bifid
Xenopus Antipodean, a novel
member of the T-box gene family that includes the Xenopus genes Xbrachyury and
Eomesodermin, is unique within the T-box family because
it is maternally expressed at a high level. Furthermore, it belongs to a rare class of maternal
mRNAs in Xenopus that are localised to the vegetal hemisphere of the egg. Low amounts of Antipodean injected into
ectoderm (animal cap cells) strongly induce pan mesodermal genes such as Xbrachyury and
ventral mesodermal genes such as Xwnt-8. Overexpression of Antipodean generates
mesoderm of ventral character, and induces muscle only weakly. This property is consistent
with the observed late zygotic Antipodean mRNA expression in the posterior paraxial
mesoderm and ventral blastopore, and its exclusion from the most dorsal mesodermal
structure, the notochord. Antipodean is induced by several molecules of the TGF-ß class, but
in contrast to Xbrachyury, is not induced by bFGF. This result suggests that the expression of these
T-box genes may be under the control of different regulatory pathways. Antipodean and Eomesodermin induce each other and both are able to
induce Xbrachyury. The early zygotic expression of Antipodean is not induced by Xbrachyury,
though to some extent this is the case later on. Considering its maternal content, Antipodean could initiate
a cascade of T-box gene activations. The expression of these genes may, in turn, sustain
one another's expression to define and maintain the mesoderm identity in Xenopus (Stennard, 1996).
An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein
(VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in
development. VegT is a nuclear protein and its C-terminal domain can activate transcription
in a yeast reporter assay, observations consistent with VegT functioning as a transcription
factor. Zygotic expression is dynamic along the dorsoventral axis, with transcripts first
expressed in the dorsal marginal zone. By the end of gastrulation, VegT is expressed
exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord.
Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory
neuron. In animal cap assays, VegT is capable of converting prospective ectoderm into
ventral lateral mesoderm. Such ectopic expression of VegT induces its own expression as
well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved.
Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain
suppresses head formation. VegT may turn out to be a localized transcription factor
that operates sequentially in several developmental pathways during embryogenesis,
including dorsoventral and posterior patterning of mesoderm (Zhang, 1996).
VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal
hemisphere of the egg and early embryo. Maternally expressed VegT controls the pattern of primary
germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different
regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only,
equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to
mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing
signals. These experiments show that maternal VegT is required for vegetal cells of the blastula to produce the
endogenous vegetal signal(s) that cause caps to form mesoderm. This represents an important departure from the popular
view that early vegetal signals cause mesoderm formation. VegT is a transcription factor and will not activate transcription
until after MBT. Thus, zygotic inducing factors downstream of VegT, not maternal signaling factors, initiate the endogenous
signal. This supports the view that mesoderm induction is a posttranscriptional event in Xenopus and that the primary
patterning event underlying it is the localization of a maternal transcription factor (Zhang, 1998).
Inhibition of fibroblast growth factor (FGF) signaling prevents trunk and tail formation in Xenopus and
zebrafish embryos. While the T-box transcription factor Brachyury (called No Tail in zebrafish) is a key
mediator of FGF signaling in the notochord and tail, the pathways activated by FGF in non-notochordal
trunk mesoderm have been uncertain. Previous studies have shown that the spadetail gene is required for non-notochordal trunk mesoderm formation: spadetail mutant embryos have major trunk mesoderm deficiencies, but relatively normal tail and notochord development. spadetail is shown to
encodes a T-box transcription factor with homologs in Xenopus and chick. The Xenopus ortholog is known as Xombi, Antipodean, VegT or BraT. Spadetail is apt to be a key
mediator of FGF signaling in trunk non-notochordal mesoderm, since spadetail expression is regulated by FGF signaling. Trunk and tail development are therefore dependent on the complementary actions of two T-box genes, spadetail and no tail. The regulatory hierarchy among spadetail, no tail and a third T-box gene, tbx6, are substantially different during trunk and tail mesoderm formation, and a genetic model is proposed that accounts for the regional phenotypes of spadetail and no tail mutants. In the tail bud spadetail acts downstream of no tail, but during gastrulation and early segmentation, when trunk mesoderm is forming, spt expression is independent of ntl function, indicating that FGF signaling must regulate these two genes independent of one another. However, as tail mesoderm begins to involute, spt expression becomes genetically downstream of ntl. It is suggested that tbx6 functions downstream of spt. tbx6 is similar to spt in residues required for DNA binding, and tbx6 expression in the trunk partially depends on spt function (Griffin, 1998).
A new zebrafish T-box-containing gene, tbx16 encodes a message that is first detected
throughout the blastoderm soon after the initiation of zygotic gene expression. Following gastrulation,
expression becomes restricted to paraxial mesoderm and later primarily to the developing tail bud. To
gain an evolutionary prospective on the potential function of this gene, its
phylogenetic relationships were analyzed to known T-box genes from other species. Zebrafish tbx16 is likely
orthologous to the chicken Tbx6L and Xenopus Xombi/Antipodean/Brat/VegT genes. Zebrafish tbx6 and mouse Tbx6 genes are paralogous to zebrafish tbx16. Despite the same name and similar expression, zebrafish tbx6 and mouse
Tbx6 genes are not orthologous to one another but instead represent relatively distant paralogs. The
expression patterns of all genes are discussed in the light of their evolutionary relationships (Ruvinsky, 1998).
Zebrafish paraxial protocadherin (papc) encodes a transmembrane cell adhesion molecule (PAPC) expressed in trunk mesoderm undergoing morphogenesis. Microinjection studies with a dominant-negative secreted construct suggest that papc is required for proper dorsal convergence movements during gastrulation. Genetic studies show that papc is a close downstream target of spadetail, a gene encoding a transcription factor required for mesodermal morphogenetic movements. The floating head
homeobox gene, acting downstream of no tail, is required in axial mesoderm to repress the expression of both spadetail and papc,
promoting notochord and blocking differentiation of paraxial mesoderm. The PAPC structural cell-surface
protein may provide a link between regulatory transcription factors and the actual cell biological behaviors
that execute morphogenesis during gastrulation (Yamamoto, 1998).
Three novel members of the T-box gene family are described in zebrafish. One of these genes,
tbx-c, was studied in detail. It is expressed in the axial mesoderm, notably, in the notochordal precursor cells
immediately before formation of the notochord and in the chordoneural hinge of the tail bud, after the
notochord is formed. In addition, its expression is detected in the ventral forebrain, sensory neurons, fin
buds and excretory system. The expression pattern of tbx-c differs from that of the other two related genes,
tbx-a and tbx-b. The developmental role of tbx-c has been analysed by overexpression of the full-length
tbx-c mRNA and a truncated form of tbx-c mRNA, which encodes the dominant-negative Tbx-c. Overexpression of tbx-c causes expansion of the midline mesoderm and formation of ectopic midline
structures at the expense of lateral mesodermal cells. In dominant-negative experiments, the midline
mesoderm is reduced with the expansion of lateral mesoderm to the midline. These results suggest that tbx-c
plays a role in formation of the midline mesoderm, particularly, the notochord. Moreover, modulation of
tbx-c activity alters the development of primary motor neurons. Results of in vitro analysis in zebrafish
animal caps suggest that tbx-c acts downstream of early mesodermal inducers (activin and ntl) and reveal an
autoregulatory feedback loop between ntl and tbx-c. These data and analysis of midline (ntl-/- and flh-/-) and
lateral mesoderm (spt-/-) mutants suggest that tbx-c may function during formation of the notochord (Dheen, 1999).
Development of the posterior body (lumbosacral region and tail) in vertebrates is delayed relative to gastrulation. In amniotes, it proceeds with the replacement of the regressed node and primitive streak by a caudal blastema-like mass of mesenchyme known as the tail bud. Despite apparent morphological dissimilarities, recent results suggest that tail development in amniotes is in essence a continuation of gastrulation, as is the case in Xenopus. However, this has been inferred primarily from the outcome of fate mapping studies demonstrating discrete, regionalized cell populations in the tail bud, like those present at gastrulation. Analysis of the tail bud distribution of several molecular markers that are expressed in specific spatial domains during chick gastrulation confirms these results. Several markers specific for axial (Gnot1 and Ch-T) and paraxial mesodermal lineages (Ch-Tbx6L) during gastrulation, are also expressed in the tail bud. Notably, expression of organizer-related markers in developing tail bud are restricted to 'caudal' components (trunk/tail organizer) (Knezevic, 1998).
The expression of representative caudal organizer-associated markers was evaluated during the transition period from streak to tail bud. Gnot1 and Tbx6L are expressed in complementary domains during gastrulation. Gnot1 is in Hensen's node and notochord and Ch-Tbx6L in primitive streak and segmental plate, while Ch-T is expressed in nascent and axial mesoderm along the entire axis. Formation of the tail bud starts in the 13 somite embryo (stage 11) as cells of Hensen's node and primitive streak begin to accumulate caudally in a bulbous mass of uniform mesenchyme. This transformation into tail bud is completed by the 24-28 somite stage (stage 15). Surprisingly, despite its uniform morphological appearance, gene expression in the forming tail bud suggests a segregation of Hensen's node- and primitive streak-derived cells. Ch-T and Ch-Tbx6L are both selectively expressed in a superficial medial to lateral ventral rim of tail bud mesenchyme, in addition to continued expression of Ch-T in the notochord and Ch-Tbx6L in the segmental plate. Gnot1 expression is detected in the formed and nascent notochord but does not extend very posteriorly from this region, as compared to Ch-T. The caudal limit of Gnot1 expression corresponds to the chordoneural hinge region (the point where caudal neural tube and notochord unite) located between the residual Hensen's node (Gnot1 and Ch-T positive) and primitive streak (Ch-T positive). Cells located within the central region of the condensing tail bud fail to express primitive streak or node-specific markers. Before the tail bud forms, the neuroectoderm at the caudal end of the neural tube/neural plate (sinus rhomboidalis) is in direct continuity with the underlying mesenchyme where the tail bud will arise. In later tailbud stages, the closed neural tube extends beneath the surface as a solid mesenchymal rod (medullary cord) in the tail bud. This medullary cord forms caudal neural tube by central cavitation and mesenchymal-epithelial conversion (Knezevic, 1998).
To determine whether the central core of marker-negative cells seen in forming tail bud are early neural progenitors of medullary cord, the pan-neural marker L5 was used (a monoclonal antibody directed against an epitope specific for early neural tissue). The central mesenchymal region that is negative for mesodermal markers stains positively for the L5 neural marker, suggesting that caudal neuroectoderm also contributes cells regionally to the early central tail bud mesenchyme prior to formation of a discrete medullary cord. Once formation of the tail bud is complete, elongation begins. During the ensuing week (stage 16-35), the tail bud elongates posteriorly, leaving behind organized tail structures proximally. Although the tail bud eventually occupies a small region at the tip of the growing tail, distinct regional domains of gene expression are still visible at these later times. Gnot1 expression continues in the chordoneural hinge region and adjacent caudal notochord, while Ch-T and Ch-Tbx6L are expressed in the ventral rim of tail bud mesenchyme. Ch-T expression also continues in chordoneural hinge and notochord, and Ch-Tbx6L in the tail segmental plate (Knezevic, 1998).
Evidence is presented that gastrulation-like ingression movements from the surface continue in the early chick tail bud and that the established tail bud retains organizer activity. This 'tail organizer' has the expected properties of being able to recruit uncommitted host cells into a new embryonic axis and induce host neural tissue with posteriorly regionalized gene expression when grafted to competent host cells that are otherwise destined to form only extra-embryonic tissue. Together, these results indicate that chick tail development is mechanistically continuous with gastrulation and that the developing tail in chick may serve as a useful experimental adjunct to investigate the molecular basis of inductive interactions operating during gastrulation, considering that residual tail organizing activity is still present at a surprisingly late stage (Knezevic, 1998).
Mouse Tbx6 codes for a 1.9-kb transcript with an open reading frame coding for a 540-amino acid protein, with a predicted molecular weight of 59 kDa. Tbx6 maps to chromosome 7 and does not appear to be linked to any known mutation. Unlike other members of the mouse T-box gene family, which are expressed in a wide variety of tissues derived from all germ layers, Tbx6 expression is quite restricted. Tbx6 transcripts are first detected in the gastrulation stage embryo in the primitive streak and newly recruited paraxial mesoderm. Later in development, Tbx6 expression is restricted to presomitic, paraxial mesoderm and to the tail bud, which replaces the streak as the source of mesoderm. Expression in the tail bud persists until 12.5 days postcoitus. Tbx6 expression thus overlaps that of Brachyury in the primitive streak and tail bud, although Brachyury is expressed earlier in the primitive streak. Brachyury is also expressed in a second domain, the node and notochord, that is not shared with Tbx6. The onset of Tbx6 expression is not affected in homozygous null Brachyury mutant embryos at 7.5 days postcoitus. However, Tbx6 expression is extinguished in mutant embryos as soon as the Brachyury phenotype becomes evident at 8.5 days postcoitus, indicating that the continued expression of Tbx6 is either directly or indirectly dependent upon Brachyury expression (Chapman, 1996).
Somites, segmented mesodermal units of the vertebrate embryo, are the precursors of adult skeletal muscle, bone and cartilage. During embryogenesis, somite progenitor cells ingress through the primitive streak, move laterally to a paraxial position (alongside the body axis) and segment into epithelial somites. Little is known about how this paraxial mesoderm tissue is specified. Mouse T-box gene Tbx6 codes for a putative DNA-binding protein. The embryonic pattern of expression of Tbx6 in somite precursor cells suggests that this gene may be involved in the specification of paraxial mesoderm. A mutation in Tbx6 profoundly affects the differentiation of paraxial mesoderm. Irregular somites form in the neck region of mutant embryos, whereas more posterior paraxial tissue does not form somites but instead differentiates along a neural pathway, forming neural-tube-like structures that flank the axial neural tube. These paraxial tubes show dorsal/ventral patterning that is characteristic of the neural tube, and have differentiated motor neurons. These results indicate that Tbx6 is needed for cells to choose between a mesodermal and a neuronal differentiation pathway during gastrulation; Tbx6 is essential for the specification of posterior paraxial mesoderm, and in its absence cells destined to form posterior somites differentiate along a neuronal pathway (Chapman, 1998).
During vertebrate embryogenesis, paraxial mesoderm gives rise to somites, which subsequently develop into the dermis, skeletal muscle, ribs and vertebrae of the adult. Mutations that disrupt the patterning of individual somites have dramatic effects on these tissues, including fusions of the ribs and vertebrae. The T-box transcription factor, Tbx6, is expressed in the paraxial mesoderm but is downregulated as somites develop. It is essential for the formation of posterior somites, which are replaced with ectopic neural tubes in Tbx6-null mutant embryos. Partial restoration of Tbx6 expression in null mutants rescues somite development, but that rostrocaudal patterning within them is defective, ultimately resulting in rib and vertebral fusions, demonstrating that Tbx6 activity in the paraxial mesoderm is required not simply for somite specification but also for their normal patterning. Somite patterning is dependent upon Notch signaling and Tbx6 is shown to genetically interact with the Notch ligand, delta-like 1 (Dll1). Dll1 expression, which is absent in the Tbx6-null mutant, is restored at reduced levels in the partially rescued mutants, suggesting that Dll1 is a target of Tbx6. The spontaneous mutation rib-vertebrae has been identified as a hypomorphic mutation in Tbx6. The similarity in the phenotypes described in this study and that of some human birth defects, such as spondylocostal dysostosis, raises the possibility that mutations in Tbx6 or components of this pathway may be responsible for these defects (White, 2003).
Tbx6 is a member of the T-box family of transcription factor genes. Two mutant alleles of this gene establish that Tbx6 is involved in both the specification and patterning of the somites along the entire length of the embryo. The null allele, Tbx6tm1Pa, causes abnormal patterning of the cervical somites and improper specification of more posterior paraxial mesoderm, such that it forms ectopic neural tubes. In this study, this allele was used to further investigate the mechanism of action of the Tbx6 gene and investigate possible genetic interactions. The developmental and differentiation potential of Tbx6tm1Pa/Tbx6tm1Pa cells was tested in ectopic sites, in vitro, and in chimeras in vivo. Cell proliferation and cell death were documented in mutant tail buds in an attempt to explain the mechanism of tail bud enlargement in the Tbx6 mutant embryos. The results indicate specific developmental restrictions on the differentiation of posterior cells lacking Tbx6, once they have traversed the primitive streak, but no restrictions in differentiation of anterior somites, or of Tbx6 null embryonic stem (ES) cells. Tbx6 null ES cells fail to populate posterior somites in chimeric embryos. To discover whether different T-box proteins interact on the same down stream targets in areas of expression overlap, potential interactions between Tbx6 and T (Brachyury) were explored in genetic crosses. The TWis mutation is epistatic to the Tbx6tm1Pa mutation and there is no apparent genetic interaction. However, homozygosity for Tbx6tm1Pa and heterozygosity for TWis mutation shows a combinatorial interaction at the phenotypic level. Thus, even in areas of expression overlap, the action of the two T-box genes appears to be independent, T affecting the production of notochord and posterior mesoderm, and Tbx6 affecting the specification of the paraxial mesoderm that does form (Chapman, 2002).
The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. This pattern is shown to be established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, it was found that the process, analyzed in the chicken, is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of bHLH transcription factor MyoR (musculin) and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart (Bothe, 2011).
Expression of Fgf and Bmp responsive molecules indicated that the anterior head mesoderm receives Fgf and Bmp signals for the first time during phase 2 when Alx4 and MyoR are upregulated. Suppression of Bmp signalling prevented, and elevated Bmp signalling advanced, Alx4 activation. Thus, Bmp is necessary and sufficient to control Alx4. MyoR, however, was repressed by suppression of either Bmp or Fgf signalling. Elevation of Bmp or Fgf signalling promoted MyoR, albeit only at the stage at which the gene is normally expressed; premature MyoR expression could only be achieved by combinatorial application of Bmp and Fgf. Thus, combined Fgf and Bmp activity is required to activate MyoR (Bothe, 2011).
Expression analysis showed that the onset of MyoR is rather sudden. The bead implantation experiments indicated that in the anterior head mesoderm, Fgf enhanced the expression of Bmp responsive genes and Bmp upregulated genes indicative of active Fgf signalling. This suggests that Bmp and Fgf reinforce each other, possibly creating the appropriate setting to activate MyoR. Studies on mouse mutants placed Pitx2 upstream of MyoR. Thus, it is conceivable that, in addition to Bmp and Fgf, the earlier activation of Pitx2 is a further prerequisite for the activation of MyoR (Bothe, 2011).
In the posterior head mesoderm, Bmp strongly suppressed Tbx1. Fgf signalling, however, was unaffected, suggesting that Bmp controls the anterior border of Tbx1 expression, possibly directly targeting Tbx1. Tbx1, by contrast, has recently been suggested to suppress Bmp signalling by preventing Smad1-Smad4 interaction. This suggests that Tbx1 indirectly controls the extension of Bmp dependent markers (Bothe, 2011).
When Bmp and Fgf signalling commences in the anterior head mesoderm, Fgf signalling levels increase significantly in the posterior domain, owing to the positive Fgf-Tbx1 feedback loop. After applying Fgf to the anterior head mesoderm, i.e. elevating the Fgf level beyond that which is normally found there, it was noticed that Pitx2 and Alx4 expression declined. Thus, although Fgf is necessary for the activation of MyoR, high Fgf levels prevent the molecular set-up of the anterior head mesoderm. This infers that, whereas Bmp controls the anterior border of the posterior head mesoderm marker, Fgf controls the posterior border of the two anterior markers Pitx2 and Alx4 (Bothe, 2011).
In phase 3, extension of MyoR and Tbx1 expression is concomitant with the spread of high-level Fgf signalling along the floor of the pharynx. Fgf application was found to accelerate the MyoR-Tbx1 spread, and suppression of Fgf signalling prevented it. This suggests that Fgf signalling is key to establishing the final head mesoderm pattern. Notably, MyoR remained sensitive to RA. In the embryo, however, the site of RA production continuously recedes posteriorly during phases 2 and 3, suggesting that the posterior extension of MyoR expression occurs at a rate set by RA (Bothe, 2011).
The anteriorly spreading Fgf signals will eventually reach the Pitx2-Alx4 domain. Both genes were negatively regulated by high Fgf levels in phases 1 and 2; yet, in phase 3 the genes remain expressed. Likewise, Tbx1 spreads anteriorly although this territory is controlled by Bmp. Notably, Fgf levels vary along the anteroposterior extent of the pharynx; at HH13, for example, Fgf signalling appears lower in the anterior compared with the posterior pharyngeal arches. Thus, it is possible that in the anterior head mesoderm, Fgf levels might remain low enough to allow Pitx2 and Alx4 expression, but rise sufficiently to override the Bmp effect on Tbx1. Conversely, the Fgf levels in the posterior head mesoderm might by so high that MyoR expression can spread, whereas Pitx2 and Alx4 remain repressed. It cannot be excluded that additional signals restrict Pitx2 and Alx4 expression. Yet, the spread of MyoR outside of the Pitx2 territory indicates that in phase 3 MyoR expression has become independent from its former upstream regulator (Bothe, 2011).
RA, Bmp and Fgf signalling play multiple roles during development. RA, in many settings, promotes cell differentiation; in the head, RA first suppresses cardiac markers to set the posterior limit of the heart field, but then specifies the sinoatrial region of the heart. Moreover, RA has the capacity to provide cells with a more posterior positional identity. Bmp is a crucial regulator of cardiac development and has been suggested to recruit head mesodermal cells into the cardiac lineage. Fgf promotes the secondary heart field and keeps cells proliferative and undifferentiated. Therefore whether the observed changes in head mesodermal marker expression occurred because of cell recruitment into cardiac lineage, premature differentiation or posteriorisation was tested. RA or Fgf treatment was found not to change cell fate or differentiation status. Bmp induced cardiac marker gene expression only when applied during phase 0. When applied in phase 1, i.e. just before Bmp signalling is normally activated in the head mesoderm, Bmp did not induce cardiac markers unless the dosage was increased. This suggests that, possibly, cardiac induction can occur from exposure to higher Bmp levels and/or longer exposure times. Taken together, this study suggests that RA, Bmp and Fgf specifically control head mesoderm patterning with the cells remaining undifferentiated and competent to enter any of the possible mesodermal lineages (Bothe, 2011).
The T-box transcription factor Tbx6 controls the expression of Mesp2, which encodes a basic helix-loop-helix transcription factor that has crucial roles in somitogenesis. In cultured cells, Tbx6 binding to the Mesp2 enhancer region is essential for the activation of Mesp2 by Notch signaling. However, it is not known whether this binding is required in vivo. This study reports that an Mesp2 enhancer knockout mouse bearing mutations in two crucial Tbx6 binding sites does not express Mesp2 in the presomitic mesoderm. This absence leads to impaired skeletal segmentation identical to that reported for Mesp2-null mice, indicating that these Tbx6 binding sites are indispensable for Mesp2 expression. T-box binding to the consensus sequences in the Mesp2 upstream region was confirmed by chromatin immunoprecipitation assays. Further enhancer analyses indicated that the number and spatial organization of the T-box binding sites are critical for initiating Mesp2 transcription via Notch signaling. A knock-in mouse was generated in which the endogenous Mesp2 enhancer was replaced by the core enhancer of medaka mespb, an ortholog of mouse Mesp2. The homozygous enhancer knock-in mouse was viable and showed normal skeletal segmentation, indicating that the medaka mespb enhancer functionally replaced the mouse Mesp2 enhancer. These results demonstrate that there is significant evolutionary conservation of Mesp regulatory mechanisms between fish and mice (Yasuhiko, 2008).
Expression patterns of Tbx2, -3, and -5 genes, homologs of Drosophila Omb, were analyzed during chick embryonic heart development. Transcripts of these
three cTbx genes were detected in overlapping patterns in the early cardiac crescent. cTbx2 and cTbx3 expression patterns
closely overlap with that of bmp2. cTbx5 expression diverges from cTbx2 and bmp2 during the elaboration and folding
of the heart tube. In comparison, cTbx2 expression overlaps significantly with that of bmp2 and bmp4 during all stages
of heart development and during later embryonic stages, suggestive of a specialized role for Tbx2 in septation. Coexpression
of cTbx2 and cTbx3 genes with bmp2 transcripts raises the possibility that these cTbx genes might be downstream bmp2
targets. Application of bmp2 selectively induces cTbx2 and cTbx3 expression in noncardiogenic embryonic tissue, and the
bmp antagonist Noggin down-regulates cTbx2 gene activity. Moreover, the appearance of murine mTbx2 is blocked in
bmp2 null mouse embryos. cTbx2 and to a lesser extent cTbx3 gene activity appears to be directed by BMPs during early
cardiogenesis (Yamada, 2000).
To further define the role of a T-box transcription factor, Tbx5, in cardiac development, its expression in the developing mouse and chick heart was examined
and this pattern was correlated with cardiac defects caused by human TBX5 mutations in Holt-Oram syndrome. Early in the developing heart, Tbx5 is uniformly
expressed throughout the entire cardiac crescent. Upon formation of the linear heart tube, Tbx5 is expressed in a graded fashion, stronger near the posterior end and
weaker at the anterior end. As the heart tube loops, asymmetric Tbx5 expression continues; Tbx5 is expressed in the presumptive left ventricle, but not the right
ventricle or outflow tract. This pattern of expression is maintained in more mature hearts. Expression in the ventricular septum is restricted to the left side and is
contiguous with left ventricular free wall expression. Trabeculae, vena cavae (inferior and superior), and the atrial aspect of the atrioventricular valves also express
high levels of Tbx5. These patterns of Tbx5 expression provide an embryologic basis for the prevalence of atrial septal defects (ostium primum and secundum),
ventricular muscular septal defects, and left-sided malformations (endocardial cushion defects, hypoplastic left heart, and aberrant trabeculation) observed in patients
with Holt-Oram syndrome (Bruneau, 1999).
Heterozygous Tbx5(del/+) mice were generated to study the mechanisms by which TBX5 haploinsufficiency causes cardiac and forelimb abnormalities seen in Holt-Oram syndrome. Tbx5 deficiency in homozygous mice (Tbx5(del/del)) decreased expression of multiple genes and caused severe hypoplasia of posterior domains in the developing heart. Surprisingly, Tbx5 haploinsufficiency also markedly decreased atrial natriuretic factor (ANF) and connexin 40 (cx40) transcription, implicating these as Tbx5 target genes and providing a mechanism by which 50% reduction of T-box transcription factors cause disease. Direct and cooperative transactivation of the ANF and cx40 promoters by Tbx5 and the homeodomain transcription factor Nkx2-5 was also demonstrated. These studies provide one potential explanation for Holt-Oram syndrome conduction system defects, suggest mechanisms for intrafamilial phenotypic variability, and account for related cardiac malformations caused by other transcription factor mutations (Bruneau, 2001).
During heart development, chamber myocardium forms locally from the
embryonic myocardium of the tubular heart. The atrial natriuretic
factor (ANF) gene is specifically expressed in this developing
chamber myocardium and is one of the first hallmarks of chamber
formation. The regulatory mechanism underlying this
selective expression has been investigated. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of
endogenous ANF gene expression. Furthermore, this fragment is
able to repress cardiac troponin I (cTnI) promoter
activity selectively in the embryonic myocardium of the
atrioventricular canal (AVC). In vivo inactivation of a T-box factor
(TBE) or NK2-homeobox factor binding element (NKE) within the
ANF fragment removes the repression in the AVC without
affecting its chamber activity. The T-box family member Tbx2,
encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 forms a complex with Nkx2.5 on the ANF TBE-NKE, and is able to repress ANF promoter activity. These data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC (Habets, 2002).
Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Isolated cardiac septal defects in a large pedigree were shown to be linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, a frame-shift mutation of GATA4 (E359del) was identified that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5 (Garg, 2003).
Extensive misexpression studies were carried out to explore the roles
played by Tbx5, the expression of which is excluded from the right
ventricle (RV) during cardiogenesis. When Tbx5 is misexpressed ubiquitously,
ventricular septum is not formed, resulting in a single ventricle. In such a
heart, left ventricle (LV)-specific ANF gene is induced. In search
of the putative RV factor(s), chick Tbx20 was found to be
expressed in the RV, showing a complementary fashion to Tbx5. In the
Tbx5-misexpressed heart, this gene is repressed. When misexpression is
spatially partial, leaving small Tbx5-negative area in the right ventricle,
ventricular septum is shifted rightwards, resulting in a small RV with an
enlarged LV. Focal expression induces an ectopic boundary of Tbx5-positive and
-negative regions in the right ventricle, at which an additional septum is
formed. Similar results were obtained from the transient transgenic mice. In
such hearts, expression patterns of dHAND and eHAND are
changed with definitive cardiac abnormalities. Furthermore,
human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and
GATA4. This activation is abrogated by Tbx20, implicating the pivotal roles
of interactions among these heart-specific factors. Taken together, these data
indicate that Tbx5 specifies the identity of LV through tight interactions
among several heart-specific factors, and highlight the essential roles of
Tbx5 in cardiac development (Takeuchi, 2003b).
Dysmorphogenesis of the cardiac outflow tract (OFT) causes many congenital heart defects, including those associated with DiGeorge syndrome. Genetic manipulation in the mouse and mutational analysis in patients have shown that Tbx1, a T-box transcription factor, has a key role in the pathogenesis of this
syndrome. Tbx1 function during OFT development have been dissected using
genetically modified mice and tissue-specific deletion, and have defined a
dual role for this protein in OFT morphogenesis. Tbx1 regulates
cell contribution to the OFT by supporting cell proliferation in the secondary heart field, a source of cells fated to the OFT. This process might be regulated in part by Fgf10, which is a
direct target of Tbx1 in vitro. Tbx1 expression is
required in cells expressing Nkx2.5 for the formation of the
aorto-pulmonary septum, which divides the aorta from the main pulmonary
artery. These results explain why aortic arch patterning defects and OFT
defects can occur independently in individuals with DiGeorge syndrome.
Furthermore, the data link the function of the secondary
heart field to congenital heart disease (Xu, 2004).
Tbx2 is a member of the T-box transcription factor gene family,
and is expressed in a variety of tissues and organs during embryogenesis. In
the developing heart, Tbx2 is expressed in the outflow tract, inner
curvature, atrioventricular canal and inflow tract, corresponding to a
myocardial zone that is excluded from chamber differentiation at 9.5 days of development. Targeted mutagenesis was used in mice to investigate Tbx2
function. Mice heterozygous for a Tbx2 null mutation appear normal
but homozygous embryos reveal a crucial role for Tbx2 during cardiac
development. Morphological defects are observed in development of the
atrioventricular canal and septation of the outflow tract. Molecular analysis
reveals that Tbx2 is required to repress chamber differentiation in
the atrioventricular canal at 9.5 dpc. Analysis of homozygous mutants also
highlights a role for Tbx2 during hindlimb digit development. Despite
evidence that TBX2 negatively regulates the cell cycle control genes
Cdkn2a, Cdkn2b and Cdkn1a in cultured cells, there is no
evidence that loss of Tbx2 function during mouse development results
in increased levels of p19ARF, p16INK4a,
p15INK4b or p21 expression in vivo, nor is there evidence for a
genetic interaction between Tbx2 and p53 (Harrelson 2004).
Birth defects, which occur in one out of 20 live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription factor TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. An allelic series of Tbx1 deficiency was generated that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription factor Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. A Fox-binding site upstream of Tbx1 has been identified that interacts with Foxa2 and is necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, a fibroblast growth factor 8 (Fgf8) enhancer was found that is dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function results in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgf10. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11 (Hu, 2004).
Tbx20, a member of the T-box family of transcriptional regulators,
shows evolutionary conserved expression in the developing heart. In the mouse,
Tbx20 is expressed in the cardiac crescent, then in the endocardium
and myocardium of the linear and looped heart tube before it is restricted to
the atrioventricular canal and outflow tract in the multi-chambered heart.
Tbx20 is required for progression from the linear
heart tube to a multi-chambered heart. Mice carrying a targeted mutation of
Tbx20 show early embryonic lethality due to hemodynamic failure. A
linear heart tube with normal anteroposterior patterning is established in the
mutant. The tube does not elongate, indicating a defect in recruitment of
mesenchyme from the secondary heart field, even though markers of the
secondary heart field are not affected. Furthermore, dorsoventral patterning
of the tube, formation of working myocardium, looping, and further
differentiation and morphogenesis fail. Instead, Tbx2, Bmp2
and vinexin alpha (Sh3d4), genes normally restricted to regions of
primary myocardium and lining endocardium, are ectopically expressed in the
linear heart tube of Tbx20 mutant embryos. Because Tbx2 is
both necessary and sufficient to repress chamber differentiation
Tbx20 may ensure progression to a multi-chambered heart by repressing
Tbx2 in the myocardial precursor cells of the linear heart tube
destined to form the chambers (Singh, 2005).
The genetic hierarchies guiding lineage specification and morphogenesis of
the mammalian embryonic heart are poorly understood. It has been shown by gene
targeting that murine T-box transcription factor Tbx20 plays a central role in
these pathways, and has important activities in both cardiac development and
adult function. Loss of Tbx20 results in death of embryos at
mid-gestation with grossly abnormal heart morphogenesis. Underlying these
disturbances is a severely compromised cardiac transcriptional program,
defects in the molecular pre-pattern, reduced expansion of cardiac progenitors
and a block to chamber differentiation. Notably, Tbx20-null embryos
show ectopic activation of Tbx2 across the whole heart myogenic
field. Tbx2 encodes a transcriptional repressor normally expressed in
non-chamber myocardium, and in the atrioventricular canal it has been proposed
to inhibit chamber-specific gene expression through competition with positive
factor Tbx5. These data demonstrate a repressive activity for Tbx20 and place it
upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical,
repressive interactions between Tbx20 and other T-box genes and factors
underlie the primary lineage split into chamber and non-chamber myocardium in
the forming heart, an early event upon which all subsequent morphogenesis
depends. Additional roles for Tbx20 in adult heart integrity and
contractile function were revealed by in-vivo cardiac functional analysis of
Tbx20 heterozygous mutant mice. These data suggest that mutations in
human cardiac transcription factor genes, possibly including TBX20,
underlie both congenital heart disease and adult cardiomyopathies (Stennard, 2005).
The establishment of chamber specificity is an essential requirement for
cardiac morphogenesis and function. Hesr1 (Hey1) and
Hesr2 (Hey2) are specifically expressed in the atrium and
ventricle, respectively, implicating these genes in chamber specification. Forced expression of Hesr1 or Hesr2 in the entire cardiac lineage of the mouse results in the reduction or loss of the atrioventricular (AV) canal. In the
Hesr1-misexpressing heart, the boundaries of the AV canal are poorly
defined, and the expression levels of specific markers of the AV myocardium,
Bmp2 and Tbx2, are either very weak or undetectable. More
potent effects were observed in Hesr2-misexpressing embryos, in which
the AV canal appears to be absent entirely. These data suggest that
Hesr1 and Hesr2 may prevent cells from expressing the AV
canal-specific genes that lead to the precise formation of the AV boundary.
These findings suggest that Tbx2 expression might be directly
suppressed by Hesr1 and Hesr2. Furthermore, the expression of
Hesr1 and Hesr2 is independent of Notch2 signaling. Taken
together, these data demonstrate that Hesr1 and Hesr2 play
crucial roles in AV boundary formation through the suppression of Tbx2 (Kokubo, 2007).
The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. Lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype (Hoogaars, 2007)
Elucidating the gene regulatory networks that govern pharyngeal arch artery (PAA) development is an important goal, as such knowledge can help to identify new genes involved in cardiovascular disease. The transcription factor Tbx1 plays a vital role in PAA development and is a major contributor to cardiovascular disease associated with DiGeorge syndrome. This study used various genetic approaches to reveal part of a signalling network by which Tbx1 controls PAA development in mice. The crucial role played by the homeobox-containing transcription factor Gbx2 downstream of Tbx1 was investigated. PAA formation requires the pharyngeal surface ectoderm as a key signalling center from which Gbx2, in response to Tbx1, triggers essential directional cues to the adjacent cardiac neural crest cells (cNCCs) en route to the caudal PAAs. Abrogation of this signal generates cNCC patterning defects leading to PAA abnormalities. Finally, it was shown that the Slit/Robo signalling pathway is activated during cNCC migration and that components of this pathway are affected in Gbx2 and Tbx1 mutant embryos at the time of PAA development. It is proposed that the spatiotemporal control of this tightly orchestrated network of genes participates in crucial aspects of PAA development (Calmont, 2009).
Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, since the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. It is hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. The expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1–/– mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, Tbx1 and Fgf8 mutants were crossed. Double heterozygous Tbx1+/–;Fgf8+/– mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1+/–;Fgf8+/+ mutants, while Tbx1+/+;Fgf8+/– animals are normal. Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. These data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. It is speculated that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1 (Vitelli, 2002).
Fgf8 is required for normal development of the nasal region. Here, a candidate approach has been used to identify genes that
are induced in chick nasal mesenchyme in response to FGF signaling. Using an explant culture system, expression of the transcription factors Tbx2, Erm, Pea3, and Pax3, but not Pax7, in nasal mesenchyme has been shown to be regulated by ectodermal signals in a stage-dependent manner. Using beads soaked in recombinant FGF protein and an FGF receptor
antagonist, it has been demonstrated that FGF signaling is necessary and sufficient for expression of Tbx2, Erm, Pea3,
and Pax3, but that it has no effect on Pax7 expression. Within the nasal mesenchyme, competence to respond to FGF signaling is initially widespread and uniform but becomes restricted to regions normally exposed to FGF at later stages of development, coincident with changes in FGF receptor expression. Finally, evidence is provided that FGF8 also regulates Erm and Pea3 expression in the nasal placodes. Together, these results identify Tbx2, Erm, Pea3, and Pax3 as downstream targets of FGF signaling in the facial area and suggest that these genes may mediate some of the effects of FGF8 during development of the nasal region (Firnberg, 2002).
The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. Chordin homozygous mutant mice, generated by targeted mutagenesis, show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally, displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis (Bachiller, 2003).
To study the interaction of Chrd with genes known to cause
DiGeorge or DiGeorge-like phenotypes in mice, the expression of
Tbx1 and Fgf8 was analyzed in Chrd mutant embryos. Tbx1 is a member of the T-box family of transcription factors. It maps within the DGS/VCFS 22q11 microdeletion in humans
and has been shown to cause DiGeorge-like phenotype upon inactivation
in mice. Expression of Tbx1 is altered in
Chrd-/- embryos. In wild-type E7.5 animals, Tbx1 is expressed in the foregut (future pharyngeal endoderm) and head mesoderm. At this stage, mutant littermates showed a clear reduction in the levels of Tbx1 expression in the same areas. The reduction in Tbx1 mRNA is equally clear in the pharyngeal region of Chrd homozygous embryos at E8.0, E8.5 and E9.0. Transverse histological sections show that at the cellular level the abundance of Tbx1 transcripts is drastically reduced in endoderm, both in the pharynx and foregut up to the level of the hepatic diverticulum. Diminution in the concentration of Tbx1 mRNA
is also evident in mesoderm, including head, splanchnic and
somatic mesoderm in the peripharyngeal region. In addition, Tbx1
expression at E9 in the mesodermal core of the first pharyngeal arch is
diffuse, extending to most of the arch, and Tbx1 transcripts are
absent from the otic vesicle (Bachiller, 2003).
Fgf8 is a secreted growth factor expressed in a variety of
tissues, including the pharyngeal endoderm and neighboring mesoderm.
During early development, Fgf8 is required for gastrulation and the establishment of the left/right axis of symmetry. At later stages of Fgf8 is required for limb and craniofacial development. Recent experiments have shown that mice with reduced Fgf8 activity present a spectrum of cardiovascular and pharyngeal defects that closely mimic DiGeorge syndrome. In addition, Fgf8 expression is abolished in the pharyngeal endoderm of Tbx1-/- mutants and both genes interact genetically during the differentiation of the pharyngeal arch arteries. At E9, Fgf8 expression in Chrd mutants is normal in the mid-hindbrain isthmus, frontonasal prominence and tail. However, in pharyngeal endoderm, Fgf8 transcript levels are drastically reduced. The reduction of Tbx1 and Fgf8 expression in Chrd-/- embryos suggest that both genes act downstream of Chrd in the same regulatory pathway. These experiments do not determine whether Chrd is required for the maintenance or for the induction of Tbx1 and Fgf8 in the pharynx and neighboring tissues. To test whether Chrd can induce Tbx1 and Fgf8, Chrd mRNA (50 pg) was injected into the ventral region of Xenopus embryos at the four-cell stage. Ventral marginal zone (VMZ) explants were dissected at early gastrula, cultured until sibling embryos reached early neurula stage, and analyzed by RTPCR. Tbx1 and Fgf8 mRNAs are expressed at high levels in whole embryos and dorsal marginal zone (DMZ) explants at this stage, and at low levels in VMZ explants. Upon microinjection, Chrd mRNA increases the levels of Tbx1 and Fgf8 in VMZ. In situ hybridization of microinjected Xenopus embryos confirmed that the Tbx1 transcripts induced by Chrd mRNA are located in pharyngeal endoderm. It is concluded that Chrd, a Bmp antagonist, can induce Tbx1 and Fgf8 expression in Xenopus embryos, and is required for full expression of these genes in the pharyngeal region of the mouse embryo (Bachiller, 2003).
During embryonic life, the initially paired pharyngeal arch arteries (PAAs) follow a precisely orchestrated program of persistence and regression that leads to the formation of the mature aortic arch and great vessels. When this program fails, specific cardiovascular defects arise that may be life threatening or mild, according to the identity of the affected artery. Fourth PAA-derived cardiovascular defects occur commonly in DiGeorge syndrome and velocardiofacial syndrome (22q11DS), and in Tbx1+/- mice that model the 22q11DS cardiovascular phenotype. Tbx1 is expressed in pharyngeal mesoderm, endoderm and ectoderm, and, in addition, it is expressed in precursors of the endothelial cells that line the PAAs, thus expanding the number of tissues in which Tbx1 is potentially required for fourth PAA development. In this study, cell fate mapping and tissue-specific gene deletion, driven by six different Cre lines, were used to explore Tbx1 gene-dosage requirements in the embryonic pharynx for fourth PAA development. Through this approach, the spatial requirements for Tbx1 have been resolved in this process, and pharyngeal epithelia is shown to be a critical tissue. It is also thereby demonstrated conclusively that the role of Tbx1 in fourth PAA development is cell non-autonomous (Zhang, 2005).
The definition of time-specific requirements for a developmental gene can pinpoint the processes with which the gene is involved and can reveal potential late functions in structures and organs that fail to develop in germline mutants. This study shows the first systematic time-course deletion, in parallel with timed cell fate mapping, of a developmentally crucial gene, Tbx1, during mouse embryogenesis. Tbx1 mouse mutants model DiGeorge syndrome, a disorder of pharyngeal and cardiovascular development. Results revealed different time requirements for the development of individual structures, as well as multiple and time-distinct roles during the development of the same organ or system. Tbx1 is required throughout pharyngeal segmentation for the regulation of endoderm expansion; thus this is the first gene implicated directly in this process. A genetic-based blueprint of crucial developmental times for organs and systems should be a valuable asset for an understanding of birth defect pathogenesis (Xu, 2005).
DiGeorge syndrome (DGS) is a common genetic disease characterized by pharyngeal apparatus malformations and defects in cardiovascular, craniofacial and glandular development. TBX1 is the most likely candidate disease-causing gene and is located within a 22q11.2 chromosomal deletion that is associated with most cases of DGS. Thus study shows that canonical Wnt-beta-catenin signaling negatively regulates Tbx1 expression and that mesenchymal inactivation of beta-catenin (Ctnnb1) in mice caused abnormalities within the DGS phenotypic spectrum, including great vessel malformations, hypoplastic pulmonary and aortic arch arteries, cardiac malformations, micrognathia, thymus hypoplasia and mislocalization of the parathyroid gland. In a heterozygous Fgf8 or Tbx1 genetic background, ectopic activation of Wnt-beta-catenin signaling caused an increased incidence and severity of DGS-like phenotypes. Additionally, reducing the gene dosage of Fgf8 rescued pharyngeal arch artery defects caused by loss of Ctnnb1. These findings identify Wnt-beta-catenin signaling as a crucial upstream regulator of a Tbx1-Fgf8 signaling pathway and suggest that factors that affect Wnt-beta-catenin signaling could modify the incidence and severity of DGS (Huh, 2010).
The organized array of smooth muscle cells (SMCs) and fibroblasts in the walls of visceral tubular organs arises by patterning and differentiation of mesenchymal progenitors surrounding the epithelial lumen. This study shows that the TBX2 and TBX3 transcription factors have novel and required roles in regulating these processes in the murine ureter. Co-expression of TBX2 and TBX3 in the inner mesenchymal region of the developing ureter requires canonical WNT signaling. Loss of TBX2/TBX3 in this region disrupts activity of two crucial drivers of the SMC program, Foxf1 and BMP4 signaling, resulting in decreased SMC differentiation and increased extracellular matrix. Transcriptional profiling and chromatin immunoprecipitation experiments revealed that TBX2/TBX3 directly repress expression of the WNT antagonists Dkk2 and Shisa2, the BMP antagonist Bmper and the chemokine Cxcl12 These findings suggest that TBX2/TBX3 are effectors of canonical WNT signaling in the ureteric mesenchyme that promote SMC differentiation by maintaining BMP4 and WNT signaling in the inner region, while restricting CXCL12 signaling to the outer layer of fibroblast-fated mesenchyme (Aydogdu, 2018).
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