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Proc Natl Acad Sci U S A. 2003 August 5; 100(16): 9410–9415. Published online 2003 July 25. doi: 10.1073/pnas.1633548100. | PMCID: PMC170932 |
Copyright © 2003, The National Academy of
Sciences Genetics An interacting network of T-box genes directs gene expression and fate in
the zebrafish mesoderm Lisa M. Goering, †‡ Kazuyuki Hoshijima, †‡ Barbara Hug, †‡ Brent Bisgrove, †§ Andreas Kispert, ¶ and David Jonah Grunwald † †Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112; and ¶Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany Received April 10, 2003. T-box genes encode transcription factors that play critical roles in
generating the vertebrate body plan. In many developmental fields, multiple
T-box genes are expressed in overlapping domains, establishing broad regions
in which different combinations of T-box genes are coexpressed. Here we
demonstrate that three T-box genes expressed in the zebrafish mesoderm, no
tail, spadetail, and tbx6, operate as a network of interacting
genes to regulate region-specific gene expression and developmental fate.
Loss-of-function and gain-of-function genetic analyses reveal three kinds of
interactions among the T-box genes: combinatorial interactions that generate
new regulatory functions, additive contributions to common developmental
pathways, and competitive antagonism governing downstream gene expression. We
propose that T-box genes, like Hox genes, often function within gene networks
comprised of related family members. T-box genes encode related transcription factors that regulate tissue
specification, morphogenesis, and cell proliferation
( 1– 3).
In addition to tissue-specific roles, T-box genes govern regional identities
within developmental fields
( 4– 6).
One puzzling aspect of T-box gene function is the recurrent finding that the
primary cellular focus of the defect seen in a T-box mutant corresponds to
only a limited portion of the expression domain of the mutated T-box gene
( 5). For example, whereas
haploinsufficiency of TBX3 results in posterior forelimb
deficiencies, the gene is expressed in the anterior and posterior margin of
the developing hind limb and forelimb
( 7,
8). Similarly, although
Brachyury orthologues are expressed throughout the nascent mesoderm
in vertebrate embryos, loss of Brachyury function blocks
differentiation of the notochord, a dorsal mesoderm tissue, but has only
limited effects on the morphogenesis of the ventrolateral mesoderm, allowing
differentiation of the full range of mesoderm cell types derived from this
tissue
( 9– 11).
These findings implicate factors that modify T-box gene function so that
individual T-box genes carry out different functions in different regions of
their expression domain
( 12– 15).
Because multiple T-box genes are expressed in overlapping patterns in many
developmental fields ( 7,
16– 19),
we hypothesized that T-box gene interactions contribute to regionalization of
T-box gene function. We examined individual and combined functions of three T-box genes, no
tail ( ntl), spadetail ( spt), and
tbx6, which are expressed exclusively in the developing zebrafish
mesoderm
( 20– 24).
The three genes are expressed in broad domains that overlap and together mark
all of the mesoderm (). We demonstrate that ntl, the zebrafish
orthologue of Brachyury, regulates different downstream targets in
different portions of its uninterrupted expression domain. Because
spt and tbx6 are coexpressed with ntl in selected
regions of the mesoderm, we analyzed whether these genes interact with
ntl to modify its function in a region-specific manner. Amacher
et al. ( 25) recently
showed that ntl and spt provide some overlapping functions
in the mesoderm. In this report, we document three kinds of interactions among
the three T-box genes: combinatorial interactions that generate new regulatory
functions, additive contributions to common developmental pathways, and
competitive antagonism governing downstream gene expression. Our findings
demonstrate that T-box genes can perform multiple region-specific functions
within a developmental field and indicate how loss of function of one T-box
gene can alter the function of other T-box genes expressed in the same
field. Isolation of ntl-Regulated Genes. A mesendoderm-specific cDNA
library was constructed by subtractive hybridization
( 26,
27): cDNA from midgastrula
embryos was depleted for sequences expressed in isolated animal cap tissue
(see Supporting Materials and Methods, which is published as
supporting information on the PNAS web site,
www.pnas.org).
Independent transformants (1,728 in all) were arrayed and analyzed by
differential hybridization screening. cDNA from WT and individually genotyped
ntlb195 mutant 70% epiboly embryos was used to generate
WT- ntl, ntl-WT-subtracted, and ntl-unsubtracted cDNA probes.
Candidate ntl-dependent sequences hybridized only with
WT- ntl probe. Whole-mount in situ hybridization was
performed on WT and mutant sibling embryos to identify genes whose expression
depended on ntl function. Genetics and Genotype Analysis. Embryos from natural spawnings were
raised at 28.5°C ( 28). WT
embryos were from the AB* line. The sptb104,
ntlb160, and ntlb195 alleles were used
( 11,
20,
24,
29). To analyze additive
effects of mutant alleles on MyoD expression, genotyping with
allele-specific primers was performed on tissue removed before in
situ hybridization (see Supporting Materials and Methods). Ectopic Gene Expression Experiments. Sequences encoding No Tail, the
No Tail DNA-binding domain (DBD) (amino acids 1–229), Tbx6, the Tbx6 DBD
(amino acids 1–240), the repressor domain of the Drosophila
Engrailed protein [amino acids 2–299
( 30)], or the activation
domain of VP16 (amino acids 19–101) were cloned into the CS2 +
and CS2 +MT plasmids
( 31). Proteins were expressed
ectopically after injection of in vitro-generated 5′-capped
mRNA (mMESSAGE mMACHINE kit, Ambion, Austin, TX) along with fluorescent
lineage tracer dye into one- to two-cell embryos. Normally cleaving embryos
with widespread dye were selected for analysis. Myc-epitope-tagged protein was
detected with the 9E10 antibody (Santa Cruz Biotechnology). To measure gene
expression in animal caps, caps were removed at 3.5 h from injected embryos
and incubated for 5 h at 28°C
( 21), and RNA expression was
detected by RT-PCR and quantified (see Supporting Materials and
Methods). Relative MyoD expression was determined as the ratio
of MyoD PCR product to cytokeratin8 PCR product for each
experimental condition. Electrophoretic Mobility-Shift Assays. T-sites were synthesized,
cloned into the SmaI site of pBluescript, purified such that all
oligomers were ≈55 bp, and radiolabeled with [32P]dATP by using
a modified Klenow fragment of DNA polymerase (Stratagene). Binding conditions
are provided as Supporting Materials and Methods. Binding reaction
products were resolved by gel electrophoresis and quantified after scanning of
autoradiograms. Free DNA signal was used to determine fraction oligo bound.
Curves represent best fits to the equation fraction oligo shifted = 1/[1 +
(Kd/[P])]. R values for the binding
curves are: Tbx6-myc-1/2T-site, 0.985; Tbx6-myc-palT-site, 0.975; No
Tail-1/2T-site, 0.986; and No Tail-palT-site, 0.970. Transactivation Assays. 293 cells were transfected by using
Lipofectamine Plus reagent (Invitrogen). T-box transcription factors were
expressed from CS2 + expression plasmids. Luciferase reporter
plasmids contained three T-sites 55 bp upstream of the promoter of –36
PRL-luc ( 32), a pGL3-derived
plasmid (Promega) containing 73 bp of the rat prolactin promoter TATA box
region. Plasmid expressing Renilla luciferase constitutively was used
as an internal reference to standardize transfection efficiency. Experiments
were performed in triplicate and repeated at least twice. Region-Specific Functions of ntl in the Mesoderm. ntl, spt,
and tbx6 are expressed exclusively in the developing zebrafish
mesoderm ( 20,
21,
23). Overlapping expression of
the three genes demarcates distinct regions of mesoderm identities
(). Before
gastrulation, at the time of mesoderm specification, the combined expression
marks two domains: dorsal mesoderm, where ntl and spt are
expressed without tbx6, and ventrolateral mesoderm, where all three
genes are present. During gastrulation, the expression patterns of the genes
are refined to generate four domains: anterior axial mesoderm, or prechordal
plate, expresses only spt; posterior axial mesoderm, or presumptive
notochord, expresses only ntl; nonaxial mesoderm generated by
involution of the ventrolateral mesoderm expresses spt and
tbx6 together; and noninvoluted ventrolateral mesoderm maintains
expression of all three genes. We hypothesized that individual T-box genes
have distinct functions in different portions of their expression domains and
that the function of each T-box gene is defined in part by the combination of
T-box genes with which it is coexpressed. To define cellular regions of ntl function, we identified genes
whose expression depends on ntl by hybridizing a mesendoderm-specific
target cDNA library with a probe enriched for sequences expressed in WT, but
not ntl mutant, gastrulae (see Materials and Methods). Two
classes of genes were recovered (). One class, exemplified by ntl-dependent gene
5 ( ntd5), was expressed solely in the presumptive notochord. A
second class, exemplified by mesogenin and wnt8
( 33), was expressed in the
noninvoluted ventrolateral mesoderm. Loss of expression of these genes was not
secondary to loss of mesoderm tissue: other markers of axial and ventrolateral
mesoderm continued to be expressed through midgastrula stages (data not shown
and refs. 21,
34, and
35). Furthermore,
mesogenin appears to be a proximal target of ntl, because
its expression is detectable within 30 min of the onset of ntl
transcription in 30% epiboly WT embryos and fails to be initiated in
ntl mutants (). Regardless of the mechanism by which the
ntl-dependent genes are regulated, the genes serve as markers of
cellular phenotype regulated by ntl. These results provide direct
evidence that ntl regulates gene expression in the ventrolateral, as
well as the dorsal, mesoderm and indicate that ntl has distinct
region-specific functions in the mesoderm. Combinatorial Interactions Between ntl and spt. mesogenin
and wnt8 are transcribed only in the portion of the ntl
expression domain where spt is also present, raising the possibility
that spt modifies ntl function in the ventrolateral
mesoderm. Analysis of spt mutant embryos indicated mesogenin
is fully dependent on spt function
(), and, thus,
both ntl and spt are required for initiating expression of
mesogenin. Because mesogenin is not transcribed where each
T-box is expressed alone, the regulation of mesogenin is a previously
uncharacterized function that ntl and spt acquire when
expressed together. spt is not the only interacting gene responsible
for directing the ventral mesoderm activities of ntl, because
wnt8 is not regulated by spt
(). ntl and spt Contribute Additive Functions to Mesoderm Development and
Gene Expression. Null mutations of ntl or spt are fully
recessive mutations that primarily affect different regions of the mesoderm
( 11,
20,
24,
29,
36). Whereas ntl
mutants lack notochord and posterior mesoderm, spt mutants produce
notochord and generate posterior mesoderm in which both somites and notochord
are formed in the tail. Conversely, spt, but not ntl, is
required for formation of trunk somites. In spt mutants, somitic
precursors fail to migrate properly and accumulate as undifferentiated
mesoderm in the tail. The two T-box genes seem to contribute in a partially
redundant manner to some aspects of mesoderm development, because double
mutants exhibit a more severe phenotype than the simple addition of the
defects present in ntl and spt mutants
( 25). To identify pathways regulated in concert by both genes, we tested whether
heterozygosity at one locus would enhance the mutant phenotype associated with
loss of the other gene. Among progeny of matings between doubly heterozygous
individuals, a previously uncharacterized phenotypic class appeared in
addition to the expected WT, ntl, spt, and ntl; spt
phenotypic classes ( Table 1).
The previously uncharacterized phenotypic class, here called
spadetail-enhanced ( spadetail-enh), was present in numbers
consistent with the interpretation that it represented embryos homozygous for
the spt mutation and heterozygous for the ntl mutation
( Table 1).
spadetail-enh embryos exhibited the surfeit of tail tissue
characteristic of spt mutants, but they were distinguishable from the
canonical spt phenotype in that they displayed a complete loss of
somites in the tail and loss of axial tissue in the trunk
().
Simultaneous phenotypic and genotypic analyses performed on individual embryos
demonstrated that the ntl mutation acted as a fully penetrant
dominant enhancer of the spt phenotype
(). | Table 1.Gene interactions between ntl and spt |
Mesoderm differentiation was analyzed in spadetail-enh embryos by
using ntl expression as a marker of notochord development and
MyoD expression as a marker of somite formation
( 23,
37). Although heterozygous
ntl/+ embryos have a WT pattern of ntl and MyoD
expression, the ntl mutation acts dominantly in a spt mutant
background. spt mutants make a complete notochord marked by
ntl expression, and they form MyoD-expressing somites in the
tail and patches of MyoD-expressing cells in the trunk. In contrast,
spadetail-enh mutants exhibit only discontinuous patches of
ntl-expressing tissue in the midline and have an almost complete lack
of MyoD-expressing tissue in the trunk and tail. Thus, loss of
spt function sensitizes both the notochord and somite pathways to the
level of ntl expression and indicates that the two T-box genes work
together to promote development of both tissues. To determine the relative quantitative contributions of ntl and
spt to mesoderm development, we measured the effects of ntl
and spt mutations on tbx6 expression
(). Absence of
spt function results in a significant but incomplete decrease in the
expression of tbx6 in early gastrula embryos
( 20). Although loss of one or
both copies of ntl in an otherwise WT genetic background does not
have a detectable effect on early expression of tbx6
( 21), loss of one copy of
ntl in a spt mutant lowers tbx6 expression to
undetectable levels. These results indicate that ntl and spt
regulate a common set of genes in the mesoderm, contributing in an additive
but unequal manner to their full expression. Antagonism of Some ntl Functions by tbx6. ntl is required
in the dorsal mesoderm to promote differentiation of the notochord and
production of the MyoD-expressing adaxial cells that flank the
presumptive notochord ()
( 11,
37). Given that the absence of
ntl's dorsal-specific functions in the ventrolateral mesoderm
correlates with the presence of tbx6, we asked whether tbx6
can antagonize the dorsal-specific functions of ntl
(). Ectopic expression of a bona fide antagonist of No
Tail, Ntl-En R-myc, in which the DBD of No Tail was fused with the
Engrailed repressor domain, suppressed posterior notochord and adaxial tissue
development ( 10). Ectopic
expression of Tbx6-myc or Tbx6 produced a similar range of phenotypes. Tbx6
acted locally to antagonize No Tail function as adaxial cells that lacked
ectopic Tbx6-myc maintained normal MyoD expression
(). Expression
of β-galactosidase or the Myc-epitope peptide had no effect on notochord
development (data not shown). In sum, ectopic expression of tbx6 acts
to antagonize ntl, resulting in a developmental syndrome expected
from hypomorphic activity of ntl. Ectopic expression of Brachyury proteins in ectodermal animal cap
cells induces MyoD expression in a dose-dependent manner
( 38). It is likely that
MyoD is regulated directly by No Tail in early zebrafish gastrulae,
because No Tail protein is physically associated with chromatin containing the
MyoD promoter at this stage (K.H., unpublished data). Coinjection of
increasing amounts of ntl-EnR-myc RNA or tbx6-myc
RNA along with a fixed amount of ntl-myc RNA inhibited No
Tail-activated MyoD expression in a dose-dependent manner
(). Comparable
amounts of tbx6-myc and ntl-EnR-myc RNA produced
similar reductions in the amount of MyoD expression, indicating that
the two proteins may have similar capacities to antagonize No Tail-dependent
activation of gene expression. Cell fate choices depend on the relative amount of ntl and
tbx6 expression in individual embryonic cells. Whereas overexpression
of either ntl or tbx6 perturbed MyoD expression in
gastrula embryos in opposite ways, overexpression of both genes together could
rescue a WT pattern of MyoD (; Table
2). The finding that the suppression of MyoD expression
in adaxial cells caused by ectopic tbx6 mRNA could be overcome by
coexpression with ntl indicates that tbx6 generally does not
suppress mesoderm formation; however, tbx6 and ntl can
interact as competitive antagonists that govern mesoderm fate in the embryo.
Furthermore, Tbx6 need not activate transcription to antagonize No Tail.
Ectopic expression of Tbx6DBD or Tbx6DBD-En R-myc, neither of which
could promote expression of reporter genes that harbored Tbx6 binding sites
( and data not
shown), suppressed the normal pattern of MyoD expression
() and
notochord development (data not shown). | Table 2.ntl and tbx6 act competitively to regulate
MyoD expression in adaxial cells |
To explain how Tbx6 might selectively suppress dorsal-specific functions of
No Tail, we hypothesized that Tbx6 can effectively antagonize No Tail at only
some target sites. Other T-box proteins are known to have overlapping but
nonidentical interactions with binding sites called T-sites
( 39). We measured the ability
of No Tail and Tbx6 to bind to and promote transcription from different forms
of the T-site ()
( 40– 42).
No Tail and Tbx6-myc demonstrated similar sequence specificity: both bound the
1/2T-site as a monomer but failed to bind the 1/2T mut-site, which
harbored a single base substitution affecting a critical contact site for
Brachyury proteins
( 41,
42)
(). However, whereas Tbx6 bound both the 1/2T- and the
palindromic palT-sites as a monomer, with identical affinity for the two kinds
of sites, No Tail exhibited considerably enhanced affinity for the palT-site,
on which it formed homodimers (). Consistent with these findings,
incubation of 1/2T-site oligomer with a mixture of Tbx6 and No Tail yielded
protein–DNA complexes containing both proteins
(), but incubation of the palT-site oligomer with the
same protein mixture yielded only No Tail homodimer protein–DNA
complexes ().
Heterodimer protein–DNA complexes were not detected in these
experiments, although mixed protein complexes could have been detected
(T6′+N′ in ). Tbx6 can compete effectively with No Tail to regulate T-site-dependent
transcription. Whereas No Tail efficiently promoted expression of reporter
genes that harbored either the palT- or 1/2T-site, Tbx6 failed to promote
expression at these sites even though the Tbx6DBD is capable of recognizing
T-sites in cells, indicated by the finding that Tbx6DBD-VP16 activated
reporter gene expression (). Coexpression of Tbx6 with No Tail at ratios ≤1:1
led to a substantial reduction of No Tail-dependent expression from the
1/2T-site reporter (). In contrast, expression of the two proteins at
similar ratios had no measurable effect on No Tail-dependent expression of the
palT reporter gene. Thus, in cells expressing the two proteins, Tbx6 can
effectively inhibit No Tail-dependent transcription at promoters that harbor
some, but not all, kinds of T-sites. T-Box Genes Function as an Interacting Network. ntl, spt,
and tbx6 function as interacting members of a network that directs
mesoderm gene expression and developmental fate in the zebrafish. The
interactions are not solely additive in that coexpression of ntl and
spt brings about new functions and tbx6 can suppress some
ntl functions. Because the interactions determine the function of a
participating T-box gene, the developmental role of any one T-box gene may
vary from cell to cell, depending on the expression of other family members.
The existence of these interactions has two implications for the analysis of
T-box gene function: (i) individual T-box genes may have multiple
region-specific functions within a single developmental field; and
(ii) loss-of-function of one T-box gene is likely to alter the
function of a second T-box gene expressed in the same morphogenetic field. As T-box genes are expressed in overlapping patterns in many developmental
fields in both vertebrate and invertebrate embryos
( 5,
43), we propose that the
formation of interacting networks is integral to the mode of action of T-box
genes, as it is for Hox gene function
( 44). Overlapping expression
of interacting T-box genes contributes to the subdivision of the zebrafish
mesoderm into smaller regional elements with distinct gene expression
patterns. The use of overlapping interactive genes is a resilient strategy for
mediating positional information to effect regionalization of a large field.
Even if embryo-to-embryo variability alters the exact breadth of a T-box
expression domain, cells are never left without identity, and neighbor
relationships among tissue precursors are preserved. Interactions Between T-Box Genes. Our results show that pairs of
T-box genes can interact combinatorially, additively, or antagonistically;
however, the data do not reveal the mechanistic basis of these interactions.
Combinatorial interactions are demonstrated by the finding that coexpression
of ntl and spt is required for mesogenin
transcription. Given the very short delay between the onset of ntl
expression and the onset of mesogenin expression, we propose that No
Tail and Spadetail interact directly, perhaps forming a heterodimeric
transcription factor with previously uncharacterized specificity. Other T-box
proteins form homodimers or heterodimers with partners of a different
transcription factor family
( 13,
41,
42). Alternatively, the
combined presence of No Tail and Spadetail may determine the cofactors
accessible to each, thereby affecting the target specificity of each T-box
protein ( 45). Amacher et al.
( 25) initially demonstrated
that ntl and spt contribute in an additive way to mesoderm
development. Our finding that the loss of a single copy of ntl acts
dominantly to enhance the spt phenotype indicates that both genes
contribute to muscle and notochord development. Furthermore, the studies of
tbx6 regulation indicate the two T-box genes have unequal and
additive effects on expression of some downstream genes, an interaction
observed between some pairs of paralogous Hox genes
( 46). Finally, Tbx6 can act as a competitive inhibitor of some No Tail functions.
Experiments in embryos and tissue culture indicate that cells can measure the
relative abundance of the two proteins. Tbx6 need not be the only factor that
suppresses ntl function ventrally, as our preliminary experiments
indicate that embryos treated with tbx6 antisense morpholino
oligonucleotides exhibit only mild axial defects. Our studies show that
differences among promoter binding sites may explain how Tbx6 can
competitively inhibit expression of some, but not all, target genes regulated
by No Tail. We have used the 1/2T- and palT-sites to model potential
interactions between Tbx6 and No Tail, realizing that the promoter sequences
mediating No Tail transcription in vivo are likely to be more
complex. Both the sequence of T-sites
( 39,
47) and the presence of
additional sequences that recruit cofactors
( 13,
14) might affect the strength
of No Tail binding to target promoters and thus affect the relative affinity
of Tbx6 and No Tail for targets. Gene Families and Gene Networks. Gene duplication events are thought
to lead to diversification of gene functions and thus to contribute to
biological diversity
( 48– 50).
One widely recognized mechanism for increasing the roles of an ancestral gene
is through diversification of the biochemical functions and/or cellular
expression patterns of individual homologous family members
( 51– 53).
Here we illustrate how interactions between homologues effectively increase
diversity of gene function. The existence of interacting networks of Hox and
T-box genes suggests that a recurrent mechanism for increasing diversity of
gene function may involve combinatorial interactions among members of a
transcription factor family. We thank G. Walter and S. Johnson for excellent technical support. S.
Amacher and B. Draper graciously shared unpublished information. This work was
supported by National Institutes of Health Grant HD37572 (to D.J.G.) and by
predoctoral fellowships from the Huntsman Cancer Institute (to L.M.G.) and the
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