs) were generated from single embryos,
stage E12.5 (Ftm littermates), and mesenchymal limb cells (MLCs) were
generated from isolated limb buds, stage E12.5. Both were maintained in DMEM
(Gibco BRL) supplemented with 10% fetal calve serum (FCS) and 1/100 (v/v)
L-glutamine (Gibco BRL) and 1/100 (v/v) pen/strep (Gibco BRL) at 37°C in
5% CO2. For Shh response analysis MLCs were incubated in DMEM
(Gibco BRL) complemented with 1% fetal calve serum (FCS) and 1/100 (v/v)
L-glutamine (Gibco BRL) and 1/100 (v/v) pen/strep (Gibco BRL) at 37°C in
5% CO2 and 1 µ/ml recombinant N-Shh protein (Sigma).
Scanning electron microscopy
Scanning electron microscopy was performed as described
(Sulik et al., 1994).
Antibodies
We generated polyclonal antibodies by immunizing rabbits with a GST-Ftm
fusion protein encompassing the Ftm-RID domain (Pineda antibody services).
Antibodies were purified with NHS sepharose (Roche) conjugated with an Ftm
protein fragment containing the RID domain. Detailed information is available
upon request.
We also used antibodies against acetylated 
-tubulin (Sigma),
-tubulin (Sigma), actin (Sigma), Nkx2.2, MNR2 (Hlxb9 - Mouse Genome
Informatics), Shh, Pax6 (monoclonal antibodies were obtained from the
Developmental Studies Hybridoma Bank developed under the auspices of the NICHD
and maintained by The University of Iowa), Pax2 (Zymed) and Olig2
(Chemicon).
In silico data
Protein sequences of mouse and human Ftm genes and of murine
Rpgrip1 were obtained from public databases (Accession number: CAC87257;
NP_056087; NP_076368). Sequences of all other Ftm orthologues were derived
from DNA contigs generated from raw sequence data (traces), including partial
gene predictions and/or partial EST sequences.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), one C2
domain, which is proposed to play a role in the binding of intermediate
filament proteins (Gallicano et al.,
1998), and a so-called RID domain. This domain was characterized
in the Ftm-related protein RPGRIP1 (retinitis pigmentosa GTPase regulator
interacting protein 1) (Boylan and Wright,
2000; Hong et al.,
2001). To analyse the function of Ftm, we generated a targeted mutation in murine ES cells by homologous recombination. The targeting construct was organized such that the mutation resulted in a truncated protein lacking all functional domains of Ftm except the first coiled-coil-domain (Fig. 1A). Ftm was not detectable in Ftm-/- embryos using antibodies raised against the C-terminus (Fig. 1B). Ftm+/- mice show no obvious phenotype. However, embryos homozygous for the Ftm mutation died around birth showing microphthalmia (eyes reduced in size) and a preaxial polydactyly in fore and hind limbs (Fig. 1C). Additionally, craniofacial structures were affected, including malformed mandibular structures and unfused maxilliary and nasal tissues (Fig. 1D). At earlier stages we found an enlargement of the pericard, exencephaly and disturbances in the establishment of left-right asymmetry (Fig. 1E,F). Thus, Ftm is required for several processes during embryonic development.
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; Lin et al.,
1999; Tsukui et al.,
1999; Liu et al.,
2001). To confirm the morphological observations at the molecular
level, we performed whole-mount in situ hybridization and analysed the
expression of left-right-specific marker genes. In wild-type embryos (2-5
somite stages), both Pitx2 and Lefty2 (Leftb) were
expressed in the left lateral plate mesoderm
(Fig. 2E; data not shown). In
Ftm-/- embryos, the laterality of this expression was
lost; both genes were expressed in a bilateral fashion, which indicates
disturbances in early events of left-right determination
(Fig. 2F; data not shown).
Thus, Ftm is required for proper establishment of left-right
asymmetry.
Loss of Ftm results in preaxial polydactyly and disturbances in Shh signalling
From E12.5 onwards, Ftm-/- embryos displayed a
broadened shape of fore- and hind-limb buds (data not shown), suggesting a
polydactylous phenotype. At later stages, this phenotype could be
characterized as preaxial polydactyly (Fig.
2G). Bone cartilage stainings revealed one to two extra digits in
fore limbs and one extra digit in hind limbs
(Fig. 2G). Known possible
causes of polydactyly are ectopic expression of Shh and/or
Hh activity, and Hh target genes Ptc1 and
Gli1 were also previously found to be upregulated
(Hui and Joyner, 1993;
Yang et al., 1998). We
therefore examined Ftm mutant limbs for ectopic expression of
Shh and Ptc1. Shh was expressed normally in the posterior
mesenchyme both, at E10.5 and E11.5 (Fig.
2H; data not shown), and no ectopic expression domain was
detectable. Likewise, Ptc1 was not ectopically expressed
(Fig. 2I), suggesting a lack of
increased Hh signalling. Moreover, Ptc1 expression in
Ftm mutant limb buds was reduced
(Fig. 2I). The reduced number
of Ptc1 transcripts in Ftm mutant limb buds could be
confirmed by RT-PCR analysis (see Fig. S1 in the supplementary material).
These data indicate a reduction of Shh signal transduction in limb buds of
Ftm-/- embryos.
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;
Dodd et al., 1998). In situ
hybridizations on cross sections revealed normal expression of Shh in the
notochord, but a loss in the ventral neural tube
(Fig. 3C). This loss was only
partial along the anteroposterior axis, as demonstrated by whole-mount in situ
hybridizations (see Fig. S2 in the supplementary material). Nevertheless, we
were not able to detect any Shh protein in the ventral neural tube
(Fig. 3K). In addition, absence
of Foxa2 expression indicated a failure of successful induction of the floor
plate (Fig. 3D). To test
whether this failure might be the result of altered Shh signalling from the
notochord, we analysed Ptc1 expression
(Fig. 3G). Interestingly, we
found a reduction of Ptc1 expression in the neural tube as well as in the
mesenchyme surrounding the notochord, thus confirming the reduction of Shh
signalling. Reduced amounts of Ptc1 expression within the neural tube were
also detectable by RT-PCR (see Fig. S1 in the supplementary material).
Absence of a functional floor plate and therefore of Shh signalling was
further investigated by analyses of ventral neural tube markers.
Nkx2.2-positive cells are normally positioned dorsal to the floor plate and
give rise to V3 interneurons (Fig.
3J). In Ftm-/- embryos, Nkx2.2-expressing
cells were strongly reduced. Only in a few cases, single scattered cells were
found (Fig. 3L, data not
shown). MNR2 expression is indicative for motoneurons, which are normally
positioned dorsolateral to the Nkx2.2 expression domains
(Fig. 3M). Like Nkx2.2, MNR2
expression was strongly reduced in Ftm mutant embryos.
MNR2-expressing cells were found in a spotted fashion also in the ventral
midline (Fig. 3O). Pax6 and
Irx3 are both expressed in V0, V1 and V2 progenitors and Pax6 is
additionally expressed in motoneuron progenitors
(Fig. 3F,N). Both genes were
found to be repressed by high amounts of Shh
(Briscoe and Ericson, 2001).
Accordingly, in Ftm-/- embryos, Pax6 and Irx3
expression domains were expanded into the most ventral part of the neural tube
(Fig. 3H,P). Thus, these
results strengthen the assumption that Hh signalling is impaired in
Ftm-/- embryos. Whether altered patterning of the neural
tube is only a consequence of the absence of the floor plate (Shh source) or
due to impairment of Hh signalling within the entire neural tube cannot be
differentiated by this analysis.
Ftm acts upstream of Gli3
Gli proteins are mediators of Shh signalling and act as transcriptional
repressors or activators. The outcome of Hh signalling is realized by the
relative amount of Gli repressor (Gli3R) to Gli activator (Gli1, Gli2), which
leads to the patterning of the neural tube
(Briscoe and Ericson, 2001).
Gli3R is known to antagonize floor plate induction and to block motoneuron
differentiation, whereas unprocessed Gli3 does not seem to have a function in
neural tube patterning (Persson et al.,
2002). Absence of Shh signalling has two consequences: firstly,
processing of Gli3 is not inhibited (more Gli3 repressor); and secondly, Gli1
and Gli2 activation is decreased (less Gli activator). Thus, in Shh mutant
embryos the loss of ventral neurons (motoneurons and several classes of
interneurons) can be alleviated by abrogating Gli3 function, as shown in
Shh-/-; Gli3-/- combined mutant embryos
(Jacob and Briscoe, 2003). If
the loss of Ftm affects Shh signalling, a reduction of Gli3 in Ftm mutant
embryos should rescue the observed mispatterning of the neural tube.
Therefore, we crossed Ftm mutant mice with Xt mice, which carry a null allele
of Gli3 (Hui and Joyner, 1993;
Büscher et al., 1998).
Ftm-/- embryos show a strong reduction of motoneurons
(MNR2-positive cells) and Nkx2.2-expressing cells
(Fig. 4D,E). In addition, Pax2
expression, which is negatively regulated by Shh, was found to be expanded
into the most ventral part, including the midline
(Fig. 4C,F). Interestingly,
combined Ftm-/-; Xt/+ embryos revealed a partial rescue of the
mispatterning in the ventral neural tube. The numbers of MNR2- and
Nkx2.2-positive cells increased, but were still reduced in comparison with the
wild type (Fig. 4G,H). In
addition, the expansion of the Pax2 domain was reduced in comparison with
Ftm-/- embryos (Fig.
4I). Most interestingly, Ftm-/-; Xt/Xt embryos showed a
nearly complete rescue of ventral neuronal subtypes. MNR2- and
Nkx2.2-expressing cells were absent from the midline and located in two
distinct lateral domains (Fig.
4J,K). Furthermore, Pax2 expression seemed to be fully restored,
positive cells were absent from the midline and the expression was restricted
to dorsal regions (Fig. 4L).
These results show that Ftm acts upstream of Gli3, and the data also confirm
that Ftm is a component of Shh signalling.
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).
In both wild-type and Ftm+/- embryos of stage E11.5, most Gli3-190
was processed into Gli3-83, leading to high amounts of Gli3-83 and low amounts
of full-length Gli3-190 (Fig.
5A). By contrast, in Ftm-/- embryos, the amount of
Gli3-190 was dramatically increased, leading to a Gli-190:Gli-83 ratio close
to 1 (Fig. 5A,B). However, the
amount of Gli3-83 seems to be equal to wild-type levels. Thus, the total Gli3
protein amount seems to be increased in Ftm-/- embryos. We
therefore analysed the levels of Gli3 RNA in mutant and wild-type embryos to
see whether the increase of Gli3 protein can be correlated with increased Gli3
RNA levels. As shown by RT-PCR analysis
(Fig. 5C), the Gli3-RNA level
was increased in Ftm-/- embryos. Quantification of relative
Gli3-RNA levels revealed a nearly twofold increase in the amount of Gli3
transcripts in Ftm-/- embryos
(Fig. 5D). As Gli3
transcription is negatively regulated by Shh
(Schweitzer et al., 2000;
Wang et al., 2000), the
increase of the number of Gli3 transcripts confirmed the impairment of Shh
signalling in Ftm-/- embryos.
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; Scholey and Anderson,
2006). Therefore, we considered the phenotypes observed in
Ftm mutant embryos to be caused by cilia defects. To test this
hypothesis, we investigated cilia in Ftm-/- embryos.
First, we examined the motile primary cilia within the node. Normally, a
single cilia protrudes from the apical surface of each cell. In contrast to
the wild type, the number of cilia in Ftm mutant embryos was found to
be reduced (Fig. 6A). Cilia are
also present in mesenchymal cells of the limb buds and on epithelial cells in
the neural tube (Huangfu and Anderson,
2005; Haycraft et al.,
2005). Staining against -tubulin, which marks the ciliary
axoneme, clearly indicated fewer cilia, both in limb buds
(Fig. 6B) and in the neural
tube (data not shown), suggesting disturbed cilia assembly and/or maintenance
in Ftm-/- embryos.
Ftm is a basal body protein, highly conserved in evolution
Interestingly, use of an antibody against Ftm revealed that Ftm localizes
to one end of the ciliary axoneme in MEFs
(Fig. 6Ci). To clarify whether
this localization is at the distal tip or at the proximal basal body of the
cilia, we analysed proteins that are indicative for the basal body, such as
-tubulin. Indeed, Ftm and -tubulin were found to be co-localized
(Fig. 6Cii). Thus, Ftm is a
component of the basal body of cilia.
As Ftm seems to be essential for cilia function in mice, we asked whether
Ftm can also be found in other species. In silico analyses revealed that Ftm
is conserved from cnidarians to humans
(Table 1; see Fig. S3 in the
supplementary material). Furthermore, Ftm shows homology to RPGRIP1, a
cilium-related protein, which is found only in higher vertebrates
(Table 1; see Fig. S3 in the
supplementary material). Strikingly, we were not able to find Ftm in
arthropods and nematodes. As it was shown for Drosophila that Hh
signalling in this arthropod member is not associated with cilia
(Huangfu and Anderson, 2006),
Ftm could be one of the proteins important for the realization of this
fundamental difference.
|
).
The question arises as to whether the loss of Ftm affected Hh signalling (e.g.
processing or nuclear transport) or is an indirect consequence of altering the
cilia structure. To address this issue, we isolated MLCs and MEFs of wild-type
and Ftm-/- embryos and measured their ability to generate
cilia, which are generated in G0 phase. The comparison of wild-type
and Ftm-/- cells (both MLCs and MEFs) in G0
phase revealed no differences in ciliogenesis: each cell developed one cilia
in both wild-type and Ftm-/- cells
(Fig. 7A, upper left part; data
not shown). Notably, we found a slight enhancement of cilia length in
Ftm-/- MLCs and MEFs (data not shown). To test whether Hh
signalling was affected in Ftm-/- cells, we measured Shh
responsiveness in G0 phase of wild-type and
Ftm-/- MLCs. Ciliated MLCs were incubated with Shh protein
and analysed for the activation of the Hh target genes Ptc1 and
Gli1. In contrast to wild type, the Shh response in
Ftm-/- MLCs was strongly reduced, indicated by decreased
Ptc1 and Gli1 expression
(Fig. 7A, lower left part;
Fig. 7B, left panels). To test
whether the Shh response is exclusively dependent on cilia, we analysed Shh
target gene induction in proliferating (non-ciliated) wild-type and
Ftm-/- MLCs (Fig.
7A,B right). Both wild-type and Ftm-/- MLCs
showed no induction of Shh target genes after stimulation with Shh
(Fig. 7A, right panels;
Fig. 7B, right panels). These
data confirm that Shh signals are exclusively transduced by cilia and that the
remaining induction of Shh targets in Ftm-/- MLCs is
cilia-dependent. Furthermore, loss of Ftm does not affect ciliogenesis and
does not completely abolish Shh signalling. However, loss of Ftm severely
interferes with the efficiency of signal transduction. Thus, Ftm is necessary
for normal cilium-related Shh signalling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Impaired Hh signalling in Ftm-/- embryos
All observed midline defects in Ftm-/- embryos (e.g.
randomized heart looping, left-lung isomerism, dorsalization of the neural
tube) can be explained by a reduction or absence of Hh signalling, similar to
the phenotypes described for Shh-/- embryos
(Chiang et al., 1996;
Tsukui et al., 1999). To
provide detailed evidence for this interpretation, we investigated patterning
of the neural tube, a structure extensively used for quantitative and
qualitative studies of Shh function
(Briscoe and Ericson, 2001;
Jacob and Briscoe, 2003).
Although Shh was expressed in the notochord throughout development we
were not able to detect Shh in the neural tube from E10.5 onwards, a
phenomenon also described for IFT mutant mice that lack cilia and therefore
functional Shh signalling (Huangfu et al.,
2003; Haycraft et al.,
2005; Liu et al.,
2005). As a consequence, the neural tube of
Ftm-/- embryos was mispatterned in a similar way to that
seen in IFT mutant embryos: the floor plate was lost, and consequently
motoneurons and V3 neurons were reduced. Furthermore, it had been shown
previously that mispatterning of the neural tube in polaris (Ift88)
and wimple (Ift172) mutant mice could partially be rescued by
depletion of Gli3 in combined mutant embryos
(Huangfu et al., 2003). The
same partial rescue was also found in Ftm-/-;
Gli3-/- combined mutant embryos, suggesting a similar
mechanism. Biochemical analyses revealed that mutations in IFT genes (e.g.
polaris) negatively influence Gli3 processing. In addition, Gli activator
function and Shh response was impaired
(Haycraft et al., 2005;
Liu et al., 2005;
May et al., 2005). Analysis of
MLCs of polaris mutant mice showed a complete loss of Shh responsiveness and a
failure of Hh target gene activation by Gli2
(Haycraft et al., 2005).
Similarly, we could show that Shh responsiveness is strongly reduced in
Ftm-/- embryos and in cultured MLCs. Therefore, in
Ftm-/- embryos, reduced activator function due to a
reduction in Shh responsiveness, in combination with remaining Gli3 repressor
function, leads to the same mispatterning of the neural tube as that in IFT
mutant mice.
In contrast to neural tube patterning, limb development is rather
indifferent to Gli activator function. Mutations in Gli1 and Gli2 have no
effect on limb patterning, whereas loss of Gli3 dramatically changes digit
number and identity. The major mediator in limb patterning is Gli3-83
(Niswander, 2003). A complete
loss of Gli3-83 causes a severe polydactyly (two to three extra digits) and
leads to a complete loss of digit identity
(Niswander, 2003). The defects
in Ftm-/- and IFT mutant embryos were much less severe. In
Ftm-/- limbs only one to two extra digits were formed,
with a clear digit identity, and in IFT mutant embryos a polydactyly
comparable to Ftm-/- embryos was described
(Haycraft et al., 2005;
Liu et al., 2005). The
polydactylous phenotype of IFT mutant mice appears to be a consequence of the
change in the ratio of Gli3-190 to Gli3-83, due to impaired Gli3 processing.
Although our western blot analysis revealed equal levels of Gli3-83 in
Ftm-/- and wild-type embryos, the ratio of Gli3-190 to
Gli3-83 is changed due to an increased amount of Gli3-190. A recent study by
Wang et al. (Wang et al.,
2007) supports this interpretation.
Ftm and cilium-related Hh signalling
Ftm and IFT proteins are both necessary for proper Hh signalling, but
strikingly they show quantitative differences in Shh responsiveness. A loss of
Ftm leads to a strong reduction of Shh responsiveness in MLCs, whereas a loss
of polaris causes a complete loss of Shh responsiveness
(Haycraft et al., 2005).
Furthermore, in contrast to polaris mutant embryos
(Haycraft et al., 2005),
Ftm-/- cells possess cilia, which are detectable by
antibodies against acetylated -tubulin. In addition, and again in
contrast to polaris mutant cells, 100% of Ftm-/- MLCs in
culture do generate cilia. Thus, Ftm is not necessary for the generation of
cilia but rather for allowing efficient cilia-dependent Shh response. As we
noticed slight changes in the architecture of Ftm-/-
cilia, Ftm might also play a role in the stability or shape of cilia, which
could have consequences on the half-life of the cilia. However, our cell
culture experiments did not give any indication for this hypothesis.
IFT function is necessary for proper Gli3 processing and thereby increases
the amount of Gli3-190 and decreases the amount of Gli3-83. A loss of Ftm
seems not to affect the efficiency of processing. In
Ftm-/- embryos the Gli3-83 level appears to be normal and
the amount of Gli3-190 is increased due to increased Gli3 expression.
Nevertheless, the consequences are the same: changes in the ratio of Gli3-190
to Gli3-83 in IFT mutants and Ftm-/- embryos result in a
reduced expression of Hh target genes (such as Ptc1), a polydactylous
phenotype and dorsalization of the neural tube
(May et al., 2005;
Haycraft et al., 2005). In
both cases Hh signalling is impaired and would be expected to lead to an
increase of Gli3 expression
(Schweitzer et al., 2000;
Wang et al., 2000). So far
this has been shown only for Ftm-negative embryos.
Ftm and its relative RPGRIP1
Sequence analysis revealed Ftm to be homologous to RPGRIP1. Mutations of
RPGRIP1 cause the human disorder retinitis pigmentosa, a degeneration of
photoreceptor cells (Mavlyutov et al.,
2002; Pawlyk et al.,
2005). RPGRIP1 functions in photoreceptor cells in the retina,
especially at the connecting cilium between the outer and inner segment.
RPGRIP1 anchors RPGR (retinitis pigmentosa GTPase regulator) to the basal body
of this connecting cilium. This interaction is necessary for cilia stability
and retroflagellar transport (Boylan and
Wright, 2000; Hong et al.,
2001; Zhao et al.,
2003). Absence of RPGRIP1 in mice and humans results in retinitis
pigmentosa, suggesting a function of this protein in maintaining the integrity
of this specialized cilium-related structure
(Pawlyk et al., 2005). The
loss of Ftm causes malformations of cilia in specific tissues such as the
node, and also leads to a reduction in cilia number. Therefore, Ftm could be
necessary for cilia maintenance in a similar fashion. However isolated primary
cells (MLCs and MEFs) showed no reduction in cilia number and only moderate
malformations of the cilia structure, which clearly shows that Ftm is not
essential for ciliogenesis. Interestingly, certain tissues, such as trachea
and olfactory epithelium, in Ftm-/- embryos show no
changes in cilia number (Ulrich Dirks and U.R., unpublished). Thus, cilia are
reduced only in structures/tissues in which morphogenetic processes take
place. This finding suggests that a fully functional cilia-coupled signalling
pathway is necessary for cilia maintenance.
Our phylogenetic analysis revealed that Ftm exists in nearly all species
from cnidarians to vertebrates. The homology constitutes from 42% in hydra to
89% in humans, implying that Ftm is highly conserved in evolution. By
contrast, RPGRIP1 is present only in vertebrates, and detailed analysis
identified Ftm as phylogenetic ancestor of RPGRIP1 (data not shown). RPGRIP1
is exclusively expressed in the eye, whereas Ftm is ubiquitously expressed,
stressing the importance of Ftm in general cilium function. Surprisingly, we
were not able to detect Ftm homologues in arthropods and nematodes. In
arthropods such as Drosophila, cilia are present only in specific
neuronal subtypes and in sperm cells (Basto
et al., 2006). The connection between Hh signalling and cilia in
arthropods has yet not been shown, suggesting that Ftm plays a specific role
in Hh signal transduction in association with cilia. As loss of Ftm leads to a
quantitative impairment of the Hh response, we speculate that a subset of
basal body proteins is essential for cilia to gain the ability to elicit a
full and robust Shh responsiveness. Ftm seems to be one of these proteins, and
thus may be used as an entry point to investigate vertebrate-specific Hh
signalling.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/14/2569/DC1
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ACKNOWLEDGMENTS |
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