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The Brachyury of Chicken, Mouse and Human

By using the quail-chicken chimera system, it has been shown that during development of the spinal cord, floor plate cells are inserted between neural progenitors giving rise to the alar plates. These cells are derived from the regressing Hensen's node or cordoneural hinge (HN-CNH). This common population of HN-CNH cells gives rise to three types of midline descendants: notochord, floor plate, and dorsal endoderm. HNF3beta, an important gene in the development of the midline structures, is continuously expressed in the HN-CNH cells and their derivatives: floor plate, notochord, and dorsal endoderm. Experiments in which the notochord was removed in the posterior region of either normal chicken or of quail-chicken chimeras in which a quail HN had been grafted demonstrate that the floor plate develops in a cell-autonomous manner in the absence of notochord. Absence of floor plate observed at the posterior level of the excision results from removal of HN-CNH material, including the future floor plate, and not from the lack of an inductive signal of notochord origin. This cell lineage analysis of gastrulation and neurulation thus leads to revision of the model previously proposed, according to which the neural plate forms first as a continuous sheet of epithelial cells in which the notochord induces the overlying ectodermal cells to become the floor plate. In fact, the neural plate forms according to a more complex process, since its midline component, the floor plate, has an embryonic origin different from its lateral ones that yield the basal and alar plates of the neural tube and the neural crest (Teillet, 1998).

The chicken Brachyury gene, Ch-T, is expressed in the epiblast close to and within the primitive streak, in early migrating mesoderm and in the notochord. In later stages Ch-T expression is found in the tail bud and in the entire notochord. The notochord expression ceases in an anterior-posterior wave when the formation of the body anlage is completed. This pattern is consistent with those reported for the expression of the mouse T gene and the T homologues of Xenopus laevis and zebrafish, suggesting that the mechanisms of embryonic pattern formation are highly conserved in all vertebrates. The N-terminal half of Ch-T shows a very high degree of sequence identity with the corresponding region of mouse T which has DNA-binding activity, and with the N-terminal half of Xenopus (Xbra) and zebrafish (Ntl) T protein. Localized activin A treatment of prestreak blastoderms results in ectopic Ch-T expression that correlates with formation of second primitive streaks or with repositioning of the site of single streak origin. These results strengthen the previous evidence that Brachyury activation is an early response to axis-inducing signals in vivo (Kispert, 1995b).

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that center on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridization revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which is called the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation (Viebahn, 2002).

In Drosophila, proliferation is reduced locally by the action of the tribbles gene (coding for a serine/threonine kinase) to enable the start of the mesoderm differentiation program. Extrapolated to the present results this would suggest that, in a mammal, epiblast cells have to leave the belt of proliferation anterior to the PGE to be able to respond to signals that initiate the Brachyury-driven mesoderm formation program such as nodal or Wnt3. Intriguingly, Brachyury expression in the presumptive area of Hensen's node, too, lies in an area of reduced proliferative activity so that the Brachyury programme in node cells may also be started by cell movement away from areas of high proliferation. In the chick, however, distinct differences in proliferative activity do not seem to exist at pre-streak stages and in the absence of regulatory control by proliferation, alternative mechanisms such as growth factors and their inhibitors (e.g. chordin) attain a more important role. In an evolutionary context, entering gastrulation using a different mechanism may be the last ontogenetic afterplay of the differences seen in egg size and cleavage patterns, for example, between birds and mammals. Together with divergent development after the phylotypic stage, this phenomenon has been known since von Baer's time as the 'bottle neck' and it seems as if initiation of gastrulation already marks the beginning of the narrow part of this neck (Viebahn, 2002).

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex (Rennebeck, 1998).

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. A detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure is reported. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridization, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures is reported. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organized at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation (Gofflot, 1999).

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, evidence is provided that the attenuation of Wnt3a signaling observed in Fgfr1-/- embryos can be rescued by lowering E-cadherin levels. It is proposed that modulation of cytoplasmic ß-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak (Ciruna, 2001).

Results from the Fgfr1 mutant expression analyses, chimeric studies, and in vitro explant experiments can be assembled into a minimal model for FGFR1 function at gastrulation. This study has defined a specific region of the primitive streak that requires FGFR1 signaling activity; this domain encompasses the paraxial and posterior embryonic mesoderm populations, but excludes the node, axial, and extraembryonic mesoderm. In the context of this domain, it is proposed that FGFR1 signaling orchestrates both the morphogenetic movement and cell fate specification events of gastrulation (Ciruna, 2001).

Beyond its morphoregulatory role at gastrulation, FGFR1 also functions in the specification of mesoderm cell fate. Chimeric analyses demonstrate that FGFR1 is required for T and Tbx6 expression in the primitive streak. The downregulation of T and Tbx6 expression in Fgfr1-/- mesoderm progenitor cells can account for both the reduction of paraxial and posterior mesoderm, and for the formation of ectopic neural tubes observed in Fgfr1 mutant and chimeric analyses. Because studies in zebrafish and Xenopus have also established the function of FGFs in T box gene regulation and posterior mesoderm specification, these results further support an evolutionarily conserved pathway for FGF signaling at gastrulation (Ciruna, 2001).

The mechanisms by which FGFR1 signaling regulates both the morphogenesis and patterning of mesoderm at gastrulation are intricately entwined. Gene dosage and chimeric analyses of Brachyury function have demonstrated that the level of T expression in progenitor cell populations influences the timing and pattern of ingression through the primitive streak. Furthermore, T box genes may also regulate cell adhesion and EMT at gastrulation. In zebrafish, the Brachyury homolog no tail, and the T box gene spadetail have both been implicated as positive regulators of Snail expression. Although regulation of mouse Snail by T has yet to be determined, it is intriguing that in late gastrula-staged Fgfr1 -/- embryos, the only observed domain of mSnail expression overlaps with an Fgfr1-independent domain of T expression at the base of the allantois. Therefore, T may positively regulate Snail expression at the primitive streak, providing another link between Brachyury expression, intercellular adhesion, and the morphogenesis of the mesodermal germ layer (Ciruna, 2001).

In addition, it is proposed that FGFR1 signaling indirectly regulates Wnt signal transduction at the primitive streak. In Fgfr1 -/- embryos, although Wnt3a is expressed in the late primitive streak, direct targets of Wnt signaling (i.e., Brachyury and the T-lacZ reporter transgene) are not activated. It is suggested that ectopic E-cadherin expression in Fgfr1 mutants attenuates Wnt3a signaling by sequestering free ß-catenin from its intracellular signaling pool, and demonstrates that forced downregulation of E-cadherin in Fgfr1 -/- explants can rescue endogenous Wnt signaling at the primitive streak. Evidence that cadherins act as regulators of ß-catenin signaling is well documented. E-Cadherin and LEF-1 bind to partially overlapping sites in the central region of ß-catenin; consequently, LEF-1 and E-cadherin form mutually exclusive complexes with ß-catenin and compete for the same intracellular signaling pool. Furthermore, overexpression of cadherins during Drosophila and Xenopus embryogenesis has been shown to phenocopy Wnt/ß-catenin signaling mutants (Ciruna, 2001).

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).

The mouse Brachyury (T) gene is required for differentiation of the notochord and formation of mesoderm during posterior development. Homozygous embryos lacking T activity do not develop a trunk and tail and die in utero. The T gene is specifically expressed in notochord and early mesoderm cells in the embryo. recent data have demonstrate that the T protein is localized in the cell nucleus and specifically binds to a palindrome of 20 bp (the T site) in vitro. The T protein activates expression of a reporter gene in HeLa cells through binding to the T site. Thus T is a novel tissue-specific transcription factor. It consists of a large N-terminal DNA binding domain (amino acids 1-229) and two pairs of transactivation and repression domains in the C-terminal protein half. T can also transactivate transcription through variously oriented and spaced T sites, a fact that may be relevant in the search for genes controlled by T protein and important in mesoderm development (Kispert, 1995a).

A characteristic abnormality of chimeras composed of wildtype and T/T (Brachyury) mutant embryonic stem cells is the aggregation and accumulation of mutant cells in the primitive streak and its descendant, the tail bud. A disproportionately high degree of mutant cells contribute to ventral neurectoderm. To demonstrate that this aberrant behaviour of mutant cells in the streak is due only to the absence of wild-type T protein and to investigate dosage effects of T function on cell deployment during gastrulation, a vector expressing T under the control of its own promoter (which results in T expression in the primitive streak but not in the notochord) was introduced into T/T mutant ES cells carrying a ubiquitous lacZ lineage marker. Four clones (TR clones) that express T appropriately in the streak and rescue abnormal chimeric morphology were recovered. In chimeras, these four clones fall into two distinct categories with respect to their ability to exit from the primitive streak and their subsequent tissue colonization profile. TR1 and TR4 descendants no longer accumulate in the tail bud and give rise to all types of mesoderm as well as colonizing ventral neurectoderm. Interestingly, TR2 and TR5 cells (which express higher levels of T protein than TR1 and TR4 in vitro) tend to exit the streak prematurely, show a marked reduction in posterior mesoderm colonization, and are virtually excluded from ventral neurectoderm. However, while descendants of all four TR clones can colonize dermomyotome at all axial levels, the parent T/T mutant cells only contribute to this tissue rostral to the forelimb bud and are completely excluded from more caudal dermomyotome. There is no evidence for transgene expression in the dorsal node at headfold and early somite stages. Lineage analysis has identified the dorsal node as the progenitor of the ventral midline neurectoderm lying caudal to the anterior midbrain. Endoderm posterior to somite 1 also shows a deficiency in TR2 and TR5 cells, similar to ventral neurectoderm. Since gut endoderm emerges from the anterior aspect of the primitive streak during gastrulation, the absence of TR2 and TR5 in the trunk and caudal gut supports the idea that cells expressing high levels of T during gastrulation cannot remain in the streak long enough to make a significant contribution to caudal gut. It is unlikely that levels of T expression in vivo specify dorsoventral position information. These results show that the abnormal aggregation of mutant cells homozygous for the Brachyury deletion ( approximately 200 kb) can be ascribed solely to the lack of wild-type T protein, as can the failure of T/T cells to colonize caudal dermomyotome. They also suggest that patterns of cell recruitment from the streak can be influenced by the level of T expression (Wilson, 1997).

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. Mutant embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells. To determine the origin of this abnormal tissue distribution, mutant and control ES cell clones were isolated which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. Prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak (Wilson, 1995).

The midline has a theoretical role in the development of left--right asymmetry, and this is supported by both genetic analyses and experimental manipulation of midline structures in vertebrates. The mouse brachyury (T) gene encodes a transcription factor that is expressed in the developing notochord and is required for normal development. T/T mice lack a mature notochord and have a dorsalised neural tube. The hearts of T/T mice were examined and consistent morphological abnormalities, resulting in ventrally displaced ventricular loops, and a 50% incidence of inverted heart situs were found. Three TGF-beta related genes, lefty-1, lefty-2 and nodal, are all expressed asymmetrically in mouse embryos, and are implicated in the development of situs. Nodal, which is normally expressed around the node and in the left lateral plate mesoderm in early somite embryos, is completely absent at this stage in T/T embryos. In contrast, lefty-1 and lefty-2, which are normally expressed in the left half of prospective floorplate and left lateral plate mesoderm, respectively, are both expressed in T/T embryos only in a broad patch of ventral cells in, and just rostral to, the node region. These results implicate the node as a source of instructive signals driving expression of nodal and lefty-2 in the left lateral plate mesoderm, these signals being required for normal looping and situs of the heart (King, 1998).

The mouse T (Brachyury) deletion causes defective mesoderm formation and notochord morphogenesis, and abnormalities in the caudal neural tube and somites. Messenger RNA levels of Evx-1 (Drosophila homolog: Even-skipped), Wnt-3a and Wnt-5a (Vertebrate homologs of Drosophila Wingless) decreases in T/T embryos late in gastrulation, although earlier expression patterns are similar to control embryos. In contrast, BMP-4 (Drosophila homolog: Decapentaplegic) and Msx-1 (Drosophila homolog: Muscle segment homeobox) expression patterns remain similar throughout the period studied. Pax-3 and Pax-6 (Drosophila homolog: Eyeless), which are expressed in specific dorsoventral domains of the neural tube, both have ventrally extended expression domains in caudal T/T neural tube. This is consistent with a missing ventral signal provided by the notochord. However, the expression of Msx-1 in the most dorsal domain of the neural tube is unaltered in T/T embryos. Pax-1 and Pax-3, which are expressed in the sclerotome and dermamyotome respectively, are expressed correctly in anterior T/T somites, although the Pax-3 expression domain is widened ventromedially. This extension into ventromedial somite domains is more pronounced caudally, supporting a function for the notochord in ventralizing somites (Rashbass, 1994).

The regulation of transcription factors HNF-3alpha and HNF-3beta has been studied during the retinoic acid-mediated differentiation of mouse P19 cells. Retinoic acid treatment converts P19 stem cells into neurons and astrocytes and gene expression of both HNF-3alpha and HNF-3beta is activated during this process. HNF-3alpha transcription is detected 2 h after addition of retinoic acid and takes place in the absence of de novo protein synthesis. This suggests that HNF-3alpha is a primary target for retinoic acid action. HNF-3alpha induction displays a biphasic profile; HNF-3alpha mRNA reaches maximal levels at 2 and 6 days postdifferentiation. Additional experiments strongly suggest that the second peak is due to HNF-3alpha induction in postmitotic neurons. In contrast, P19 stem cells do not contain any detectable HNF-3alpha mRNA. According to these studies, the retinoic acid-mediated induction of HNF-3alpha occurs at the level of transcriptional initiation and is conferred by distal promoter sequences. In comparison to HNF-3alpha, HNF-3beta induction is a subsequent event and detectable levels of HNF-3beta mRNA materialize approximately 1 day after addition of retinoic acid to P19 stem cells. Time course studies firmly demonstrate that HNF-3beta mRNA peaks at about 2 days postdifferentiation and then declines to virtually unreadable levels. This temporal pattern is consistent with HNF-3beta being a secondary target for retinoic acid. In analogy to HNF-3alpha, HNF-3beta activation also takes place at the level of transcriptional initiation. Recent studies implicate HNF-3alpha and HNF-3beta in early mammalian neurogenesis (Jacob, 1997)

A novel murine and human gene has been identified encoding a putative transcription factor related to the Brachyury (T) gene that is expressed only in postmitotic cells. T-brain-1 (Tbr-1) mRNA is largely restricted to the cerebral cortex, where during embryogenesis it distinguishes domains that may give rise to paleocortex, limbic cortex, and neocortex. Tbr-1 and Id-2 expression in the neocortex have discontinuities that define molecularly distinct neocortical areas. Tbr-1 expression is analyzed in the context of the prosomeric model. Topological maps are proposed for the organization of the dorsal telencephalon (Bulfone, 1995).

Murine Sp5 encodes a protein having a C-terminal C2H2 zinc finger domain closely related to that of the transcription factor Sp1. In vitro, DNA binding studies show that Sp5 binds to the GC box, a DNA motif present in the promoter of a very large number of genes, including Brachyury, and recognized by members of the Sp1 family. However, outside of its DNA binding domain, Sp5 has little homology with any other member of the Sp1 family. In contrast to the ubiquitous expression of Sp1, Sp5 exhibits a remarkably dynamic pattern of expression throughout early development. This is suggestive of a role in numerous tissue patterning events, including gastrulation and axial elongation; differentiation and patterning of the neural tube, pharyngeal region, and somites; and formation of skeletal muscle in the body and limbs. Mice homozygous for a targeted mutation in Sp5 show no overt phenotype. However, the enhancement of the T/1 phenotype in compound mutant mice (Sp5lacZ/Sp5lacZ,T/1) indicates a genetic interaction between Sp5 and Brachyury. These observations are consistent with a role for Sp5 in the coordination of changes in transcription required to generate pattern in the developing embryo (Harrison, 2000).

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with ß-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a was genetically examined. Analysis of the expression of Brachyury in Lef1-/-:Tcf1-/- mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicates that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. The expression of an activated form of LEF-1, containing the ß-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation (Galceran, 2001).

During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. The chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T-/- null mutant limb buds reveals disrupted AER morphogenesis. These data also suggest that FGF and WNT signals may operate both upstream and downstream of T. During limb induction, WNT signals maintain high Fgf10 expression in prospective limb and FGF10 activates ectodermal Wnt3a and Fgf8 expression, initiating AER formation. AER signals subsequently also maintain mesodermal Fgf10 expression. T transcripts are first clearly detected at stage 15, at the onset of Wnt3a and Fgf8 activation in the ectoderm. Both the ability of WNT3a and FGF8 to induce T expression, and the ability of T to increase subridge expression of Fgf10 early after misexpression suggest that T may be a component of the mesodermal response to developing AER signals that maintains high Fgf10 apically and thereby also maintains the forming AER, establishing a regulatory loop between ectoderm and mesoderm. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelial-mesenchymal regulatory loop involved in maintenance of a mature functioning AER (Liu, 2003).

Table of contents

brachyenteron: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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