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First published online 23 February 2005
doi: 10.1242/dev.01723
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1 Department of Developmental Biology, University Freiburg, Germany
2 Department of Molecular Biology, Princeton University, NJ 08544-1014,
USA
3 Skirball Institute, New York University School of Medicine, NY 10016,
USA
4 Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254,
USA
* Author for correspondence (e-mail: zoltan{at}zfin.org)
Accepted 25 January 2005
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Adenohypophysis, Anterior neural plate border, Cell fate specification, Cell lineage, Distal-less, Patched, Smoothened, Sonic hedgehog, Ventral ectoderm, Zebrafish
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Dasen and Rosenfeld, 1999a;
Dasen and Rosenfeld, 1999b).
In mammals and birds, the anterior pituitary forms from Rathke's pouch, an
invagination of oral ectoderm beneath the ventral forebrain, whereas in
zebrafish, the anterior pituitary derives from a placode that forms between
the anterior hypothalamus and ventral ectoderm
(Glasgow et al., 1997).
Although several studies analyzed anterior pituitary precursor cells
(Houart et al., 1998;
Kozlowski et al., 1997;
Whitlock and Westerfield,
2000), the precise origin of precursor cells is not well
understood. Most studies suggest a dual origin for anterior pituitary; it
derives either from the anterior neural ridge or from the non-neural ectoderm
(Couly and Le Douarin, 1988;
Dasen and Rosenfeld, 1999b;
Eagleson et al., 1995;
Eagleson and Harris, 1990;
Kawamura and Kikuyama, 1992;
Kondoh et al., 2000;
Sheng and Westphal, 1999).
Several transcription factors demarcate the presumptive pituitary and lens
placodal field. Presumptive lens ectoderm in Xenopus expresses
Otx2, Pax6 and Sox3, and in zebrafish, expression of
dlx3b demarcates olfactory
(Whitlock and Westerfield,
2000; Zygar et al.,
1998) and otic placode precursor cells
(Akimenko et al., 1994;
Liu et al., 2003;
Solomon and Fritz, 2002). We
recently showed that dlx3b, dlx4b, sox9a and fibroblast growth factor
(Fgf) signaling interact to specify the zebrafish otic placode
(Liu et al., 2003). Because
dlx3b and dlx4b are also expressed around the anterior
border of the neural plate, these factors might also play a role in formation
of the anterior pituitary.
Analysis of zebrafish Hedgehog pathway mutants, you-too
(gli2) (Karlstrom et al.,
1999), iguana (Kondoh
et al., 2000) and smu (smoothened)
(Varga et al., 2001), suggests
that Hedgehog signaling is required for anterior pituitary placode
specification. The observations that zebrafish Hedgehog pathway mutants form
ectopic lenses at the expense of pituitary and that early Rathke's pouch
expresses lens Delta-crystallin in chick suggest that these apparently
unrelated tissues might arise from common precursors and that Hedgehog may be
required to specify the pituitary lineage
(Karlstrom et al., 1999;
Kondoh et al., 2000;
Varga et al., 2001). Other
studies suggest that pituitary and lens cells `transdifferentiate' in Hedgehog
pathway mutants (Kondoh et al.,
2000). In zebrafish, overexpression of Shh suggests that Hedgehog
signaling is sufficient to pattern the anterior pituitary and induce pituitary
specific gene expression (Herzog et al.,
2003; Sbrogna et al.,
2003). In mouse, Shh is thought to regulate cell proliferation and
cell-type specification (Treier et al.,
2001) indirectly (Takuma et
al., 1998).
Several homeodomain transcription factors may control specification of
pituitary cell types in a combinatorial manner and are likely targets of
signaling interactions (Dasen and
Rosenfeld, 1999a). Lhx3, for example, is expressed initially in
all cells of Rathke's pouch, is required for proliferation of ventral cell
types (Bach et al., 1995;
Zhadanov et al., 1995), and
controls gene expression in thyrotrope and gonadotrope cells in synergy with
recently identified Pitx genes (Bach et
al., 1997; Tremblay et al.,
1998). The Pitx family members belong to a
Bicoid-related subclass of homeobox genes. Pitx1 binds to
and transactivates a cis-acting element required for
proopiomelanocortin (Pomc)
(Lamonerie et al., 1996) and
other pituitary-specific gene expression, including Lhx3 and
prolactin (Szeto et al.,
1999; Tremblay et al.,
1998). In mouse, lens and mesencephalon, but not anterior
pituitary, express Pitx3. Mutations in Pitx3 lead to
dominant cataracts and malformations of the anterior eye mesenchyme and its
derivatives (Nunes et al.,
2003; Semina et al.,
2000). In Xenopus, stomodeum, lens and pituitary express
Xpitx3 (Pommereit et al.,
2001).
To understand whether Hedgehog acts directly on pituitary and lens placode precursor cells and whether Hedgehog is sufficient to induce placode formation, we used lineage tracers to define the locations and gene expression patterns of pituitary and lens precursor cells precisely. We then analyzed placode specification in Hedgehog gain- and loss-of-function experiments.
Our results indicate that by the end of gastrulation, pitx3 expression demarcates an equivalence domain of cells that gives rise to lens and pituitary. This domain overlaps partially with the dlx3b and dlx4b expression domains at the anterior neural plate border. pitx3 is required at bud stage for pituitary pre-placode formation, whereas dlx3b restricts pituitary placode size during somitogenesis. During mid-somitogenesis, Hedgehog signaling is necessary for pituitary placode specification and is sufficient to induce expression of pituitary genes in pitx3 expressing pre-placode. Hedgehog signaling is also sufficient to block pitx3 expression in presumptive lens precursors and subsequent lens tissue differentiation. We suggest that a non-Hedgehog signal initially induces an unspecified pitx3 expressing pre-placodal field around the anterior border of the neural plate. Later, during mid-somitogenesis Hedgehog signaling specifies pituitary characteristics in the placode that forms from the midline of the pre-placode.
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Materials and methods |
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|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) zebrafish
(Danio rerio) embryos were obtained and maintained with standard
procedures (Westerfield, 2000)
at 28.5°C. Developmental stages described by standard terms
(Kimmel et al., 1995) or hours
post fertilization (h). Gene symbols according to ZFIN
(http://zfin.org).
Cloning of pitx3 cDNA and phylogenetic analysis
Mouse and human Pitx2 homeodomain sequences were used to design degenerate
PCR primers: PitxF1, 5' CYAGCCAGCAGCTSCASGAGCTGGA 3'; PitxR1,
5' AGGCCCTTGGCDGCCCARTTGTTGTA 3'. These primers amplified an
275 bp fragment from a 16- to 18-somite zebrafish cDNA library (B. Appel)
that we cloned and used as probe to screen the cDNA library. A full-length
cDNA was obtained and subcloned into pCS2+ vector (Accession Number AY525643).
Parsimony and nearest neighbor joining phylogenetic analyses were performed
with Paup (Sinauer Associates), ClustalX
(Thompson et al., 1997)
(ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/)
and NJPlot software (M. Gouy, UMR CNRS 5558, Université Lyon).
Single cell labeling, lineage tracing and fate mapping
To predict the locations of cells relative to morphological landmarks and
gene expression domains in live, unlabeled embryos, we performed double in
situ hybridization on 10-15 size-selected (590±5 µm diameter),
staged embryos as described (Varga et al.,
1999) and averaged gene expression domains of pitx3 and
dlx3b. Single-cell labeling with vital fluorescent dyes and cell fate
mapping were conducted (Varga et al.,
1999) using a 10x10 grid reticule (19 mm diameter, KR406;
Zeiss) in the ocular (10x) of a fixed stage microscope (Zeiss Axioscope)
and a differential amplifier (A-M Systems, Neuroprobe Amplifier 1600). At
prim-5 stage (24 h), the descendants of the labeled progenitor cells were
analyzed using fluorescence and confocal laser scanning microscopy (Zeiss LSM
510). Serial 1.6 µm optical sections (0.5 µm overlap between consecutive
sections) were projected onto 2D images using Zeiss Software.
Whole-mount mRNA in situ hybridization, lineage tracer detection and immunofluorescence
Embryos were fixed and hybridized with one or two mRNA probes
(Hauptmann and Gerster, 1994;
Varga et al., 1999). We used
mRNA probes for pitx3 and dlx3b as landmarks for the
prospective placodal field and mRNA probes for pomc, ptc1, ptc2, nkx2.2,
lhx3, bmp2b, shh, eyes absent1 (eya1), foxb1.2 and
pax6a. Fast Red (Boehringer, Sigma) was the substrate for alkaline
phosphatase color reaction to detect one mRNA probe and the lineage tracer and
NBT and BCIP (Boehringer Mannheim) were used to detect the other mRNA probe.
This allowed us to detect fluorescein labeled mRNA probe and
fluorescein-dextran injected cells at the same time. We labeled lens fiber
cells with zl-1 (Trevarrow et al.,
1990) monoclonal antibody (Zebrafish International Resource
Center) as described (Varga et al.,
2001).
Photoactivation of fluorescein
To label groups of precursor cells, we injected one-cell stage embryos with
1% DMNB-caged fluorescein (Molecular Probes) in 0.2 M KCl. At bud stage, we
uncaged cells in regions of the dlx3b and pitx3 expression
domains that include lens or pituitary precursors. UV light directed through a
pinhole diaphragm, 40 µm diameter or larger, was used for
photoactivation and we detected uncaged fluorescein at prim-5 stage using
fluorescence and confocal microscopy (Zeiss LSM510), or antibody labeling in
combination with in situ hybridization as described above.
Morpholino antisense oligonucletide and mRNA injection
To study the roles of pitx3, dlx3b and dlx4b in pituitary
and lens formation, we injected 5-8 ng of morpholino antisense
oligonucleotides into one-cell stage embryos: pitx3 morpholino
5'-AGGTTAAAATCCATCACCTCTACCG-3'; dlx3b morpholino,
5'-ATGTCGGTCCACTCATCCTTAATAA-3'; dlx4b morpholino,
5'-GCCCGATGATGGTCTGAGTGCTGC-3'
(Liu et al., 2003). Per
embryo, we injected 8-10 ng of 8 µg/µl (in 0.2 M KCl)
pitx3 morpholino; 5-7 ng each dlx3b and dlx4b
morpholinos; and 5 ng each pitx3, dlx3b and dlx4b
morpholinos. Per embryo, we injected pitx3:eGFP (600 pg);
5
pitx3:eGFP (600 pg); shh (10-100
pg); and dominant-negative Protein Kinase A
(dnPKA:GFP, 1 ng). mRNAs were synthesized in vitro (mMessage
Machine Kits, Ambion). The embryos were analyzed morphologically at bud (10
h), prim-5 and long-pec (48 h) stages, and fixed in 4% paraformaldehyde. We
analyzed 16 µm cryosections to identify cells doubly labeled with
photoactivated fluorescein and lim3 mRNA probe.
To ensure that the pitx3 morpholinos block translation of
pitx3 mRNA, we co-injected pitx3 mRNA (see Fig. S1A-D in the
supplementary material), pitx3:eGFP mRNA that encodes the normal
5' UTR of pitx3 fused to the eGFP open reading frame
(Fig. S1E-G), or a 5'UTR that contains five modified bases
(5pitx3:eGFP) (see Fig. S1H,I in the supplementary
material).
Cell transplantation
Wild-type embryos were injected with dnPKA:GFP mRNA and
rhodamine-dextran (2.5%). The dnPKA mRNA and rhodamine-injected
embryos were used as donors at dome stage and we transplanted 10 to 20 cells
from the animal pole to wild-type host animal poles. Donor and host embryos
were maintained as pairs in 24-well dishes in penicillin and streptomycin (1%
each) in Danieau's solution. At prim-5 stage, donors were analyzed for loss of
lens phenotype that resulted from dnPKA expression; hosts were
analyzed only if the donor sibling lacked both lenses.
|
Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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|
;
Herzog et al., 2003;
Sbrogna et al., 2003). From
the 21-somite stage (19.5 h) through prim-10 stage (27 h), lens, pituitary,
head mesenchyme and ventral diencephalic cells express pitx3
(Fig. 1G-J) as well as
mesenchymal cells of the presumptive first branchial arch
(Fig. 1K,L). In trunk somites,
presumptive muscle pioneers express pitx3
(Fig. 1M). At long-pec (48 h)
and protruding-mouth (72 h) stages, ventral diencephalic cells and lens fiber
cells downregulate pitx3 expression
(Fig. 1N). In addition, we find
that presumptive nucleus coeruleus cells in the first rhombomere
(Holzschuh et al., 2001) and
tegmental cells express pitx3
(Fig. 1O). At protruding-mouth
(72 h) stage, pituitary cells and the primordia of the first and second
mandibular arches express pitx3
(Fig. 1P).
|
). Because Pax6 has been shown to regulate both lens and eye
development (Ashery-Padan and Gruss,
2001; Kondoh,
1999; Zygar et al.,
1998), we did not use pax6a and pax6b as neural
plate landmarks or in functional assays. However, because dlx3b has
been shown to demarcate prospective olfactory
(Kondoh, 1999;
Whitlock and Westerfield,
2000; Zygar et al.,
1998) and otic placode cells
(Akimenko et al., 1994;
Liu et al., 2003), we compared
pitx3 expression with dlx3b expression
(Fig. 2E,F) and find that the
expression domains overlap significantly
(Fig. 2F).
To understand the spatial relationship between Hedgehog-producing cells and
placodal precursors, we analyzed where pitx3-positive cells are
located relative to median shh-expressing cells. Because polster
cells express hgg1 at bud stage just beneath the superficial
pitx3-expressing ectoderm (Fig.
2D), we also used hgg1 expression to correlate the
locations of median neural plate border cells, ventral ectoderm and
shh expression. At bud stage, we find that prechordal plate cells and
cells in the midline of the neural plate express shh
(Fig. 2G). Thus, the median
ectodermal cells that express pitx3 near the polster, are near
Hedgehog-secreting cells in the median neural plate
(Fig. 2G), suggesting that
these cells are in a position to receive significant levels of Hedgehog
signal. We also analyzed other genes that demarcate ventral ectoderm and the
border of the neural plate such as bmp2b
(Fig. 2G) and eya1
(Fig. 2H). However, we did not
use the eya1 expression domain to establish the pituitary and lens
fate map, because eya1 expression at the anterior neural plate border
overlaps with the dlx3b expression domain
(Fig. 2H) that gives rise to
olfactory placode (Whitlock and
Westerfield, 2000). Although eya1 is expressed in the
olfactory placode at prim-5 (24 h) stage, it is downregulated in lens during
late somitogenesis (Sahly et al.,
1999).
Pituitary precursor cells are mis-specified in smoothened mutant embryos
We have previously shown that smoothened mutant embryos that
cannot transduce Hedgehog signals lack pituitary and instead form ectopic
lenses (Varga et al., 2001)
(see Fig. S2 in the supplementary material). This could occur because median
pituitary precursors are mis-specified and form lens at the expense of
pituitary or, alternatively, because lens precursors aberrantly invade the
midline.
To distinguish between these possibilities, we fate mapped the cells that give rise to pituitary and lens in wild-type and smoothened mutant embryos. We labeled cells at bud stage, using pitx3 and dlx3b as landmarks for lens and pituitary precursors, and analyzed their progeny at prim-5 stage.
In wild-type embryos, we find that pitx3- and dlx3b-expressing cells at the midline of the anterior neural plate border contribute to pituitary (Fig. 3B, and red dots in 3A) or olfactory placodes (Fig. 3A, yellow dots). Precursors closer to non-neural ectoderm mainly form pituitary cells (Fig. 3A, red dots) or ventral ectoderm (Fig. 3A, green dots), and cells closer to the neural plate have a greater tendency to contribute to olfactory placode (Fig. 3A, yellow dots).
|
Lens cells derive from lateral positions in the pitx3 and
dlx3b expression domains (Fig.
3A, blue dots). They intermingle with precursors of non-neural
ectoderm on the ventral side (Fig.
3A, green dots) and with olfactory placode precursors closer to
the neural plate (Fig. 3A,
yellow dots). Thus, we find that lens precursors
(Fig. 3A, blue dots) are
usually farther from the neural plate and express pitx3, whereas
dlx3b-expressing cells closer to the neural plate mainly contribute
to olfactory placode (Fig. 3A,
yellow dots). The large degree of intermingling of these two placodal cell
fates is consistent with the overlap of the two gene expression domains and
indicates that placodal fields have not yet resolved by this developmental
stage (Whitlock and Westerfield,
2000).
In smoothened mutant embryos, lateral pitx3- and dlx3b-expressing cells also give rise to lens (Fig. 3E,F). In the midline, however, in the region that corresponds to the location of pituitary precursors in wild-type embryos (Fig. 3A,B), precursors (dark blue dots) give rise to daughter cells in ectopic (Fig. 3G), distorted (Fig. 3H) and fused lenses (Fig. 3I). Thus, in the absence of Hedgehog signal transduction, median placode precursors are mis-specified and form lens instead of pituitary.
Lens precursor cells do not invade the midline in smoothened mutant embryos
We tested whether lens precursors invade the midline of the anterior neural
plate border in smoothened mutant embryos by photoactivating
DMNB-caged fluorescein in lens precursors in wild types and mutants. Because
large numbers of cells are labeled, this approach allows us to track cell
movements that might have been missed previously with single cell labeling.
When we uncage fluorescein in the anterior midline of the pitx3
expression domain at bud stage (Fig.
4A,B), we find that pituitary and skin are labeled at prim-5 stage
in wild-type embryos (Fig. 4E; arrowhead). In smoothened mutant embryos, however, when we uncage in
the anterior midline, the ectopic median lens
(Fig. 4F; arrowhead) and skin
are later labeled (Fig. 4B,F).
When we uncage in cells farther lateral in the pitx3 and
dlx3b expression domain (Fig.
4C,D), we find that in both wild-type and smoothened
mutant embryos, skin, lens, olfactory placode, telencephalon and retina are
labeled later (Fig. 4G,H).
Typically, almost the entire retina associated lens is labeled in wild-type
and mutant embryos, whereas in smoothened mutant embryos, the ectopic
median lens is never labeled (Fig.
4H; arrowhead). Our results indicate that in smoothened
mutant embryos, lens precursors do not invade the midline aberrantly and that
apparently the entire pitx3 expression domain gives rise to lens.
Thus, lateral cells contribute to the retinal lens and median cells that form
pituitary in wild-type embryos are mis-specified and form ectopic lens in
smoothened mutants.
|
), the
Hedgehog target gene nkx2.2 and pomc
(Herzog et al., 2003), a gene
expressed in corticotropes and melanotropes. We find that `knock-down' of
pitx3 causes reduction or complete loss of lim3, nkx2.2 and
pomc expression in pituitary (Fig.
5A-F), indicating that pitx3 is required for pituitary
cell specification. We find that pitx3 is required in a
dose-dependent manner; high doses of morpholinos (8-10 ng/embryo) lead to
complete loss of pituitary placode (Fig.
5D-F), whereas less morpholino (2-4 ng/embryo) leads to a smaller
placode, and variably reduced or lost expression of lim3, nkx2.2 and
pomc (Fig. 5D, inset).
By contrast, injection of dlx3b and dlx4b morpholinos
increases both pituitary placode size and cell number
(Fig. 5G-I), indicating that
dlx3b and dlx4b function as negative regulators in pituitary
placode development. To test epistatic relationships between pitx3
and dlx3b-dlx4b, we injected embryos with all three
morpholinos. We find that expression of lim3, nkx2.2 and
pomc is reduced or entirely lost at prim-5 stage
(Fig. 5J-L), comparable with
embryos injected with pitx3 morpholinos alone. Together, these
observations suggest that pitx3 is epistatic to dlx3b and
dlx4b, and that Dlx3b-Dlx4b is required to restrict pituitary cell
number and placode size. By contrast, Pitx3 is required for pituitary placode
formation and cell type specification.
|
).
Thus, our results suggest that at the end of gastrulation, pituitary
precursors require Pitx3 function for pitx3 expression and subsequent
pituitary pre-placode formation, whereas Dlx3b and Dlx4b act on lens
precursors at bud stage and on the median pre-placode later, during
somitogenesis.
|
Hedgehog overexpression blocks lens formation and induces ectopic expression of pituitary genes
Loss of Hedgehog signal transduction results in mis-specification of
pituitary precursor cells and subsequent ectopic lens formation
(Fig. 3) (Karlstrom et al., 1999;
Varga et al., 2001).
Conversely, overexpression of shh suppresses lens formation
(Barth and Wilson, 1995) and
increases the number of ventral, anterior pituitary cell types
(Herzog et al., 2003;
Sbrogna et al., 2003).
Consistent with these previous observations, we find that increasing amounts
(10-100 pg/embryo) of synthetic shh mRNA block lens tissue formation
(Fig. 7A-D) and, in parallel,
downregulate lens pitx3 expression
(Fig. 7E-G,I-K). Although
pitx3 expression expands in branchial arches
(Fig. 7K) and into the
epithalamus (not shown), the size of the pituitary placode as such is
unaffected (Fig. 7I-K, insets).
Interestingly, at high levels of shh mRNA (100 pg/embryo),
prospective ventral ectoderm at bud stage
(Fig. 7H) and cells covering
the retina at prim-5 stage (Fig.
7L) upregulate pitx3 expression in a pattern reminiscent
of normal bud stage pitx3 expression
(Fig. 7E, compare with
Fig. 7H,L). This observation
suggests that pitx3-expressing cells respond to Hedgehog
differentially, depending on their position in the embryo and the source of
Hedgehog signal. During normal development, median cells might acquire
pituitary characteristics because of their proximity to Hedgehog signal,
whereas in lateral regions absence of Hedgehog signal might favor lens
specification. Thus, at the end of gastrulation, pitx3-expressing
precursor cells might be competent to respond to Hedgehog signal directly, or
by Hh induced, secondary signaling interactions.
|
;
Sbrogna et al., 2003).
Co-injection of shh mRNA (100 pg per embryo) and pitx3 MO (8
ng per embryo) completely eliminates lim3 expression from median and
ectopic locations (Fig. 7O)
suggesting that pitx3 is required for pituitary cell specification
when Hedgehog is overexpressed. In response to high levels of Hedgehog,
ectopic pomc-expressing cells are scattered on top of the forebrain
and retina (Fig. 7Q,R) and in
some embryos, form small, flattened clusters of cells that resemble miniature
lenses (Fig. 7R, arrowheads).
This observation supports the idea that lens precursors form in their normal
positions but express pomc in response to shh. We further tested the possibility that Hedgehog induces pituitary gene expression in lens precursors by co-injecting caged-fluorescein and shh mRNA (100 pg per embryo) into one-cell stage embryos. We uncaged fluorescein in prospective lens and skin precursors (Fig. 7S) and analyzed these embryos by in situ hybridization with probes for lim3 and by antibody labeling to detect caged-fluorescein. We find that shh mRNA-injected embryos lack lens and have ectopic lim3-expressing cells near the eyes doubly labeled with fluorescein (16/16 embryos; Fig. 7T, arrowheads). Because lens is entirely absent in these embryos, doubly labeled cells indicate that (at least some) lens/nasal precursors express pituitary genes in response to shh mRNA injection. Thus, loss of lens tissue and ectopic pitx3, lim3 and pomc expression suggest that Hedgehog signaling suppresses lens development and induces gene expression characteristic of the pituitary cell lineage in derivatives of pitx3 expressing lens precursors.
Hedgehog acts indirectly to regulate pituitary and lens fates
To test whether pitx3-expressing cells are competent to respond to
Hedgehog and, as a result, express genes characteristic of pituitary, we
analyzed whether shh induces ptc genes in pituitary or lens
precursors. At bud stage, ptc1 is expressed in median neural plate
cells (Fig. 8A,C), but not in
pitx3-expressing cells at the anterior neural plate border that give
rise to lens or pituitary. In embryos injected with shh mRNA (100
pg/embryo), pituitary and lens precursors that express pitx3, but not
dlx3b, do not upregulate ptc1
(Fig. 8B,D). By contrast, cells
throughout the neural plate and dlx3b-expressing neural plate border
cells, upregulate ptc1 in response to shh injection
(Fig. 8D). Thus, even though
Hedgehog induces pituitary gene expression, is required for pituitary
specification, and is sufficient to block lens formation, placodal precursor
cells that express only pitx3, do not express ptc1 and
ptc2 (data not shown) in response to shh mRNA injection.
|
;
Sbrogna et al., 2003) during
mid-somitogenesis when pituitary and lens placodes form. However, the
observations that shh overexpression fails to upregulate ptc
expression in pituitary precursors at bud stage or to affect median placode
size suggest that Hedgehog acts indirectly on pituitary and lens precursors at
bud stage. To test this idea, we injected one-cell stage donor embryos with
biotin-rhodamine dextran and dnPKA that activates the Hedgehog
signaling cascade cell autonomously. We then transplanted groups of cells from
these donor embryos into uninjected blastula stage hosts. We find that
ptc1 is upregulated before the 18-somite stage in transplanted cells
on top of the eye vesicle (Fig.
8E). However, at prim-5 stage, we find that donor derived cells
that express dnPKA contribute to lens
(Fig. 8F) and do not express
pituitary genes (not shown), even though all corresponding donor embryos
completely lack lens. We also find that dnPKA-expressing cells
contribute to lens, regardless of whether large groups
(Fig. 8F) or only a few cells
(not shown) have been transplanted. Thus, even though overexpression of
Hedgehog blocks lens formation and induces ectopic pituitary gene expression
(Fig. 7), cell-autonomous
activation of the Hedgehog signaling cascade does not induce pituitary marker
genes or prevent cells from contributing to lens. Because injected mRNA is
degraded during the first day of development, this result supports the
hypothesis that Hedgehog signaling acts indirectly on lens precursors during
early stages of development. Presumably, block of lens formation and induction
of ectopic pituitary cells in shh mRNA-injected embryos is an event
downstream of Hedgehog signaling due to changes in other cellular interactions
and signaling systems regulated by Hedgehog. |
Discussion |
|---|
|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
) and function to limit the size of the pituitary placode
(Figs 5,
6). Both Pitx3 and Dlx3b-Dlx4b
are required for pituitary and lens specification and differentiation
(Fig. 5; see Fig. S3 in the
supplementary material). Our results support the idea that pitx3
demarcates an equivalence domain, because in the absence of Hedgehog
signaling, median Pitx3-expressing cells are mis-specified and contribute to
ectopic or medially fused lenses (Kondoh
et al., 2000; Sbrogna et al.,
2003; Varga et al.,
2001). In addition, we show that Hedgehog signaling induces genes
in lens precursors characteristic of pituitary placode, further supporting the
view that pitx3-expressing precursor cells have equivalent potential
to form lens or pituitary (Figs
7,
8). Our results also indicate
that cell fate depends on the level of Hedgehog signal, even though lens and
pituitary precursors are not competent to respond to Hedgehog at the end of
gastrulation (Figs 7,
8). Pituitary precursors do not
express Ptc genes at bud stage, and lens precursors form lens in spite of
cell-autonomous activation of the Hedgehog signaling pathway. Thus,
overexpression of Hedgehog might lead to activation of other mechanisms that
affect patterning and specification of the placodal field. We suggest that
initially an unspecified placodal field is formed, in a Hedgehog independent
manner, mediated by Pitx3, Dlx3b and Dlx4b interaction. The placodal field is
subsequently subdivided into an outer, pitx3-expressing pre-placode
with lens epithelial cell character and an inner,
dlx3b-dlx4b-expressing olfactory pre-placode
(Fig. 9). During
mid-somitogenesis, Hedgehog is sufficient and required to induce pituitary
genes in the outer pre-placode, and thus directs it to form median pituitary
and lateral lens placodes.
|
;
Gage et al., 1999).
Pitx1 expression is required for development of Rathke's pouch and
activation of pituitary pomc and prl expression
(Szeto et al., 1999;
Tremblay et al., 1998).
However, midbrain dopaminergic neurons, eye mesenchyme and lens, but not
pituitary, express Pitx3. Mouse aphakia mutants and mice
lacking functional Pitx3 promoter elements fail to develop
differentiated lenses (Nunes et al.,
2003; Semina et al.,
2000) but have no apparent pituitary defects. Consistent with
these studies, human fetal mesencephalon, but not anterior pituitary,
expresses PITX3
(Pellegrini-Bouiller et al.,
1999). By contrast, the Xenopus ortholog,
Xpitx3, is expressed in presumptive stomodeum, lens and pituitary
(Pommereit et al., 2001); and
our results show that, in zebrafish, pitx3 is also expressed in
prospective lens and pituitary and that embryos injected with pitx3
morpholinos fail to differentiate primary lens fiber cells or pituitary
placode (Figs 5,
6).
pitx3 expression demarcates an equivalence domain with transient lens character in non-neural ectoderm
Our results provide a genetic mechanism to explain the placode forming
competence of non-neural ectoderm. In Xenopus, apparently all
non-neural head ectoderm, including the midline, has a transient lens-forming
bias (Henry and Grainger,
1987; Zygar et al.,
1998). We show that median placodal cells are mis-specified and
give rise to lens in smoothened mutant embryos
(Fig. 3). This suggests that
the pitx3 expression domain demarcates an equivalence domain at the
end of gastrulation, because the placodal field gives rise to a single (fused)
placode with lens character. Formation of ectopic and fused lenses in
smoothened mutants further indicates that Hedgehog signaling from
neural plate puts an end to the lens-forming capacity of median non-neural
ectoderm and specifies pituitary fate instead. Consistent with this idea,
overexpression of Shh suppresses lens formation
(Barth and Wilson, 1995) and
induces pituitary genes in lens derivatives (Figs
7,
8).
We also show that loss of Hedgehog signal transduction leads to increased
olfactory placode size (see Fig. S1 in the supplementary material). In lateral
regions of the placodal field, we find that lens and olfactory precursor cell
populations intermingle only where the pitx3 and dlx3b
expression domains overlap. By contrast, median pitx3 and
dlx3b domains overlap significantly and olfactory and pituitary
precursors intermingle to a high degree in this region, presumably because the
placodal fields of olfactory and pituitary placode have not segregated from
each other by bud stage (Whitlock,
2004). In smu mutant embryos, olfactory and ectopic lens
precursor cells intermingle near the midline. Although we do not find
increased numbers of olfactory precursor cells in smu mutants
(Fig. 3B), which might provide
an explanation for the larger olfactory placodes, we can not exclude the
possibility that median precursor cells have an equivalent potential to
contribute to lens, pituitary and olfactory placode before segregation of
median placodal fields. Nevertheless, our lineage analysis of median,
pitx3-positive precursor cells strongly supports an equivalence
domain for at least lens and pituitary fates.
Pitx3, Dlx3b and Dlx4b regulate pituitary placode size at different stages of development
Our observations provide evidence for a novel mechanism that regulates the
size of the pituitary placode. We find that Pitx3 is required for pituitary
placode specification and formation (Fig.
5D-F) at the end of gastrulation (Figs
5,
6). Previous studies have
implicated Dlx3b and Dlx4b in olfactory and otic placode formation
(Whitlock and Westerfield,
1998; Whitlock and
Westerfield, 2000; Solomon and
Fritz, 2002; Liu et al.,
2003; Hans et al.,
2004). We find that at bud stage, Pitx3, Dlx3b and Dlx4b are
expressed in partially overlapping domains, and that reducing Dlx3b and Dlx4b
increases the size of the pituitary placode and prevents lens fiber cell
specification (Fig. 5; see Fig.
S3 in the supplementary material). Our results indicate that Dlx3b-Dlx4b and
Pitx3 may not interact directly, because loss of Dlx3b and Dlx4b functions
leads to downregulation of pitx3 expression in lens precursors at bud
stage, but does not affect pituitary pitx3 expression. Similarly, the
size of the pituitary pre-placode increases during mid-somitogenesis when
Dlx3b and Dlx4b function is reduced (Fig.
6). Because Dlx3b and Dlx4b are required for olfactory placode
formation, the enlargement of the pituitary pre-placode in their absence
further supports the idea that intermingled olfactory and pituitary precursors
at bud stage might have equivalent potential to form nose or pituitary. This
possibility is also supported by split pituitary and olfactory fates that
arise from single precursor cells that express both pitx3 and
dlx3b (Fig. 3).
We suggest that the initial overlap between the pitx3, dlx3b and
dlx4b expression domains in the placodal field may eventually result
in an outer pre-placodal domain, demarcated by pitx3 expression
(Fig. 9) and fated to give rise
to pituitary and lens, and in an inner dlx3b and dlx4b
positive domain, fated to give rise to olfactory placode
(Whitlock and Westerfield,
2000). Initially, the entire outer pre-placodal domain may have
potential to form lens (Kamachi et al.,
1998; Ueda and Okada,
1986). Observations in chick that the early anterior pituitary
anlage expresses the lens protein, 
-crystalline
(Kamachi et al., 1998;
Ueda and Okada, 1986) are
consistent with this interpretation. Pax6 and other transcription factors are
thought to mediate the competence of head ectoderm to form lens in response to
inductive signals (Kondoh,
1999). Our results show that pitx3 is required for
specification of lens (see Fig. S3 in the supplementary material) and
pituitary placodes (Fig. 5).
Moreover, pitx3 is required for lim3 gene expression during
normal development. Therefore, we suggest that Pitx3, in addition to Pax6 and,
presumably, other factors, mediates the response of placode precursor cells
and placodal cells to signaling interactions.
Non-Hedgehog signaling establishes the lens-pituitary placodal field, but Hedgehog induces pituitary character in the median placode
Previous studies showed that loss of Hedgehog signaling results in a
conversion of pituitary to lens (Karlstrom
et al., 1999; Kondoh et al.,
2000; Varga et al.,
2001) and have led to the generally accepted hypothesis that a
major function of anterior midline Hedgehog expression is to specify the
pituitary placode and prevent it from forming a median lens. The observation
that ectopic, cell non-autonomous activation of Hedgehog signaling induces
pituitary gene expression in at least some lens precursors is consistent with
this interpretation. However, we find that overexpression of Shh does not
increase pituitary placode size and that in smoothened mutants, a
median placode (albeit of lens character) still forms. This indicates that
Hedgehog signaling is neither sufficient nor required for median placode
formation. Nevertheless, changes in Hedgehog signaling affect pituitary cell
proliferation and specification in mouse and zebrafish
(Herzog et al., 2003;
Sbrogna et al., 2003;
Treier et al., 2001). We
suggest (Fig. 9) that formation
of the lens and pituitary pre-placode is independent of Hedgehog signaling;
however, once the placode forms, Hedgehog from ventral forebrain specifies
pituitary character in the median placode
(Herzog et al., 2003;
Sbrogna et al., 2003;
Treier et al., 2001). We
further suggest that non-Hedgehog signaling interactions from neighboring
tissues specify or block lens character in the lateral placodes, because
cell-autonomous activation of the Hedgehog pathway does not prevent lens
formation (Fig. 8). Several
lines of evidence suggest that BMP is required and sufficient to promote lens
fiber cell differentiation in lens epithelial cells
(Belecky-Adams et al., 2002;
Faber et al., 2002). Thus,
localized BMP, together with Hedgehog signaling, may mediate the subsequent
differentiation of unspecified pre-placodal cells into lens fiber cells or
hormone-producing pituitary cells.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/7/1579/DC1
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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