|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 12 April 2006
doi: 10.1242/dev.02352
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 MRC Centre for Developmental Neurobiology, New Hunt's House, King's College
London, London SE1 9RT, UK.
2 The FIRC Institute of Molecular Oncology, Via Adamello, 16, 20139 Milano,
Italy.
3 CEINGE Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples,
Italy.
4 Institute of Genetics and Biophysics-ABT, Via G. Marconi 12, 80125 Naples,
Italy.
5 Zebrafish Neurogenetics, GSF-Research Center for Environment and Health,
Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D.85764
Neuherberg, Germany.
6 Institute of Virology, Technical University-Munich, Trogerstrasse 4b, D-81675
Munich, Germany.
* Author for correspondence (e-mail: corinne.houart{at}kcl.ac.uk)
Accepted 9 March 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Cerebellum, Fgf8, Otx2, Isthmic organiser
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Jessell and Sanes, 2000;
Lumsden and Krumlauf, 1996).
Understanding how these organisers influence their surrounding is a current
challenge. As yet, two such local organisers have been described
(Houart et al., 1998;
Wilson and Houart, 2004;
Wurst and Bally-Cuif, 2001).
One of these, the isthmic organiser (IsO), placed at the interface between
mesencephalon (or midbrain) and metencephalon (comprising rhombomeres 1 and 2;
r1, r2), is necessary and sufficient for the development of these structures
[also called mes/met domain (Liu and
Joyner, 2001; Raible and
Brand, 2004; Wurst and
Bally-Cuif, 2001)]. The patterning activity of the isthmus was
initially demonstrated in avian embryos by transplantation experiments
inducing either transformation of part of the caudal forebrain into an ectopic
midbrain (Alvarado-Mallart et al.,
1990; Martinez et al.,
1991), or re-fating of some of the posterior hindbrain into
ectopic cerebellum [a major derivative of the metencephalon
(Martinez et al., 1995)].
Genetic deletions and mutations of different IsO mouse genes cause the loss of
mes/met derivatives (Chi et al.,
2003; Lun and Brand,
1998; McMahon and Bradley,
1990; Schwarz et al.,
1997; Wurst et al.,
1994) and demonstrate that the IsO controls proliferation,
maintenance and regionalisation of the mes/met domain. Fgf8, a secreted
signalling molecule expressed at the IsO, can mimic the inductive effects of
IsO grafts, by regulating cell proliferation and controlling gene expression
around the isthmus (Crossley et al.,
1996). In addition, like the IsO grafts, local misexpression of
Fgf8 can induce the development of ectopic or enlarged cerebella
(Sato et al., 2001;
Sato and Nakamura, 2004;
Suzuki-Hirano et al., 2005).
However, although transcriptional targets of Fgf signal have been isolated
[i.e. feedback-induced antagonists such as Spry4, Pea3, Erm, Sef or Mkp3
(Furthauer et al., 2002;
Furthauer et al., 2001;
Kawakami et al., 2003;
Roehl and Nusslein-Volhard,
2001)], the cellular events directly requiring Fgf8 signalling are
yet to be understood. Fgf8-/- mouse embryos fail to
gastrulate, making it impossible to readily study Fgf8 contribution to the IsO
(Meyers et al., 1998;
Sun et al., 1999). By
contrast, the analysis of a variety of fgf8 conditional inactivations
showed the need for Fgf8 in isthmus formation
(Meyers et al., 1998). More
recently, Fgf8 lack-of-function studies in zebrafish
(Jaszai et al., 2003;
Reifers et al., 1998) and mice
[conditional Fgf8 mutation inside the mes/met domain
(Chi et al., 2003)] revealed
that the loss of cerebellum in these mutants is preceded by a fate
transformation of presumptive r1 cells into otx2-expressing
mesencephalic progenitors. In the zebrafish, the lack of gastrulation defect
in fgf8-null embryos is explained by the presence of maternal Fgf8
protein, allowing the embryo to gastrulate. The zygotic protein present in
ace/fgf8-/- is fully inactive, and no Fgf activity is
detectable in the MHB of ace/fgf8-/- mutants
(Furthauer et al., 2001) (this
study).
Thus, a growing body of evidence present the restriction of the
otx2 expression domain as one of the early functions of Fgf8 inside
the IsO (Martinez et al.,
1999; Sato et al.,
2001; Sato and Nakamura,
2004; Suzuki-Hirano et al.,
2005). Molecular studies, using Fgf8 bead implantation rostral to
the isthmus, suggest that the formation of an ectopic midbrain is always
associated with a very small cerebellum around the Fgf8 source. These tiny
cerebella are thought to be formed by local repression of otx2
transcripts, creating an artificial boundary of otx2 expression that
would allow formation of an IsO able to repolarise the area
(Martinez et al., 1999). The
local repression of Otx2 is suggested to be necessary for the induction of
fgf8 in the host cells surrounding the beads. These
fgf8-positive host cells are, in turn, required for the formation of
an ectopic cerebellum (Martinez et al.,
1999).
We addressed the relationship between Fgf signal and Otx function in the
zebrafish. Reduced level of Otx proteins in Otx2+/-;
Otx1-/- or Otx2+/-; Otx1+/-
mutant mouse embryos previously unravelled Otx function in positioning the IsO
(Acampora et al., 1997;
Suda et al., 1997). We
obtained hypomorphic Otx lack-of-function (OtxH) embryos using morpholinos
against two of the three zebrafish Otx genes. These embryos retain enough Otx
function to gastrulate normally but they subsequently show a transformation of
the presumptive midbrain territory into an extended r1, resulting in an
anterior shift of the isthmus and formation of a bigger cerebellum. In
Otx2+/-; Otx1-/- mutant mice, the same
transformation was attributed to a rostrally expanded fgf8 expression
domain (Acampora et al., 1997;
Suda et al., 1997). Although
such an expansion is also observed in OtxH embryos, we show that zebrafish
embryos lacking both Otx and Fgf8 functions maintain a transformation of the
midbrain tissue into r1 and retain the ability to differentiate cerebellar
cell types. This indicates that both early r1 territory and cerebellar cell
types are developing in absence of Fgf8, as long as Otx function is also
abrogated. This is not due to persistence or upregulation of other Fgf
proteins, as we show that OtxH embryos lacking fgf8 function have no
Fgf activity in the presumptive mes/met and that OtxH embryos still develop r1
territory and cerebellar granule cell precursors following complete inhibition
of all Fgf signals. Together, our work shows that Fgf8 is not instructive
neither in the formation of the r1 territory nor for the differentiation of
cerebellar cell identities. Fgf signalling activity is, however, crucial for
proliferation, fusion and folding of the cerebellar anlage.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Data
presented were obtained from analysis of Kings College London wild-type and of
homozygous acerebellar/fgf8-/- embryos
(Reifers et al., 1998)
maintained at 28°C on a 14 hour light/10 hour dark cycle.
Whole-mount in situ hybridisation and immunochemistry were performed using
standard procedures (Tallafuss and
Bally-Cuif, 2003; Westerfield,
2000), details available upon request. The Engrailed antibody 4D9
was kindly provided by Simon Hughes.
Fgf signal inhibition
SU5402 (20 µM, Calbiochem) was added on embryos kept at 31°C from
50% epiboly stage until fixation. Efficiency of the treatment on wild-type
embryos is measured by the absence of isthmic constriction similar to the one
observed in ace embryos. A typical reduction in the length of the
yolk extension and the tail is also seen after SU5402 treatment
(Fig. 7E,F) but not in
fgf8-/- embryos, as several other Fgfs are expressed and
have redundant functions in tail bud. Loss of spry4 expression after
1 hour of incubation at 31°C was used to confirm treatment efficiency (not
shown).
Morpholinos and injection
MOs (Gene Tools) were designed against otx1 (GenBank Accession
Number BC045290), otx2 (NM_131251) and otx1-like (D26174),
diluted in Danieau's media and injected at the one- to four-cell stages.
Embryos received 1.2 ng of each MO (0.133 mM, 0.133 pmol/embryo). Morpholinos
were as follows: Otx1 MO1-Fluo (complementing bases 417-441),
5'-TGAGGTATGACATCATGCTAGAGGC-3'; Otx2 MO-Fluo (complementing bases
261-285), 5'-GTTGCTTGAGATACGACATCATGCT-3'; Otx1-like MO-Fluo
(complementing bases 225-249), 5'-GAGGTATGACATCATTGTTGAGCCC-3';
Otx1 MO2-Bare (complementing bases 375-379),
5'-GATCTTGCCAATAGTTCGATTTCCC-3'; Otx1MO3-Bare (complementing bases
350-374), 5'-CCGATGTTGCAGTTTGACGAAGGAC-3'.
Most of our morpholinos are labelled with fluorescein to facilitate the
selection of equally injected embryos. Under these conditions, we observed the
phenotype described here in around 98% of the injected embryos, while we
observed a range of milder phenotypes in 
5% of all injected embryos in
the absence of selection. Each experiment was repeated two to six times, with
highly similar results.
To control for the specificity and efficiency of our MOs, we cloned all
three full-length Otx cDNAs, including 150 bp of their 5' sequence. As
previously reported in Xenopus, overexpression of Otx after RNA
injection leads to cell movement defects during gastrulation (otx1
RNA 80% affected, n=52; otx2 RNA 88% affected,
n=44; otx1-like RNA, 88.2% affected, n=34) (see
also Bellipanni et al., 2000).
The vast majority of embryos injected with a given Otx RNA and its
corresponding MOs showed a rescue [otx1RNA+Otx1-1 MO, 80.6%
of wild type (n=31); otx2 RNA+otx2-1 MO, 63.8% of
wild type (n=40); otx1-like RNA+ otx1like-1 MO,
60.4% of wild type (n=43).
As previously reported, fgf8-/- mutant larvae are of two different phenotypic classes. Type A larvae (65%, n=78) show a tectum slightly altered in its shape but not affected in its length, whereas type B (35%) larvae are overall retarded most probably as a secondary effect of variably abnormal blood circulation. Some of those embryos (generally 10%) with oedema were removed from our analysis of 5-day-old embryos.
Western blot
Total extracts were prepared from heads of E10.5 mouse or prim-5 zebrafish
(10 embryos), processed for standard western blot assay and probed with Otx
antibody (Mallamaci et al.,
1996) (1/10000). For detection at bud stage, nuclear extracts were
prepared from 50 wild-type or OtxH embryos. Coomassie staining was carried out
after transfer to compare proteins levels.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Acampora et al., 2001). To
date, three homologues have been described in zebrafish
(Mercier et al., 1995). The
third zebrafish gene, previously called otx3 is renamed
otx1-like as it has a high percentage of similarity with otx1.
otx1 and otx2 are both located on chromosome 17 and
otx1-like is on chromosome 1 (Ensembl database). The most likely
scenario leading to this zebrafish genomic organisation is that the single
ancestral Otx gene found in cephalochordates was first tandemly duplicated
(Williams and Holland, 1998).
Then the two genes were separated in higher vertebrates while an additional
whole genome duplication in teleosts led to four Otx gene copies, with a
subsequent loss of an otx2-like gene.
Gene duplication may have been followed by functional diversification of
the different paralogues in zebrafish. To define the function of the three
paralogues, we used a battery of morpholino antisense oligomers (MOs)
(Nasevicius and Ekker, 2000)
targeting zebrafish Otx genes. Only the double knock-down of both
otx1-like and otx2 led to a severe defect in the embryonic
brain. Western blot analyses, using a polyclonal Otx2 antibody recognising
vertebrate Otx1 and Otx2, showed that residual Otx proteins were detectable in
these embryos at bud stage, but that the proteins were lost by the end of
somitogenesis (Fig. 1A). We
called these double knock-down embryos OtxH morphant embryos (H standing for
`hypomorph'). Brain morphology, first analysed in prim-20 live embryos, showed
that the telencephalon, olfactory placodes and hypothalamus are present but
the epiphysis is missing in OtxH embryos. The patterning defect is also
evident caudally where the isthmic constriction is absent. Only one uniform
dorsal structure can be observed (arrow in
Fig. 1C) instead of a tectum
and cerebellum present in wild type embryos
(Fig. 1B). As the single Otx2
and Otx1-like morphants have no visible defect, our data indicate that
zebrafish otx1-like and otx2 genes carry a redundant
function during early development.
Transformation of the midbrain into rhombomere 1 in OtxH morphants
Severe reduction of Otx proteins triggers transformation of the
mesencephalic anlage into metencephalic derivatives in mouse. We confirmed, by
analyzing mbx and mab21L2, two markers expressed in midbrain
and pretectal areas (Kawahara et al.,
2002; Kudoh and Dawid,
2001), that this area is dramatically reduced as early as the
six-somite stage (Fig. 1F,G;
data not shown) and absent by the end of somitogenesis
(Fig. 1H,I; data not shown) in
OtxH embryos. In contrast to this result, genes expressed across the entire
mes/met domain, such as engrailed (En), her5 and
pax2.1, maintain their expression in OtxH embryos until
mid-somitogenesis (data not shown). Among those, En is the only one
steadily expressed in the whole mes/met domain during development and is still
partially maintained in prim-5 OtxH embryos
(Fig. 1J,K).
|
). From
early somite stage, efnb2a r1 domain is reproducibly expanded in OtxH
embryos (Fig. 2A,B). In order
to assess whether this efnb2a domain is indeed derived from mes/met
descendants, we took advantage of the her5pac:egfp line expressing
the green fluorescent protein (GFP) under the control of the her5
regulatory regions (Tallafuss and
Bally-Cuif, 2003). her5 is specifically induced in the
entire mes/met domain during late gastrulation (and completely absent in the
rest of the hindbrain). As GFP is a very stable protein, it is able to follow
temporally the progeny of the mes/met domain. In her5pac:egfp
embryos, the metencephalic precursors co-express GFP and efnb2a,
while the mesencephalic cells express only GFP
(Fig. 2A,C). In
OtxH/her5pac:egfp embryos, GFP is always co-expressed with
efnb2a (Fig. 2B,D),
showing that the OtxH entire presumptive mes/met domain is transformed into an
extended metencephalon.
|
; Jaszai et al.,
2003) and the cerebellar granule cells
(Wingate and Hatten, 1999).
The LC is a small group of noradrenergic (NA) neurons, known to be induced by
coordinated isthmic Fgf and dorsal midline BMP signals
(Guo et al., 1999). In
agreement with their commitment as NA lineage, LC neurons express two homeobox
genes, phox2a and phox2b. In 20-somite stage OtxH embryos,
this phox2a-expressing population, located in the dorsal
metencephalon, has at least doubled (arrows in
Fig. 2E,F). The origin of
cerebellar granule neurons has been microsurgically fate-mapped to r1 in chick
(Wingate and Hatten, 1999),
and specifically to the embryonic rhombic lip, a specialised proliferative
epithelium arising at the interface between the neural plate and the roof
plate of the IVth ventricle. To further characterise the nature of the
expanded r1 area in morphant embryos, we analysed the expression of markers of
cerebellar differentiation. We find that the expanded r1 expresses
atoh1a and atoh1b, two granule cell markers
(Adolf et al., 2004;
Koster and Fraser, 2001)
(Fig. 2G-J;
Fig. 4A,B). Finally, zebrin,
marker for cerebellar Purkinje cells also shows an expanded population of
these cells in OtxH embryos (see Fig.
5I,J). The OtxH transformed mes/met region is therefore
differentiating into a large LC and cerebellum. However, the morphology of the
enlarged cerebellum is abnormal, mainly owing to an absence of fusion
(Fig. 5F,N;
Fig. 6F). Some level of
disorganisation is also detected in the Purkinje cell layers
(Fig. 5J; see also Fig. S1I in
the supplementary material).
|
) -
thereby defining, from early somitogenesis onwards, the midbrain and hindbrain
part of the isthmus. In OtxH embryos, an initial residual ventromedial
wnt1 expression is rapidly lost (by the six-somite stage, not shown).
Expression then reappears at the 20-somite stage in some dorsal cells located
anterior to the expanded r1 (likely diencephalic) and within the roof plate of
the posterior neural tube (Fig.
3A,B). Conversely, fgf8 is improperly activated in the
morphants, from the one-somite stage onwards, in a broad area inside the
anterior part of the presumptive mes/met domain, while expression in ventral
r2 and r4, and within dorsal forebrain appears unaltered
(Fig. 3C-F).
We therefore conclude that, in fish, as in mouse
(Acampora et al., 1997;
Suda et al., 1997), a
decreased level of Otx activity leads to the loss of wnt1 and the
expansion of fgf8 signal inside the mes/met area. In mouse, this
fgf8 upregulation was proposed as the major cause for the local
transformation of the midbrain into r1.
Cerebellar cell fates develop in fgf8-/- mutant embryos when Otx function is lowered
Fgf signal is thought to be necessary and sufficient to instruct r1-derived
fates. OtxH embryos provide the unique opportunity to test this hypothesis.
Unlike OtxH embryos, the fgf8-/- mutant [acerebellar,
ace (Reifers et al.,
1998)] lacks cerebellum because of a progressive transformation of
the anterior r1 into mesencephalon (Jaszai
et al., 2003). Thus, embryos with lowered level of Otx proteins in
an fgf8-/-/ace background are expected to lack both
msencephalon and r1. Zebrafish fgf8-/-embryos were
injected with the combination of otx1-like and otx2
morpholinos (referred to as fgf8-/-; OtxH embryos). As
previously reported, atoh1a is consistently missing from the upper
rhombic lip in fgf8-/- mutant embryos
(Jaszai et al., 2003) and
atoh1a-expressing cells from the lower rhombic lip are not able to
contribute to the cerebellar system
(Koster and Fraser, 2001). To
our surprise, in fgf8-/-; OtxH embryos, an upper rhombic
lip cell population is restored, resembling the one seen in OtxH embryos
(Fig. 4B,D), albeit reduced in
size.
To test whether cells from the presumptive posterior rhombic lip may possibly migrate anteriorly to regenerate a cerebellar granule cell population in fgf8-/-; OtxH embryos, we used the stability of GFP protein to follow the origin of the upper rhombic lip cells in fgf8-/-; her5pac:egfp; OtxH embryos. We consistently observe co-localisation of atoh1a and GFP on parasagittal cryostat sections of fgf8-/-; her5pac:egfp; OtxH prim5 brains, ruling out the possibility of cell migration from posterior GFP-negative rhombomeres (Fig. 4N,O). In her5pac:egfp wild-type embryos, a subpopulation of GFP-positive cells is co-expressing atoh1a, confirming that, as in higher vertebrates, zebrafish granule cells are born within an r1 territory characterised by her5 expression. This atoh1a and GFP co-expressing population is absent in fgf8-/- embryos (Fig. 4K,L) and is present in fgf8-/-; OtxH (Fig. 4N,O), as well as in OtxH embryos (Fig. 4H,I).
Differentiation of cerebellar cells is further assessed using pax6
and reelin staining granule cells (Fig.
5A-H) (Costagli et al.,
2002) and Zebrin/AldolaseC marking Purkinje cells
(Fig. 5I-L; see Fig. S1 in the
supplementary material) (Lannoo et al.,
1991; Miyamura and Nakayasu,
2001) in 5-day-old embryos. These two cell populations are
expanded in OtxH embryos (Fig.
5B, n=30 embryos; Fig.
5F, n=16; Fig.
5J, n=36), and are absent in fgf8-/-
(except the rare zebrin-positive cells in 10% of mutants,
Fig. 5C, n=12;
Fig. 5G, n=2,
Fig. 5K, n=8). All
pax6, reelin and zebrin-positive cell populations are rescued in
fgf8-/-; OtxH embryos
(Fig. 5D, n=10;
Fig. 5H, n=4;
Fig. 5L, n=12).
|
|
Finally, we analysed development of the r1-derived LC neurons. In fgf8-/-; OtxH embryos [analysed at both the 15-somite stage (n=85) and prim-5 stage (n=52); data not shown], phox2a expression is never found, showing that LC fate requires fgf8, whether or not Otx function is lost.
Fgf signalling is not instructive for the establishment of the r1 territory and differentiation of cerebellar identities
The presence of cerebellar differentiation in absence of Fgf8 in our OtxH
morphants may mean that Fgf activity is required to maintain Otx expression
away from r1 territory but is not necessary for any other step in cerebellar
cell fate determination. However, a more trivial possibility is that some Fgf
activity, carried out by other Fgfs in the MHB, may rescue cerebellar
differentiation in the fgf8-/-; OtxH embryos. To address
this possibility, we measured Fgf activity in the MHB region. As the best
indicator of Fgf gene activity is the expression of transcriptional targets,
three of them were analysed in the four genetic contexts studied. As
illustrated in Fig. 6I-P (and
in Fig. S2 in the supplementary material), erm, spry4 and
pea3 are highly expressed in wild-type MHB from bud stage onwards,
but are never induced there in fgf8-/- and
fgf8-/-; OtxH embryos, indicating a complete absence of
Fgf activity in this region. Finally, treating batches of
fgf8-/- embryos with the Fgfr inhibitor SU5402
(Mohammadi et al., 1997) does
not worsen the fgf8-/- midbrain phenotype (data not
shown), further supporting a complete absence of Fgf signalling in the MHB of
fgf8-/- mutants. The differentiation of cerebellar cell
fates in our double loss-of-function embryos therefore occurs in absence of
any Fgf activity in the MHB from at least bud stage onwards.
|
Gbx2 is not strictly required for cerebellar differentiation
In higher vertebrates, two major transcription factors establish the
presumptive mesencephalon and metencephalon [respectively, Otx2
(Martinez-Barbera et al.,
2001) and Gbx2 (Wassarman et
al., 1997)]. In fgf8-/- embryos, metencephalon
precursors erroneously express otx2, indicating a posterior shift of
mesencephalon identity (Jaszai et al.,
2003; Tallafuss and
Bally-Cuif, 2003), which we found can be revealed as early as the
eight-somite stage (Fig. 7E,F).
Complementarily, rostral expansion of r1 territory in OtxH embryos also starts
at early somitogenesis, which may possibly be induced by rostral expansion of
Gbx2 expression. Testing this possibility, we found that formation of
cerebellar tissue, in fgf8-/-; OtxH embryos, occurs
independently of presence of gbx2 (arrows in
Fig. 7G-J). Zebrafish
gbx1 and gbx2 are sequentially expressed in r1; and it has
been suggested that gbx1 is the functional homologue of mouse Gbx2
(Rhinn et al., 2003).
Zebrafish gbx1 expression is switched off in the wild-type mes/met
area at a stage preceding the onset of mispatterning in OtxH embryos and is
not altered in these embryos, precluding its involvement in the process
studied here. Conversely, gbx2 expression is expanded anteriorly in
OtxH embryos, as expected following the anterior shift of the r1 territory. As
previously reported in fish and mouse (Chi
et al., 2003; Rhinn et al.,
2003), we confirm that maintenance of gbx2 expression is
dependent upon Fgf activity. However, we observed that loss of Otx function in
fgf8-/-; OtxH embryos is unable to rescue gbx2
expression (Fig. 7J). As these
embryos develop cerebellar fates, this implies that from the six-somite stage
onwards, gbx2 is not required for cerebellum cell differentiation in
the absence of Otx function. Supporting this, a mouse conditional knockout
showed that cerebellum still develops after gbx2 inactivation at 9.5
dpc (Li et al., 2002).
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Suda et al., 1997). It is
worth noting that OtxH zebrafish embryos, like Otx2+/-;
Otx1-/- mice, present a transformation of retinal pigment
epithelium into an enlarged retina (Fig.
2C and data not shown)
(Martinez-Morales et al.,
2001). This confirms a conserved role of Otx homeobox proteins
across different species. Taking advantage of these results, we analysed, for the first time, the mes-metencephalic phenotype of embryos lacking both Otx and Fgf functions in the MHB. We show that these embryos develop r1 identity and differentiate several cerebellar cell fates but lack the locus coeruleus. Thus, early r1 territory and cerebellar cell identities are induced in absence of Fgf signalling as long as Otx function is also removed. Our results therefore challenge the current model in which Fgf activity from the IsO is providing the information required for induction of cerebellar cell identities. If Fgf signalling is not instructive for cerebellar identity; it is, however, required to maintain the r1 compartment free of Otx protein, stabilizing compartment boundaries; it is also required not only for the proliferation of cerebellar precursor cells but also for cerebellar dorsoventral organisation and dorsal fusion.
Cerebellar identities can differentiate in the absence of Fgf signalling activity
Fgf8 gain- and loss-of-function (Sato
et al., 2001; Sato and
Nakamura, 2004; Suzuki-Hirano
et al., 2005; Trokovic et al.,
2003) have led to a prevalent model in which Fgf8 signalling
instructs cerebellar development. More recently, studies in fish, chick and
ascidians propose a primary role for Fgf8 in repressing Otx expression, in
vertebrates Otx2 in turn represses r1 and cerebellar identity
(Hudson et al., 2003;
Jaszai et al., 2003;
Martinez et al., 1999;
Sato et al., 2001;
Sato and Nakamura, 2004;
Suzuki-Hirano et al., 2005). A
mouse Fgf8 conditional null mutation producing a truncated protein similar to
the one present in zebrafish ace has been reported
(Chi et al., 2003). This
shortened protein is also completely inactive in mouse, and embryos that carry
this conditional knockout allele (which removes Fgf8 function inside the
En1-expressing territory) show, like their zebrafish counterparts, a
posterior shift of otx2 expression at the 12-somite stage
(Chi et al., 2003;
Jaszai et al., 2003).
Here, we report that Fgf signalling is dispensable for cerebellar cell
differentiation in the absence of Otx function. As we argue above, Otx2 is
never expressed in presumptive r1. In chick and mouse, the posterior boundary
of otx2 expression defines the caudal limit of the presumptive
midbrain and, hence, the anterior limit of the cerebellar anlage
(Millet et al., 1996;
Zervas et al., 2004). In fish
too, three arguments strongly suggest that otx2 is never expressed in
prospective r1 territory. First, the posterior limit of otx2
expression at late gastrulation is always anterior to that of her5,
which maps to the posterior part of r1
(Tallafuss and Bally-Cuif,
2003; Tallafuss et al.,
2001). Second, the gap between the posterior boundary of
otx2 expression and the anterior limit of the hoxb1b domain
(earliest marker defining r3/r4 boundary)
(Prince et al., 1998) is
constant between 60% epiboly and the end of gastrulation (see Fig. S1 in the
supplementary material). And third, gbx1 expression only overlaps
with otx2 at 60% epiboly, well before both the sharpening of the r1
boundary and the onset of fgf8 expression in r4
(Reifers et al., 1998;
Rhinn et al., 2003). Finally,
the onset of fgf8 expression in r1 is taking place during late
gastrulation, long after the formation of the Otx expression domain in fish
(Maves et al., 2002;
Reifers et al., 1998;
Rhinn et al., 2003) and mouse
(Martinez-Barbera et al.,
2001; Garda et al.,
2001). All together, the existing data all strongly suggest that
Fgf8 has no role in defining otx2 posterior boundary initially but
maintains it during somitogenesis. Hence, we propose that the function of Fgf
signalling from the IsO is neither to induce r1 nor cerebellar cell identities
but to prevent a later propagation of Otx expression inside the r1 territory,
thereby maintaining a set of developmental decisions required to maintain r1,
and preventing repression of cerebellar identities by Otx.
If it is not Fgf, which is the key molecule responsible for the switch to a
cerebellar fate? Targeted mutations of either otx2 or hoxa2
resulted in either a rostral or a caudal expansion of the cerebellum,
respectively (Acampora et al.,
1997; Gavalas et al.,
1997). Moreover, hoxa2 mis-expression experiments in r1
showed cell-autonomous inhibition of granule cell fate
(Eddison et al., 2004).
Therefore, in Fgf misexpression experiments, the formation of an ectopic
cerebellum would rather occur by local downregulation of the cerebellum
repressors otx2 and/or hoxa2
(Irving and Mason, 1999;
Martinez et al., 1999), rather
than by direct induction. A member of the iroquois homeodomain gene
family, irx2 has been recently suggested as a specific inducer of
chick cerebellar identity downstream of Fgf8 signal
(Matsumoto et al., 2004).
Exclusively in presence of Fgf activity, activated phosphorylated Irx2 forces
the rostral hindbrain towards cerebellar fate. Although not discussed by the
authors, the possibility of irx2 attributes cerebellar identity
independently from any Fgf activity cannot be excluded. Indeed, iro1b
and iro2a are both expressed in r1 during early to mid-somitogenesis
(Lecaudey et al., 2005). In
our four conditions, both genes are still expressed in r1 of
fgf8-/-; OtxH embryos, showing that iro1b and
iro2a are responding like other r1 markers studied (see Fig. S3 in
the supplementary material). However, presence of these genes transcripts in
both the mesencephalic and metencephalic compartment confirms that any
r1-specific function of Iroquois should be studied at the protein level.
Fgf8 is directly required for proliferation and morphogenesis of the cerebellum and specification of the locus coeruleus
There are great variations in cerebellum morphology among teleosts and the
zebrafish cerebellum (mainly lateral and vestibular) is one of the simplest.
The corpus cerebelli and valvula cerebelli are thought to be related to the
mouse vermis and flocculi, respectively; and the corpus cerebelli, which
received inputs from the vestibular apparatus, is believed to be the most
primitive part of the cerebellum
(Wullimann et al., 1996).
In our experiments, lack of Otx function affects the dorsal midline fusion
that normally occurs during the early stage of cerebellar development. Based
on an unexpected increase in isthmic structure in
En1+/Otx2LacZ mouse embryos, it has been suggested that
otx2-positive cells are crossing the otx2 boundary to form a
substratum region (called velum) required for midline fusion
(Louvi et al., 2003). Even if,
in zebrafish, the valvula cerebelli forms directly adjacent to the
mesencephalon - without the presence of a recognizable velum - our results are
compatible with a function of Otx in cerebellar dorsal fusion. Indeed, the
absence of fusion in OtxH embryos suggests that Otx-expressing cells may be
involved in midline fusion in fish.
This absence of fusion is exacerbated in absence of Fgf signalling.
Phenotypic analysis of fgf8-/-; OtxH zebrafish embryos
shed some light on the Fgf function in cerebellar morphogenesis.
fgf8-/-; OtxH cerebellar tissue undergoes a severe lateral
displacement. In particular, the granule cells of the eminentia granularis
normally spread dorsoventrally in wild-type and OtxH embryos
(Fig. 5A,B,E,F) but are only
seen in the ventral area in fgf8-/-; OtxH embryos
(arrowheads in Fig. 5D,H).
Finally, in fgf8-/-; OtxH, the Purkinje cells loose their
layered organisation and the ventral axonal projections are much reduced. Such
projection has not yet been described in zebrafish and the only ventral
projection described in adult teleost is thought to come from a specific
subpopulation of caudal lobe Purkinje cells
(Lannoo et al., 1991). Absence
or disturbed axonal projections in the fgf8-/-; OtxH
cerebellum could therefore be due to either a defect in pathfinding, a delay
in neuronal maturation or loss of a specific subpopulation of the Purkinje
cell population. We therefore conclude that Otx activity is primarily required
for cerebellar fusion, while Fgf8 activity is necessary for cerebellum folding
and cellular organisation (but not differentiation) of the cerebellum.
The mechanisms that underlie cerebellar dorsoventral organisation and late
cerebellar morphogenesis are also starting to be uncovered in mouse. Recent
mouse studies reveal the importance of Hedgehog signalling for the cerebellar
morphogenesis and its dorsoventral organisation
(Sotelo, 2004;
Wechsler-Reya and Scott,
1999). Our next challenge is therefore to understand how Fgf and
Hh signals coordinate these events and what the downstream effectors are that
they regulate in these processes.
Finally, our findings confirm the central role of Fgf in proliferation. In
mouse, mis-expression of fgf8 under the wnt1 promoter
revealed its mitogenic activity on mesencephalic cells
(Lee et al., 1997) and partial
fgf8 lack of function reduces proliferation of the medial cerebellar
anlage (Xu et al., 2000). This
effect on proliferation is detected from prim-22 in
fgf8-/- embryos, and reducing Otx levels in this context
does not affect the proliferation rate, indicating that Otx is not involved in
this process.
Conversely, cell death is not more frequent in absence of Fgf function, although the dying cells are more often found at the roof plate of the enlarged midbrain. By contrast, cell death is increased in OtxH mes/met area starting at 20-somite stage, most probably owing to the lack of Wnt1 shown to be crucial for cell survival. This phenotype is exacerbated in absence of Fgf, probably revealing cooperation between these two signals. This result explains the reduced size (52% the size of OtxH; Fig. 4B,D) of the rescued granule cells population in fgf8-/-; OtxH. At earlier stages, the overall size of the Her5-GFP territory is not dramatically different in double mutant embryos, neither is the expanded efnb2a territory in the SU-treated morphants, showing that there is no significant apoptosis during the first half of somitogenesis. Together, reduced proliferation and increased cell death explain the relatively variable number of rescued differentiated cells observed at 5 dpf in fgf8-/-; OtxH.
Our study supports previous studies
(Guo et al., 1999; Lam, 2003)
showing that the IsO Fgf signalling is strictly required for the induction
and/or early survival of LC tyrosin hydroxylase-positive precursors. Our data
also indicate that loss of Otx function is not able to rescue LC neurons in
fgf8-/-mutant. This result suggests that, together with
Bmp, Fgf is directly required to induce LC identity.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/10/1891/DC1
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Acampora, D., Avantaggiato, V., Tuorto, F. and Simeone, A. (1997). Genetic control of brain morphogenesis through Otx gene dosage requirement. Development 124,3639 -3650.[Abstract]
Acampora, D., Gulisano, M., Broccoli, V. and Simeone, A. (2001). Otx genes in brain morphogenesis. Prog. Neurobiol. 64,69 -95.[CrossRef][Medline]
Acampora, D., Annino, A., Puelles, E., Alfano, I., Tuorto, F. and Simeone, A. (2003). OTX1 compensates for OTX2 requirement in regionalisation of anterior neuroectoderm. Gene Expr. Patterns 3,497 -501.[Medline]
Adolf, B., Bellipanni, G., Huber, V. and Bally-Cuif, L. (2004). atoh1.2 and beta3.1 are two new bHLH-encoding genes expressed in selective precursor cells of the zebrafish anterior hindbrain. Gene Expr. Patterns 5,35 -41.[CrossRef][Medline]
Alvarado-Mallart, R. M., Martinez, S. and Lance-Jones, C. C. (1990). Pluripotentiality of the 2-day-old avian germinative neuroepithelium. Dev. Biol. 139, 75-88.[CrossRef][Medline]
Bellipanni, G., Murakami, T., Doerre, O. G., Andermann, P. and Weinberg, E. S. (2000). Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos. Dev. Biol. 223,339 -353.[CrossRef][Medline]
Chi, C. L., Martinez, S., Wurst, W. and Martin, G. R.
(2003). The isthmic organizer signal FGF8 is required for cell
survival in the prospective midbrain and cerebellum.
Development 130,2633
-2644.
Cooke, J., Moens, C., Roth, L., Durbin, L., Shiomi, K., Brennan, C., Kimmel, C., Wilson, S. and Holder, N. (2001). Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain. Development 128,571 -580.[Abstract]
Costagli, A., Kapsimali, M., Wilson, S. W. and Mione, M. (2002). Conserved and divergent patterns of Reelin expression in the zebrafish central nervous system. J. Comp. Neurol. 450, 73-93.[CrossRef][Medline]
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380,66 -68.[CrossRef][Medline]
Echevarria, D., Vieira, C., Gimeno, L. and Martinez, S. (2003). Neuroepithelial secondary organizers and cell fate specification in the developing brain. Brain Res. Brain Res. Rev. 43,179 -191.[CrossRef][Medline]
Eddison, M., Toole, L., Bell, E. and Wingate, R. J. (2004). Segmental identity and cerebellar granule cell induction in rhombomere 1. BMC Biol. 2, 14.[Medline]
Furthauer, M., Reifers, F., Brand, M., Thisse, B. and Thisse, C. (2001). sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish. Development 128,2175 -2186.[Medline]
Furthauer, M., Lin, W., Ang, S. L., Thisse, B. and Thisse, C. (2002). Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell Biol. 4, 170-174.[CrossRef][Medline]
Garda, A. L., Echevarria, D. and Martinez, S. (2001). Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer. Mech. Dev. 101,111 -118.[CrossRef][Medline]
Gavalas, A., Davenne, M., Lumsden, A., Chambon, P. and Rijli, F. M. (1997). Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning. Development 124,3693 -3702.[Abstract]
Guo, S., Brush, J., Teraoka, H., Goddard, A., Wilson, S. W., Mullins, M. C. and Rosenthal, A. (1999). Development of noradrenergic neurons in the zebrafish hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a. Neuron 24,555 -566.[CrossRef][Medline]
Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature 391,788 -792.[CrossRef][Medline]
Hudson, C., Darras, S., Caillol, D., Yasuo, H. and Lemaire,
P. (2003). A conserved role for the MEK signalling pathway in
neural tissue specification and posteriorisation in the invertebrate chordate,
the ascidian Ciona intestinalis. Development
130,147
-159.
Irving, C. and Mason, I. (1999). Regeneration of isthmic tissue is the result of a specific and direct interaction between rhombomere 1 and midbrain. Development 126,3981 -3989.[Abstract]
Jaszai, J., Reifers, F., Picker, A., Langenberg, T. and Brand,
M. (2003). Isthmus-to-midbrain transformation in the absence
of midbrain-hindbrain organizer activity. Development
130,6611
-6623.
Jessell, T. M. and Sanes, J. R. (2000). Development. The decade of the developing brain. Curr. Opin. Neurobiol. 10,599 -611.[CrossRef][Medline]
Kawahara, A., Chien, C. B. and Dawid, I. B. (2002). The homeobox gene mbx is involved in eye and tectum development. Dev. Biol. 248,107 -117.[CrossRef][Medline]
Kawakami, Y., Rodriguez-Leon, J., Koth, C. M., Buscher, D., Itoh, T., Raya, A., Ng, J. K., Esteban, C. R., Takahashi, S., Henrique, D. et al. (2003). MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. Cell Biol. 5, 513-519.[CrossRef][Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Koster, R. W. and Fraser, S. E. (2001). Direct imaging of in vivo neuronal migration in the developing cerebellum. Curr. Biol. 11,1858 -1863.[CrossRef][Medline]
Kudoh, T. and Dawid, I. B. (2001). Zebrafish mab21l2 is specifically expressed in the presumptive eye and tectum from early somitogenesis onwards. Mech. Dev. 109, 95-98.[CrossRef][Medline]
Lam, C. S., Sleptsova-Friedrich, I., Munro, A. D. and Korzh, V. (2003). SHH and FGF8 play distinct roles during development of noradrenergic neurons in the locus coeruleus of the zebrafish. Mol. Cell. Neurosci. 22,501 -515.[Medline]
Lannoo, M. J., Ross, L., Maler, L. and Hawkes, R. (1991). Development of the cerebellum and its extracerebellar Purkinje cell projection in teleost fishes as determined by zebrin II immunocytochemistry. Prog. Neurobiol. 37,329 -363.[Medline]
Lecaudey, V., Anselme, I., Dildrop, R., Ruther, U. and Schneider-Maunoury, S. (2005). Expression of the zebrafish Iroquois genes during early nervous system formation and patterning. J. Comp. Neurol. 492,289 -302.[CrossRef][Medline]
Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997). Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain. Development 124,959 -969.[Abstract]
Li, J. Y., Lao, Z. and Joyner, A. L. (2002). Changing requirements for Gbx2 in development of the cerebellum and maintenance of the mid/hindbrain organizer. Neuron 36, 31-43.[CrossRef][Medline]
Liu, A. and Joyner, A. L. (2001). Early anterior/posterior patterning of the midbrain and cerebellum. Annu. Rev. Neurosci. 24,869 -896.[CrossRef][Medline]
Louvi, A., Alexandre, P., Metin, C., Wurst, W. and Wassef,
M. (2003). The isthmic neuroepithelium is essential for
cerebellar midline fusion. Development
130,5319
-5330.
Lumsden, A. and Krumlauf, R. (1996). Patterning
the vertebrate neuraxis. Science
274,1109
-1115.
Lun, K. and Brand, M. (1998). A series of no isthmus (noi) alleles of the zebrafish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125,3049 -3062.[Abstract]
Mallamaci, A., Di Blas, E., Briata, P., Boncinelli, E. and Corte, G. (1996). OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area. Mech. Dev. 58,165 -178.[CrossRef][Medline]
Martinez, S., Wassef, M. and Alvarado-Mallart, R. M. (1991). Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en. Neuron 6,971 -981.[CrossRef][Medline]
Martinez, S., Marin, F., Nieto, M. A. and Puelles, L. (1995). Induction of ectopic engrailed expression and fate change in avian rhombomeres: intersegmental boundaries as barriers. Mech. Dev. 51,289 -303.[CrossRef][Medline]
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L. and Martin, G. R. (1999). FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126,1189 -1200.[Abstract]
Martinez-Barbera, J. P., Signore, M., Boyl, P. P., Puelles, E.,
Acampora, D., Gogoi, R., Schubert, F., Lumsden, A. and Simeone, A.
(2001). Regionalisation of anterior neuroectoderm and its
competence in responding to forebrain and midbrain inducing activities depend
on mutual antagonism between OTX2 and GBX2.
Development 128,4789
-4800.
Martinez-Morales, J. R., Signore, M., Acampora, D., Simeone, A.
and Bovolenta, P. (2001). Otx genes are required for tissue
specification in the developing eye. Development
128,2019
-2030.
Matsumoto, K., Nishihara, S., Kamimura, M., Shiraishi, T., Otoguro, T., Uehara, M., Maeda, Y., Ogura, K., Lumsden, A. and Ogura, T. (2004). The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation. Nat. Neurosci. 7,605 -612.[CrossRef][Medline]
Maves, L., Jackman, W. and Kimmel, C. B. (2002). FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129,3825 -3837.[Medline]
McMahon, A. P. and Bradley, A. (1990). The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62,1073 -1085.[CrossRef][Medline]
Mercier, P., Simeone, A., Cotelli, F. and Boncinelli, E. (1995). Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish. Int. J. Dev. Biol. 39,559 -573.[Medline]
Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18,136 -141.[CrossRef][Medline]
Millet, S., Bloch-Gallego, E., Simeone, A. and Alvarado-Mallart, R. M. (1996). The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridisation and chick/quail homotopic grafts. Development 122,3785 -3797.[Abstract]
Miyamura, Y. and Nakayasu, H. (2001). Zonal distribution of Purkinje cells in the zebrafish cerebellum: analysis by means of a specific monoclonal antibody. Cell Tissue Res. 305,299 -305.[Medline]
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh,
B. K., Hubbard, S. R. and Schlessinger, J. (1997). Structures
of the tyrosine kinase domain of fibroblast growth factor receptor in complex
with inhibitors. Science
276,955
-960.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Prince, V. E., Moens, C. B., Kimmel, C. B. and Ho, R. K. (1998). Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. Development 125,393 -406.[Abstract]
Raible, F. and Brand, M. (2004). Divide et Impera - the midbrain-hindbrain boundary and its organizer. T
