|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 4 July 2007
doi: 10.1242/dev.02872
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Institute for Developmental Biology, University of Cologne, Gyrhofstrasse 17,
50923 Köln, Germany.
2 Skirball Institute of Biomolecular Medicine, New York University School of
Medicine, New York, NY 10016, USA.
3 Max-Planck-Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Author for correspondence (e-mail:
klaus.rohr{at}uni-koeln.de)
Accepted 24 May 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Thyroid, Zebrafish, hands off (han, hand2), acerebellar, Fibroblast growth factors, fgf8, Fate mapping, Heart
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
);
endothelial cells (Lammert et al.,
2001); and the notochord (Kim
et al., 1997) are involved in induction of the pancreas from the
endoderm. For both liver and lung primordia, cardiac tissue adjacent to the
endodermal layer has been identified as a source of inducing signals
(Gualdi et al., 1996;
Jung et al., 1999;
Serls et al., 2005).
Understanding the processes specifying different domains of the primitive gut
can provide insight into congenital defects in humans and into regenerative
responses to tissue damage.
Whereas increasing details of the inductive steps in the lung, liver and
pancreas are emerging, our knowledge of such early developmental processes in
thyroid development is scarce. As an anterior derivative of the primitive gut,
the thyroid primordium buds from the ventral midline of the primitive pharynx
(reviewed in De Felice and Di Lauro,
2004). During a relocation process, the primordium loses contact
with the pharynx, adopting a species-specific position in the hypopharyngeal
mesenchyme. In many vertebrates, including mice and man, the primordium
bifurcates in the neck area, leading to its final bilobed shape, whereas, in
zebrafish and other teleosts, thyroid tissue forms an elongated strand of
tissue along the ventral aorta (Wendl et
al., 2002). Development of the thyroid is comparable between fish
and mammals on the molecular level. The thyroid-specific transcriptional
programme, including the transcription factors Nkx2.1 (also known as Nk2.1a
and Titf1a/TITF1), Pax8 and Hhex, is conserved with respect to expression
patterns and function between zebrafish and mouse
(Elsalini et al., 2003;
Wendl et al., 2002). In this
study, we use zebrafish as a model to investigate the initiation of thyroid
development.
In mouse development, induction of lung and liver by cardiac mesoderm was
anticipated because of the close association of cardiac mesoderm with lung and
liver primordia (Gualdi et al.,
1996; Jung et al.,
1999; Serls et al.,
2005). Similarly, early mouse thyroid markers start to be
expressed in the primitive pharynx adjacent to the aortic sac
(Fagman et al., 2005)-the
cardiac region that gives rise to the embryonic outflow tract and cervical
arteries. This spatial correlation appears to be conserved in zebrafish, in
which initial thyroidal nk2.1a expression starts, at 24 hours
post-fertilisation (hpf), adjacent to the outflow tract of the heart
(Rohr and Concha, 2000).
Earlier in development, during the zebrafish somitogenesis stages, the
anterior lateral plate mesoderm (aLPM), from which the heart later develops,
as well as the endoderm, converge in parallel processes to the midline
(Keegan et al., 2004;
Warga and Nusslein-Volhard,
1999). Parallel development and close association of both tissues
is reflected in a functional relationship. The endoderm is necessary for
normal cardiac morphogenesis and, in its absence, the converging halves of the
aLPM fail to fuse (Alexander and Stainier,
1999).
Fibroblast growth factors (FGFs) constitute a large family of signalling
molecules that have been shown to act in multiple ways on endoderm-derived
organ development. FGF1 and FGF2 are crucial for induction of lung and liver
in mammals (Jung et al., 1999;
Serls et al., 2005), and
tissue explant assays suggest cardiac tissue to be the source of the signals.
Moreover, in tissue-explant assays, these FGFs act in a
concentration-dependent manner, with high concentrations required for lung,
and lower concentrations for liver, induction
(Serls et al., 2005). However,
an exclusive role is not supported by the phenotype of FGF1/FGF2
double-knock-out mice, which are viable
(Miller et al., 2000b).
In this study, we show that the zebrafish mutant hands off
(han, hand2) has severe defects in early thyroid development. The
han locus encodes the bHLH transcription factor Hand2
(Yelon et al., 2000). Research
on this mutant so far has concentrated on defects correlating with known sites
of han expression, including the cardiac mesoderm, the fin buds and
the pharyngeal arches (Angelo et al.,
2000; Miller et al.,
2003; Yelon et al.,
2000). Two han alleles have been isolated,
hans6, which has a deletion spanning a maximum of 100 kb,
including the han locus, and hanc99, which has an
insertion in the han locus. hans6 is a null
mutation, and homozygotes exhibit a stronger phenotype than
hanc99 mutants (Yelon
et al., 2000). A role of han in thyroid development has
not been described before and represents a novel aspect in thyroid research.
In grafting experiments, we show that the han gene is required in the
surrounding tissue for thyroid development. Further studies suggest that it is
han-expressing anterior plate mesoderm or cardiac mesoderm that is
crucial for thyroid specification. We further show that FGF signalling is
required for thyroid development in zebrafish, and that FGF-coated beads are
able to restore thyroid development in hans6 mutants.
Thus, our study provides a first step towards understanding the role of
surrounding tissue during thyroid specification.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Rohr and Concha, 2000;
Wendl et al., 2002). We used
the hans6, hanc99
(Yelon et al., 2000) and
aceti282a (fgf8ti282a)
(Brand et al., 1996) alleles.
The identity of homozygotes was possible based on morphology. In addition, the
identity of homozygous han mutant embryos was always confirmed by
MF20 immunostaining visualising myocardial morphology.
Embryonic manipulation
As the lineage tracer for grafting experiments, we injected biotin-dextran
(10,000 Mr, 5 mg/ml; Molecular Probes) into zebrafish embryos and
detected biotin-labelled donor-derived cells after in situ hybridisation using
the ABC kit (Vector Laboratories). In grafted embryos, the peroxidase reaction
against biotin-dextran was carried out in DAB medium containing 67 mM
NiCl2, resulting in black donor-derived cells. This first reaction
was followed by MF20 immunostaining using normal DAB medium, resulting in
brown staining of the myocardium.
For fate mapping of thyroid precursor cells, photoactivation of caged
fluorescein was essentially carried out as described previously
(Keegan et al., 2004).
Morpholino oligonucleotides targeted against endothelin 1 (edn1-MO)
(Miller and Kimmel, 2001),
lockjaw (tfap2a; 3.1-MO)
(Knight et al., 2003) and
foxi1 (foxi1-MO) (Mackereth et
al., 2005), as well as an unspecific control morpholino (Gene
Tools), were used as described previously. Implantation of beads was performed
as described (Reifers et al.,
2000) in low-melting-agarose-embedded embryos. Beads (45 µm
Microspheres, Polysciences) were soaked overnight in 250 µg/ml human
recombinant FGF1 (Sigma, St Louis, USA) or 100 µg/ml human recombinant FGF2
(Roche, Indianapolis, USA), in both FGFs together, or in 250 µg/ml mouse
recombinant FGF8b (R&D Systems, Minneapolis, USA) or 250 µg/ml BSA, all
dissolved in PBS.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In zebrafish, thyroid markers, such as nk2.1a, hhex and
pax2.1, start being expressed in presumptive thyroid precursor cells
in the endoderm at around 24 hpf (Elsalini
et al., 2003; Rohr and Concha,
2000). In 24-28 hpf hans6 mutants, expression
of pax2.1 and hhex was always absent
(Fig. 1E-H). nk2.1a
expression was absent at this stage in most mutants, but, in about 10% of
mutants, could be detected in a few endodermal cells
(Fig. 1I-K), indicating that
the thyroid phenotype is variable or not fully penetrant. Later, at 55 hpf,
expression of nk2.1a, pax2.1 and hhex was not detectable in
any domain that would indicate a thyroid in hans6 (data
not shown). In some clutches, occasional faint nk2.1a expression in
few cells of the hypopharyngeal area indicated that some thyroid cells might
be specified in hans6 mutants (barely detectable; data not
shown).
The differentiation marker slc5a5, encoding the sodium iodide
symporter (NIS), is expressed exclusively in thyroid follicle cells from about
40 hpf (Alt et al., 2006).
slc5a5 expression was usually not detectable in
hans6 mutants at 60 hpf
(Fig. 1L,M), although we found
a strongly reduced expression domain in 23 out of 258 homozygous specimen (9%;
Fig. 1N). Taken together, the
absence of the thyroid gland in hans6 mutants can be
explained by the lack of the early primordium in most specimens. In a small
proportion of homozygous embryos, a reduced number of thyroid precursor cells
were present and started the differentiation programme, but eventually failed
to form a mature gland. Because it is hardly conceivable that slc5a5
is expressed in the complete absence of thyroid-specific developmental genes,
we assume that, in some mutants, remaining low levels of developmental genes
are sufficient to initiate slc5a5 expression in some cells, but not
sufficient for all aspects of terminal differentiation.
To find out whether increased cell death might be responsible for the absence of the thyroid primordium in hans6 mutants, we carried out TUNEL assays. However, we did not observe visibly increased cell death in the pharyngeal endoderm or in the area where the thyroid would develop [tested at the 16-somite stage (ss), 20 hpf and 24 hpf; data not shown]. It should be noted that the thyroid primordium is very small, so it is possible to miss its few precursor cells undergoing cell death.
In hans6 mutants, the deletion might affect the expression of a neighbouring locus, and so we tested the second available, hypomorphic hanc99 allele for thyroid defects. Here, from the beginning of detectable marker gene expression, the thyroid primordium was reduced in size, albeit not absent (Fig. 1O-Q). The reduced size of the primordium persisted during development and resulted in a smaller differentiated gland at the larval stages (Fig. 1Q). The fact that thyroid development in hanc99 mutants followed a similar, albeit less-severe, phenotypic trend to that of hans6 mutants confirms that it is the han locus in the hans6 deletion that is involved in thyroid development. Furthermore, this observation suggests a dosage-sensitive requirement for Hand2 during thyroid development.
han is expressed in tissues including and surrounding the site of initiation of thyroid development
We reinvestigated han expression in the area in which thyroid
markers start to be expressed. Here, han was expressed in the heart
tube and the roots of the first pair of branching arteries, and, in addition,
in the neural crest-derived mesenchyme of the pharyngeal arches
(Fig. 2A,B). Furthermore,
strong han expression was detectable in a set of bilateral cells at
the border between the first and second arch, on the same anteroposterior
(a-p) level as thyroid marker expression, but more lateral. These cells were
directly adjacent to tyrosine-hydroxylase-positive cells called
arch-associated neurons (AANs; Fig.
2A-D), which are presumably the precursor cells of the carotid
bodies (Holzschuh et al.,
2001). It is likely that these two bilateral and distinct groups
of han-expressing cells form part of the carotid bodies.
|
|
Fate mapping of thyroid precursor cells reveals their close association to the aLPM
Earlier fate-mapping studies have shown that both endoderm and cardiac
mesoderm converge medially to the embryonic axis during the somitogenesis
stages (Keegan et al., 2004;
Warga and Nusslein-Volhard,
1999). Comparison of han expression with the endodermal
marker foxa3 (fkd2) shows that, at the 8 ss, bilateral
stripes of endodermal cells are distributed with aLPM cells in a partially
overlapping fashion, with some endodermal cells being closer to the midline
(Fig. 3A). We wanted to know
where prospective thyroid precursor cells are located in relation to the
han expression in the aLPM.
For this fate-mapping approach, we injected caged fluorescein into embryos at the one-cell stage and photoactivated the fluorescein dye at around the 8 ss. This stage was chosen because it is when the endoderm has not yet reached a position ventral to the neural tube during its convergence movements and is therefore accessible for photoactivation. As landmarks along the a-p axis, we used the posterior end of the eye, the midbrain-hindbrain boundary (MHB) and the anterior tip of the notochord to subdivide the aLPM into four zones (Fig. 3B). Nomarski optics allowed for visualisation of the aLPM edge. During photoactivation, we targeted cells along the medial border of the aLPM in zone 1 to zone 4 (Fig. 3C-E) and probed their contribution to the thyroid at 55 hpf.
|
). To get a rough estimation of han expression in relation to the MHB during subsequent somitogenesis stages, we compared the expression of han with that of pax2.1, a MHB marker. han expression in the aLPM had its anterior border on the level at the MHB throughout the somitogenesis stages (Fig. 3H-M). Thus, han expression in the cardiac mesoderm is always ventral to the MHB. Even if thyroid precursors are only roughly associated with the MHB along the a-p level, it is likely that the han-expressing cardiac mesoderm is continuously close to thyroid precursors throughout the somitogenesis stages.
Grafted wild-type cells can restore thyroidal nk2.1a expression in han s6 mutants in a non-cell-autonomous manner
To find out whether han is cell-autonomously required in the
endoderm for initial thyroid development or is required non-cell-autonomously
in adjacent structures, we created genetic mosaics by the transplantation of
wild-type donor cells into hans6 mutant hosts. We first
tested whether wild-type grafted cells are capable of expressing han
in the hans6 environment. Embryos were fixed at the 12 ss,
when han is broadly expressed in the anterior lateral plate mesoderm.
In hans6 mutant embryos, han expression was
completely missing because of the deletion, but single wild-type cells ending
up in the region of the anterior lateral plate mesoderm expressed han
(Fig. 4A-C).
For analysis of thyroid development, embryos were fixed at 55 hpf and processed for nk2.1a in situ hybridisation. Homozygotes were identified by MF20 immunohistochemistry visualising heart muscle, which is strongly reduced in hans6 mutants. Unequivocal identification of mutants was possible because grafted wild-type cells did not restore hans6 heart morphology. Out of 87 homozygous hans6 hosts (from 327 hosts in total) that received grafted wild-type cells, 75 did not show any sign of a thyroid at 55 hpf. However, 12 embryos (13.8%) showed a strong nk2.1a expression domain in the pharyngeal epithelium or in the pharyngeal mesenchyme (Table 1, Fig. 4D-G). The position of these nk2.1a domains resembled normal nk2.1a expression in the thyroid primordium. In the remaining homozygotes from the same clutches, which were fixed as controls, thyroidal nk2.1a expression was consistently not detectable at 55 hpf. As mentioned earlier, we occasionally observed faint nk2.1a expression in homozygotes of other clutches, but expression was weaker and restricted to a smaller domain. We therefore conclude that wild-type cells can restore nk2.1a expression in hans6 mutants, or can increase weak levels of expression that are otherwise below detection, or can prolong initially present expression to unusually late time points.
|
|
;
Trinh et al., 2005;
Yelon et al., 2000).
Therefore, grafted cells cannot be unequivocally classified as belonging to
either of these structures. To determine whether cardiac or branchial arch
tissue is required for thyroid development, we eliminated han
expression in the pharyngeal arches and putative carotid bodies by morpholino
knock-down of upstream genes.
Thyroid development is independent of han expression in pharyngeal arches and arch-associated cells
As was previously demonstrated, han expression in the branchial
arches depended on the expression of endothelin 1 (edn1) and
tfap2a (Knight et al.,
2003; Miller and Kimmel,
2001; Miller et al.,
2000a; Piotrowski et al.,
2003). Correspondingly, double morpholino knock-down of these two
genes eliminated han expression in the pharyngeal arches completely
(Fig. 5A-D). By contrast,
han expression in the putative carotid bodies, heart and endoderm was
unaffected in these double morphants. Thus, edn1/tfap2a
double morphants can serve as a model for thyroid development in the absence
of pharyngeal arch han expression.
nk2.1a and slc5a5 expression is essentially normal in edn1 and tfap2a single morphants as well as in edn1/tfap2a double morphants (Fig. 5E, and data not shown), indicating that thyroid development does not depend on han expression in the pharyngeal arches.
|
). We
found that, in foxi1 morphants, not only the AANs, but also the
adjacent han-expressing cells located between the first and second
pharyngeal arches are missing (Fig.
5F). Nevertheless, foxi1 morphants had a normal thyroid
(Fig. 5G), indicating that
han expression in arch-associated cells is not required for thyroid
development. These data suggest that the remaining other site of detectable
han expression, the cardiac mesoderm, is required for thyroid
development. To date, it is not possible to ablate this tissue specifically,
and so its role in thyroid development awaits further experimental
confirmation.
FGFs are candidate signalling factors in thyroid development
We focused on FGFs as putative downstream factors of han in
thyroid development, because they have been shown to act downstream of
han in tooth development (Abe et
al., 2002) and are known to play roles in cardiac development
(Reifers et al., 2000). In
zebrafish ace mutant embryos, the fgf8 gene is disrupted
(Reifers et al., 1998). In
this mutant, a reduced size of the thyroid primordium at early stages
(Fig. 6A-F) and a reduced
number of follicles after differentiation
(Fig. 6G,H) indicates that
fgf8 is required for normal thyroid development.
FGFs constitute a large family of signalling molecules
(Ornitz and Itoh, 2001;
Thisse and Thisse, 2005), and
the loss of a specific FGF might be compensated for by the overlapping
expression of other family members. The FGF-receptor blocker su5402 is an
excellent tool to eliminate FGF signalling completely in specific time
windows, and has also been used to narrow down the temporal requirement of FGF
in zebrafish heart development (Reifers et
al., 2000). We treated zebrafish embryos at various stages with 10
µM su5402 for a time window of 2 hours and tested at 36 hpf for
nk2.1a expression and at 60 hpf for slc5a5 expression.
Higher concentrations of su5402 lead to severe malformations, making analysis
of thyroid development questionable, so that we confined our analysis to 10
µM concentrations only.
In general, su5402 treatment during the somitogenesis stages was sufficient to eliminate thyroidal nk2.1a and slc5a5 expression in around 70% of embryos (Fig. 6I-M). The uniform result for different time windows (Fig. 6M) suggests that washing su5402 out after 2 hours of treatment was probably inefficient, or that FGF signalling is continuously required for thyroid development. Treatment starting at 30 hpf, after the thyroid primordium is induced, affects the thyroid less efficiently, but still eliminates the gland in about 20% of embryos. This could be the result of a reduced influence of FGFs on later thyroid development, but could also be due to a limited potential of the chemical to diffuse into older embryos. su5402 treatment at the somitogenesis stages did not visibly affect endoderm development on the level of foxa2 (axial) expression at 30 hpf (data not shown), indicating that it is not a severe reduction of endoderm that causes the absence of the thyroid. In conclusion, su5402 treatment did not enable precise definition of the time window in which FGF signalling is required for thyroid development. Nevertheless, the drug treatments confirm that FGF signalling is required for thyroid specification and for subsequent differentiation. Furthermore, these data indicate that FGF signalling is not only required during the early steps of thyroid development, as indicated by the reduced early thyroid primordium in ace mutants, but also during later steps, after 30 hpf.
FGFs restore thyroid development in han s6 mutants
To test a possible role of FGFs downstream of han in thyroid
development, we implanted beads soaked with recombinant FGF protein into
hans6 embryos and analysed subsequent thyroid
differentiation based on the level of slc5a5 expression. We chose
recombinant mouse FGF8 and, in addition, recombinant human FGF1 and FGF2. FGF1
and FGF2 signals from the cardiac mesoderm are suspected to be involved in
liver induction in mice (Jung et al.,
1999; Serls et al.,
2005) and are therefore also good candidates as having a role
downstream of han. Using the MHB as a landmark, beads were embedded
into the embryo within proximity to the endoderm. Implantation was carried out
at the 14-18 ss, because the reduced size of the early thyroid primordium in
ace mutants suggests a specific role of FGFs in thyroid development
before or around the onset of thyroid marker expression. Numbers of untreated
hans6 mutants with residual slc5a5 expression
were not significantly different compared to hans6 mutants
that received BSA-soaked control beads
(Fig. 7A,B). The implantation
of beads soaked in FGF proteins, however, resulted in significantly increased
numbers of hans6 embryos expressing slc5a5 (BSA
control compared to FGF8: X2=3.99, P=0.046; FGF1:
X2=4.49, P=0.034; FGF2:
X2=5.03, P=0.024; FGF1+FGF2:
X2=8.00, P=0.004). Interestingly, all three FGFs
were able to restore slc5a5 expression to a similar percentage. Thus,
on the protein level, these different FGFs and probably also other members of
the family can replace each other functionally. In summary, recombinant FGF
protein is able to rescue the thyroid in hans6 mutants by
restoration of slc5a5 expression, showing that FGFs act downstream or
in parallel to han in the differentiation of this gland
(Fig. 7C).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
By contrast, the genetics of thyroid specification are poorly understood.
Neither the factors involved in induction, nor in defining the competence of
endodermal cells to become thyroid have been identified. Our grafting
experiments show that han is required for thyroid development in a
cell non-autonomous manner. Because han encodes a transcription
factor, it is conceivable that the Hand2 protein is necessary for the
development of certain tissues (most probably lateral plate mesoderm or heart)
in the vicinity of the endoderm. Thyroid development, in turn, depends on the
proper development of these surrounding structures. Such an indirect role
could also account for the incomplete penetrance of the
hans6 phenotype at early stages of thyroid development,
and for the hanc99 phenotype, in which both heart and
thyroid are less severely affected. If the development of adjacent tissue is
impaired, local sources of signalling molecules, such as FGFs, are not
necessarily completely abolished. Interestingly, final thyroid differentiation
leading to hormone (T4) production always failed in hans6
mutants, despite initial nk2.1a expression in some embryos, and
despite occasional later expression of the differentiation marker
slc5a5. It is possible that reduced nk2.1a levels are not
sufficient for normal differentiation. Alternatively, the severely abnormal
heart and pharyngeal arches in hans6 mutants might
influence later thyroid development independently of the early steps.
|
Because it is unknown exactly when han-expressing tissue is
required for thyroid specification, at least two different scenarios are
possible. Our fate mapping indicates that aLPM is continuously close to
thyroid progenitors in the converging endoderm and, in
hans6 mutants, normal expansion of the aLPM fails during
the somitogenesis stages. Thus, one possible model is that the aLPM signals to
the endoderm, specifying thyroid precursors early during the somitogenesis
stages. Alternatively, because cardiac development is severely disrupted in
hans6 mutants, it could also be that cardiac structures
such as heart muscle or the outflow tract are required for thyroid
specification. In this model, interactions between heart and pharyngeal
endoderm would occur later than in the first model, just before the onset of
thyroid markers, at around 24 hpf. Because the aLPM is considered to give rise
to cardiac structures, both models are similar in that they support a central
role of heart development in thyroid specification. Moreover, in both
han and ace mutants, defects in cardiac morphogenesis
(Reifers et al., 2000;
Yelon et al., 2000) are
correlated with a similar thyroid phenotype.
Interactions between cardiac and thyroid development are further supported
by human syndromes. In DiGeorge (22q11) syndrome, caused by variable deletions
on chromosome 22 in humans, congenital heart defects occur. In conjunction, an
increased risk of congenital thyroid defects has been described
(Bassett et al., 2005).
Similarly, in human patients suffering from congenital hypothyroidism, an
increased incidence of congenital heart defects has been observed
(Olivieri et al., 2002).
Furthermore, ectopic thyroid tissue can occasionally be found in human cardiac
tissue (Casanova et al.,
2000).
|
), tissue
that is continuously close to thyroid precursors and therefore a candidate for
being the source of FGF signals. A search for FGFs acting together with
fgf8 and probably redundantly in thyroid development has not been
successful as yet (T.W., D.A. and K.B.R., unpublished observations). For
instance, morpholino knock-down of other zebrafish FGFs (Fgf1, Fgf2, Fgf3) in
conjunction with Fgf8 did not lead to a more-severe thyroid phenotype.
Genes encoding downstream factors or modifiers of the intracellular
signalling cascade of Fgf8, such as spry2, spry4 or sef
(also known as il17rd-Zebrafish Information Network)
(Furthauer et al., 2002), were
not found to be expressed at visible levels in the thyroid or in the endoderm
at corresponding stages (T.W., D.A. and K.B.R., unpublished observations),
suggesting that Fgf8 is unlikely to signal directly to the pharyngeal
endoderm. It is therefore possible that further, unknown factors link FGF
signalling and thyroid development.
FGFs have been implicated to play a role in thyroid development previously.
In mouse embryos deficient for the FGF receptor 2 IIIb, multiple defects in
organogenesis occur, including dysgenesis of the thyroid
(Revest et al., 2001). A
similar phenotype of FGF10 knock-out mice suggests that FGF10 is a major
ligand acting via FGF receptor 2 IIIb
(Ohuchi et al., 2000).
However, initial thyroid development still occurs in the absence of the
receptor 2 IIIb isoform (De Felice and Di
Lauro, 2004), suggesting that FGF activity via this isoform is not
responsible for early specification of the thyroid. Taken together, it can be
anticipated that several FGFs act at different time points in thyroid
development.
han and FGFs: a novel link in thyroid development
In hans6 mutants, FGF proteins are able to restore
thyroid differentiation, therefore acting downstream or in parallel of
han in thyroid development (Fig.
7C). fgf8 expression in the aLPM appears to be normal in
hans6 mutants (J.J.S. and D.Y., unpublished data),
indicating that here fgf8 expression does not depend on Hand2. Thus,
fgf8 rather acts in parallel to Hand2 in thyroid development, and it
is possible that morphogenetic changes in hans6 mutants
alter the temporal or spatial relation of FGF-expressing tissue to pharyngeal
endoderm.
In the bead-implantation experiments, the thyroid was never restored at the wrong level along the a-p axis. This argues against inductive activity of FGFs, which would be likely to cause ectopic primordia. In particular, because slc5a5 expression is seen in a percentage of untreated mutants, we would expect a second thyroid in some FGF-bead-implanted embryos, which was not the case. Alternatively, we favour the possibility that FGFs act permissively in thyroid development, together with other signals. Our su5402 data, as well as the abovementioned mouse data, suggest that FGFs are also, and probably continuously, required for later thyroid differentiation, at which point structures in addition to the aLPM or cardiac tissues might act as a source.
Taken together, han and ace mutants represent two models that shed light on the role of the surrounding tissue in thyroid specification. Our study identifies the aLPM or cardiac structures to be key in this process. It will be interesting to analyse the role of Hand transcription factors as well as FGF signals and their downstream pathway components with respect to congenital thyroid defects in humans, in particular in those cases where they are associated with congenital heart defects.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: MRC Centre for Developmental Neurobiology, King's College
London, London SE1 1UL, UK
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abe, M., Tamamura, Y., Yamagishi, H., Maeda, T., Kato, J., Tabata, M. J., Srivastava, D., Wakisaka, S. and Kurisu, K. (2002). Tooth-type specific expression of dHAND/Hand2: possible involvement in murine lower incisor morphogenesis. Cell Tissue Res. 310,201 -212.[CrossRef][Medline]
Alexander, J. and Stainier, D. Y. (1999). A molecular pathway leading to endoderm formation in zebrafish. Curr. Biol. 9,1147 -1157.[CrossRef][Medline]
Alt, B., Reibe, S., Feitosa, N. M., Elsalini, O. A., Wendl, T. and Rohr, K. B. (2006). Analysis of origin and growth of the thyroid gland in zebrafish. Dev. Dyn. 235,1872 -1883.[CrossRef][Medline]
Angelo, S., Lohr, J., Lee, K. H., Ticho, B. S., Breitbart, R. E., Hill, S., Yost, H. J. and Srivastava, D. (2000). Conservation of sequence and expression of Xenopus and zebrafish dHAND during cardiac, branchial arch and lateral mesoderm development. Mech. Dev. 95,231 -237.[Medline]
Bassett, A. S., Chow, E. W., Husted, J., Weksberg, R., Caluseriu, O., Webb, G. D. and Gatzoulis, M. A. (2005). Clinical features of 78 adults with 22q11 Deletion Syndrome. Am. J. Med. Genet. A 138,307 -313.[Medline]
Brand, M., Heisenberg, C. P., Jiang, Y. J., Beuchle, D., Lun, K., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. A. et al. (1996). Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain. Development 123,179 -190.[Abstract]
Casanova, J. B., Daly, R. C., Edwards, B. S., Tazelaar, H. D.
and Thompson, G. B. (2000). Intracardiac ectopic thyroid.
Ann. Thorac. Surg. 70,1694
-1696.
De Felice, M. and Di Lauro, R. (2004). Thyroid
development and its disorders: genetics and molecular mechanisms.
Endocr. Rev. 25,722
-746.
Elsalini, O. A. and Rohr, K. B. (2003). Phenylthiourea disrupts thyroid function in developing zebrafish. Dev. Genes Evol. 212,593 -598.[Medline]
Elsalini, O. A., von Gartzen, J., Cramer, M. and Rohr, K. R. (2003). Zebrafish hhex, nk2.1a and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors. Dev. Biol. 263,67 -80.[CrossRef][Medline]
Fagman, H., Andersson, L. and Nilsson, M. (2005). The developing mouse thyroid: embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation. Dev. Dyn. 235,444 -455.[CrossRef]
Furthauer, M., Lin, W., Ang, S. L., Thisse, B. and Thisse, C. (2002). Sef is a feedback-induced antagonist of Ras/MAPK-mediated FGF signalling. Nat. Cell Biol. 4, 170-174.[CrossRef][Medline]
Gualdi, R., Bossard, P., Zheng, M., Hamada, Y., Coleman, J. R.
and Zaret, K. S. (1996). Hepatic specification of the gut
endoderm in vitro: cell signaling and transcriptional control.
Genes Dev. 10,1670
-1682.
Guo, S., Wilson, S. W., Cooke, S., Chitnis, A. B., Driever, W. and Rosenthal, A. (1999). Mutations in the zebrafish unmask shared regulatory pathways controlling the development of catecholaminergic neurons. Dev. Biol. 208,473 -487.[CrossRef][Medline]
Holzschuh, J., Ryu, S., Aberger, F. and Driever, W. (2001). Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech. Dev. 101,237 -243.[CrossRef][Medline]
Jung, J., Zheng, M., Goldfarb, M. and Zaret, K. S.
(1999). Initiation of mammalian liver development from endoderm
by fibroblast growth factors. Science
284,1998
-2003.
Keegan, B. R., Meyer, D. and Yelon, D. (2004).
Organization of cardiac chamber progenitors in the zebrafish blastula.
Development 131,3081
-3091.
Kim, S. K., Hebrok, M. and Melton, D. A. (1997). Notochord to endoderm signaling is required for pancreas development. Development 124,4243 -4252.[Abstract]
Knight, R. D., Nair, S., Nelson, S. S., Afshar, A., Javidan, Y.,
Geisler, R., Rauch, G. J. and Schilling, T. F. (2003).
lockjaw encodes a zebrafish tfap2a required for early neural crest
development. Development
130,5755
-5768.
Kumar, M., Jordan, N., Melton, D. and Grapin-Botton, A. (2003). Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev. Biol. 259,109 -122.[CrossRef][Medline]
Lammert, E., Cleaver, O. and Melton, D. (2001).
Induction of pancreatic differentiation by signals from blood vessels.
Science 294,564
-567.
Mackereth, M. D., Kwak, S. J., Fritz, A. and Riley, B. B.
(2005). Zebrafish pax8 is required for otic placode induction and
plays a redundant role with Pax2 genes in the maintenance of the otic placode.
Development 132,371
-382.
Miller, C. T. and Kimmel, C. B. (2001). Morpholino phenocopies of endothelin 1 (sucker) and other anterior arch class mutations. Genesis 30,186 -187.[CrossRef][Medline]
Miller, C. T., Schilling, T. F., Lee, K., Parker, J. and Kimmel, C. B. (2000a). sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development. Development 127,3815 -3828.[Abstract]
Miller, D. L., Ortega, S., Bashayan, O., Basch, R. and Basilico,
C. (2000b). Compensation by fibroblast growth factor 1 (FGF1)
does not account for the mild phenotypic defects observed in FGF2 null mice.
Mol. Cell. Biol. 20,2260
-2268.
Miller, C. T., Yelon, D., Stainier, D. Y. and Kimmel, C. B.
(2003). Two endothelin 1 effectors, hand2 and bapx1, pattern
ventral pharyngeal cartilage and the jaw joint.
Development 130,1353
-1365.
Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S. and Itoh, N. (2000). FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multiorgan development. Biochem. Biophys. Res. Commun. 277,643 -649.[CrossRef][Medline]
Olivieri, A., Stazi, M. A., Mastroiacovo, P., Fazzini, C.,
Medda, E., Spagnolo, A., De Angelis, S., Grandolfo, M. E., Taruscio, D.,
Cordeddu, V. et al. (2002). A population-based study on the
frequency of additional congenital malformations in infants with congenital
hypothyroidism: data from the Italian Registry for Congenital Hypothyroidism
(1991-1998). J. Clin. Endocrinol. Metab.
87,557
-562.
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2, REVIEWS3005.
Piotrowski, T., Ahn, D. G., Schilling, T. F., Nair, S.,
Ruvinsky, I., Geisler, R., Rauch, G. J., Haffter, P., Zon, L. I., Zhou, Y. et
al. (2003). The zebrafish van gogh mutation disrupts tbx1,
which is involved in the DiGeorge deletion syndrome in humans.
Development 130,5043
-5052.
Reifers, F., Bohli, H., Walsh, E. C., Crossley, P. H., Stainier, D. Y. and Brand, M. (1998). Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125,2381 -2395.[Abstract]
Reifers, F., Walsh, E. C., Leger, S., Stainier, D. Y. and Brand, M. (2000). Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar). Development 127,225 -235.[Abstract]
Revest, J. M., Spencer-Dene, B., Kerr, K., De Moerlooze, L., Rosewell, I. and Dickson, C. (2001). Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev. Biol. 231,47 -62.[CrossRef][Medline]
Rohr, K. B. and Concha, M. L. (2000). Expression of nk2.1a during early development of the thyroid gland in zebrafish. Mech. Dev. 95,267 -270.[CrossRef][Medline]
Serls, A. E., Doherty, S., Parvatiyar, P., Wells, J. M. and
Deutsch, G. H. (2005). Different thresholds of fibroblast
growth factors pattern the ventral foregut into liver and lung.
Development 132,35
-47.
Thisse, B. and Thisse, C. (2005). Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev. Biol. 287,390 -402.[Medline]
Trinh, L. A., Yelon, D. and Stainier, D. Y. (2005). Hand2 regulates epithelial formation during myocardial differentiation. Curr. Biol. 15,441 -446.[CrossRef][Medline]
Warga, R. M. and Nusslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Development 126,827 -838.[Abstract]
Wendl, T., Lun, K., Mione, M., Favor, J., Brand, M., Wilson, S.
W. and Rohr, K. B. (2002). pax2.1 is required for the
development of thyroid follicles in zebrafish.
Development 129,3751
-3760.
Yelon, D., Ticho, B., Halpern, M. E., Ruvinsky, I., Ho, R. K., Silver, L. M. and Stainier, D. Y. (2000). The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development 127,2573 -2582.[Abstract]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
