Download Evidence that FGF8 signalling from the midbrain

959
Development 124, 959-969 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV9525
Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates
growth and polarity in the developing midbrain
Scott M. K. Lee1, Paul S. Danielian1, Bernd Fritzsch2 and Andrew P. McMahon1
1Department
2Department
of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
of Biomedical Sciences, Creighton University, Omaha, Nebraska 68178, USA
SUMMARY
The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular
markers and in the cytoarchitectonic differentiation of the
tectum, where cells cease proliferating and differentiate in
a rostral to caudal progression. Tissue grafting experiments
have implicated cell signalling by the mesencephalicmetencephalic (mid-hindbrain) junction (or isthmus) in
orchestrating these events. We have explored the role of
Wnt-1 and FGF8 signalling in the regulation of mesencephalic polarity. Wnt-1 is expressed in the caudal mesencephalon and Fgf8 in the most rostral metencephalon. Wnt1 regulates Fgf8 expression in the adjacent metencephalon,
most likely via a secondary mesencephalic signal. Ectopic
expression of Fgf8 in the mesencephalon is sufficient to
activate expression of Engrailed-2 (En-2) and ELF-1, two
genes normally expressed in a decreasing caudal to rostral
gradient in the posterior mesencephalon. Ectopic
expression of Engrailed-1 (En-1), a functionally equivalent
homologue of En-2 is sufficient to activate ELF-1
expression by itself. These results indicate the existence of
a molecular hierarchy in which FGF8 signalling establishes
the graded expression of En-2 within the tectum. This in
turn may act to specify other aspects of A-P polarity such
as graded ELF-1 expression. Our studies also reveal that
FGF8 is a potent mitogen within the mesencephalon: when
ectopically expressed, neural precursors continue to proliferate and neurogenesis is prevented. Taken together our
results suggest that FGF8 signalling from the isthmus has
a key role in coordinately regulating growth and polarity
in the developing mesencephalon.
INTRODUCTION
The vertebrate mesencephalon exhibits many aspects of rostrocaudal polarity. Anatomically there is a pronounced rostrocaudal developmental gradient of cytoarchitectonic differentiation in the chick tectum (dorsal mesencephalon) (LaVail and
Cowan, 1971) which appears to be present in the dorsal mesencephalon (colliculi) (Altman and Bayer, 1981a,b) of
mammals as well (for simplicity we will use the general term
‘tectum’ in reference to the chick tectum and the mammalian
colliculi). As a consequence, neural precursors cease cell
division and differentiate first in the most rostral tectum, while
precursors in more caudal regions, close to the isthmus, are still
dividing and expanding. Additionally, the rostral and caudal
regions of the tectum acquire different sets of afferent inputs
from the retina: axons extending from ganglion cells in the
temporal retina innervate the rostral tectum, while the caudal
tectum receives axons from the nasal retina (DeLong and
Coulombre, 1965; Crossland et al., 1974).
Clues as to the molecular regulation of mesencephalic
polarity are provided by the observations that many molecules
are expressed in gradients across the tectum. First En-2, a transcription factor, is expressed in a rostrocaudally increasing
gradient across the tectum. (Davis and Joyner, 1988; Davis et
al., 1988; Gardner et al., 1988; Patel et al., 1989; Gardner and
Barald, 1992; Millet and Alvarado-Mallart, 1995). Addition-
While vertical and planar signals which occur at gastrulation
impart an initial anterior-posterior (A-P) pattern onto the
central nervous system (CNS) subsequent interactions between
adjacent cell populations within the neural plate are required
to refine this pattern (Bally-Cuif and Wassef, 1995; Kelly and
Melton, 1995). The role of cellular interactions between
adjacent regions in the establishment of axial pattern has been
intensively studied in the developing brain. For example, interspecies grafting experiments between quail and chick embryos
have revealed that the mesencephalic-metencephalic junction
(hereafter refered to as the isthmus) acts as an organizing center
(Bally-Cuif and Wassef, 1995; Joyner, 1996). Grafts of isthmic
tissue into the caudal diencephalon induce the formation of
ectopic mesencephalic structures in the host (Gardner and
Barald, 1991; Itasaki et al., 1991; Martinez et al., 1991; BallyCuif et al., 1992; Marin and Puelles, 1994), whereas grafts into
the myelencephalon induce ectopic cerebellar structures
(Martinez et al., 1995). These results suggest that signals from
the isthmus may normally be involved in the induction of mesencephalic cell fates rostrally and cerebellar fates caudally.
Cell interactions somewhat later in development may also
regulate rostrocaudal polarity within a specific brain region.
Key words: mesencephalon, polarity, Fgf8, Engrailed, ELF-1, Wnt-1,
cell proliferation, pattern formation, CNS
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S. M. K. Lee and others
ally, ELF-1 and RAGS, two ligands for EPH-like receptor
tyrosine kinases are expressed in rostrocaudally increasing
gradients across the caudal tectum and may function to inhibit
temporal axon ingrowth and/or to attract nasal axons (Cheng
and Flanagan, 1994; Cheng et al., 1995; Drescher et al., 1995;
Nakamoto et al., 1996). Ectopic high level expression of En
genes in the rostral tectum leads to a perturbation of retinal
axon guidance such that temporal axons rarely enter the tectum
and nasal axons often arborize ectopically (Friedman and
O’Leary, 1996; Itasaki and Nakamura, 1996; Logan et al.,
1996). A molecular basis for this result was provided by the
observations that ELF-1 and RAGS are ectopically expressed
in these experiments (Logan et al., 1996) and that ectopic
expression of ELF-1 alone is sufficient to inhibit ingrowth of
temporal axons into the tectum (Nakamoto et al., 1996).
Ectopic high level rostral expression of En also abolishes the
rostrocaudal gradient of cytoarchitectonic development (Logan
et al., 1996). These results support a model in which the graded
expression of En genes across the rostrocaudal axis of the
tectum is involved in conferring positional specification to
these cells.
A role for graded En expression in determining tectal
polarity is further supported by grafting experiments in the
chick. If the rostral mesencephalon is inverted, En-2 expression
is reprogrammed in the grafted tissue to form a gradient consistent with its new environment (Martinez and AlvaradoMallart, 1990; Itasaki et al., 1991). Subsequently, a normal
mesencephalon develops from the grafted tissue (Matsuno et
al., 1991), demonstrating that mesencephalic rostrocaudal
polarity is plastic at the time of the experiment (2 days of incubation) and implicating signals from adjacent tissue in mesencephalic rostrocaudal patterning and En-2 regulation. Additionally, ectopic mesencephalic structures resulting from
isthmic grafts into the diencephalon display both an En-2
gradient and anatomical polarity which are the mirror image of
those in the endogenous mesencephalon (Alvarado-Mallart et
al., 1990; Itasaki and Nakamura, 1992; Itasaki et al., 1991;
Martinez et al., 1991; Marin and Puelles, 1994). Together, these
data implicate isthmic derived signals in the establishment of
mesencephalic rostrocaudal polarity and imply that such
signals may act via the regulation of En expression.
Recent experiments have demonstrated the importance of
FGF8 in mesencephalic development. FGF8 is expressed in
cardiac mesoderm underlying the presumptive mes/met region
(Heikinheimo et al., 1994; Crossley and Martin, 1995;
Mahmood et al., 1995). Application of beads soaked in FGF8
results in ectopic mesencephalic development suggesting that
FGF8 may normally play a role in mesencephalic induction
(Crossley et al., 1996a) (see Discussion). Fgf8 is also expressed
in the neural plate itself, in the rostral metencephalon, from 3somites (Mahmood et al., 1995), with expression refining to a
tight band of cells immediately caudal to the isthmus by 16somites (Heikinheimo et al., 1994; Crossley and Martin, 1995;
Mahmood et al., 1995; note that the region of isthmus used in
the above mentioned grafting experiments includes Fgf8expressing cells). Thus FGF8 may also play a role in regulating the rostrocaudal polarity of the mesencephalon.
We have explored the role of cell signalling at the isthmus
in establishing rostrocaudal polarity in the mesencephalon. By
analyzing Wnt-1 mutants we show that a mesencephalic
derived signal(s) is required to maintain Fgf8 expression at the
isthmus. To address the possibility that FGF8 regulates mesencephalic polarity we have used the Wnt-1 enhancer to
generate a new domain of Fgf8 expression at the dorsal midline
of the mesencephalon. Our results are consistent with FGF8
signalling playing a central role in regulating growth and
polarity in the developing mesencephalon.
MATERIALS AND METHODS
Production and genotyping of Wnt-1 mutant embryos
Wnt-1 mutant embryos were obtained by intercrossing mice heterozygous for a Wnt-1 null mutation (McMahon and Bradley, 1990).
Yolk sac DNA was used for genotyping as described by McMahon et
al. (1992).
Fgf8 misexpression construct
To ectopically express Fgf8 under the control of the Wnt-1 enhancer
we utilized a variant of the previously described pWEXPZ vector
(Danielian and McMahon, 1996), here designated pWEXPZ2. Each
contains a Wnt-1 minigene upstream of the 5.5 kb 3-prime located
Wnt-1 enhancer sequences. In pWEXPZ2 an approximately 800 base
pair region in the Wnt-1 3-prime UTR has been deleted. This has no
effect on the previously reported expression pattern regulated by the
Wnt-1 enhancer (Echelard et al., 1993, 1994). The WEXPZ2-Fgf8
construct was obtained by cutting pWEXPZ2 with EcoRV and
inserting an end filled EcoRI-XhoI cut piece of the Fgf8 cDNA
(Mahmood et al., 1995). This cDNA corresponds to Fgf8 variant 4 of
Crossley and Martin (1995).
Production of Fgf8 transgenic embryos
Transgenic mice were generated essentially as described by Echelard
et al. (1993). Linearized DNA was purified by agarose gel electrophoresis followed by electroelution into 1× TAE and concentrated
using the Wizard DNA Cleanup System (Promega).
C57BL/6J×CBA/J F1 mice were used as embryo donors. Injected
embryos were cultured overnight in M-16 medium following microinjection, and transferred at the 2-cell stage to pseudopregnant recipient
SWB females. Noon of the day of the transfer was considered to be
0.5 d.p.c. For some experiments, pregnant females were injected
intraperitoneally with 50 µg/g body weight of 5-bromo-2′ deoxyuridine (BrdU; Sigma) 1.5 hours prior to killing. Staged embryos were
collected at 10.5 d.p.c., 12.5 d.p.c., 13.5 d.p.c., 14.5 d.p.c., and 18.5
d.p.c. into PBS and photographed using an Olympus SZH stereo photomicroscope on Kodak EPY 64T color slide film.
The genotype of embryos was determined by PCR analysis of
genomic DNA extracted from embryonic yolk sacs with care taken to
avoid cross-contamination. The primers used were directed against the
lacZ sequences present in the transgene construct.
5′ primer: 5′-TTTAACGCCGTGCGCTGTTCG-3′
3′ primer: 5′-GATCCAGCGATACAGCGCGTC-3′
The PCR program used was as follows: cycle 1: 94°C, 3 minutes;
cycle 2: 94°C, 45 seconds; cycle 3: 55°C, 45 seconds; cycle 4: 72°C,
1 minute; cycle 5: 35 times to cycle 2; cycle 6: 72°C, 5 minutes. PCR
products were visualized on ethidium bromide stained agarose gels.
Of a total of 366 embryos obtained between 10.5 d.p.c. and 18.5
d.p.c., 126 (34.4%) were transgenic. Of these 68 (54.0%) showed the
reported brain phenotype.
Histological analysis
Embryos were fixed in 4% paraformaldehyde in PBS for 18-24 hours
at 4°C while 18.5 d.p.c. brains were fixed for 18-24 hours in Bouin’s
fixative at room temperature. Paraformaldehyde-fixed tissue was
transferred into 70% ethanol and stored at 4°C. Bouin’s-fixed tissue
was washed extensively in 70% ethanol for 2-4 days with subsequent
storage at 4°C. For embedding, samples were dehydrated through a
FGF8 signalling in the midbrain
series of graded ethanols and embedded in paraffin wax (Fibrowax,
BDH). Embryos were serially sectioned at 6-7 µm in the sagittal
plane. Representative sections were stained with haemotoxylin and
eosin and photographed on a Leitz DMRD photomicroscope using
Kodak 64T film. Selected remaining sections were used for the
immunohistochemical detection of BrdU incorporation.
Immunohistochemical detection of BrdU
Immunohistochemical detection of BrdU was performed as described
by Dickinson et al. (1994). Photographs of representative sections
were taken on a Leitz DMRD photomicroscope using Kodak 64T film.
In situ hybridization
Embryos at 10.5-13.5 d.p.c. were fixed in 4% paraformaldehyde in
PBS overnight at 4°C, washed in PBS, dehydrated through a series of
graded methanols, and stored at −20°C. For 12.5 and 13.5 d.p.c.
embryo brains were dissected prior to in situ hybridization. Wholemount in situ hybridization was performed as described by Parr et al.
(1993) and modified according to Knecht et al. (1995). Digoxigenin
probes were synthesized using the Digoxigenin RNA Labeling Kit
(Boehringer Mannheim). ELF-1 expression was detected by the RAP
in situ technique using a Mek4-AP probe as described by Cheng and
Flanagan (1994); Cheng et al. (1995). Stained embryos and brains
were transferred to 80% glycerol and photographed using a Leica
Wild M10 photomicroscope using Kodak 64T film.
For all markers analyzed a minimum of three transgenic embryos
were analyzed and identical results where always observed.
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abolished (Fig. 1E,F), even though the metencephalon is still
intact at this time (McMahon et al., 1992; Serbedzija et al.,
1996). Thus, the initiation of Fgf8 expression occurs independently of Wnt-1. However, Wnt-1, or more likely a secondary
mesencephalic derived signal, is required for the maintenance
of Fgf8 expression (see Discussion).
Ectopic expression of Fgf8 in the mesencephalon
and caudal diencephalon
To directly explore the role of FGF8 signalling in establishing
mesencephalic rostrocaudal polarity we ectopically expressed
Graphics
All composites were generated by scanning slides into Adobe
Photoshop v3.0, and transferred to Canvas v3.5 for labeling, all on a
Power Macintosh.
RESULTS
A midbrain derived signal maintains metencephalic
Fgf8 expression
Studies of Wnt-1 mutants indicate that Wnt-1 signalling plays
a key role in mes/met development. Wnt-1 is broadly expressed
within the presumptive mesencephalon at neural plate stages
(1-somite), but expression becomes refined to a narrow band
of cells immediately anterior to the isthmus, as well as much
of the dorsal central nervous system (CNS) following neural
tube closure (16-somites; Wilkinson et al., 1987; McMahon et
al., 1992; Parr et al., 1993). In embryos homozygous for a Wnt1 null mutation mesencephalic development proceeds normally
up to the 5-somite stage, but by 14 somites most of the mesencephalon is absent. The metencephalon is lost subsequently,
most likely reflecting a requirement of mesencephalic derived
signals for metencephalic development (McMahon and
Bradley, 1990; Thomas and Capecchi, 1990; McMahon et al.,
1992; Serbedzija et al., 1996).
Fgf8 is expressed in a narrow band of cells within the
isthmus immediately caudal to the Wnt-1-expressing cells from
3-somites until at least 14.5 d.p.c.(Heikinheimo et al., 1994;
Crossley and Martin, 1995; Mahmood et al., 1995). To investigate the possibility that Fgf8 expression is dependent on Wnt1 or, more generally, on any mesencephalic derived signals, we
analyzed Fgf8 expression in Wnt-1 mutant embryos. Fgf8
expression in Wnt-1 mutants was indistinguishable from that
in the wild type up to 6-somites (Fig. 1A,B). However, by 9somites expression is markedly reduced (Fig. 1C,D) and by 14somites metencephalic Fgf8 expression was completely
Fig. 1. Fgf8 expression in Wnt-1 mutant embryos. (A,B) Fgf8
expression appears identical in the mes-met region of wild-type (A)
and mutant (B) embryos at the 6-somite stage, indicating that Wnt-1
is not required for activation of Fgf8 expression. (C,D) Comparison
of wild-type (C) and mutant (D) 9-somite embryos reveals that by
this time mesencephalon development is reduced in Wnt-1 mutants.
Fgf8 expression in the metencephalon (arrow) is also markedly
reduced. (E) By 15 somites Fgf8 expression in wild-type embryos is
restricted to a narrow band of cells immediately posterior to the
isthmus. (F) In a 14-somite mutant embryo the metencephalon is still
present (arrow) but metencephalic Fgf8 expression is completely
abolished.
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S. M. K. Lee and others
Fgf8 in the rostral mesencephalon of mouse embryos using the
Wnt-1 enhancer (Echelard et al., 1994). The Wnt-1 enhancer
directs expression throughout the presumptive mesencephalon
at neural plate stages (1-somite). Expression rapidly becomes
refined to, and stably maintained in, a narrow ring of cells
immediately anterior to the isthmus as well as much of the
dorsal central nervous system (CNS) by the time that neural
tube closure in the brain is complete (16-somites; Wilkinson et
al., 1987; McMahon et al., 1992; Parr et al., 1993). Consequently, this expands the domain of Fgf8 expression into the
dorsal aspect of the mesencephalon and caudal diencephalon
coincident with the initiation of a distinct rostrocaudal polarity
in mesencephalic development. Because we expected that
ectopic expression of Fgf8 would cause postnatal lethality,
founder (generation 0 [G0])
embryos were examined for aberrant
phenotypes.
A characteristic phenotype was
observed in 54% of transgenic
embryos. At 10.5 d.p.c. these
embryos exhibited an overgrowth of
the mesencephalon and most caudal
diencephalic segment, prosomere 1
(p1) (nomenclature of Rubenstein et
al., 1994), (Fig. 2A,B) together with
the formation of a constriction at the
boundary of the midbrain and diencephalon (arrows in 2B,D), which
was more pronounced by 12.5 d.p.c.
(Fig. 2C,D). Analysis at 18.5 d.p.c.
showed that transgenic brains
displayed a dramatic hyperplasia of
the mesencephalon and caudal diencephalon which partially overgrew
the cerebellum and telencephalon
(compare Fig. 2E,G with F,H). The
volume of the mesencephalon of
transgenic embryos was as much as
3 times greater than that of sibling
non-transgenic controls (Figs 2, 4).
In addition to this consistent
phenotype, four 10.5 d.p.c. embryos
appeared to have more widespread
and non-specific defects most likely
resulting from widespread activation
of the transgene. These were not
studied further.
Histological analysis of the
Fgf8 transgenic phenotype
Sagittal sections through 12.5 d.p.c.
transgenic embryos were examined
histologically to identify the
anatomical consequences of ectopic
Fgf8
expression.
Mid-sagittal
sections of 12.5 d.p.c. embryos
revealed a rostrocaudal expansion of
the dorsal mesencephalon and p1,
together with an expansion of the
ventral mesencephalon and mammillary region, and enlargement of
the third ventricle (Fig. 3A,B). We also observed an apparent
reduction in the medial thalamus, which most likely is
secondary to the expansion of the third ventricle (Fig. 3B). In
all cases the overgrowth correlated with the presence of the
transgene and the characterized expression pattern of the Wnt1 enhancer (Echelard et al., 1993, 1994). Analysis of more
lateral regions revealed the remainder of the diencephalon had
formed essentially normally (Fig. 3C,D). Interestingly, lateral
regions of the expanded rostral mesencephalon and p1
consisted of only a thin ventricular zone and very few differentiated cell types, unlike wild-type embryos which have
undergone considerable differentiation in the rostral mesencephalon by this time (compare Fig. 3C,D). This suggests that
the morphological expansion may therefore result from a main-
Fig. 2. Views of WEXPZ2-Fgf8 transgenic embryos and brains revealing an overgrowth of the
mesencephalon and caudal diencephalon. (A-D) Lateral views of wild-type (A,C) and transgenic
(B,D) embryos at 10.5 d.p.c. (A,B) and 12.5 d.p.c. (C,D). The mesencephalon and most caudal
diencephalic segment, p1, are expanded in the transgenics, leading to a large increase in the
flexion of the brain by 12.5 d. p. c. (compare C and D). Additionally, an ectopic constriction is
present between the rostral mesencephalon and p1 (arrows in B and D). (E-H) Lateral (E,F) and
dorsal (G,H) views of 18.5 d.p.c. wild-type (E,G) and transgenic (F,H) brains in which the
mesencephalon-p1 region is overgrown. The expansion in the mediolateral dimension is greater
rostrally than caudally, probably due the fact that the Wnt-1 enhancer drives expression of Fgf8 to
a broad dorsal region of p1 and only to a very narrow dorsally restricted band of cells in the
mesencephalon. cb, cerebellum; ch, cerebral cortex; i, isthmus; ms, mesencephalon; mt,
metencephalon; my, myelencephalon; p1, prosomere 1 (pretectum); t, telencephalon.
FGF8 signalling in the midbrain
tenance of proliferative ventricular zone cells and a concomitant reduction in neuronal differentiation.
Consistent with this suggestion, expression of Fgf8 leads, by
18.5 d.p.c., to a severe reduction of differentiated cell types in
the dorsal mesencephalon and caudal diencephalon and an
even greater expansion of the third ventricle than that observed
at 12.5 d.p.c. (Fig. 4A,B). As FGF8-soaked beads have been
shown to induce the formation of an ectopic mesencephalon in
the caudal diencephalon of chick embryos (Crossley et al.,
1996a), we examined the diencephalon for evidence of mesencephalic transformation. In such experiments the ectopic
mesencephalon forms at the expense of the dorsal thalamus and
pretectum of the host, and duplicated cranial nerves III and IV
are formed ventrally. In our transgenics the pretectum is significantly overgrown but both the dorsal and ventral parts of
the thalamus are present and the habenulopeduncular tract has
formed close to its normal location (Fig. 4C,D). Additionally,
a detailed histological analysis of four 18.5 d.p.c. trangenic
brains revealed no evidence for duplications of mesencephalic
cranial nerves III or IV (Fig. 4 and data not shown). This was
further confirmed by placing DiI into the orbit of 14.5 d.p.c.
transgenic embryos. While we were consistently able to label
the oculomotor, trochlear and abducens projections to their
normal locations we never observed any ectopic projections to
more rostral positions (data not shown). Based on these observations and molecular analysis (see below) we conclude that
the continued ectopic expression of
Fgf8 using the Wnt-1 regulatory
element leads to a massive
expansion of neural precursor cells
at the expense of differentiated cell
types in both mesencephalon and
caudal diencephalon, but is not sufficient to induce the formation of
ectopic mesencephalic structures.
The differences between our results
and the bead experiments may
reflect variability in the experimental paradigm, species differences in
the competence to respond to FGF8
at different developmental stages,
or alternatively may represent differences in the activities of FGF8
isoforms (see Materials and
Methods, Crossley et al., 1996a,
and MacArthur et al. 1995a,b).
Fgf8 expression enhances
the proliferation of precursor
cells in the mesencephalon
and diencephalon
The observed ventricular zone
expansion in regions expressing
Fgf8 suggests that FGF8 signalling
leads to enhanced or prolonged
mitotic activity in these regions. To
investigate this issue further we
directly assayed cell proliferation
by monitoring the incorporation of
bromodeoxyuridine (BrdU) at 14.5
d.p.c. In the mesencephalon of wild
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type embryos only the most caudal regions of the tectum, close
to the isthmus, retain a thick ventricular zone with a large
number of dividing precursor cells (Fig. 5A,C). Note that in
this region almost the entire neuroepithelium consists of mitotically active ventricular zone cells and few differentiated cell
types are present (Fig. 5C). More rostrally the converse is true,
there are very few mitotically active precursor cells and a thick
layer of differentiated cell types has formed (Fig. 5E). In
contrast, transgenic embryos have a thick layer of mitotically
active precursor cells along the entire rostrocaudal extent of the
expanded midbrain-p1 ventricle (Fig. 5B,D,F). A corresponding reduction in the number of differentiated cell types is also
observed (compare Fig. 5E with F). The expanded mammillary
recess of the third ventricle present in transgenic brains also
contains a layer of mitotically active cells (data not shown). In
summary, we observed a strict correlation between ectopic
Fgf8 expression, increased neural precursor cell proliferation,
and a reduction of neural differentiation.
Characterization of rostrocaudal patterning in
embryos ectopically expressing Fgf8 in the rostral
mesencephalon and caudal diencephalon
In all transgenic embryos analyzed between 10.5 and 13.5
d.p.c. Fgf8 is ectopically expressed in a ring of cells rostral to
the isthmus, along the dorsal and ventral midlines of the mesencephalon, in a dorsally restricted transverse stripe in p1,
Fig. 3. Histological analysis of 12.5 d.p.c. WEXPZ2-Fgf8 transgenic embryos. Sections were
stained with haematoxylin and eosin. A and C are sections through the wild-type embryo shown in
Fig. 2C, A is mid-sagittal and C more lateral. B and D are sections through the transgenic embryo
shown in Fig. 2D, B is mid-sagittal and D is more lateral. Mid-sagittal sections reveal a
rostrocaudal expansion of the dorsal mesencephalon and most caudal diencephalic segment,
prosomere 1 (p1), together with an expansion of the ventral mesencephalon and mammillary
region, and enlargement of the third ventricle (compare A and B). An apparent reduction in the
medial thalamus is most likely secondary to the expansion of the third ventricle. In all cases, the
overgrowth correlates with expression of the transgene. Analysis of more lateral regions reveals
that the remainder of the diencephalon formed essentially normally (compare C and D), consistent
with the fact that transgene expression is largely limited to medial regions. cb, cerebellum; mr,
mammilary region; ms, mesencephalon; p, pituitary gland; my, myelencephalon; p1, prosomere 1;
t, telencephalon; th, thalamus; III, third ventricle.
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S. M. K. Lee and others
Fig. 4. Histological analysis of 18.5 d.p.c.
WEXPZ2-Fgf8 transgenic brains. Sections
were stained with haematoxylin and eosin.
A and C are sections through the wild-type
embryo shown in Fig. 2E,G, A is midsagittal and C more lateral. B and D are
sections through the transgenic embryo
shown in Fig. 2F,G, B is mid-sagittal and D
is more lateral. Transgenic brains display a
reduction in differentiated cell types in the
dorsal mesencephalon and caudal
diencephalon and a concomitant expansion
in the number of precursor cells, which
leads to a large expansion of the effected
regions tangential to the ventricular surface
(compare A,C with B,D). In some regions,
which are likely expressing Fgf8, the roof
of the mesencephalon is only a few cell
layers thick (* in B). Additionally, the third
ventricle is expanded as observed at 12.5
d.p.c. (B). Note that while the thalamus
exhibits some dismorphology, likely a
secondary consequence of the expanded
third ventricle, it appears to be
anatomically rather normal and the
habenulopeduncular tract is present (compare C and D). Transgenic brains also display a consistent reduction in the cerebellum. cb, cerebellum;
ch, cerebral cortex; ht, habendopenduncular tract; mr, mammilary region; ms, mesencephalon; pt, pretectum; my, myelencephalon; th,
thalamus; III, third ventricle.
along the dorsal myelencephalon and spinal cord, and in the
mammillary region (Fig. 6A,B and data not shown), a pattern
identical to Wnt-1 itself and to that of a lacZ reporter driven
by the same Wnt-1 enhancer (Echelard et al., 1993; Echelard
et al., 1994). We never observed an ectopic Wnt-1 expression
domain nor any apparent increase in the levels of Wnt-1
expression in transgenic embryos analyzed between 10.5 and
13.5 d.p.c. (compare Fig. 6C with D). The dorsal mesencephalic stripe of Wnt-1 expression however appears consistently to be slightly broader in transgenics. This most likely
reflects an expansion of the entire mesencephalon along the
mediolateral axis by this time (compare Fig. 6C with D).
The transgenic phenotype does not result from the formation
of an ectopic isthmus organizing center in the rostral mesencephalon or caudal diencephalon. First, we never observed
ectopic expression of En-1, Pax-2, or Pax-5 in transgenic
embryos examined between 10.5 and 13.5 d.p.c. (Fig. 7A,B
and data not shown). All three of these genes are normally
expressed in overlapping domains centered on the isthmus
(Davis and Joyner, 1988; Davis et al., 1988; Nornes et al.,
1990; Adams et al., 1992; Asano and Gruss, 1992; Puschel et
al., 1992; Urbanek et al., 1994). Secondly, expression of PaxFig. 5. BrdU incorporation in transgenic brains at 14.5 d.p.c. A is a
section through a mid-sagittal region of a wild-type control and B is
a section through a corresponding mid-sagittal region of a transgenic
embryo. C,D,E,F are higher magnification views of the regions
indicated in A and B. Cells that have incorporated BrdU appear black
and they are the only stained cells in these photomicrographs. In the
wild-type mesencephalon (A) high mitotic activity is limited to the
most caudal region (C) and few mitotic cells are observed rostrally
(E). In contrast, the expanded dorsal mesencephalon and p1 of
transgenic brains contains a thick ventricular zone of mitotically
active cells along its entire rostrocaudal extent (B,D,F).
6, which is normally expressed in p1 (Walther and Gruss, 1991;
arrow in Fig. 7C) is maintained in p1 of Fgf8 transgenic
embryos (arrow in Fig. 7D). Thus, these cells, while morphologically abnormal, maintain a p1 identity.
Our results are more consistent with FGF8 signalling playing
a later role in the regulation of rostrocaudal polarity. In wild type
embryos En-2 expression in the mes/met region is highest at the
isthmus and decreases both rostrally and caudally (Davis and
FGF8 signalling in the midbrain
Fig. 6. Fgf8 and Wnt-1 expression in the mesencephalon of
transgenic brains. Whole-mount in situ hybridization analysis of
Fgf8 (A,B) or Wnt-1 (C,D) expression in isolated 13.5 d.p.c. brains.
(A,B) The WEXPZ2-Fgf8 transgene drives expression of Fgf8 along
the dorsal midline of the mesencephalon, in a broad dorsal domain of
p1, and into the ventral mesencephalon (which is not visible in this
view; B) whereas endogenous Fgf8 expression is only detected at the
mes-met junction at this time (A). Comparison of Wnt-1 expression
in wild-type (C) and transgenic (D) brains reveals that Wnt-1 is not
ectopically activated by Fgf8. The dorsal mesencephalic stripe of
Wnt-1 expression in transgenics (D) may be broader than in the wildtype (C), but this is likely due to a mediolateral expansion of the
entire mesencephalon, and not ectopic Wnt-1 activation. Accordingly,
the WEXPZ2-Fgf8 expression domain is also consistently broader
(B) than the wild-type Wnt-1 domain (C).
965
Fig. 7. Analysis of En-1 and Pax-6 expression in transgenic brains.
Isolated brains of (A,B) 12.5 d.p.c. (En-1) or (C,D) 13.5 d.p.c. (Pax6) were stained as whole mounts. En-1 expression is limited to the
caudal midbrain in both wild-type (A) and transgenic (B) brains.
Pax-6 is expressed in the dorsal part of p1 in wild-type brains (arrow
in C) and this expression domain is maintained in transgenic brains
(arrow in D).
contained within the domain of ectopic En-2 expression (Fig.
8B,C,E,F). Note that scattered clumps of En-2 and ELF-1
expressing cells are always observed in dorsal p1 (Fig.
8B,C,E,F). These may be mislocated mesencephalic cells which
crossed the disrupted mesencephalon-p1 boundary. However,
we can not rule out the possibility that FGF8 signalling induced
En-2 and ELF-1 expression in a subset of p1 cells.
Ectopic En expression activates ELF-1 in the rostral
Joyner, 1988; Davis et al., 1988), however En-2 expression was
mesencephalon
observed in a very broad dorsal domain all along the rostrocauIn the chick tectum ectopic high level expression of En-1 or Endal axis of the mesencephalon of transgenic embryos (Fig. 8A2 in the rostral mesencephalon leads to ectopic activation of ELFC). Interestingly, En-2 is not activated by ectopic expression of
1 (Logan et al., 1996). To test whether FGF8 activation of ELFFgf8 in the dorsal diencephalon (see below), but is activated in
1 in our transgenics is mediated by Engrailed genes we examined
the dorsal myelencephalon (white arrows in Fig. 8B,C). This
argues that the competence to
express En-2 in response to dorsal
FGF8 signalling has a rostral limit at
the
mesencephalon-diencephalon
boundary, and that FGF8 signalling
can regulate En-2 expression in the
mesencephalon and metencephalon.
To investigate the possibility that
Fgf8 is a more general regulator of
posterior mesencephalon development we examined the expression of
ELF-1, a ligand for the Mek-4 and
Sek-1 receptor tyrosine kinases
which is implicated in the regulation
of the retinal-tectal connection
(Cheng and Flanagan, 1994). In
transgenic brains, ELF-1 is ectopically activated in a broad stripe
along the dorsal mesencephalon and Fig. 8. En-2 and ELF-1 are ectopically expressed in the rostral mesencephalon of transgenics.
Isolated 12.5 d.p.c. brains were stained as whole mounts using either in situ hybridization to detect
in a transverse stripe at the mesen- En-2 or the RAP in situ technique to detect ELF-1. In wild-type brains mesencephalic expression of
cephalon-p1 boundary (Fig. 8D-F), En-2 (A) and ELF-1 (D) is limited to caudal regions. In contrast, En-2 is expressed throughout the
suggesting that all mesencephalic alar region of the entire mesencephalon in transgenic brains (B,C). Similarly, ELF-1 is ectopically
cells within a certain distance of expressed in a very broad dorsal domain along the entire rostrocaudal extent of the mesencephalon
FGF8 signal are induced to express and in a transverse stripe (limited to alar regions) in the rostral mesencephalon of transgenic brains
ELF-1. The domain of ectopic mes- (E,F). Note that clumps of En-2- and ELF-1- (black arrows in B,D) expressing cells are also present
encephalic ELF-1 expression is in dorsal p1.
966
S. M. K. Lee and others
Fig. 9. Ectopic expression of En-1 activates ectopic expression of
ELF-1. In a transgenic mouse line which expressed En-1 under the
control of the Wnt-1 enhancer En-1 is ectopically expressed in the
dorsal mesencephalon and p1 at 12.5 d.p.c. (A, and compare with
Fig. 7A). ELF-1 expression is ectopically activated along the dorsal
midline of the entire mesencephalon of transgenic brains from this
line but not in dorsal p1 (B). The arrow in B indicates the rostral
limit of high level dorsal ELF-1 expression in wild-type brains
(compare with Fig. 8D).
ELF-1 expression in transgenic mice expressing En-1 (En-1 and
En-2 are functionally interchangeable; Hanks et al., 1995) under
the control of the Wnt-1 enhancer. Transgenic embryos from this
line express En-1 in the typical Wnt-1 expression pattern
(Danielian and McMahon, 1996; Fig. 9A). In these transgenic
embryos ELF-1 expression was ectopically activated in a narrow
strip of cells along the dorsal midline of the anterior mesencephalon (Fig. 9B), most likely in only those cells that also
express En-1 (compare Fig. 9A with B). Interestingly, En-1
expression in the dorsal diencephalon was not sufficient to
activate ELF-1 expression in this region. Since Fgf8 expression
is normal in this line of mice (Danielian and McMahon, 1996,
data not shown) ectopic En expression is sufficient to activate
expression of ELF-1 independently of FGF8 signalling.
DISCUSSION
We have provided evidence that an interaction between mesencephalic and metencephalic cells is required for the localized
expression of Fgf8 at the isthmus. Continuous ectopic
expression of FGF8 along the dorsal mesencephalon results in
a posteriorization of much of the tectum. These results imply
that the localized production of FGF8 signal at the isthmus
serves to regulate many aspects of rostrocaudal polarity across
the tectum.
FGF8 signalling regulates rostrocaudal polarity in
the mesencephalon
The normal expression of En genes in a rostrocaudally increasing gradient across the mesencephalon (Davis and Joyner, 1988;
Davis et al., 1988; Gardner et al., 1988; Patel et al., 1989;
Gardner and Barald, 1992; Millet and Alvarado-Mallart, 1995)
and the ability of ectopic high level En expression to caudalize
the rostral tectum (Friedman and O’Leary, 1996; Itasaki and
Nakamura, 1996; Logan et al., 1996) implies that En expression
level acts to confer positional specification on cells along the
rostrocaudal axis of the tectum. In our experiments the ectopic
expression of Fgf8 along the dorsal midline of the mesencephalon leads to ectopic activation of En-2 expression in a
broad dorsal domain. This implies that the localized production
of FGF8 at the isthmus is normally involved in establishing the
graded pattern of En-2 expression across the mesencephalon
and thus in regulating the rostrocaudal specific expression of
En-2 target genes. Indeed, in normal development Fgf8
expression is initiated just prior to the onset of En-2 expression
(Davis and Joyner, 1988; Davis et al., 1988; Heikinheimo et al.,
1994; Crossley and Martin, 1995; Mahmood et al., 1995) consistent with FGF8 activating En-2 expression.
A putative En target, ELF-1, is indeed activated in the
rostral mesencephalon of Fgf8 transgenic embryos. The
domain of ectopic ELF-1 expression is always contained
within the domain of ectopic En-2 expression and the ectopic
expression of En-1 (En-1 and En-2 are functionally interchangeable) is sufficient to activate ectopic ELF-1 expression
in the mesencephalon. The simplest interpretation of these
results is that FGF8 signalling indirectly regulates ELF-1
expression via regulation of En-2. Together with the recently
published observation that ectopic expression of En-1 in the
rostral tectum of the chick is sufficient to activate ectopic
expression of ELF-1 (Logan et al., 1996), these results imply
that Engrailed proteins act additively to generate the graded
expression of ELF-1 in the tectum.
Normal En-2 expression in the metencephalon is the mirror
image of mesencephalic En-2 expression. Expression is highest
rostrally and forms a caudally decreasing gradient (Davis and
Joyner, 1988; Davis et al., 1988; Gardner et al., 1988; Patel et
al., 1989; Gardner and Barald, 1992; Millet and AlvaradoMallart, 1995). Our results indicate that expression of Fgf8 in
the dorsal myelencephalon is sufficient to activate expression
of En-2 in this brain region as well. This result is reminiscent
of the ability of isthmus (Fgf8 expressing tissue) grafted into
the myelencephalon to induce En-2 expression in adjacent host
tissue (Martinez et al., 1995). This implies that FGF8 signalling from the isthmus may act on both rostrally and caudally
adjacent cells of the mes/met region to establish a broad
domain of graded En-2 expression.
It should be pointed out that while our gain-of-function
experiments strongly implicate En-2 in the regulation of mesencephalic rostrocaudal polarity, En-2 mutant mice seem to
have a normal mesencephalon and display only minor defects
in cerebellar foliation (Millen et al., 1994; Millen et al., 1995).
A likely explanation is provided by the fact that En-1 and En2 are expressed in overlapping domains centered on the
isthmus such that the domain of En-2 expression is only
slightly broader than the domain of En-1 expression (Davis and
Joyner, 1988; Davis et al., 1988; McMahon et al., 1992). Additionally, En-1 expression initiates at 1-somite, prior to initial
expression of En-2 at 5-somites. Therefore, En-1 and En-2 are
likely to serve redundant functions, an idea supported by rescue
of the En-1 mutant phenotype by expression of En-2 under the
control of the En-1 promoter (Hanks et al., 1995). Thus it is
likely to be the additive concentration of En protein a tectal
cell expresses which specifies its rostrocaudal position.
Interestingly, we have never observed the ectopic expression
of En-1 in Fgf8 transgenic embryos, suggesting the existence
of a distinct mechanism which is responsible for establishing
the rostrocaudal domian of En-1 expression. Consistent with
this explanation, normal activation of En-1 (Davis and Joyner,
1988; McMahon et al., 1992) precedes the onset of Fgf8
expression in the brain (Mahmood et al., 1995).
FGF8 mediated regulation of neural proliferation and
neurogenisis
An important aspect of tectal polarity is the rostrocaudal
FGF8 signalling in the midbrain
gradient of cytodifferentiation (LaVail and Cowan, 1971;
Altman and Bayer, 1981a,b). Rostral regions differentiate first,
have a greater overall thickness, and manifest a more advanced
laminar structure than caudal regions, which retain mitotic
activity in neural precursors late in development. Our studies
show that ectopic expression of Fgf8 in transgenic mice leads
to a dramatic overproliferation in all areas of the dorsal mesencephalon and diencephalon expressing the transgene. The
continued expression of Fgf8 leads to a large expansion of the
ventricular zone and subsequently to the perdurance of a large
population of dividing ventricular zone cells. This phenotype
is complemented by an apparent reduction in the number of
differentiated cell types formed. These data are consistent with
a model in which FGF8 is a potent mitogen for neural precursor
cells and suggest that FGF8 signalling also has a role in regulating the rate of neural differentiation from dividing precursor
cells. In this model, as the mesencephalon increases in size, the
most rostral cells may come to lie beyond the effective range
of FGF8 signal produced by the isthmus and as a result these
cells cease dividing and differentiate, leading to the normal rostrocaudal cytoarchitectonic developmental gradient. It is interesting to note that widespread ectopic expression of En in the
tectum disrupts the gradient of differentiation as well (Logan
et al., 1996). Thus, the widespread activation of En-2 in our
transgenics may be responsible for a retardation of cytoararchitectonic differentiation. However, as the widespread
expression of En does not lead to overgrowth (Logan et al.,
1996) we conclude that the mitogenic role of FGF8 we observe
is independent of En.
Several lines of evidence indicate that FGFs may regulate
neural proliferation. For example bFGF (FGF2) has been
shown to act as a potent mitogen for precursor cells of many
CNS regions in vitro (Gensburger et al., 1987; Temple and
Qian, 1995). In these experiments bFGF acts primarily to
increase the number of precursor cells in the culture without
promoting the production of differentiated cell types. Interestingly, as FGF2 and FGF8 behave similarly in various assays,
such as their ability to induce ectopic limb development in the
flank of chick embryos (Cohn et al., 1995; Crossley et al.,
1996b; Vogel et al., 1996), these two factors may have similar
activities in the developing brain. We propose that the local
production of FGF8 at the isthmus may be responsible for the
known gradient of growth and differentiation along the rostrocaudal axis of the tectum. As development proceeds and the
tectal neuroepithelium expands along the rostrocaudal axis,
cells progressively become positioned further away from the
isthmic source of FGF8 and thus beyond its mitogenic
influence, leading to a cessation of mitosis and subsequent
differentiation. Interestingly, despite the fact that Fgf8 is
expressed in the myelencephalon and spinal cord of transgenic
embryos we observe no apparent growth phenoytpe in these
regions. This suggests a polarity in the responsiveness of cells.
Caudal to the isthmus neural cells (including those normally
expressing Fgf8) are not competent to respond mitogenically
to FGF8 signalling, although they do clearly activate En-2 transcription in response to ectopic FGF8.
Our experiments have identified two overlapping yet
distinct domains of competence for two unique responses to
FGF8 signalling. The ability of FGF8 to induce En-2
expression in our experiments extends at least as far caudally
as the myelencephalon and has a rostral limit at the mesen-
967
cephalon-p1 boundary. In contrast, the mitogenic response to
FGF8 signalling extends from p1 (the most rostral domain
tested) to a caudal limit at the isthmus. The mesencephalon is
competent for both responses.
Mes/met pattern formation and the midbrain
organizer
Our results have only addressed the role of isthmic-derived
FGF8 signalling in regulating mesencephalic polarity.
However, it is interesting to consider these experiments in the
light of other work that has implicated FGF8 in mesencephalon
induction.
The ability of FGF8 protein coated beads to induce ectopic
mesencephalon formation in chick embryos suggests the
involvement of FGF8 in normal mesencephalon induction
(Crossley et al., 1996a). If FGF8 signalling normally has a role
in mesencephalon induction it seems unlikely that the normal
source of FGF8 is from the brain, as expression is not initiated
in the mes/met region until 3-somites (Mahmood et al., 1995).
This is after the regional activation of several genes in the presumptive mes/met region including Pax-2, Wnt-1 and En-1
(Davis and Joyner, 1988; McMahon et al., 1992; Rowitch and
McMahon, 1995). Fgf8 is also expressed in the cardiogenic
mesoderm (Heikinheimo et al., 1994; Crossley and Martin,
1995; Mahmood et al., 1995) and it has been suggested that
vertical FGF8 signalling may induce mesencephalon formation
in the overlying neural plate (Crossley et al., 1996a). However,
Pax-2 expression is initiated at 7.75 d.p.c. in the presumptive
mes/met domain (Rowitch and McMahon, 1995), before Fgf8expressing cardiogenic mesodermal cells underlie the presumptive mesencephalon. This suggests that mesencephalon
development initiates prior to FGF8 signalling.
Our data argue strongly that following mesencephalon
induction, FGF8 is involved in a signalling loop which regulates
the rostrocaudal polarity of the mesencephalon. It is possible
that one of the targets of this pathway (e.g. En) may play an
earlier role in mesencephalic specification. Consequently, its
activation at earlier stages in responsive tissue may trigger mesencephalon development. Thus, the issue of whether FGF8
plays a direct role in mesencephalon induction remains open.
Analysis of Wnt-1 mutants indicates that the maintenance of
Fgf8 expression in the rostral metencephalon is dependent on
mesencephalic derived factors. Because mesencephalon development is initiated normally in these mutants we can not rule
out the possibility that mesencephalic derived factors might
also be required to initiate Fgf8 expression. Recent studies in
which Fgf8 expression is maintained in Wnt-1 mutants argue
that it is not Wnt-1 directly which maintains metencephalic
Fgf8 expression, but rather a secondary mesencephalic derived
signal(s) (Danielian and McMahon, 1996). Our results suggest
that FGF8 signalling in turn activates En-2 expression in the
caudal mesencephalon and rostral metencephalon. The fact that
Fgf8 expression initiates at 3-somites (Mahmood et al., 1995)
and En-2 expression shortly thereafter, at 5-somites (Davis and
Joyner, 1988; Davis et al., 1988; McMahon et al., 1992),
suggests that isthmic derived FGF8 signalling may actually be
involved in En-2 activation. Consensus sites for Pax-2,5, or 8
have been shown to be required for proper activation of an En2 minimal enhancer and are therefore implicated in the regulation of En-2 (Song et al., 1996). However, we have observed
continued expression of En-2 in the absence of ectopic Pax-2
968
S. M. K. Lee and others
or Pax-5 expression. Thus, the relative importance of Pax-2/5
and FGF8 signalling in regulating En-2 expression during
normal development remains unclear. Once established, the
En-2 expression gradient then acts to regulate midbrain
polarity through the regulation of genes such as ELF-1.
While our data strongly implicate FGF8 signalling in the
regulation of growth and polarity in the mesencephalon, lossof-function experiments are required to directly assess the
requirement for Fgf8 in regulating these processes. Additionally, it will be interesting to determine the range of FGF8 signalling from the isthmus and whether FGF8 acts directly to
regulate mesencephalic development or whether secondary,
FGF8 induced signals are involved.
We thank Bianca Klumpar for sectioning; Clive Dickson for the
Fgf8 cDNA; John Flanagan for Mek4-AP supernatant; Alex Joyner
and Peter Gruss for in situ probes; Andreas Kispert and Terry
Yamaguchi for helpful comments on an earlier version of the manuscript; and especially Elisa Marti for many valuable discussions.
These studies were supported by a grant from the NIH to A. P. M.,
and by a postdoctoral fellowship from the Human Frontiers Science
Program to P. S. D.
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(Accepted 23 December 1996)