1 Max-Planck Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg,
Germany
2 Max-Planck Institute for Developmental Biology, Spemannstraße 35, 72076
Tübingen, Germany
3 University of Freiburg, Department of Biology I, Hauptstraße 1, 79104
Freiburg, Germany
Author for correspondence (e-mail:
hammerschmid{at}immunbio.mpg.de)
Accepted 21 April 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Fgf3, Pituitary, Adenohypophysis, Neurohypophysis, Zebrafish, Lia, Cell survival, Cell specification, Apoptosis, Sonic Hedgehog
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The neurohypophysis is of neuroectodermal origin and derives from the
infundibulum, an evagination of the ventral diencephalon. By contrast, the
adenohypophysis is a derivative of the non-neuronal, placodal ectoderm. It is
initially located at the anterior neural ridge (ANR) and becomes part of the
oral roof ectoderm, from where it invaginates toward the presumptive
neurohypophysis/infundibulum, forming Rathke's pouch. This co-development of
neuro- and adenohypophysis is governed by various signaling processes between
infundibulum and adenohypophyseal placode (for reviews, see
Treier and Rosenfeld, 1996;
Burgess et al., 2002
;
Scully and Rosenfeld, 2002
).
For example, the infundibulum generates at least three members of the
fibroblast growth factor (Fgf) family, Fgf8, Fgf10 and Fgf18
(Norlin et al., 2000
;
Ohuchi et al., 2000
).
Gain-of-function studies with fgf8 transgenic mice or pituitary
explants treated with Fgf8 beads suggest that Fgf signaling promotes the
proliferation and differentiation of dorsal cell types of the adenohypophysis
(corticotropes and melanotropes), antagonized by Bmp2, which promotes ventral
cell fates (thyrotropes, gonadotropes, somatotropes and lactotropes)
(Ericson et al., 1998
;
Treier et al., 1998
).
Consistent results were obtained in loss-of-function studies, showing that
treatment of embryonic day (E) 10 pituitary transplants with the Fgf receptor
inhibitor SU5402 (Mohammadi et al.,
1997
) selectively blocks the proliferation and generation of
dorsal cell types (Norlin et al.,
2000
). Together, the data suggest a patterning and cell type
selective role for Fgf signaling. By contrast, knockout mice deficient in
Fgf10 (Ohuchi et al., 2000
) or
the Fgf receptor 2 isoform IIIB (Fgfr2IIIb)
(De Moerlooze et al., 2000
)
display a poorly formed Rathke's pouch, with numerous apoptotic cells
throughout the entire pouch. These defects are already apparent at E10,
suggesting an earlier and more general role for Fgf signaling during mouse
pituitary development, affecting pouch morphogenesis and the survival and
proliferation of all adenohypophyseal progenitor cells.
Here, we describe mutant analyses to uncover the role of Fgf3 during
adenohypophysis development in the zebrafish. Recent work has revealed both
striking similarities and differences in pituitary development between the
different vertebrate classes. Thus, we found that despite differences in the
morphogenesis and architecture of their pituitary glands, the role of Sonic
Hedgehog during induction, growth and patterning of the adenohypophyseal
anlage appears to be largely conserved between zebrafish and mouse
(Herzog et al., 2003;
Sbrogna et al., 2003
). To
isolate additional genes required for zebrafish pituitary development, we
conducted a screen for ENU-induced mutations affecting adenohypophyseal
production of Gh (Herzog et al.,
2004
). One gene identified in this screen is the
pituitary-specific transcription factor Pit1. pit1 mutant zebrafish
are semi-viable and show very similar phenotypic traits to those of their
human and mouse counterparts (Nica et al.,
2004
), strengthening the value of the zebrafish system as a
clinically relevant model organism. Here, we show that another identified
gene, limabsent (lia), encodes zebrafish Fgf3
(Kiefer et al., 1996
). Using
antisense-mediated inactivation, fgf3 has recently been proposed to
be necessary for craniofacial development
(David et al., 2002
;
Walshe and Mason, 2003b
) and,
in partial redundancy with fgf8, telencephalic patterning
(Shanmugalingam et al., 2000
;
Fürthauer et al., 2001
;
Shinya et al., 2001
;
Walshe and Mason, 2003a
),
hindbrain patterning (Liu et al.,
2003a
; Maves et al.,
2002
; Walshe et al.,
2002
) and otic placode induction
(Phillips et al., 2001
;
Leger and Brand, 2002
;
Maroon et al., 2002
;
Liu et al., 2003a
). Our mutant
analyses confirm most, but not all, of the proposed Fgf3-unique roles.
During pituitary development, Fgf3 is primarily required to promote early specification steps and the subsequent survival of adenohypophyseal cells. However, we find that morphogenesis and growth of the pituitary anlage do not depend upon Fgf3. In addition, we show that the source of Fgf3 is cells of the ventral diencephalon, presumably encompassing the presumptive neurohypophysis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of constructs, mRNA synthesis and microinjection
For expression constructs, full-length wild-type and mutant fgf3
cDNAs were amplified via RT-PCR with primers containing EcoRI and
XbaI restriction sites, and cloned into pCS2+
(Rupp et al., 1994). Capped
RNA was prepared with the Message Machine kit (Ambion). shh RNA was
prepared, and RNA was injected as described
(Herzog et al., 2003
).
In-situ hybridization, Alcian Blue stainings, Acridine Orange stainings
Whole-mount in-situ hybridizations were carried out as previously described
(Hammerschmidt et al., 1996;
Herzog et al., 2003
). For
fgf3 in-situ probe synthesis, plasmid pCRII-fgf3 was
linearized with KpnI and transcribed with T7 RNA polymerase. In
addition, riboprobes of the following cDNAs were used: dlx2, dlx3
(Akimenko et al., 1994
),
eya1 (Sahly et al.,
1999
), emx1 (Morita
et al., 1995
), gh, pomc, prl
(Herzog et al., 2003
),
hgg (Thisse et al.,
1994
), krox20 (Oxtoby
and Jowett, 1993
), lim3
(Glasgow et al., 1997
),
nkx2.1a (Rohr and Concha,
2000
), pit1 (Nica et
al., 2004
), shh
(Krauss et al., 1993
),
spry4 (Fürthauer et al.,
2001
).
To visualize craniofacial cartilage, 120 hours postfertilization (hpf)
embryos were stained with Alcian Blue (Sigma) as described
(Schilling et al., 1996).
For detection of apoptotic cells, dechorionated live embryos were incubated for 10 minutes in 5 µg/ml Acridine Orange (Sigma) in embryo medium (E3), followed by fluorescent microscopy (Zeiss axiophot) and photography.
Single cell tracing experiments
To study the fate of pituitary precursor cells, single cells in the
prospective pituitary forming region were injected in tailbud-stage embryos
with 3% rhodamine dextran (3 kD) in 0.2 M KCl, using an AM-Systems 1600
Neuroprobe amplifier, and the underlying mesodermal polster as a morphological
landmark (Varga et al., 1999)
(S. Dutta and Z.M.V., unpublished). Fates of labeled daughter cells were
analyzed between 24 and 30 hpf, using conventional fluorescence microscopy, or
a Zeiss LSM 510 confocal microscope. Individual embryos were genotyped after
photography.
SU5402 treatment
Embryos were incubated at 28.5°C in 12 or 20 µM SU5402 containing
E3, prepared from a 3 mM SU5402 (Calbiochem, S72630) stock solution in DMSO.
Control embryos were incubated in E3 medium with the corresponding amount of
DMSO. After treatment, embryos were washed five times with E3/DMSO and
transferred to fresh E3.
Cell transplantations
For transplanting wild-type cells into offspring of heterozygous mutants,
wild-type embryos were injected with a 1% biotin-dextrane, 0.5% fluorescein
dextrane solution. Homotopic transplantations of presumptive telencephalic or
diencephalic cells were carried out at the shield stage. At 32 hpf, chimeras
were fixed and analyzed by in-situ hybridization, followed by anti-biotin
stainings with the Vectastain Elite ABC kit (Vector Laboratories) to stain
transplanted cells. The tails of chimeras (containing only recipient-derived
cells) were genotyped, as described above.
Bead implantations
Heparin-coated acrylic beads (Sigma, H-5263) were washed twice in PBS, and
incubated with 0.5 µg/µl recombinant human FGF3 (R&D Systems,
1206-FG) or 0.5 µg/µl BSA overnight at 4°C. At 19 hpf, offspring
from a cross of two t24149/+ carriers were dechorionated, embedded in 1% low
melting agarose on agarose-coated petri dishes, and covered with Ringer's
solution. Using a fine tungsten needle and forceps, a bead was implanted
behind the eye and pushed to the front of the head. After implantation,
embryos were left on the plates for recovery for 2 hours at 28.5°C, then
carefully taken out of the agarose and incubated for another 5 hours until
fixation at 26 hpf.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR amplification and sequencing of fgf3 cDNA from the four
different lia alleles revealed three missense mutations and one
nonsense mutation, leading to exchanges or deletions of highly conserved amino
acid residues of Fgf3 protein (Fig.
1A). In the fgf3t26212 allele, a
T-to-A transition leads to the replacement of an isoleucine residue at amino
acid position 76 of the protein by an asparagine (I76N). In
fgf3t24149, a G-to-A transition causes the exchange of
glutamate 138 to lysine (E138K). Both mutations generate RFLPs, which were
used to confirm the linkage between the lia mutations and the
fgf3 gene (no recombination in 436 meioses). In
fgf3t21142, tyrosine 148 is exchanged to a cysteine
(T148C), due to an A-to-G transition, while in the
fgf3t24152 allele, a G-to-A transition introduces a
premature stop codon after amino acid residue 177. This results in a deletion
of the last 79 amino acids of the protein, including 40 amino acids of the
central core of 140 amino acids that is highly conserved among the different
Fgf family members (Powers et al.,
2000).
|
fgf3 mutants display defects during craniofacial and otic vesicle development
Several recent publications describe fgf3 and combinatorial
fgf3 and fgf8 loss-of-function studies, using antisense
morpholino oligonucleotide technology (see Introduction). The combinatorial
activity of fgf3 and fgf8 was reported to be required for
otic vesicle formation, telencephalon and hindbrain patterning, and
craniofacial development. Most of these processes were also affected upon
single loss of fgf3; however, the effects were more moderate or more
restricted, pointing to partial redundancy of Fgf3 and Fgf8.
During craniofacial development, Fgf3 from the pharyngeal endoderm was
reported to be required for the specification and survival of chondrogenic
neural crest cells of the gill arches (pharyngeal arches 3-7)
(David et al., 2002), while the
crest cells forming mandibular and hyoid (pharyngeal arches 1 and 2) are under
the redundant control of Fgf3 and Fgf8
(Walshe and Mason, 2003b
).
Consistent with this notion, Alcian Blue stainings of fgf3 mutants at
120 hpf revealed an almost complete loss of the cartilage of the gill arches,
while the cartilage of mandibular and hyoid was present
(Fig. 2G,H). These defects are
anticipated by a progressive reduction in the number of dlx2-positive
chondrogenic neural crest cells of arches 3-7, starting around 26 hpf, while
the two anterior dlx2 domains, forming cartilage of arches 1 and 2,
appear normal (Fig. 2C,D). In
addition, the posterior dlx2 domain fails to subdivide into the
streams corresponding to the different gill arches
(Fig. 2C,D). Similarly, the
pharyngeal endoderm remains unsegmented, as reflected by the fgf3
expression pattern (Fig.
2E,F).
|
fgf3 expression during adenohypophysis development
By contrast to the processes mentioned above, no role of Fgf3 during
adenohypophysis development had been described so far. We carried out double
in-situ hybridizations to investigate fgf3 expression relative to the
developing adenohypophysis. At the end of gastrulation (10 hpf), fgf3
is expressed in the telencephalon, adjacent to the placodal cells of the
anterior neural ridge (ANR) that are marked by dlx3 expression
(Fig. 3A). The placodal
ectoderm itself is devoid of fgf3 transcripts. Similarly, the
progressing anterior dorsal mesoderm, called polster and marked by the
expression of hgg, lacks fgf3 expression
(Fig. 3B). Median ventral
regions of the neuroectoderm, marked by the expression of shh
(Fig. 3C) (cf.
Varga et al., 1999), might
show some weak and diffuse fgf3 staining
(Fig. 3B,E). However, strong
and distinct diencephalic fgf3 expression can first be detected from
the 18-somite stage onward (18 hpf), after fgf3 expression in the
telencephalon has ceased (Fig.
3F). Within the diencephalon, fgf3 expression is
restricted to the ventralmost cell layers, as revealed with double-stainings
using the hypothalamic marker nkx2.1a
(Fig. 3I,L). This tuberal
region of the posterior-ventral hypothalamus is thought to correspond to the
presumptive infundibulum of higher vertebrates, which gives rise to the
neurohypophysis (Fürthauer et al.,
2001
; Mathieu et al.,
2002
). It is in close contact with the adenohypophyseal cells,
which at 26 hpf are located in a horseshoe-like pattern around the anterior
and lateral borders of the fgf3 expression domain
(Fig. 3J,M). During further
development, the lateral-posterior cells of the adenohypophyseal anlage
converge to the midline (Nica et al.,
2004
) and become located underneath the fgf3-expressing
cells of the presumptive infundibulum (Fig.
3N,O). By contrast, the anteriormost cells of the adenohypophysis,
as characterized by the expression of prolactin, are in some distance
from the fgf3-positive cells (Fig.
3K).
|
Fgf3 is required for the early steps of adenohypophyseal specification
As a first step to characterize the adenohypophyseal defects of
fgf3 mutants, we carried out in-situ hybridizations at various stages
of development. Placodal ectoderm starts to be specified toward the end of
gastrulation, indicated by the expression of eya1, dlx3 or
pitx3. All of them are expressed throughout the entire placodal
field, while marker genes specific for the different placodes along the
anterior-posterior axis of the embryo (adenohypophysis, olfactory, lense, otic
etc.) only come up later. At tailbud stage (10 hpf), fgf3 mutants
display normal expression of eya1
(Fig. 4A,B), dlx3 and
pitx3 (data not shown), indicating that Fgf3 is not required for the
earliest steps of general placodal specification. Similar unaltered expression
patterns were found in fgf3 and fgf8 (ace)
(Reifers et al., 1998) double
mutants, and in embryos treated with the Fgf receptor inhibitor SU5402 from
mid-through late-gastrula stages
(Mohammadi et al., 1997
) (data
not shown), indicating that early placodal development is independent of Fgf
signaling in general.
|
The infundibular region itself appears to be less affected by the
fgf3 mutations. Infundibular cells of mutant embryos lack expression
of the Fgf target gene and autocrine feedback antagonist sprouty4
(spry4) (Fig. 4I,J)
(Fürthauer et al., 2001).
However, fgf3 mutants display normal infundibular expression of
fgf3 at 36 hpf (Fig.
2E,F) and later (48 hpf, 72 hpf, data not shown), indicating that
infundibular cells are maintained, and that in contrast to Fgf8
(Reifers et al., 1998
;
Heisenberg et al., 1999
), Fgf3
is not required for the maintenance of its own expression.
Like the infundibulum, which is part of the tuberal, posterior-ventral
hypothalamus, other regions of the ventral diencephalon appear to develop
independently of Fgf3, too. This is indicated by the unaltered expression of
shh (Fig. 4G,H), a
marker for the anterior-dorsal hypothalamus
(Mathieu et al., 2002), and by
the normal expression of pomc in endorphin-synthesizing hypothalamic
neurons of mutant embryos (Fig.
4K,L).
Adenohypophyseal development is driven by Fgf3 from the diencephalon
As described above, adenohypophyseal cells are exposed to Fgf3 signals from
different sources (telencephalon, diencephalon) and at different stages of
development (end of gastrulation, midsegmentation)
(Fig. 3). To determine the
source of Fgf3 required for adenohypophyseal development, we generated mosaic
embryos, transplanting presumptive telencephalic or diencephalic cells from
early gastrula wild-type donor embryos into the same regions of fgf3
mutant recipients (Fig. 5B). We
found that wild-type cells in the ventral region of the diencephalon rescued
lim3 expression in adjacent adenohypophyseal cells of fgf3
mutants (Fig. 5E,F;
n=9). By contrast, even largest clones of wild-type cells in the
telencephalon of fgf3 mutants failed to rescue lim3
expression (Fig. 5G,H;
n=8). This indicates that it is Fgf3 from the ventral diencephalons
that is required in a non-cell autonomous fashion to instruct adenohypophyseal
specification in the underlying placodal ectoderm.
|
|
In sum, these experiments indicate that adenohypophysis development depends on Fgf3 during stages when it is normally expressed in the presumptive infundibular region.
Shh cannot rescue adenohypophyseal development but can induce ectopic pituitary cells in other placodal regions of fgf3 mutants
In addition to Fgfs, the diencephalon is known to be the source of other
crucial signals driving adenohypophysis development. One of the identified
diencephalic signals governing pituitary induction, patterning and growth in
mouse and zebrafish is Sonic Hedgehog (Shh) from the anterior-dorsal
hypothalamus (Treier et al.,
2001; Herzog et al.,
2003
; Sbrogna et al.,
2003
) (see Fig.
4G). In light of results obtained for other developmental
processes such as limb/pectoral fin development, proposing a role of Fgf
signaling to activate shh expression
(Fischer et al., 2003
), we
wanted to investigate whether neurohypophyseal Fgf3 might regulate
adenohypophyseal development via Shh. For this purpose, fgf3 mutant
embryos were injected with shh mRNA. However, shh-injected
fgf3 mutants still lacked endogenous pomc and prl
expression in the adenohypophyseal domain
(Fig. 7B), indicating that Shh
cannot compensate for the loss of Fgf3 and suggesting that Fgf3 acts in
parallel to, rather than upstream of, Shh.
|
Unspecified adenohypophyseal cells of fgf3 mutants die by subsequent apoptosis
In Hedgehog signaling pathway mutants, the adenohypophysis is trans-fated
to lens material (Kondoh et al.,
2000; Varga et al.,
2001
). Several experiments were carried out to investigate the
fate of non-specifying adenohypophyseal cells in fgf3 mutant embryos.
At 24 hpf, the ANR region of mutant embryos appeared morphologically normal,
despite the lack of lim3 expression
(Fig. 4E,F). Using Nomarski
optics, the adenohypophysis of wild-type embryos became visible as a distinct
organ at approximately 25 hpf (Fig.
8A). An organ of similar size and shape, and at the same position,
was also present in fgf3 mutants at 25 hpf
(Fig. 8B). However, by 28 hpf
and 29 hpf, the size of the adenohypophysis in mutants had become
progressively smaller (Fig.
8D,H), while in wild-type siblings, it was similar to the size at
25 hpf (Fig. 8C,G).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early and late roles of Fgf signaling during adenohypophyseal development?
The mouse infundibulum expresses at least three different Fgfs,
Fgf10 of the Fgfr2IIIb-binding subgroup, and Fgf8 and
Fgf18 of the IIIc group (Maruoka
et al., 1998). The requirement of Fgf8 signaling for
adenohypophyseal development has not been addressed as yet, due to early
embryonic lethality of Fgf8 mutant embryos
(Meyers et al., 1998
;
Sun et al., 1999
). However,
Fgf10 mutants are viable until birth, and their phenotype suggests an
essential role of Fgf10 affecting all adenohypophyseal cell types before
definitive pouch formation (E10) (Ohuchi
et al., 2000
). Furthermore, results obtained upon treatment of E10
pouch explants with the Fgf receptor inhibitor SU5402 suggest an additional
later role of Fgf signaling to pattern the definitive pouch along its
dorsoventral axis (Norlin et al.,
2000
). In zebrafish, our expression pattern analyses even suggest
a third, much earlier, phase of Fgf signaling, which might occur during late
gastrulation stages, when adenohypophyseal progenitor cells are located at the
ANR and in close proximity to fgf3- and fgf8-expressing
cells of the presumptive telencephalon
(Fig. 3). Our rescue
experiments via transplantation of wild-type cells or implantation of Fgf3
beads, as well as our experiments with temporally controlled blockage of Fgf
signaling by SU5402, indicate that the later Fgf3 from the ventral
diencephalon is absolutely necessary for adenohypophysis formation. Whether
the earlier Fgf3 from the telencephalon is required, too, is less clear. The
rescue of lim3 expression by diencephalic wild-type cells or late
Fgf3 bead implants is only partial, never yielding lim3 expression in
the entire adenohypophyseal domain. Similarly, early SU5402 treatment of
embryos during gastrulation and early segmentation stages also leads to a
strong reduction of adenohypophyseal marker gene expression, which, however,
could be a secondary consequence of the effect of SU5402 on hypothalamus
development. In sum, we can neither prove, nor rule out, an involvement of
early telencephalic Fgf3 signaling on adenohypophysis development.
In addition to this general role affecting all pituitary cell types, Fgf3
signaling might also have a cell type-discriminating function to pattern the
zebrafish adenohypophysis, similar to the proposed morphogenetic role of Fgf8
in mouse. As in mouse, the different cell types of the zebrafish
adenohypophysis display a differential distribution along the dorsoventral
axis of the anlage (Liu et al., 2003b;
Nica et al., 2004), with
dorsal cells being closer to the infundibulum than ventral cells. In addition,
the zebrafish adenohypophysis is patterned along its antero-posterior axis
(Herzog et al., 2003
), with
posterior cell types being closer to the infundibular Fgf3 domain than
anterior cells. In conclusion, different adenohypophyseal cell types might
indeed be exposed to different doses of Fgf3 signals. It is currently unclear
whether this has any effects on the patterning of the adenohypophysis. This
question cannot be addressed via regular analyses of fgf3 mutants,
due to the earlier requirement of Fgf3 signaling for all adenohypophyseal cell
types. Approaches allowing temporally controlled blockage of Fgf signaling
have to be entertained, such as late applications of the inhibitor SU5402.
The subsequent effects of Fgf3 signaling on cell specification and cell survival
During mouse adenohypophysis development, Fgf signaling is supposed to have
diverse effects on target cells, regulating organ morphogenesis and
patterning, cell proliferation, cell survival and cell specification (see
Introduction). Here, we investigated how these different effects might be
correlated, studying the time course with which the different phenotypic
aspects arise during adenohypophyseal development of fgf3 mutant
zebrafish. First defects are concerned with early adenohypophysis-specific
specification steps. While the general specification of the placodal ectoderm
at the interphase of neural and non-neural ectoderm (10-12 hpf) (for a review,
see Baker and Bronner-Fraser,
2001) occurs normally in mutant embryos, later (18.5 hpf) they
fail to initiate expression of the adenohypophyseal-specific marker genes
lim3 and pit1 in the ANR, the anteriormost region of the
placodal domain. This, however, neither affects the proliferation of
adenohypophyseal progenitor cells nor further pituitary morphogenesis. At 25
hpf, fgf3 mutants display a pituitary gland of relatively normal size
and shape. Also, we failed to detect any differences in BrdU incorporation
studies (W.H. and M.H., unpublished data). However, we found dramatic
apoptosis of adenohypophyseal cells after organ formation, starting at 25 hpf,
and leading to a complete loss of the organ within 5 hours.
In conclusion, it appears that Fgf3 signaling primarily induces the
activation of genes involved in early steps of adenohypophyseal specification,
subsequently affecting cell survival. Pituitary morphogenesis and growth is
affected even later, most probably as a consequence of the death of
adenohypophyseal cells. However, it still remains largely unclear how
Fgf3-induced cell specification and cell survival processes are correlated.
pit1 and lim3 might be direct target genes of Fgf3
signaling, as their expression appears to be initiated shortly after the onset
of fgf3 expression in the presumptive neurohypophysis (18 hpf versus
18.5 hpf). This would be consistent with the proposed role of Fgf8 in
activating Lhx3 expression in mouse
(Takuma et al., 1998;
Ericson et al., 1998
). In
mouse, Pit-1 and Lhx3 have been shown to be directly involved in the
transcriptional activation of hormone-encoding genes
(Anderson and Rosenfeld, 2001
;
Lamolet et al., 2001
;
Liu et al., 2001
), explaining
why zebrafish fgf3 mutants fail to initiate prl and
pomc expression at 24 hpf. It remains unclear whether the subsequent
death of adenohypophyseal cells between 25 hpf and 30 hpf is a default
consequence of their failed specification/differentiation, or whether Fgf3
activates the transcription of additional genes, particularly regulating cell
survival.
Differential effects of Fgf and Shh signaling
In midsegmentation zebrafish embryos, such survival factors appear also to
be present in other tissues, where they are generated in response to other or
additional Fgf signals. This is indicated by our observation that Shh can
induce the formation of viable lactotropes and corticotropes in ectopic
placodal locations in an Fgf3-independent, but SU5402-sensitive, manner.
According to their position, the intermediate domains of ectopic
prl-positive cells in shh-injected embryos most probably
represent trans-fated lense precursor cells, which might survive due to the
redundant function of Fgf3 and Fgf8 from the optic stalk. In
shh-injected embryos, this optic stalk is laterally enlarged
(Macdonald et al., 1995), and
therefore close to the trans-fated placodal lense precursor cells.
The shh overexpression studies further show that Fgf3 does not simply act via an activation of shh, as Shh is not capable of rescuing pituitary cell types in the adenohypophyseal region itself. Rather, Fgf3 and Shh appear to act in parallel and to have rather distinct effects. Thus, ectopic Shh can induce the expression of adenohypophyseal genes in ectopic locations, whereas Fgf3 cannot (Fig. 7; W.H. and M.H., unpublished observations). Furthermore, Shh has mitogenic activity, indicated by the reduced growth of the adenohypophysis in shh mutants without any sign of increased cell death, whereas the adenohypophysis of fgf3 mutants shrinks due to loss of cells by apoptosis.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: University of Sheffield, Department of Biomedical Science,
Firth Court, Western Bank, Sheffield S10 2TN, UK
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akimenko, M.-A., Ekker, M., Wegner, J., Lin, W. and Westerfield, M. (1994). Combinatorial expression of three zebrafish genes related to distalless: part of a homeobox gene code for the head. J. Neurosci. 16,3475 -3486.
Anderson, B. and Rosenfeld, M. G. (2001). POU
domain factors in the neuroendocrine system: lessons from developmental
biology provide insights into human disease. Endocrinol.
Rev. 22,2
-35.
Baker, C. V. H. and Bronner-Fraser, M. (2001). Vertebrate cranial placodes I: embryonic induction. Dev. Biol. 232,1 -61.[CrossRef][Medline]
Burgess, R., Lunyak, V. and Rosenfeld, M. G. (2002). Signaling and transcriptional control of pituitary development. Curr. Opin. Genet. Dev. 12,534 -539.[CrossRef][Medline]
David, N. B., Saint-Etienne, L., Tsang, M., Schilling, T. F. and
Rosa, F. M. (2002). Requirement for endoderm and FGF3 in
ventral skeleton formation. Development
129,4457
-4468.
De Moerlooze, L., Spencer-Dene, B., Revest, J.-M., Hajihosseini,
M., Rosewell, I. and Dickson, C. (2000). An important role
for the IIIb isoform of fibroblast growth factor 2 (FGFR2) in
mesenchymal-epithelial signaling during mouse organogenesis.
Development 127,483
-492.
Ericson, J., Norlin, S., Jessell, T. M. and Edlund, T.
(1998). Integrated FGF and BMP signaling controls the progression
of progenitor cell differentiation and the emergence of pattern in the
embryonic anterior pituitary. Development
125,1005
-1015.
Fischer, S., Draper, B. W. and Neumann, C. J.
(2003). The zebrafish fgf24 mutant identifies an
additional level of Fgf signaling involved in vertebrate forelimb initiation.
Development 130,3515
-3524.
Fürthauer, 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]
Fürthauer, M., Thisse, C. and Thisse, B.
(1997). A role for FGF-8 in the dorsoventral patterning of the
zebrafish gastrula. Development
124,4253
-4264.
Geisler, R. (2002). Mapping and cloning. In Zebrafish, Practical approach, vol.261 (ed. C. Nüsslein-Volhard and R. Dahm), pp.175 -212. Oxford, UK: Oxford University Press.
Glasgow, E., Karavanov, A. A. and Dawid, I. B. (1997). Neuronal and neuroendocrine expression of lim3, a LIM class homeobox gene, is altered in mutant zebrafish with axial signaling defects. Dev. Biol. 192,405 -419.[CrossRef][Medline]
Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., van
Eeden, F. J. M., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P.,
Heisenberg, C.-P. et al. (1996). dino and
mercedes, two genes regulating dorsal development in the zebrafish
embryo. Development 123,95
-102.
Heisenberg, C.-P., Brennan, C. and Wilson, S. W.
(1999). Zebrafish aussicht mutant embryos exhibit
widespread overexpression of ace (fgf8) and coincident
defects in CNS development. Development
126,2129
-2140.
Herzog, W., Sonntag, C., Walderich, B., Odenthal, J., Maischein,
H.-M. and Hammerschmidt, M. (2004). Genetic analysis of
adenohypophysis formation in zebrafish. Mol.
Endocrinol. 18,1185
-1195.
Herzog, W., Zeng, X., Lele, Z., Sonntag, C., Ting, J.-W., Chang, C.-Y. and Hammerschmidt, M. (2003). Adenohypophysis formation in the zebrafish and its dependence on Sonic Hedgehog. Dev. Biol. 254,36 -49.[CrossRef][Medline]
Kiefer, P., Strähle, U. and Dickson, C. (1996). The zebrafish Fgf-3 gene: cDNA sequence, transcript structure and genomic organization. Gene 168,211 -215.[CrossRef][Medline]
Kondoh, H., Uchikawa, M., Yoda, H., Takeda, H., Furutani-Seiki, M. and Karlstrom, R. O. (2000). Zebrafish mutations in Gli-mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage. Mech. Dev. 96,165 -174.[CrossRef][Medline]
Koshida, S., Shinya, M., Nikaido, M., Ueno, N., Schulte-Merker, S., Kuroiwa, A. and Takeda, H. (2002). Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish. Dev. Biol. 244,9 -20.[CrossRef][Medline]
Krauss, S., Concordet, J.-P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[Medline]
Lamolet, B., Pulichino, A.-M., Lamonerie, T., Gauthier, Y., Brue, T., Enjalbert, A. and Drouin, J. (2001). A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104,849 -859.[CrossRef][Medline]
Leger, S. and Brand, M. (2002). Fgf8 and Fgf3 are required for zebrafish ear placode induction, maintenance and inner ear patterning. Mech. Dev. 119,91 -108.[CrossRef][Medline]
Liu, D., Hsin, C., Maves, L., Yan, Y.-L., Morcos, P. A.,
Postlethwait, J. and Westerfield, M. (2003a). Fgf3 and Fgf8
dependent and independent transcription factors are required for otic placode
specification. Development
130,2213
-2224.
Liu, J., Lin, C., Gleiberman, A., Ohgi, K. A., Herman, M. A.,
Huang, H., Tsai, M. J. and Rosenfeld, M. G. (2001). Tbx19, a
tissue-selective regulator of POMC gene expression. Proc. Natl.
Acad. Sci. USA 98,8674
-8679.
Macdonald, R., Barth, K. A., Xu, Q., Holder, N., Mikkola, I. and
Wilson, S. W. (1995). Midline signalling is required for Pax
gene regulation and patterning of the eyes.
Development 121,3267
-3278.
Mansour, S. L., Goddard, J. M. and Capecchi, M. R.
(1993). Mice homozygous for a targeted disruption of the
proto-oncogene int-2 have developmental defects in the tail and inner
ear. Development 117,13
-28.
Maroon, H., Walshe, J., Mahmood, R., Kiefer, P., Dickson, C. and
Mason, I. (2002). Fgf3 and Fgf8 are required together for
formation of the otic placode and vesicle. Development
129,2099
-2108.
Maruoka, Y., Ohbayashi, N., Hoshikawa, M., Itoh, N., Hogan, B. L. M. and Furuta, Y. (1998). Comparison of the expression of three highly related genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74,175 -177.[CrossRef][Medline]
Mathieu, J., Barth, A., Rosa, F. M., Wilson, S. W. and
Peyriéras, N. (2002). Distinct and cooperative roles
of Nodal and Hedgehog signals during hypothalamic development.
Development 129,3055
-3065.
Maves, L., Jackman, W. and Kimmel, C. B. (2002). Fgf3 and Fgf8 mediate rhombomere 4 signaling activity in the zebrafish hindbrain. Development 129,3825 -3837.[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]
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B.
D., Tarpley, J. E., DeRose, M. and Simonet, W. S. (1998).
Fgf-10 is required for both limb and lung development and exhibits striking
functional similarity to Drosophila branchless. Genes
Dev. 12,3156
-3161.
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.
Morita, T., Nitta, H., Kiyama, Y., Mori, H. and Mishina, M. (1995). Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain. Neurosci. Lett. 198,131 -134.[CrossRef][Medline]
Ng, J. K., Kawakami, Y., Büscher, D., Raya, Á., Itoh, T., Koth, C. M., Esteban, C. R., Rodríguez-León, J., Garrity, D. M., Fishman, M. C. et al. (2002). The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129,5161 -5170.[Medline]
Nica, G., Herzog, W., Sonntag, C. and Hammerschmidt, M.
(2004). Zebrafish pit1 mutants lack three pituitary cell
types and develop severe dwarfism. Mol. Endocrinol.
18,1196
-1209.
Norlin, S., Nordström, U. and Edlund, T. (2000). Fibroblast growth factor signaling in required for the proliferation and patterning of progenitor cells in the developing anterior pituitary. Mech. Dev. 96,175 -182.[CrossRef][Medline]
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). Fgf10 acts as a major ligand for Fgf receptor 2 IIIb in mouse multi-organ development. Biochem. Biophys. Res. Com. 277,643 -649.[CrossRef][Medline]
Ornitz, D. M., Xu, J. S., Colvin, J. S., McEwen, D. G.,
MacArthur, C. A., Coulier, F., Gao, G. X. and Goldfarb, M.
(1996). Receptor specificity of the fibroblast growth-factor
family. J. Biol. Chem.
271,15292
-15297.
Oxtoby, E. and Jowett, T. (1993). Cloning of the zebrafish Krox-20 (Krx-20) and its expression during hindbrain development. Nucleic Acids Res. 21,1087 -1095.[Abstract]
Phillips, B. T., Bolding, K. and Riley, B. B. (2001). Zebrafish fgf3 and fgf8 encode redundant functions required for otic placode induction. Dev. Biol. 235,351 -365.[CrossRef][Medline]
Powers, C. J., McLeskey, S. W. and Wellstein, A.
(2000). Fibroblast growth factors, their receptors and signaling.
Endocr. Relat. Cancer 7,165
-197.
Reifers, F., Böhli, H., Walsh, E. C., Crossley, P. H.,
Stanier, D. Y. R. and Brand, M. (1998). Fgf8 is
mutated in zebrafish acerebellar (ace) mutants and is required for maintenance
of midbrain-hindbrain boundary development and somitogenesis.
Development 125,2381
-2395.
Rohr, K. B. and Concha, M. L. (2000). Expression of nk2.1a during early development of the thyroid gland in zebrafish. Mech. Dev. 95,267 -270.[CrossRef][Medline]
Rupp, R. A. W., Snider, L. and Weintraub, H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8,1311 -1323.[Abstract]
Sahly, I., Andermann, P. and Petit, C. (1999). The zebrafish eya1 gene and its expression during embryogenesis. Dev. Genes Evol. 209,399 -410.[CrossRef][Medline]
Sbrogna, J. L., Barresi, M. J. F. and Karlstrom, R. O. (2003). Multiple roles for Hedgehog signaling in zebrafish pituitary development. Dev. Biol. 254, 19-35.[CrossRef][Medline]
Schilling, T. F., Piotrowski, T., Grandel, H., Brand, M.,
Heisenberg, C. P., Jiang, Y. J., Beuchle, D., Hammerschmidt, M., Kane, D. A.,
Mullins, M. C. et al. (1996). Jaw and branchial arch mutants
in zebrafish I: branchial arches. Development
123,329
-344.
Scully, K. M. and Rosenfeld, M. G. (2002).
Pituitary development: regulatory codes in mammalian organogenesis.
Science 295,2231
-2235.
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itho, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21,138 -141.[CrossRef][Medline]
Shanmugalingam, S., Houart, C., Picker, A., Reifers, F.,
Macdonald, R., Barth, A., Griffin, K., Brand, M. and Wilson, S. W.
(2000). Ace/Fgf8 is required for forebrain commissure formation
and patterning of the telencephalon. Development
127,2549
-2561.
Shinya, M., Koshida, S., Sawada, A., Kuroiwa, A. and Takeda,
H. (2001). Fgf signaling through MAPK cascade is required for
development of the subpallial telencephalon in zebrafish embryos.
Development 128,4153
-4164.
Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R.
(1999). Targeted disruption of Fgf8 causes failure of cell
migration in the gastrulating mouse embryo. Genes Dev.
13,1834
-1846.
Takuma, N., Sheng, H. Z., Furuta, Y., Ward, J. M., Sharma, K.,
Hogan, B. L. M., Pfaff, S. L., Westphal, H., Kimura, S. and Mahon, K. A.
(1998). Formation of Rathke's pouch requires dual induction from
the diencephalon. Development
125,4835
-4840.
Thisse, C., Thisse, B., Halpern, M. E. and Postlethwait, J. H. (1994). goosecoid expression in neuroectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas. Dev. Biol. 164,420 -429.[CrossRef][Medline]
Treier, M. and Rosenfeld, M. G. (1996). The hypothalamic-pituitary axis: codevelopment of two organs. Curr. Opin. Cell Biol. 8,833 -843.[CrossRef][Medline]
Treier, M., Gleiberman, A. S., O'Connell, S. M., Szeto, D. P.,
McMahon, J. A., McMahon, A. P. and Rosenfeld, M. G. (1998).
Multistep signaling requirements for pituitary organogenesis in vivo.
Genes Dev. 12,1691
-1704.
Treier, M., O'Connell, S., Glieberman, A., Price, J., Szeto, D.
P., Burgess, R., Chuang, P. T., McMahon, A. P. and Rosenfeld, M. G.
(2001). Hedgehog signaling is required for pituitary gland
development. Development
128,377
-386.
Varga, Z. M., Wegner, J. and Westerfield, M.
(1999). Anterior movement of ventral diencephalic precursors
separates the primordial eye field in the neural plate and requires
cyclops. Development
126,5533
-5546.
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y. L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128,3497 -3509.[Medline]
Walshe, J., Maroon, H., McGonnell, I. M., Dickson, C. and Mason, I. (2002). Establishment of hindbrain segmental identity requires signaling by Fgf3 and Fgf8. Curr. Biol. 12,1117 -1123.[CrossRef][Medline]
Walshe, J. and Mason, I. (2003a). Unique and
combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain
development. Development
130,4337
-4349.
Walshe, J. and Mason, I. (2003b). Fgf signaling is required for formation of cartilage in the head. Dev. Biol. 264,522 -536.[CrossRef][Medline]
Woo, K. and Fraser, S. E. (1995). Order and
coherence of the fate map of the zebrafish nervous system.
Development 121,2595
-2609.