An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning
Justin Gage Crump1,*,
Lisa Maves1,
Nathan D. Lawson2,
Brant M. Weinstein3 and
Charles B. Kimmel1
1 Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254,
USA
2 Program in Gene Function and Expression, University of Massachusetts Medical
School, Worcester, MA 01605, USA
3 Laboratory of Molecular Genetics, NICHD/NIH, Bethesda, MD 20892, USA
*
Author for correspondence (e-mail:
gage{at}uoneuro.uoregon.edu)
Accepted 18 August 2004
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SUMMARY
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Fibroblast growth factor (Fgf) proteins are important regulators of
pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and
Fgf3 function in zebrafish have demonstrated a role for Fgfs in the
differentiation and survival of postmigratory neural crest cells (NCC) that
give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an
earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation
of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show
that pharyngeal pouches form by the directed lateral migration of discrete
clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the
migration of pharyngeal endodermal cells is disorganized and pouches fail to
form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3
are required in the neural keel and cranial mesoderm during early somite
stages to promote first pouch formation. In addition, we show that animals
doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages
and the more posterior branchial cartilages. By examining early pouch and
later cartilage phenotypes in individual animals hypomorphic for Fgf function,
we find that alterations in pouch structure correlate with later cartilage
defects. We present a model in which Fgf signaling in the mesoderm and
segmented hindbrain organizes the segmentation of the pharyngeal endoderm into
pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the
pharyngeal endoderm into pouches is critical for the later patterning of
pharyngeal cartilages.
Key words: Pouch, Pharyngeal endoderm, Cartilage, Neural crest, Segmentation, Fgf8, Fgf3, acerebellar, GFP, Zebrafish
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Introduction
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The cartilages and bones that form the skeleton of the face and, in
mammals, the middle ear, are derived from a specialized population of
ectomesenchyme, the cranial neural crest
(Le Douarin, 1982
;
Weston et al., 2004
). Cranial
neural crest cells (NCC) originate adjacent to neural ectoderm and migrate in
three streams (mandibular, hyoid and branchial) to form seven pharyngeal
arches. Segmentation of NCC into distinct streams is coupled to the
segmentation of the hindbrain into rhombomeres (R1-7)
(Kontges and Lumsden, 1996
).
NCC that contribute to the formation of the mandibular arch delaminate
adjacent to posterior midbrain-R2 and do not express Hox genes, whereas NCC of
the hyoid and branchial arches originate next to R4 and R6-R7, respectively,
and are Hox-positive (Schilling and
Kimmel, 1994
; Trainor and
Krumlauf, 2001
).
Fibroblast growth factors (Fgfs) are a family of extracellular signaling
molecules that have been implicated in diverse facets of vertebrate
craniofacial development. Fgf8 from the oral surface ectoderm induces patterns
of gene expression in adjacent mandibular mesenchyme, subdividing the
mandibular arch into rostral odontogenic and caudal skeletogenic fields
(Tucker et al., 1999
) and
controlling the position of the jaw joint
(Wilson and Tucker, 2004
). In
addition to functions in mandibular arch development, Fgfs have roles in the
development of cartilages derived from the hyoid and branchial arches and in
the formation of pharyngeal pouches. Pouches are outpocketings of the
pharyngeal endoderm that interdigitate with the crest-derived pharyngeal
arches. Fgf8neo/ mice, which are hypomorphic for
Fgf8, display a range of craniofacial abnormalities that include
reductions in cartilages and bones derived from all pharyngeal arches and
disorganized endodermal pouches (Abu-Issa
et al., 2002
). Likewise, in the zebrafish acerebellar
(ace) mutant, a strong loss-of-function mutation of fgf8
(hereafter referred to as fgf8), hyoid cartilage is
reduced and pouches are misshaped (Draper
et al., 2001
; Reifers et al.,
1998
; Roehl and
Nusslein-Volhard, 2001
).
Increasing evidence suggests that signals from the pharyngeal endoderm
pattern the bones and cartilages of the pharyngeal arches. Analysis of
casanova (cas) mutant zebrafish, which make no endoderm
(Alexander et al., 1999
),
suggest that endoderm is required for the development of all pharyngeal
cartilages (David et al.,
2002
). In tbx1 (van gogh) mutant zebrafish,
pharyngeal pouches are largely absent and cartilages are misshaped and fused
with those of adjacent arches (Piotrowski
and Nusslein-Volhard, 2000
), suggesting that pouches contribute to
the segmentation of NCC into distinct arches. In addition, transplantation
experiments in chick show that foregut endoderm is both necessary and
sufficient to induce the shape and orientation of pharyngeal skeletal elements
(Couly et al., 2002
). One role
of pharyngeal endoderm may be to locally promote the survival of skeletogenic
NCC (Crump et al., 2004
). Fgf3
has been shown to be required in pouch endoderm for the survival of
skeletogenic NCC of the hyoid and branchial arches
(David et al., 2002
;
Nissen et al., 2003
), and a
similar NCC survival-promoting role for endodermal Fgf8 has been proposed but
not proven in zebrafish (Walshe and Mason,
2003a
). Clearly, understanding how pharyngeal endoderm develops is
critical for understanding the later development of the pharyngeal
skeleton.
In this study, we investigate an earlier role for Fgfs in the formation of
pharyngeal pouches. Whereas in fgf8 animals
pharyngeal pouches are variably misshaped, we find that reducing both Fgf8 and
Fgf3, by injecting fgf8 animals with an
fgf3 morpholino (fgf3-MO), leads to a complete failure of
pouch formation. We use time-lapse microscopy to show that pharyngeal pouches
form by the directed lateral migration of periodic clusters of endodermal
cells. In fgf8; fgf3-MO animals,
pharyngeal endodermal cells are present but their lateral migration is
disorganized and discrete pouches fail to form. We use the Fgf
receptor-inhibiting drug SU5402 to show that Fgf signaling is required during
early somite stages for first pouch formation. At these stages, fgf8
is expressed in the head in lateral mesoderm
(Reifers et al., 2000
) and in
midbrain-hindbrain boundary (MHB)-R2 and R4 domains of the hindbrain
(Maves et al., 2002
;
Reifers et al., 1998
).
Starting at 4-somites (11.3 hours post-fertilization), fgf3
expression overlaps fgf8 expression in neural MHB and R4 domains
(Maves et al., 2002
;
Walshe et al., 2002
), and
mesodermal and neural Fgf expression domains are in close proximity to
developing pharyngeal endoderm. At later stages, fgf3 and, to a
lesser extent, fgf8 are expressed in the pharyngeal pouches
(David et al., 2002
;
Walshe and Mason, 2003a
).
However, we use mosaic analysis to show that Fgfs are required in mesodermal
and neural domains, and not in the pharyngeal endoderm, to rescue pharyngeal
arch structure in fgf8; fgf3-MO animals.
Thus, we find an essential, Fgf-dependent function of the brain and head
mesoderm in controlling segmentation of the pharyngeal endoderm into
pouches.
In addition to their requirement in pouch formation, we find that Fgf8 and
Fgf3 have redundant, essential functions in pharyngeal cartilage development.
In fgf8; fgf3-MO animals, hyoid and
branchial cartilages are largely absent and mandibular cartilages are reduced.
By imaging pouch structure early and cartilage structure later in individual
sides of animals in which Fgf signaling has been manipulated, we find that
altered pouches correlate with later rearrangements of the cartilage pattern.
This analysis suggests that pharyngeal pouch structure is a critical
determinant of the pharyngeal cartilage pattern. We present a model in which
an earlier function of Fgfs in pouch formation, in addition to their
well-documented role as pouch-secreted survival factors later in development,
contributes to the diversity of craniofacial phenotypes seen in fgf8
mutants.
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Materials and methods
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Zebrafish lines and morpholinos
Zebrafish (Danio rerio) were raised and staged as previously
described (Kimmel et al.,
1995
; Westerfield,
1995
). Time (hpf) refers to hours post-fertilization at
28.5°C. The wild-type line used was AB. Homozygous
acerebellarti282a (ace) mutant embryos were
scored by their loss of the cerebellum or loss of midbrain pax2a
expression (Brand et al., 1996
;
Reifers et al., 1998
).
fli1-GFP albino transgenic fish are the same as
TG(fli1:EGFP)y1; albb4
(Lawson and Weinstein, 2002
)
and H2A.F/Z:GFP transgenic fish are as described
(Pauls et al., 2001
).
The fgf8 MOs E2I2 and E3I3
(Draper et al., 2001
) were
used at 0.5 mg/ml each. To generate fgf8;
fgf3-MO embryos, we pressure-injected fgf8
embryos at the one-cell stage with 5 nl of the fgf3B (1.0 mg/ml) +
fgf3C (0.25 mg/ml) MO combination. As previously described
(Maves et al., 2002
), this
fgf3 MO dose gave highly reproducible
fgf8; fgf3-MO phenotypes, scored as either
the lack of ears or the lack of R5 krox20 staining.
Phenotypic analysis
Alcian Green staining was performed as described
(Miller et al., 2003
). For
flat-mount dissections, Alcian-stained animals were digested for 1 hour in 8%
trypsin at 37°C and transferred to 100% glycerol. Cartilages were
dissected free from surrounding tissues with fine stainless-steel insect pins
and photographed using a Zeiss Axiophot 2 microscope. Image background was
cleaned up with Adobe Photoshop. For immunocytochemistry, embryos were
prepared as described (Maves et al.,
2002
). Antibodies were used at the following dilutions: rabbit
anti-GFP, 1:1000 (Molecular Probes); Zn8, 1:400
(Fashena and Westerfield,
1999
; Trevarrow et al.,
1990
), goat anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor
568, both 1:300 (Molecular Probes).
The following cDNA probes were used: dlx2
(Akimenko et al., 1994
);
krox20 (Oxtoby and Jowett,
1993
); pax2a (Krauss
et al., 1991
); axial
(Odenthal and Nusslein-Volhard,
1998
); nkx2.7 (Lee et
al., 1996
); pea3
(Brown et al., 1998
). Probe
syntheses and whole-mount in-situ hybridizations were performed as previously
described (Hauptmann and Gerster,
1994
; Jowett and Lettice,
1994
).
SU5402 treatment
fli1-GFP embryos were manually dechorionated and incubated in 40
µl of EM with 0.4 mM SU5402 (Calbiochem) in agar wells. SU5402 was diluted
from a 40 mM stock in DMSO. After 1- or 4-hour incubations, embryos were
washed vigorously in EM. For 4-hour incubation experiments, sibling controls
were fixed and processed for in-situ hybridizations with pea3.
Transplantations
Transplant techniques were as described
(Maves et al., 2002
). For
endoderm transplants, donor embryos were injected at the 1-cell stage with an
`Alexa568' mixture of 2% Alexa Fluor 568 dextran and 3% lysine-fixable biotin
dextran (10,000 Mr, Molecular Probes) along with activated
Taram-A receptor (TAR*) RNA prepared according to David et al.
(David et al., 2002
). At 40%
epiboly (ca. 4 hpf) donor TAR* tissue was moved to the margins of
fgf8; fgf3-MO; fli1-GFP host
embryos. For neural and mesodermal transplants, donor embryos were injected at
the 1-cell stage with Alexa568. For neural transplants, donor tissue was taken
from the animal cap at shield stage (ca. 6 hpf) and moved to a position
approximately two germ ring widths from the margin and 70° from dorsal in
fgf8; fgf3-MO; fli1-GFP hosts
(Maves et al., 2002
). For
mesodermal transplants, donor tissue was taken from the margin at 50% epiboly
(ca. 5 hpf) and moved to the margins of fgf8;
fgf3-MO; fli1-GFP hosts
(Kimmel et al., 1990
). All
hosts were screened using a fluorescence stereomicroscope at 34 hpf, and only
hosts with substantial, tissue-specific contributions to the pharyngeal
endoderm, hindbrain or cranial mesoderm were used for subsequent analysis. In
addition, the mesodermal transplant technique produced six embryos with
contributions to both the hindbrain and cranial mesoderm. In order to control
for variability in the effectiveness of the fgf3-MO, only
fgf8; fgf3-MO; fli1-GFP hosts in
which the ear was missing in at least one side were used for the analysis. In
control transplants, mutant siblings in which donor tissue did not contribute
to head tissues, we never observed the presence of an ear on only one side. At
34 hpf, confocal images of select host embryos were analyzed for pharyngeal
arch structure. Rescue of arch structure was scored as complete if the first
pouch and mandibular and hyoid arches were indistinguishable from wild type,
and rescue was scored as partial for all other embryos with arch structure
subjectively more organized than in fgf8;
fgf3-MO; fli1-GFP controls.
Time-lapse analysis and confocal imaging
For the endoderm movies (see Movies 3-6 in supplementary material),
pharyngeal endoderm was labeled by transplanting donor tissue injected with a
mixture of Alexa568 and TAR* into GFP hosts. Embryos were selected
for large fractions of labeled donor endoderm and bright GFP fluorescence
using a Leica MZ FLIII fluorescence stereomicroscope. After manual
dechorionation and anesthetization with buffered
ethyl-m-aminobenzoate methane sulfanate (MESAB)
(Westerfield, 1995
), embryos
were transferred to 0.2% agarose in embryo media (EM) with 10 mM HEPES and
MESAB and then mounted onto a drop of 3% methylcellulose on a rectangular
coverslip with three superglued #1 square coverslips on each side. A ring of
vacuum grease was added around the embryo to make an airtight seal upon
addition of the top coverslip. A heated stage kept the embryos at 28.5°C.
Approximately 80 µm Z-stacks at 2 µm intervals were captured every 6
minutes using a Zeiss LSM5 Pascal confocal fluorescence microscope. At each
time point, Z-stacks were projected with maximum intensity onto a single
plane. Time-lapse recordings were further processed with Adobe Premiere. For
single time point confocal sections, embryos were mounted without vacuum
grease.
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Results
|
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Fgf8 and Fgf3 are essential for the formation of pharyngeal pouches and most pharyngeal cartilages
As previously reported (Roehl and
Nusslein-Volhard, 2001
), fgf8 zebrafish
had incompletely penetrant and expressive defects in the formation of
pharyngeal pouches and cartilages (Fig.
1B,B',F and especially
Fig. 2I,J). Since the
fgf8 phenotype is relatively mild, we wondered if
other Fgfs were partially redundant with Fgf8 in patterning pharyngeal arches.
Fgf3 was a good candidate, as it has been shown to act redundantly with Fgf8
to pattern the posterior hindbrain, forebrain and ear
(Maroon et al., 2002
;
Maves et al., 2002
;
Phillips et al., 2001
;
Walshe et al., 2002
;
Walshe and Mason, 2003b
). In
addition, Fgf3 has been shown to play a role in zebrafish pharyngeal arch
development (David et al.,
2002
; Nissen et al.,
2003
). Although fgf3-MO animals had largely normal
pouches (Fig. 1C,C')
(David et al., 2002
), we found
that in fgf8; fgf3-MO animals no pouches
were made (Fig. 1D,D').
In addition, we found that Fgf8 and Fgf3 acted redundantly to promote
pharyngeal cartilage development. In fgf3-MO animals, the
ceratobranchial (CB) cartilages, which derive from the branchial arches
located posterior to the hyoid arch, were largely absent
(David et al., 2002
;
Nissen et al., 2003
). However,
whereas hyoid cartilages of fgf3-MO and
fgf8 animals were relatively mildly affected at 4
days (Fig. 1F,G), nearly all of
the hyoid and CB cartilages in fgf8;
fgf3-MO animals were absent (Fig.
1H). Although reduced in size, mandibular cartilages were
patterned correctly in fgf8; fgf3-MO
animals (inset to Fig. 1H).

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Fig. 1. Fgf8 and Fgf3 have redundant functions in the formation of pharyngeal
pouches and cartilages. (A-D) Confocal micrographs are merged, lateral views
of cranial NCC (green: anti-GFP antibody) and endodermal pouches (red: Zn8
antibody) at 34 hpf; A'-D' show just the red channel. (A,A')
In wild-type fli1-GFP animals, the NCC-containing pharyngeal arches
are numbered 1-7 (A) and the pouches are numbered p1-p5 (A'). A few
hours later, the sixth pouch will form and arches 6 and 7 will separate to
form the final arrangement of seven arches. (B,B')
fgf8; fli1-GFP animals have variable defects in
pouch structure (arrowhead in B' denotes a misshapen first pouch).
Whereas fgf3-MO; fli1-GFP animals have normal pouches
(C,C'), fgf8; fgf3-MO;
fli1-GFP animals lack all pouches (D,D'), although pharyngeal
endoderm is still present (white line in D'). The Zn8 antibody also
recognizes cranial sensory ganglia (dots in A-C, A'-C'). (E-H)
Ventral whole-mount views show Alcian-stained pharyngeal cartilages at 4 days.
As shown for wild type (E), M and PQ cartilages are derived from the
mandibular, or first, arch; CH and HS are hyoid, or second, arch cartilages,
and CB1-5 cartilages are formed from the five most posterior branchial arches.
fgf8 animals have relatively mild defects in
pharyngeal cartilages (F), and in fgf3-MO animals CB cartilages are
lost and hyoid cartilages are misshapen (G). However, in
fgf8; fgf3-MO animals, nearly all CB and
hyoid cartilages are absent and mandibular cartilages are reduced in size (H).
The inset to H is a flat-mount preparation of fgf8;
fgf3-MO cartilages showing that, although reduced in size, M and PQ
cartilages retain their distinctive shapes. In E-H, asterisks denote the
position of the midline neurocranium that is still present in
fgf8; fgf3-MO animals. Anterior is to the
left in all panels. M, Meckel's; PQ, palatoquadrate; CH, ceratohyal; HS,
hyosymplectic; CB, ceratobranchial. Scale bars: 50 µm in A-D; 100 µm in
E-H.
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Fig. 2. Correlated first pouch and hyoid cartilage defects in animals reduced for
Fgf8. Confocal projections of fli1-GFP-labeled pharyngeal arches in
living wild-type (A) and fgf8-MO (C,E,G) animals at 28 hpf. By this
stage of wild-type development, the mandibular (1), hyoid (2), and three
branchial arches (3, 4, 5-7) have formed. Pouches are labeled p1-p4 (A), and
white arrowheads mark the positions of the first pouch. In fgf8-MO;
fli1-GFP animals, variable phenotypes include shape changes in the
first pouch (C,E), ectopic pouches (arrow in G), and reductions of more
posterior pouches (C,E,G). (I) Confocal micrograph of a fixed
fgf8; fli1-GFP animal with a similar
ectopic pouch phenotype (arrow) to the fgf8-MO; fli1-GFP
animal in G; Zn8 staining (red) confirms that the
non-fli1-GFP-expressing region is probably an ectopic endodermal
pouch. (B,D,F,H,J) Flat-mount preparations of Alcian-stained mandibular and
hyoid cartilages at 4 days. As labeled in the wild-type example (B), M and PQ
are mandibular (1) and CH and HS are hyoid (2) cartilages. (C and D, E and F,
G and H) Paired images of individual animals imaged live for fli1-GFP
early and subsequently stained for cartilage. Variable hyoid cartilage defects
(D,F,H) are correlated with earlier first pouch defects (C,E,G) in individual
fgf8-MO; fli1-GFP animals. In H, the black arrowhead marks
an apparent ectopic hyoid cartilage that correlates with the ectopic pouch in
G. (J) Similar ectopic cartilages (black arrowhead) were seen in some
fgf8; fli1-GFP animals. Anterior is to the
left and dorsal is up. M, Meckel's; PQ, palatoquadrate; CH, ceratohyal; HS,
hyosymplectic. Scale bar: 50 µm.
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Since fgf8; fgf3-MO animals have both
pouch and cartilage defects, we wondered whether defects in pouch development
could be responsible for later cartilage losses. In order to study
correlations between pouch and cartilage defects in individual animals, we
took advantage of the fact that partially reducing Fgf function with an
fgf8 morpholino (fgf8-MO), or genetically with the
fgf8 mutation, causes variably penetrant and expressive phenotypes.
We imaged pharyngeal arch structure in live fgf8-MO;
fli1-GFP and fgf8; fli1-GFP
embryos at 28 hpf and subsequently raised individuals to 4 days to examine
cartilage. The fli1-GFP transgene
(Lawson and Weinstein, 2002
)
labels cranial NCC shortly after ventrolateral migration and perdures as cells
differentiate into the pharyngeal cartilage elements. fli1-GFP also
marks the developing vasculature but is not expressed in the pharyngeal
mesoderm or endoderm (except for early, transient expression in the second
pouch; see below). At pharyngula stages, the endodermal pouches are evident as
non-fli1-GFP-expressing regions separating the
fli1-GFP-expressing NCC of the pharyngeal arches (black in
Fig. 2A). In individual sides
of fgf8-MO; fli1-GFP and fgf8;
fli1-GFP animals we found a correlation between early arch structure
and alterations of the hyoid cartilage pattern
(Fig. 2C-J and
Table 1). In all sides with
abnormal first pouch morphology we observed hyoid cartilage alterations later.
In some cases, the first pouch was `deformed'
(Fig. 2C), invading hyoid NCC
territory, and this arch phenotype was most often linked to a complete loss of
the dorsal hyomandibular cartilage element
(Fig. 2D). In other cases, the
first pouch was `shifted' to a more posterior position
(Fig. 2E), and this `shift' was
correlated with changes in the shape and position of dorsal hyoid cartilage
(Fig. 2F). In the most striking
example of pouchcartilage shape correlations, a small ectopic pouch, in
addition to the normal first pouch, formed in the middle of the hyoid NCC
territory (Fig. 2G), and in
these same sides an ectopic cartilage element developed later in the hyoid
arch (Fig. 2H). To confirm that
the non-GFP-expressing regions observed in live animals probably corresponded
to misshapen and ectopic pouches, and not another non-GFP-expressing tissue
such as mesoderm, we fixed and stained fgf8 animals
displaying similar GFP phenotypes with the endoderm-labeling Zn8 antibody
(Trevarrow et al., 1990
). In
numerous examples, non-GFP expressing regions in
fgf8 animals, of similar shapes and positions to
those observed in the live analysis, were found to be Zn8-positive (red in
Fig. 1B and
Fig. 2I; and data not shown).
Lastly, whereas the majority of fgf8 animals with
normal arch morphology early had no defects in the hyoid cartilage pattern
later, we observed graded reductions of the hyomandibular cartilage element in
some sides with no pouch defects (Table
1). Thus, Fgf8 is likely to have other functions in cartilage
development, in addition to its role in controlling pouch development.
Nonetheless, we conclude that, in contrast to simple cartilage losses, the
alterations in hyoid cartilage shape and position observed in a subset of
animals reduced for Fgf8 are most tightly correlated with early changes in
pouch morphology.
Posterior pharyngeal pouch defects underlie reduced arch segmentation in fgf8-MO animals
After NCC migration the third, most posterior, NCC mass segments into the
five branchial, or gill-bearing, arches from which the five CB cartilages
subsequently develop (Fig. 3A).
It has been previously shown that the segmentation of NCC into distinct arches
requires the segmentation of the pharyngeal endoderm into pouches
(Piotrowski and Nusslein-Volhard,
2000
). In animals reduced for Fgf8, we observed a range of CB
cartilage phenotypes that suggests defects in the segmentation of NCC into
distinct branchial arches. These phenotypes include reductions in the number
of CB cartilages (Fig. 3B) and
fusions of cartilages of adjacent segments
(Fig. 3C); incompletely formed
CB cartilages were also observed (Fig.
3C). In addition, as reported for fgf8
animals (Roehl and Nusslein-Volhard,
2001
), more posterior pharyngeal pouches were reduced and
disorganized in fgf8-MO; fli1-GFP animals
(Fig. 2C,E,G). We investigated
whether the CB cartilage defects seen in fgf8-MO animals might be
secondary to posterior pouch formation defects that result in reduced
branchial NCC segmentation.

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Fig. 3. Fgf8 is required for segmentation of the branchial arches. Bilateral
flat-mount dissections of Alcian-stained pharyngeal cartilages at 4 days. As
shown for wild type (A), M and PQ are mandibular (1) cartilages, CH and HS are
hyoid (2) cartilages, and CB1-5 cartilages are formed from the five most
posterior branchial arches (3-7). Note the teeth (*) on the CB5
cartilages. fgf8 animals have variable CB cartilage
defects, which include reduced CB number (only 4 CBs per side in B),
incompletely formed CB cartilages (arrowhead in C), and fusions between
adjacent cartilages (arrow in C). In a representative
fgf8; fli1-GFP clutch (n=172)
there was an average of 3.9 CB cartilages per side; 6% of sides had fusions of
adjacent cartilages and 2% had incomplete cartilages. (D-K) Time-lapse
recordings of wild-type fli1-GFP; H2A.F/Z:GFP (D-G, and see Movie 1
in supplementary material) and fgf8-MO; fli1-GFP (H-K, and
see Movie 2 in supplementary material) animals show the cellular basis of
branchial arch segmentation. In wild-type animals, branchial arches form as
pouches separate the branchial NCC mass into segments in an AP wave of
development. At the beginning of Movie 1 (5-somites, 12 hpf), H2A.F/Z:GFP
labels the nuclei of NCC that are migrating ventrolaterally in two streams
anterior to, and one stream posterior to, the developing ear. After
fli1-GFP initiates in NCC of the pharyngeal arches, selected
projections from Movies 1 and 2 show the subdivision of each successive
branchial arch (arch 3 at 20 hpf: D,H; arch 4 at 28 hpf: E,I; arch 5 at 34
hpf: F,J; and arches 6 and 7 at 38 hpf: G,K). Pouches are labeled p1-p6, and
white arrowheads in D-G indicate the developing vasculature that also
expresses fli1-GFP. The white arrows in panels I-K and Movie 2 (in
supplementary material) refer to arches 4 and 5, which fail to separate
completely in this fgf8-MO animal. Similar cell behaviors were seen
in three time-lapse recordings of wild-type animals, and variable defects were
observed in four time-lapse recordings of fgf8-MO; fli1-GFP
animals. Anterior is to the left in all panels. A-C are ventral views, and
dorsal is up and slightly to the right in D-K. M, Meckel's cartilage; PQ,
palatoquadrate; CH, ceratohyal; HS, hyosymplectic; CB, ceratobranchial. Scale
bar: 50 µm.
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In order to understand the cellular basis of NCC segmentation, we made
time-lapse recordings of pharyngeal arch development in wild-type (see Movie 1
in supplementary material) and fgf8-MO (see Movie 2 in supplementary
material) animals. Time-lapse recordings were made of wild-type animals
expressing both the pan-nuclear H2A.F/Z:GFP and the NCC-expressing
fli1-GFP (12-38 hpf; Movie 1). In wild-type animals, NCC migrated in
three streams to ventrolateral regions, where they contributed to the
formation of the pharyngeal arches. Starting at 12 hpf (5-somites),
H2A.F/Z:GFP-labeled NCC were seen migrating in two streams (mandibular and
hyoid) anterior to, and one stream (branchial) posterior to, the developing
otic vesicle. By around 16 hpf, the NCC finished their migration and began to
express fli1-GFP as they condensed to form the arch masses. Shortly
after the initiation of fli1-GFP expression in NCC, the first
branching of the branchial mass occurred as the third pouch was formed
(Fig. 3D). Over the next 20
hours, the fourth, fifth and sixth pouches formed in an anterior-posterior
(AP) wave of development, and by 38 hpf the branchial mass had been subdivided
into the five segments from which the CB cartilages would arise
(Fig. 3E-G).
By contrast, in the fgf8-MO; fli1-GFP example shown (see
Movie 2 in supplementary material), one fewer branchial segment formed. We
found that in this animal the reduced number of segments was due to the
failure of the fourth pouch to develop. While the third pouch (i.e. the first
pouch to subdivide the branchial mass) formed normally
(Fig. 3H), the fourth pouch
initiated outgrowth yet failed to fully subdivide the branchial mass into a
new segment (Fig. 3I). By 38
hpf the fifth and sixth pouches had fully formed, but the fourth pouch had
retracted, and what in wild-type animals would have been the second and third
posterior branchial segments had fused together, resulting in one less
branchial segment (Fig. 3J,K).
Consistent with this animal forming one less branchial segment, we found that
one less CB cartilage developed (data not shown). These results suggest that
at least some, and possibly all, of the CB cartilage defects seen in
fgf8 animals are the result of a failure of
posterior endodermal pouches to form properly and segment branchial NCC into
discrete arches.
Fgf signaling is required during early somite stages for first pouch development
In order to determine when Fgfs act to control pouch development, we
inhibited Fgf signaling at different times of development using the Fgf
receptor antagonist SU5402. As extended (24-hour) treatment of embryos with
SU5402 causes widespread death of NCC
(David et al., 2002
), we
performed shorter treatments of SU5402 in order to dissociate requirements for
Fgf signaling in pharyngeal pouch formation from those in NCC survival. After
addition of SU5402 for 1- to 4-hour periods, followed by a washout, embryos
were scored for pouch defects at 34 hpf. In the case of 4-hour treatments, we
examined effectiveness of inhibition of Fgf signaling by expression of the Ets
factor, pea3, in treated siblings. pea3 is a downstream
target of Fgf signaling (Roehl and
Nusslein-Volhard, 2001
), and we observed partial-to-complete
inhibition of pea3 expression during the treatment and gradual
recovery after washout (data not shown, summarized in
Fig. 4 legend). In embryos
treated with SU5402 from 10 to 14 hpf, we found that the first pouch was
specifically lost in 39% of embryos (Fig.
4B) and misshapen in another 26% of embryos (data not shown). In
addition, when animals with first pouch defects were raised to 4 days, we
observed specific losses of hyoid cartilage
(Fig. 4E). By contrast, SU5402
treatments from 6 to 10 hpf and 14 to 18 hpf had lesser effects on pouch
development (Fig. 4G). We found
a similar temporal requirement for Fgf signaling using 1-hour SU5402
treatments (Fig. 4C,H). The
highest penetrance of first pouch shape defects was seen when 1-hour SU5402
treatments began between 9 and 13 hpf. Interestingly, later (>20 hpf)
treatments with SU5402 produced variable defects in the development of more
posterior pouches but not the first pouch
(Fig. 4F), consistent with more
posterior pouches forming later in an AP temporal wave of development. In
summary, we find that Fgf signaling has a peak requirement in first pouch
development from 10 hpf (tailbud) to 14 hpf (10-somites).

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Fig. 4. Fgf signaling is required during early somite stages for first pouch and
hyoid cartilage development. (A-C) Confocal micrographs show Zn8-labeled
pharyngeal pouches (red) and GFP-labeled NCC (green) in fli1-GFP
animals at 34 hpf. Cranial sensory ganglia (dots) also stain with Zn8. In the
wild-type animal (A), an arrowhead marks the first pouch. (B) The first pouch
is variably absent (arrowhead) in fli1-GFP animals upon treatment
with the Fgf receptor antagonist SU5402 from 10-14 hpf. The absence of the
first pouch is selective, as more posterior pouches form normally. (C)
Treatment with SU5402 for 1-hour periods starting from 9-13 hpf (a 10.5-11.5
hpf treatment is shown) produce subtler shape changes of the first pouch
(arrowhead). (D,E) Flat-mount preparations of Alcian-stained pharyngeal
cartilages at 4 days. In wild-type (D), mandibular M and PQ, hyoid CH and HS,
and branchial CB1-5 cartilages are labeled. In those animals in which 10-14
hpf SU5402 treatment caused losses of the first pouch early, the HS cartilage
was selectively absent later (E). Although M, PQ and CH cartilages are reduced
in size, posterior CB cartilages are relatively unaffected. (F) Whereas later
treatments with SU5402 (a 20-21 hpf treatment is shown) do not affect first
pouch development (arrowhead), they do occasionally disrupt the formation of
more posterior pouches (arrow shows an unsegmented branchial NCC mass). (G)
Quantitation of first pouch defects after 4-hour treatments with SU5402. The
percentages of animals with first pouch losses, in black, and misshapen first
pouches, in gray, are plotted. n6-10 hpf=49,
n10-14 hpf=99, n14-18 hpf=21. First
pouch loss after 10-14 hpf treatment is statistically significant using Tukey
HSD test. (H) The percentage of fli1-GFP animals having first pouch
defects (primarily shape changes) plotted against the start time of 1 hour
treatments with SU5402. n5.5 hpf=17, n7
hpf=14, n8 hpf=24, n9 hpf=14,
n10.5 hpf=21, n11 hpf=18,
n12 hpf=26, n13 hpf=26,
n14 hpf=22, n16 hpf=26,
n20 hpf=24, n24 hpf=31. The period of
strongest effect is from 9 hpf (90% epiboly) to 13 hpf (8-somites). In order
to assess the efficiency of inhibition of Fgf signaling, and the recovery
after washout, we fixed sibling controls and examined pea3
expression, a downstream effector of Fgf signaling, at 0 and 4 hours after
washout. We know that SU5402 is at least partially being washed out as
omission of the washout step leads to severe necrosis of animals. For 4-hour
incubation experiments, the levels of pea3 in individual animals,
relative to those in similarly staged untreated controls, were as follows:
6-10 hpf, 6/8 reduced at 10 hpf, 11/13 reduced and 2/13 absent at 14 hpf;
10-14 hpf, 5/13 reduced and 8/13 absent at 14 hpf, 6/13 reduced and 7/13
absent at 18 hpf; 14-18 hpf, 5/12 reduced and 7/12 absent at 18 hpf. As
pea3 levels were similarly reduced at 18 hpf in 10-14 hpf and 14-18
hpf treatments, yet only 10-14 hpf treatments cause first pouch defects, we
conclude that Fgf signaling is required from 10-14 hpf for first pouch
development. However, these experiments do not exclude additional requirements
for Fgf signaling at later times. Anterior is to the left and dorsal is up. M,
Meckel's; PQ, palatoquadrate; CH, ceratohyal; HS, hyosymplectic; SU, SU5402.
Scale bar: 50 µm.
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Neural and mesodermal requirement for Fgfs in pharyngeal pouch formation
As Fgf8 and Fgf3 are expressed in neural and mesodermal tissues at early
somite stages, and then in the pharyngeal endoderm at later stages, we
investigated which Fgf sources are required for pouch formation. Using
transplantation techniques (see Materials and methods), we introduced
wild-type tissues at pre-shield stages (<6 hpf) into
fgf8; fgf3-MO; fli1-GFP embryos and then
assayed for rescue of first pouch development based on GFP-labeled arch
structure. First, we found that wild-type endoderm was not able to rescue
first pouch structure (Fig.
5B-B'') compared with control contralateral sides that
did not receive transplants (Fig.
5A-A''). Wild-type mesoderm
(Fig. 5C-C'') or
neural tissue (Fig.
5D-D'') alone was able to only partially rescue pouch
and arch structure in less than half of fgf8;
fgf3-MO; fli1-GFP hosts (Fig.
5F). By contrast, transplantation of wild-type neural and
mesodermal tissues together completely rescued pouch structure in 3/6 embryos,
and partial rescue was seen in another 2/6 embryos
(Fig. 5E-E''). As
neural tube transplants alone were sufficient to rescue the ear and cerebellar
defects of fgf8; fgf3-MO embryos
(Fig. 5D'''),
yet failed to completely rescue pouch structure
(Fig. 5D'), we conclude
that the requirements for both neural and mesodermal tissues is not simply a
function of restoring neural structure in fgf8;
fgf3-MO embryos. Thus, Fgf8 and Fgf3 are required additively in both the
neural tube and mesoderm, but not the endoderm, to promote morphogenesis of
the pharyngeal endoderm into pouches.

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Fig. 5. Fgfs are required in neural and mesodermal tissues for first pouch
formation. Labeled wild-type tissues (red: A''-E'')
were transplanted into fgf8; fgf3-MO;
fli1-GFP animals from 4-6 hpf, and GFP-expressing NCC (green:
A'-E') were examined at 34 hpf for rescue of pharyngeal arch
structure, a proxy for pouch structure. A'-E' and
A''-E'' are confocal projections and are merged in
A-E. A'''-E''' are individual confocal
sections from A-E and include the Nomarski channel. (A-A''')
As transplantations generally contribute donor tissue unilaterally, we used
contralateral non-recipient sides of fgf8;
fgf3-MO; fli1-GFP animals as negative controls for rescue
(the red staining in A,A'' represents a comparatively small amount
of donor tissue that has crossed the midline). In control sides, only
mandibular (1) and a few unidentified (?) NCC are evident (A'), and the
ear is missing (A'''). (B-B''') Wild-type
endoderm (e) fails to rescue pharyngeal arch structure (B') and the ear
(B'''). As seen in B'', wild-type endoderm does
not segment into pouches in fgf8; fgf3-MO;
fli1-GFP hosts. Wild-type mesoderm (m, C-C''') or
wild-type neural tissue (n, D-D''') only partially rescues
pharyngeal arch structure in a fraction of animals. In the non-rescued
mesodermal example shown (C'), pharyngeal (1 +?) NCC remain unsegmented,
revealing a lack of pouches. The neural example shown (D') represents
what we scored as partial rescue of arch structure. There is an increase in
the amount and organization of NCC, but they are not segmented into ordered
pharyngeal arches as in wild-type animals. The lack of rescue of arch
structure by wild-type neural tissue is striking, as other structures such as
the MHB blood vessel (asterisk in D'), the neural flexure (white
arrowhead in D''), and the ear (black arrowhead in
D''') are rescued by neural tissue. By contrast, wild-type
mesoderm did not rescue the ear (C''').
(E-E''') Both wild-type mesoderm and neural tissue are
required together to completely rescue pharyngeal arch structure, and hence
pouches, in fgf8; fgf3-MO;
fli1-GFP animals. In this example, a morphologically normal first
pouch (p1, arrow) and mandibular (1) and hyoid (2) arches are clearly seen
(E'). Some of the more posterior pouches (E', note the
segmentation of the branchial (3+) NCC mass) and the ear (black arrowhead in
E''') are rescued as well. The identification of mesoderm in
the transplants was based on the lack of colocalization with the neural crest
marker fli1-GFP in confocal sections, and in this example by the
characteristic morphology of the mesodermal cores of the pharyngeal arches
(Kimmel et al., 2001 ) (F)
Quantitation of pharyngeal arch rescue by wild-type tissues is plotted as
percentage of host sides with complete (black) or partial (gray) rescue of
arch structure. nendoderm=34,
nmesoderm=11, nneural=30,
nneural+mesoderm=6. Complete rescue by neural and
mesodermal tissue (neur. + meso.) and partial rescue by mesoderm or neural
tissue were statistically significant using Tukey HSD test. In addition, no
rescue was seen by wild-type neural crest, a tissue that does not express
either Fgf8 or Fgf3 (data not shown). Anterior is to the left and dorsal is
up. Scale bar: 50 µm.
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Fgf8 and Fgf3 are required for the subsequent development, and not the generation, of pharyngeal endoderm and NCC
In order to test whether the lack of pouches is due to a general reduction
in pharyngeal endoderm, we examined early markers of pharyngeal endoderm in
fgf8; fgf3-MO animals. In wild-type
embryos, the first pouch began to form around 16 hpf (see below). At 18 hpf,
nkx2.7 and axial normally were expressed in lateral
pharyngeal endoderm, in particular the first pouch and regions where the
second and more posterior pouches would form
(Fig. 6A,E). In
fgf3-MO animals, nkx2.7 and axial expression was
similar to that seen in wild-type animals
(Fig. 6C,G), whereas in
fgf8 animals nkx2.7 and axial
were present but first pouch staining was variably absent
(Fig. 6B,F). Strikingly, in
fgf8; fgf3-MO animals, these markers
revealed that a significant amount of pharyngeal endoderm was present, yet
there was no clear evidence of pouches
(Fig. 6D,H). In addition, as
assayed by axial staining, we saw no differences in the amount of
pharyngeal endoderm at an earlier stage (10 hpf) between
fgf8; fgf3-MO and wild-type animals (data
not shown). These results suggest that Fgf8 and Fgf3 act to promote the
segmentation of pharyngeal endoderm into pouches and not endoderm
generation.

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Fig. 6. Pharyngeal endoderm and cranial NCC defects in animals lacking Fgf8 and
Fgf3. nkx2.7 (A-D) and axial (E-H) label pharyngeal endoderm
during early pouch morphogenesis stages (18 hpf). nkx2.7 and
axial are in blue, and, in E-H, krox-20 in red labels R3 and
R5. In wild-type animals (A,E), the first pouch (p1: arrows) has formed
anterior to R3, and a more posterior endodermal mass that will give rise to
the remaining pouches (black lines) is situated adjacent to R4-R6 territory.
The first pouch is variably lost in fgf8 animals
(asterisk in B, question mark in F). Whereas pharyngeal endoderm develops
normally in fgf3-MO animals (C,G), in
fgf8; fgf3-MO animals (D,H) pharyngeal
endoderm is present as a single anterior mass (black line) and no pouches are
evident. (I-P) dlx2, in blue, labels cranial NCC; in red (I-L),
pax2a labels the MHB and krox-20 labels R3 and R5. (I) In 18
hpf wild-type animals, mandibular (1), hyoid (2), and branchial (3) NCC
streams give rise to seven pharyngeal arches. (M) At 33 hpf, the third
branchial stream has generated arches 3-5 and arches 6 and 7 have yet to
separate. In fgf8 (J,N) and fgf3-MO (K,O)
animals, the migration and coalescence of NCC to form the pharyngeal arches is
largely normal. In fgf8; fgf3-MO animals,
the mandibular (1) stream is disorganized and hyoid and branchial streams are
fused together (2/3) at 18 hpf (L). By 33 hpf (P), nearly all hyoid and
branchial NCC are absent, and mandibular (1) NCC are present but reduced.
Anterior is to the left in all panels. A-D are dorsal views, and E-P are
lateral views. R3 and R5, rhombomeres 3 and 5; MHB, midbrain-hindbrain
boundary; p1, first pouch. Scale bar: 50 µm.
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Similarly, in order to understand the losses of the NCC-derived cartilages
in fgf8; fgf3-MO animals, we examined the
development of NCC in doubly reduced animals. In 18 hpf wild-type embryos,
dlx2 expression marked three postmigratory NCC masses: the
mandibular, hyoid and branchial primordia
(Fig. 6I). In fgf3-MO
and fgf8 embryos, the three dlx2-positive
masses resembled those in wild-type animals
(Fig. 6J,K). By contrast, in
fgf8; fgf3-MO embryos, only two
dlx2-positive masses were present
(Fig. 6L). Based on their
positions with respect to the R3 domain of the hindbrain, we interpreted these
two masses as a disorganized mandibular mass and a single fused mass
incorporating hyoid and branchial NCC. As hyoid NCC are generated adjacent to
R4 and branchial NCC develop adjacent to R6-R7 domains
(Schilling and Kimmel, 1994
;
Trainor and Krumlauf, 2001
),
the NCC fusions are probably due to the absence of intervening R5-R6 territory
in fgf8; fgf3-MO embryos
(Maves et al., 2002
;
Walshe et al., 2002
). We
observed similar dlx2 phenotypes at 12 hpf (data not shown). By 33
hpf, dlx2 labeled the mandibular and hyoid arches and four branchial
segments in wild-type animals (Fig.
6M). In fgf3-MO and fgf8
animals, dlx2 expression was only mildly reduced compared with
wild-type controls (Fig. 6N,O).
However, dlx2 expression in fgf8;
fgf3-MO animals revealed that by 33 hpf most NCC were absent except
for a reduced mandibular population (Fig.
6P). We observed similar NCC losses in
fgf8; fgf3-MO animals based on the NCC
expression of the fli1-GFP transgene
(Fig. 1D). Moreover, the
selective disappearance of hyoid and branchial NCC between 18 hpf and 33 hpf
was consistent with the later specific losses of the hyoid and branchial
cartilages in fgf8; fgf3-MO animals. In
conclusion, we found requirements for Fgf8 and Fgf3 in both the early
organization (12-18 hpf) and the later survival (33 hpf) of NCC.
Pharyngeal pouches form by the lateral migration of endodermal cells
Understanding the role of Fgfs in pouch formation requires a detailed
knowledge of the cell behaviors underlying pouch development in wild-type
animals. Surprisingly, little is known about the mechanism of pouch formation
in any species. In order to investigate the morphogenesis of pouch endoderm
directly, we made time-lapse recordings of Alexa568-labeled developing
endoderm in H2A.F/Z:GFP; fli1-GFP zebrafish (10-30 hpf: see Movies 3,
4 in supplementary material). As labeled endoderm was generated by a
combination of TAR* induction and transplantation techniques (see
Materials and methods for details), a large fraction, but not all, of the
endodermal cells can be seen in the recordings. H2A.F/Z:GFP labels the nucleus
of every cell (Pauls et al.,
2001
) and helps to identify landmarks, whereas fli1-GFP
labels postmigratory NCC. At 10 hpf of wild-type development, Alexa568-labeled
pharyngeal endodermal cells were scattered along the surface of the yolk
(Fig. 7A,A'), a
distribution that closely resembled endodermal axial expression at
this time (Reiter et al.,
2001
). Over the next 6 hours, endodermal cells underwent a medial
migration and became increasingly packed together near the midline
(Fig. 7B,B' show a 14 hpf
intermediate stage). Shortly after medial migration, endodermal cells that
would give rise to the first pouch began to extend thin cytoplasmic processes
and migrate back out laterally (18 hpf:
Fig. 7C,C' and more
clearly in Movie 4). At this time, the first postmigratory NCC began to turn
on fli1-GFP. During the next few hours, endodermal cells continued to
migrate laterally in a directed fashion, and by 22 hpf the first pouch was
nearly fully formed (Fig.
7D,D'). In addition, clusters of endodermal cells situated
periodically along the AP axis migrated laterally to form progressively more
posterior pouches in an AP wave of development. By 30 hpf in this recording,
the positions of the first three pouches were clearly seen relative to the
fli1-GFP-labeled NCC of the arches
(Fig. 7E,E'). In three
wild-type recordings, the lateral migration of endodermal cells was observed
to underlie the formation of all labeled pouches. We conclude that the
directed lateral migration of periodically spaced endodermal cells is the
mechanism that generates the pharyngeal pouches.

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Fig. 7. Pharyngeal pouches form by an Fgf-dependent lateral migration of endodermal
cells. (A-E) Representative still images from a time-lapse confocal recording
of pharyngeal pouch and arch development in wild-type animals (see Movies 3, 4
in supplementary material). Pharyngeal endoderm has been labeled in red
(A'-E'; merged with GFP in A-E) by transplanting TAR*
endoderm into a fli1-GFP; H2A.F/Z:GFP host at 4 hpf; this technique
leads to mosaic labeling of endoderm in the host animal. In green, H2A.F/Z:GFP
allows the nuclei of every cell to be seen, and fli1-GFP marks NCC of
the pharyngeal arches. In wild-type development, endodermal cells are spread
out in a monolayer over the surface of the yolk at 10 hpf (A,A').
Concomitant with medial ectodermal movements to form the neural keel,
endodermal cells migrate medially and begin to aggregate (14 hpf: B,B').
Shortly after medial migration, individual endodermal cells destined to become
the first pouch (arrow in C') then migrate back out laterally (C: 18
hpf). As seen in Movie 4, pouch endodermal cells extend cytoplasmic processes
laterally during migration. At the same time, cranial NCC begin to condense
and express fli1-GFP. By 22 hpf, the first two pouches (D':
p1,p2) have formed and interdigitate three NCC-containing pharyngeal arches
(D: 1-3). Although in this example most second pouch cells are not labeled in
red, their development can still be observed based on transient expression of
fli1-GFP (asterisks in C,D). Also, the characteristic flexure of the
neural keel near the MHB is visible by H2A.F/Z:GFP (arrowhead in D). At 30
hpf, three pouches (E': p1-p3) and four arches (E: 1-4) are well
developed. (F-J) Representative still images from a time-lapse recording of
pharyngeal development in an animal reduced for Fgf8 and Fgf3 (see Movies 5, 6
in supplementary material). Similar cell behaviors were seen in two separate
recordings. Labeled endoderm (red) was generated by transplantation of
wild-type TAR* endoderm into fgf8;
fgf3-MO; H2A.F/Z:GFP animals (see text for discussion of experimental
rationale, including how wild-type and mutant endoderm probably behave
similarly in a mutant host). In fgf8;
fgf3-MO animals, the generation (F,F') and medial migration
(G,G') of pharyngeal endoderm is normal. However, by 18 hpf, the lateral
migration of endodermal cells is delayed (H,H'). Also, the migration of
endodermal cells is disorganized, with cytoplasmic processes not being
oriented laterally as in wild-type animals (arrows in Movie 6 in supplementary
material). By 22 hpf lateral endodermal cells form an extended anterior mass
(white line in I'). A confirmation of the
fgf8; fgf3-MO phenotype is the lack of a
neural flexure (arrowhead in I), increased cell death at the MHB, and the lack
of an ear (data not shown). By 30 hpf, pharyngeal endoderm has not segmented
into discrete pouches and remains a single anterior mass (white line in
J'). Although animals did not carry the fli1-GFP transgene,
reduced mandibular (1) and possibly hyoid (2?) arches are visible as
condensations of H2A.F/Z:GFP-expressing cells. The views are dorsolateral with
anterior to the left. Scale bar: 50 µm.
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Fgfs are required for the segmentation and directed migration of endodermal cells that form pouches
In order to understand the cellular basis for the lack of pouches in
animals reduced for Fgf8 and Fgf3, we made time-lapse recordings of pharyngeal
endoderm development in fgf8; fgf3-MO;
H2A.F/Z:GFP animals (10-30 hpf: see Movies 5, 6 in supplementary material).
For technical reasons, we visualized pharyngeal endoderm by transplanting
labeled, TAR*-induced endoderm from wild-type donors into
fgf8; fgf3-MO; H2A.F/Z:GFP hosts (see
Materials and methods). However, as we knew that wild-type endoderm failed to
make pouches in an fgf8; fgf3-MO
background, we inferred that the endoderm defects we describe here would be
the same as in non-mosaic fgf8; fgf3-MO
animals. At 10 hpf, we saw a similar distribution of pharyngeal endodermal
cells over the surface of the yolk as in wild-type animals
(Fig. 7F,F'), consistent
with our earlier finding that fgf8;
fgf3-MO animals had no defect in 10 hpf endodermal axial
expression. In addition, the medial migration and subsequent compaction of
endodermal cells near the midline was largely normal in
fgf8; fgf3-MO animals
(Fig. 7G-H'). However, by
18 hpf we saw defects in the lateral migration of endodermal cells
(Fig. 7H,H'). Although
endodermal cells extended cytoplasmic processes in
fgf8; fgf3-MO animals, these processes
were not always directed laterally and often retracted (see Movie 6 in
supplementary material). By 22 hpf, endodermal cells had failed to migrate
laterally and discrete pouches were not seen
(Fig. 7I,I'). Moreover,
putative pouch endodermal cells did not align into regularly spaced arrays and
by 30 hpf had formed a single, unsegmented clump of cells laterally
(Fig. 7J,J'). We conclude
that Fgf8 and Fgf3 are not required for the initial generation or medial
migration of pharyngeal endodermal cells. Instead, Fgfs have later functions
in the directed lateral migration and regular spacing of pharyngeal endodermal
cells along the AP axis, processes critical for the formation of discrete
pouches.
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Discussion
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Pharyngeal pouches form by an Fgf-dependent lateral migration of endodermal cells
We have demonstrated an essential role for Fgf signaling in the formation
of pharyngeal pouches. Whereas fgf8 animals had
variable defects in pouch formation, animals reduced for both Fgf8 and Fgf3
lacked all pharyngeal pouches. In addition, transient inhibition of Fgf
signaling with the Fgf receptor antagonist SU5402 caused similar defects in
pouch formation. This essential function of Fgfs in pouch formation is
probably conserved among vertebrates, as mice hypomorphic for Fgf8 or
Fgf receptor 1 (FgfR1) have similar pouch defects to those
in fgf8 zebrafish
(Abu-Issa et al., 2002
;
Trokovic et al., 2003
).
Previous to this study, little was known about the cellular behaviors
underlying pouch formation. Pharyngeal pouches arise as lateral branches of
the foregut endoderm. Branching morphogenesis is a common theme in
organogenesis, and various cellular mechanisms, such as clefting and cell
migration, have been implicated in branch generation
(Chuang and McMahon, 2003
;
Ghabrial et al., 2003
;
Sakai et al., 2003
). By
directly imaging pouch formation in developing zebrafish embryos, we showed
that cell migration is the mechanism that drives the lateral branching of the
pharyngeal endoderm into pouches. After coalescence of pharyngeal endoderm
near the ventral midline, subsets of endodermal cells migrated laterally at
periodic intervals along the AP axis to form pouches. As cells could be seen
extending cytoplasmic processes laterally during migration, we propose that
chemotactic or substrate cues in the local environment guide pouch endodermal
cells to lateral positions.
Several lines of evidence indicate that the lack of pouches in
fgf8; fgf3-MO animals is probably due to a
defect in the later morphogenesis of pouch endoderm. Based on the expression
of endodermal markers such as axial and nkx2.7, pharyngeal
endoderm was present in fgf8; fgf3-MO
animals, although we do not know if mediolateral patterning of the endoderm
was completely normal. Whereas axial and nkx2.7 expression
suggest that pouch formation was defective at early stages in
fgf8; fgf3-MO animals, a lack of pouch
formation was clearly seen later using the Zn8 antibody or transplant
techniques to label endoderm. A role for Fgfs in the morphogenesis of pouch
endoderm was most evident in time-lapse recordings of endodermal development
in fgf8; fgf3-MO embryos. Whereas the
generation, medial migration and coalescence of endodermal cells were largely
normal, we saw defects in both the later lateral migration and AP positioning
of pharyngeal endodermal cells in fgf8;
fgf3-MO embryos. The migration of endodermal cells was delayed, and
the thin cytoplasmic processes characteristic of migrating cells were
disorganized in fgf8; fgf3-MO embryos.
Thus, in the absence of Fgfs, putative pouch endodermal cells had the ability
to migrate but could not orient themselves along the mediolateral axis. In
addition, whereas in wild-type embryos pouch endodermal cells migrated
laterally at periodic AP positions, in fgf8;
fgf3-MO embryos endodermal cells remained a continuous mass occupying
the anterior pharyngeal region. We propose that Fgf signaling may regulate
both the migration and AP positioning of pouch endodermal cells, and future
experiments are needed to elucidate the extent to which these processes are
interrelated.
How do neural and mesodermal Fgfs pattern pharyngeal pouches?
Our transplantation experiments demonstrated that Fgf8 and Fgf3 are
required additively in the neural keel and head mesoderm to rescue first pouch
formation in fgf8; fgf3-MO embryos.
Consistent with this, inhibition of Fgf signaling from tailbud (10 hpf) to
10-somites (14 hpf), stages at which Fgf8 and Fgf3 are expressed in the neural
keel and lateral mesoderm, blocked first pouch formation. An attractive model
is that signals from the neural keel help to pattern both the pharyngeal
endoderm and premigratory NCC (Fig.
8A). Such a strategy would link the two sources of pharyngeal
segmentation, segmentation of NCC into distinct streams and segmentation of
the endoderm into pouches, to the earlier segmentation of the hindbrain.
Intriguingly, mandibular NCC originate from ectomesenchyme adjacent to the MHB
and are Hox-negative, and the first pharyngeal pouch develops in the vicinity
of MHB-R2 and is also Hox-negative (Hunt
et al., 1991
; Miller et al.,
2004
). By contrast, the second pouch and hyoid NCC develop
adjacent to R4 and are both Hox-positive. Whereas development of the first two
pouches connects to segmental expression of Fgf in the hindbrain, it is less
clear how development of the more posterior pouches would be regulated. All
pouches were lost in animals lacking both Fgf8 and Fgf3, and transient
inhibition of Fgf signaling, at times later than those used to inhibit first
pouch formation, disrupted the formation of more posterior pouches. However,
pouches three through six develop at stages when Fgfs are no longer expressed
in the hindbrain and head mesoderm. Further analysis will be required to
determine how similar the functions of Fgfs in posterior pouch formation are
to those described here for first pouch formation.

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|
Fig. 8. Fgf8 and Fgf3 as positional determinants of pharyngeal segmentation. Model
of pharyngeal segmentation in wild type (A,B) and animals lacking Fgf8 and
Fgf3 (C,D). This model is based on fgf8 expression
(Reifers et al., 2000 ) and
fgf3 expression (Maves et al.,
2002 ) at 13 hpf (8-somites). A and C are lateral views with
anterior to the left and dorsal up, and B and D are cross-sectional views at
the level of R2. (A,B) In wild type, Fgf8 protein, dark blue, is produced in
neural MHB-R2 and R4 domains and in the lateral mesoderm. Fgf3 protein, light
blue, is co-produced with Fgf8 in the MHB and R4 (striped domains represent
overlap). Mandibular (1) NCC (Hox negative: light green) are generated
adjacent to MHB-R2 territory, whereas hyoid (2) and branchial (3) NCC (Hox
positive: dark green) have their origins at R4 and R6-R7 axial levels,
respectively. Likewise, the first (p1) endodermal pouch (Hox negative: red)
develops ventrolateral to R2, and the second (p2) and more posterior (p3+)
pouches (Hox positive: wine) form ventrolateral to R4 and R6-R7, respectively.
The ear (black circle with two dots) develops adjacent to R5. (B) A
cross-sectional view shows that during lateral migration pouch endodermal
cells are in close proximity to Fgf-expressing ventral neural keel and lateral
mesoderm. Pharyngeal pouches would form where Fgf expression in the hindbrain
coincides with Fgf8 expression in the underlying lateral mesoderm. (C,D) In
fgf8; fgf3-MO animals, NCC and pharyngeal
endoderm are generated but subsequent morphogenesis is defective. Pharyngeal
endoderm remains unsegmented and hyoid and branchial NCC streams fuse. The
structure of the hindbrain is also defective in animals lacking Fgf8 and Fgf3.
MHB, R1, R5 and R6 regions fail to develop, and R2 and R3 are reduced in size
(Brand et al., 1996 ;
Walshe et al., 2002 ;
Maves et al., 2002 ;
Reifers et al., 1998 ). As Fgfs
are required in both neural and mesodermal tissues to promote the formation of
pouches, Fgf signaling may link early neural and mesodermal patterning to
segmentation of the pharyngeal endoderm. In a direct model, Fgfs from the
lateral mesoderm and ventral hindbrain act as chemoattractants to promote the
lateral migration of pouch endodermal cells (B). In animals lacking Fgf8 and
Fgf3, pouch endodermal cells would fail to get the appropriate cues to migrate
laterally (D). Alternatively, in an indirect model, Fgfs function to regulate
the structure of, and gene expression in, the hindbrain and lateral mesoderm.
In the absence of Fgf signaling, guidance cues for pouch endodermal migration
would be reduced or absent. LM, lateral mesoderm; MHB,
midbrainhindbrain boundary; R, rhombomere.
|
|
Although we showed that Fgfs are essential for pouch morphogenesis, our
results do not distinguish between direct and indirect functions of Fgfs in
pouch outgrowth. For example, Fgfs from the lateral mesoderm may act directly
as chemoattractants for the lateral migration of pharyngeal endodermal cells.
In Drosophila, the branching of the trachea also requires Fgf
signaling (Klambt et al.,
1992
; Sutherland et al.,
1996
), and tracheal cells have been shown to migrate toward
ectopic sources of Fgf (Sato and Kornberg,
2002
). Alternatively, Fgfs may influence pouch formation
indirectly by regulating patterning of the hindbrain and lateral mesoderm. By
16 hpf, pharyngeal endodermal cells in close proximity ventrally to the neural
keel and medially to lateral mesoderm begin to migrate to form the pouches
(Fig. 8B). However, we could
block first pouch formation by inhibiting Fgf signaling from 10-14 hpf,
although we cannot rule out that there is a delay between addition of the drug
and effective inhibition of Fgf signaling. In one model, the role of Fgfs
would be to establish segmental signals in the hindbrain that control the
later lateral migration of endodermal cells at periodic positions along the AP
axis. Consistent with this, animals reduced for Fgf8 and Fgf3 have hindbrain
defects that include losses of MHB and segments R1, R5 and R6, and reductions
in size of additional rhombomeres (Walshe
et al., 2002
; Maves et al.,
2002
). If pouch endodermal cells are responding to segmental cues
in the hindbrain for their migration, the reduced size and segmentation of the
hindbrain may explain why endodermal cells did not migrate at discrete AP
positions and ultimately formed compressed clumps in
fgf8; fgf3-MO animals
(Fig. 8C,D). In addition, as
fgf8 but not fgf3-MO zebrafish have
defects in MHB structure (Reifers et al.,
1998
), a lack of early Fgf8-dependent MHB signals might explain
why fgf8 but not fgf3-MO embryos had
variable first pouch defects. Similarly, Fgf8 has been shown to control both
the gene expression profile and morphogenesis of the lateral head mesoderm
(Reifers et al., 2000
). Thus,
Fgfs might promote pouch formation indirectly by controlling the positioning
and expression of pouch guidance factors in the hindbrain and lateral
mesoderm. Future experiments that address where Fgf signaling is required, for
example by manipulating Fgf receptor function in the endoderm and other
tissues, should help to clarify how directly Fgfs act to control pouch
formation.
Pharyngeal pouches pattern cartilages of the hyoid and branchial arches
In fgf8; fgf3-MO animals, no pouches
formed and little or no cartilage was made from the Hox-expressing NCC of the
hyoid and branchial arches. Similarly, transient inhibition of Fgf signaling
during early somite stages led to correlated losses of both the first pouch
and dorsal hyoid cartilage. However, in fgf8;
fgf3-MO animals, mandibular cartilages, which are derived from NCC
that do not express Hox genes, were less affected. By contrast, cas
mutant zebrafish lack all endoderm and are missing cartilages derived from all
pharyngeal arches (David et al.,
2002
), implying that Fgf-independent endodermal signals pattern
cartilages of the mandibular arch. As pharyngeal endoderm was present but not
segmented into pouches in fgf8; fgf3-MO
animals, we conclude that the outpocketing of the pharyngeal endoderm to form
pouches is a critical event that allows endoderm to induce cartilage in
Hox-positive NCC.
By analyzing individual sides of fgf8 animals,
we found a correlation between early changes in pouch structure and later
rearrangements of the cartilage pattern. In our studies of
integrin
5 mutant zebrafish, we found that the first
pouch promotes the local compaction and survival of NCC that give rise to
specific regions of dorsal hyoid cartilage
(Crump et al., 2004
). In
fgf8 mutant sides in which the first pouch was shifted in position or
an ectopic pouch formed in presumptive hyoid NCC territory, we observed
similar positional shifts of dorsal hyoid cartilage or ectopic cartilage
elements. These correlations are consistent with the abnormal first pouch
promoting hyoid cartilage development in abnormal locations. In other cases a
deformed first pouch invaded NCC territory that, based on our previous fate
mapping (Crump et al., 2004
),
normally forms dorsal hyoid cartilage, and this deformed pouch was correlated
with a subsequent loss of dorsal hyoid cartilage. In addition, defects in the
formation of more posterior pouches were correlated with losses and fusions of
the ceratobranchial cartilages. Thus, whereas early pouch defects are largely
predictive of later cartilage alterations, the precise interpretation of the
resultant cartilage defects in fgf8 animals is
complicated by the fact that Fgf8 probably has multiple functions in
pharyngeal cartilage development.
Our finding that pharyngeal pouches were essential sources of patterning
information for the cartilages of the Hox-expressing hyoid and branchial
arches is consistent with work in chicken showing that different types of
foregut endoderm interact with Hox-expressing versus non-Hox-expressing NCC to
specify cartilage pattern. In these studies, involving the transplantation and
ablation of endoderm domains at stages prior to pouch morphogenesis, anterior
endoderm can respecify cartilage pattern when transplanted adjacent to
Hox-negative, but not Hox-positive, NCC
(Couly et al., 2002
). However,
more posterior endoderm can respecify cartilages derived from Hox-positive NCC
(Ruhin et al., 2003
). Based on
our analysis of Fgf function in zebrafish, we predict that the posterior
endoderm domains that induce cartilage from Hox-positive NCC in chicken will
include endodermal regions that form pharyngeal pouches during later
embryogenesis.
Lastly, what are the pouch-derived factors that promote cartilage
development? Recent evidence suggests that Fgfs themselves are expressed later
in the pouches and promote the survival of skeletogenic NCC. Studies in
zebrafish have shown that Fgf3 is required in the pharyngeal endoderm for the
survival of hyoid and branchial NCC (David
et al., 2002
; Nissen et al.,
2003
). As Fgf8 is also expressed, albeit less strongly, in the
pouches, it has been proposed that Fgf8 may act redundantly with Fgf3 as an
endoderm-derived NCC survival factor
(Walshe and Mason, 2003a
).
Interestingly, we did observe graded reductions of dorsal hyoid cartilage in
some fgf8 animals that had normal first pouches,
consistent with Fgf8 also having a role in the later survival of NCC.
Moreover, the lack of hyoid and branchial cartilages in
fgf8; fgf3-MO animals is most consistent
with a survival defect of postmigratory Hox-positive NCC. Based on
dlx2 expression, hyoid and branchial NCC are present early but
disappear later in fgf8; fgf3-MO animals.
However, as pouches do not form in fgf8;
fgf3-MO animals, the NCC survival defects are probably due in part to
there being no pouches to secrete survival factors such as Fgf8 and Fgf3. As
has been observed in tooth and lung development
(Chuang and McMahon, 2003
;
Jernvall and Thesleff, 2000
),
it is becoming apparent that Fgfs also have multiple, temporally distinct
functions during pharyngeal ontogeny. We propose that, in addition to a later
function as pouch-derived survival factors, an essential early function of
Fgfs in endodermal pouch morphogenesis may help explain the diversity of
craniofacial phenotypes seen in fgf8- animals.
 |
ACKNOWLEDGMENTS
|
---|
We thank John Dowd and the UO Fish Facility for abundant help with raising
fish; Jose Campos-Ortega for providing the H2A.F/Z:GFP line before
publication; Craig T. Miller, Le Trinh, Nick Osborne and Tom Schilling for
helpful discussions, especially about endoderm; and Johann Eberhart for
comments on the manuscript. J.G.C. is an O'Donnell Fellow of the Life Sciences
Research Foundation. L.M. was supported by a fellowship from the Damon
Runyon-Walter Winchell Cancer Research Fund. Research is funded by NIH grants
DE13834 and HD22486.
 |
Footnotes
|
---|
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/131/22/5703/DC1
 |
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