optomotor blind is homologous to mouse Brachyury. The central region of OMB is homologous to the N-terminal half of the Brachyury protein. This conserved domain has a general DNA binding affinity but has no significant protein sequence similarity to recognized DNA binding motifs (Pflugfelder (1992b). Information about vertebrate Brachyury and its role in mesoderm formation is found at the T-related gene site. The T-box family has a slightly greater sequence affinity to Drosophila Optomotor blind than to Drosophila Brachyenteron (T-related gene) and consequently information about the T-box family is found in the Omb site.

In Drosophila, the T-box genes optomotor-blind (omb) and H15 have been implicated in specifying the development of the dorso-ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. This study extends the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe-shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene, two paralogous genes were identified in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm-H15-1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear (Prpic, 2005).

Transcription factors of the T-domain family regulate many developmental processes. A new member of the Tbx2 subfamily, coquillette, has been isolated from the sea urchin. Coquillette has a late zygotic expression whose localization is dynamic: at the blastula stage it is restricted to the aboral side of most of the presumptive ectoderm and endoderm territories and from gastrulation on, to the aboral-most primary mesenchyme cells. Perturbation of coquillette function delays gastrulation and strongly disorganizes the skeleton of the larva. Coquillette is sensitive to alteration of the oral-aboral (OA) axis and goosecoid, which controls oral and aboral fates in the ectoderm, is identified as a probable upstream regulator. Coquillette appears to be an integral part of the patterning system along the OA axis (Croce, 2003).

The first phase of coquillette expression occurs for a large part in the presumptive ectoderm. In this territory, several regulators of fate along the OA axis have been identified, including goosecoid. Goosecoid is a transcriptional repressor that promotes oral fate and represses aboral fate. A number of observations suggest that goosecoid may control coquillette. (1) Both goosecoid and coquillette expression begin at about the same time, at the swimming blastula stage. (2) Many genes that are restricted to the aboral ectoderm are initially activated throughout the ectoderm. Coquillette, however, is expressed only in the aboral ectoderm from the earliest time its expression can be detected, indicating that it is downstream of or simultaneous with oral-aboral specification. (3) The expression domains of goosecoid and coquillette are opposite one another. (4) Overexpression of goosecoid suppresses coquillette expression (Croce, 2003).

Studies on ascidians (phylum Urochordata) provided the first evidence for localized determinants in animal development. The destruction of particular blastomeres leads to the specific loss of muscle derivatives. Lineage studies have established a tight correlation between the distribution of yellow crescent and muscle differentiation in Styela. The yellow crescent becomes localized to the vegetal cytoplasm shortly after fertilization and is ultimately inherited by the two B4.1 blastomeres that form most of the tail muscles in the ascidian tadpole. A new T-box gene resembling Drosophila Optomotor blind, CiVegTR, that fulfils the criteria of the classic muscle determinant, has been isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago (Erives, 2000).

A Ciona ortholog of the Drosophila Snail repressor (Ci-sna) was found to repress a 434-bp notochord-specific enhancer in the promoter region of the Ciona Brachyury gene. Ci-sna is expressed in the developing tail muscles, where it is important for restricting Ci-Bra expression to the developing notochord. Ci-sna is activated early during muscle specification (32-cell stage), at the time when maternal determinants first activate zygotic genes. The present study identifies a 262-bp enhancer from the Ci-sna 5' flanking region that is sufficient to mediate expression in derivatives of the B4.1 blastomeres. This enhancer contains two conserved sequence motifs that are also present in the regulatory regions of muscle-specific genes in the distantly related ascidian Halocynthia. One of the motifs corresponds to a specialized E-box sequence (CAACTG), whereas the other contains conserved residues recognized by different T-box DNA binding proteins (GT-GNNA). Mutations in either motif diminish or abolish the expression driven by otherwise normal Ci-sna/lacZ transgenes (Erives, 2000).

Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. The zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. (1) The gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). (2) The mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. (3) Dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail (Hug, 1997).

Two bilaterally symmetric eyes arise from the anterior neural plate in vertebrate embryos. An interesting question is whether both eyes share a common developmental origin or do they originate separately. The expression pattern of a new gene ET, a homolog of Drosophila optomotor blind and more distantly related to Drosophila T-related gene reveals that there is a single retina field in Xenopus which resolves into two separate primordia, a suggestion supported by the expression pattern of the Xenopus Pax-6 gene. Lineage tracing experiments demonstrate that retina field resolution is not due to migration of cells in the median region to the lateral parts of the field. Removal of the prechordal plate (a mesodermal tissue) leads to formation of a single retina both in chick embryos and in Xenopus explants. Transplantation experiments in chick embryos indicate that the prechordal plate is able to suppress Pax-6 expression. These provide direct evidence for the existence of a single retina field, indicate that the retina field is resolved by suppression of retina formation in the median region of the field, and demonstrate that the prechordal plate plays a primary signaling role in retina field resolution. A similar origin to bilaterality of the forebrain is suggested. Sonic hedgehog (See Drosophila Hedgehog) is expressed in prechordal mesoderm. Abnormality in human shh has been implicated as the underlying cause of a subset of holoprosencephaly. (Li, 1997 and references).

One novel T-box gene (Ch-TbxT) becomes restricted to the axial mesoderm lineage and is a potential candidate for complementing or extending Brachyury function in the anterior axis (formation of the head process, prechordal plate). The other gene (Ch-Tbx6L), together with chick T, appears to mark primitive streak progenitors before gastrulation. As cells leave the primitive streak, Ch-Tbx6L becomes restricted to the early paraxial mesoderm lineage and could play a role in regulating somitogenesis. Ch-Tbx6L is equally divergent from the two major classes of T-box genes identified to date, sharing about 50% amino acid identity with both Brachyury and Drosophila OMP in the T-box region (Knezevic, 1997).

A human gene (TBX2) exhibits strong sequence homology within a putative DNA binding domain to the Drosophila Optomotor-blind (OMB) gene and lesser homology to the DNA binding domain of the murine brachyury or T gene. Unlike omb, which is expressed in neural tissue, or T, which is not expressed in adult animals, TBX2 is expressed primarily in adult in kidney, lung, and placenta as multiple transcripts of between approximately 2 and 4 kb. At least part of this transcript heterogeneity appears to be due to alternative polyadenylation. This is the first reported human member of a new family of highly evolutionarily conserved DNA binding proteins, the Tbx or T-box proteins. The human gene has been mapped to chromosome 17q23, a region frequently altered in ovarian carcinomas (Campbell, 1995).

Sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes establishes a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, a search was carried out for the human homolog, Hs-TBX2, within a region of synteny (similar position on homologous regions of chromosomes of two species) conservation on Chr 17q. Dm-Omb polymerase chain reaction (PCR) primers were used to amplify a 137 bp product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product shows 89% DNA sequence identity and 100% peptide sequence identity to the corresponding T-box segment of Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggests that the putative human TBX2 and mouse Tbx2 are true homologs. TBX2 is expressed in fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels are seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation (Law, 1995).

Two key melanocyte-specific elements termed MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but they are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in the strong up-regulation of TRP-1 promoter activity in melanoma cells. Repression mediated by MSEu and MSEi also operates in melanocytes. Both MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is the binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as the repressor of TRP-1 expression: both MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase (microphthalmia) or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoters, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter, and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo cannot rule out, the evidence presented suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member (Carreira, 1998).

Sea urchin LvTbx2/3 has been cloned and characterized. Nuclear localization of LvTbx2/3, as indicated by a polyclonal antibody, initiates at the mesenchyme blastula stage and protein is present into the pluteus stage. Localization is asymmetric throughout this period and costaining with marker genes indicates that an asymmetric distribution exists about the oral/aboral (O/A) axis. Asymmetric distribution of LvTbx2/3 is observed in the aboral territories of all three germ layers. In the skeletogenic mesoderm lineage, LvTbx2/3 expression is dynamic because expression appears initially in all skeletogenic mesenchyme cells (PMCs) but, subsequently, becomes refined solely to the aboral cells during skeletogenesis. To determine if the aboral expression of LvTbx2/3 is linked between germ layers, and to place LvTbx2/3 in the sequence of events that specifies the O/A axis, the effects of a series of perturbations to O/A polarity on LvTbx2/3 expression in each germ layer were examined. Preventing the nuclear localization of ß-catenin, pharmacological disruption of the O/A axis with NiCl2, overexpression of BMP2/4 and disruption of the extracellular matrix all block LvTbx2/3 expression in all germ layers. This indicates that expression of LvTbx2/3 in the aboral territories of each germ layer is a common aspect of O/A specification, downstream of the molecular events that specify the axis. Furthermore, blocking the nuclear localization of ß-catenin, overexpression of BMP2/4 and disruption of the extracellular matrix also prevents the oral (stomodael) expression of LvBrachyury (LvBrac) protein, indicating that the O/A axis is established by a complex series of events. Last, the function of LvTbx2/3 in the formation of the O/A axis was characterized by examining the phenotypic consequences of ectopic expression of LvTbx2/3 mRNA on embryonic development and the expression of marker genes that identify specific germ layers and tissues. Ectopic expression of LvTbx2/3 produces profound morphogenetic defects in derivatives of each germ layer with no apparent loss in specification events in these tissues. This indicates that LvTbx2/3 functions as a regulator of morphogenetic movements in the aboral compartments of the ectoderm, endoderm and mesoderm (Gross, 2003).

T-box gene function in C. elegans

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, a C. elegans lineage was studied that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. It has been shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. This study examined tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, the T-box protein TBX-2 was identified as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage (Singhvi, 2008).

Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis

For over a century, muscle formation in the ascidian embryo has been representative of 'mosaic' development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, this study has identified the mechanisms through which Ciona Macho1 (Ci-Macho1, a divergent member of the Zic family) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and the cross-regulatory interactions have been unveiled between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). These representative examples were used to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos (Kugler, 2010).

Ci-macho1 postplasmic mRNA is relocated after fertilization by the cortical centrosome-attracting body (CAB). As cleavage proceeds, in both Halocynthia and Ciona Ci-macho1 mRNA becomes progressively restricted to a narrow region of the embryo, the B7.6 blastomeres; however, the Macho1 protein is generally believed to persist in an unlocalized form, and to be distributed to all descendants of the B4.1 cells. Studies in Halocynthia show that for the proper formation of other lineages that also derive from the B4.1 cells, such as mesenchyme and endoderm, the function of Macho1 needs to be actively suppressed by FGF and BMP signaling pathways. Similar mechanisms are also likely responsible for the functional suppression of zygotically expressed Ci-Macho1 in the Ciona CNS, considering that Ci-FGF16/19/20 is expressed in the Ciona CNS through tailbud stages and is required for neural development (Kugler, 2010).

Misexpression experiments described in this study suggest that no such restraining mechanism is present in notochord cells before the early tailbud stage. In fact, at early developmental stages the ectopic activation of both Ci-Tbx6b and Ci-Tbx6c was seen in notochord precursors of both lineages in Bra>macho embryos, whereas at the mid-tailbud stage only the ectopic activation of Ci-Tbx6b was observed, and it was confined to a subset of mesenchyme cells. These cells are most likely descendants of the B7.3 blastomere, a 64-cell stage precursor of both secondary notochord and mesenchyme cells. The differential competence of the notochord to respond to Ci-Macho1 might be explained by the requirement for temporally and spatially localized co-factors and/or transcriptional intermediaries. Alternatively, as in the case of the CNS, Ci-Macho1 might be functionally suppressed in the notochord of tailbud embryos by the activation of the FGF signaling pathway, as suggested by the observation that Ci-FGFR is expressed in the notochord beginning at the early tailbud stage. These mechanisms might also account for the relatively mild phenotype that was observed in embryos carrying the Bra>macho transgene, whereby the notochord is still able to form, even in transgenic embryos where mosaic incorporation is minimal (Kugler, 2010).

Using in vivo transient transgenic assays, a 2.4 kb CRM upstream of Ci-Tbx6b was identified that is able to faithfully recapitulate the muscle expression of this gene. The temporal muscle activity of the 2.4 kb CRM represents the composite read-out of early- and late-acting cis-regulatory sequences, which interpret maternal and zygotic information. The Ci-Tbx6b CRM contains a distal region which functions as the repository of the temporal information necessary to recapitulate the early expression pattern previously reported for Ci-Tbx6b. When this distal region is deleted, muscle activity is not lost, but its onset is considerably delayed. Sequence inspection and point-mutation analyses suggested that this early-acting distal region might be controlled by maternal Ci-Macho1, because three putative binding sites for this factor are present in this sequence. These sites were found to be bound in vitro by Macho1 and their concomitant mutation was found to be sufficient to cause the same delay in the onset of transcriptional activity that was observed when the entire fragment encompassing them was deleted. Together, these observations provide a mechanistic cis-regulatory explanation to the results of the misexpression assay, as well as to previous results showing that overexpression of Ci-Macho1 is sufficient to induce ectopic expression of Ci-Tbx6b and that, likewise, Hr-Macho-1 is able to ectopically induce Hr-Tbx6, among other muscle genes. It is noteworthy that in Ciona, Ci-ZicL cooperates with Ci-Macho1 to promote muscle development; this zygotic zinc-finger transcription factor is related to Ci-Macho1 and recognizes a similar consensus binding site in vitro. Interestingly, one of the three Ci-Macho1-binding sites that were characterized in the Ci-Tbx6b CRM, namely site 'C', contains permutations of the published ZicL consensus site that are compatible with binding in vitro. If this site is bound in vivo by either transcription factor, then this would explain the observation that Ci-Tbx6b is still weakly expressed in Ci-Macho1 morphant embryos, whereas its expression is no longer detectable in Ci-Macho1 and Ci-ZicL double-morphants (Kugler, 2010).

Within the 2.4 kb CRM, a 266 bp proximal region is able to direct transcription only from neurulation onwards, thus acting as a late muscle enhancer. Sequence analysis of this region revealed the presence of an imperfect CREB-binding site, a T-box-binding site (generic sequence: TNNCAC) partly matching the core consensus sequence previously reported for Ci-Tbx6b/c, and an 'AC'-core E-box. Both CREB-binding sites and AC-core E-boxes have been previously shown to be necessary for muscle activity of other muscle CRMs; however, in this case, only the T-box site substantially contributes to the muscle activity, qualitatively and quantitatively. Through EMSA, it was shown that this T-box site is bound in vitro by both Ci-Tbx6b and Ci-Tbx6c (Kugler, 2010).

Originally isolated in a subtractive screen aimed to identify genes downstream of Ci-Bra, the CiFCol1 gene attracted interest because of its sustained muscle expression, which begins around mid-gastrulation, and because its upstream region is enriched in T-box-binding sites (Kugler, 2010).

Dissection of a 2.2 kb genomic fragment located upstream of the transcription start site of CiFCol1 revealed the presence of discrete CRMs active in all the tissues where CiFCol1 is expressed. In particular, this 2.2 kb fragment harbors two distinct muscle CRMs: a distal CRM containing two generic E-boxes and depleted of T-box-binding sites and Ci-Macho1-binding sites, and a proximal CRM containing four clustered T-box-binding sites, some of which are bound weakly in vitro by the Ci-Tbx6b protein, and a low-affinity Ci-Macho1-binding site. The heterogeneity of these sequences is reflected by the temporal activity of the two CRMs, because the distal one, which does not contain any apparent T-box-binding sites, is activated later than the proximal one, which is enriched in these motifs. In particular, the distal CiFCol1 muscle CRM is active in a small subset of muscle precursors from the 110-cell stage to the neurula stage, and only by the early tailbud stage does its territory expand to encompass all muscle cells. Afterwards, it remains active in the majority of muscle cells. Therefore, the spatial range of action of this CRM in the muscle seems to be controlled by an activator(s) functioning from neurulation onwards. The presence of two E-boxes in this sequence prompted an investigation of the possible involvement of transcription factors of the bHLH family in the regulation of this CRM. It was found that neither mutation of the E-boxes nor misexpression, individual or combined, of two bHLH transcription factors, Ci-MRF and Ci-paraxis had any detectable effect, thus leaving the identification of the late activator(s) to future investigations (Kugler, 2010).

Conversely, the proximal CiFCol1 muscle CRM is ignited early in most muscle cell precursors, starting from the 32-cell stage, but its activity fades by the mid-tailbud stage. It is concluded that the additive activity of the two CRMs is probably responsible for the sustained expression of CiFCol1 in muscle cells (Kugler, 2010).

Interestingly, misexpression of Ci-Macho1, Ci-Tbx6b or Ci-Tbx6c in notochord cells all result in ectopic activation of CiFCol1 in this territory. Although it is not possible to rule out that this might be attributable to the low-affinity Ci-Macho1-binding site in the CiFCol1 early CRM, given the late onset of CiFCol1 muscle expression it seems more likely that Ci-Macho1 activates expression of CiFCol1 indirectly, through Ci-Tbx6b. To test this hypothesis the response of the CiFCol1 proximal muscle CRM to the misexpression of Ci-Tbx6b was monitored in notochord cells. It was found that misexpression of Ci-Tbx6b caused the ectopic activation of the CiFCol1 proximal muscle CRM in the notochord, whereas misexpression of Ci-Tbx6c did not have any effect. It is concluded that the ectopic activation of CiFCol1 seen in notochord cells of embryos carrying the Bra>Tbx6c construct might occur indirectly, via the activation of Ci-Tbx6b expression by Ci-Tbx6c (Kugler, 2010).

Finally, no ectopic activation was observed when the distal CiFCol1 muscle CRM was co-electroporated with either construct, consistent with the lack of Tbx6b/c-binding sites in its sequence (Kugler, 2010).

By analyzing the cis-regulatory sequences that mediate the response to Ci-Macho1 and its mediators, this study has begun to provide sharper insights into the molecular mechanisms controlling cell-autonomous muscle development in the ascidian embryo. Given the large number of genes that respond to Ci-Tbx6b and Ci-Tbx6c, it is conceivable that the mechanisms of transcriptional regulation that control the CRMs presented in this study might be shared by several other muscle genes. This hypothesis is supported by the abundance of putative Tbx6b/c-binding sites in muscle CRMs identified (Kugler, 2010).

Although the early cell-fate determination mediated by Macho-like proteins in muscle cells has been described so far as an ascidian-specific mechanism, transcription factors of the Zic family, of which Macho, ZicL and related proteins represent a diverged branch, are known to be required for shaping the body plan of widely different animals. In addition, Tbx6-related proteins in Ciona appear to be part of an evolutionarily conserved kernel that is employed for the specification and differentiation of paraxial mesoderm in several other chordates, including mouse, Xenopus and zebrafish. Hence, the elucidation of the cis-regulatory mechanisms used by these transcription factors to modulate expression of their target genes should provide insights on the inner workings of other model systems in which cis-regulatory elements are less tractable, including higher chordates (Kugler, 2010).

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT (Saunders, 2014).

T-box family gene mutation

The specificity of the Xenopus T box proteins Xbra, VegT and Eomesodermin resides in the DNA-binding domain, or T box. Binding site selection experiments show that the three proteins bind the same core sequence, but they select paired sites that differ in their orientation and spacing. Lysine 149 of Xbra is conserved in all Brachyury homologs (it is also conserved in the Drosophila Xbra homolog Optomotor-blind), while the corresponding amino acid in VegT and Eomesodermin is asparagine. Mutation of this amino acid to lysine changes the inductive abilities of VegT and Eomesodermin to resemble those of Xbra (Conlon, 2001).

The data indicate that the different inducing activities of Xbra, VegT and Eomesodermin are mostly defined by their T boxes. Comparison of the presumed protein-DNA contact points of the three proteins, based on the crystal structure of the Xbra T box, suggest that lysine 149 of Xbra might be important in defining functional specificity. Mutation of the corresponding asparagine residue in VegT and Eomesodermin to lysine causes the modified proteins to behave more like Xbra, in that they can not induce high levels of Pintallavis or chordin and they can not activate goosecoid at all (Conlon, 2001).

The mechanism by which a single amino acid substitution might change the specificity of the T box proteins is unclear. This difficulty is compounded because position 149 of Xbra contacts the phosphate backbone of DNA and is not predicted to make a base-specific contact. Indeed, Xbra, VegT and Eomesodermin select the same core sequence. One possibility is that position 149 affects the affinity of protein-DNA interactions, but this is unlikely because even the highest levels of Xbra fail to activate anterior markers such as goosecoid. Another suggestion is that position 149 of Xbra might alter target specificity through protein-protein interactions, as occurs in Sox proteins and homeobox proteins. Consistent with this proposal, it has been demonstrated that the transcriptional activity of the T box protein Tbr-1 is altered by its association with the guanylate kinase CASK/LIN-2. Moreover, classical genetic studies carried out on the mouse Brachyury allele TC are consistent with the presence of a Brachyury interacting protein. However, no interacting protein has been yet identified for Xbra, VegT or Eomesodermin (Conlon, 2001).

The TBX5 transcription factor is required for normal cardiogenesis, and human TBX5 mutations cause congenital heart defects. Previous studies have shown that TBX5 can localize to cellular nuclei during embryogenesis and have suggested that altered nuclear localization may contribute to disease pathogenesis. Current analyses suggest that TBX5 nuclear localization is not uniform during organogenesis. To determine the biochemical mechanisms underlying TBX5 nuclear import, site-directed mutagenesis of human TBX5 was performed. Two distinct nuclear localization signals were identified in TBX5, one monopartite and one bipartite. While each is insufficient to promote complete TBX5 nuclear localization, they act cooperatively to do so. These sequences are evolutionarily conserved and have cognates in other T-box gene family members (Collavoli, 2003).

TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study, functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization are described for the mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5 in vivo and in vitro. Immunofluorescent staining shows that wild type TBX5 is localized completely into the nucleus, but mutants are localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations investigated in this study are functional mutations with a spectrum of defects ranging from decreases in DNA-binding activity and transcriptional activation to the dramatic reduction of interaction between TBX5 and NKX2.5, and loss of synergy in transcriptional activation between these two proteins, as well as impairment in the nuclear localization of TBX5. These defects are likely central to the pathogenesis of Holt-Oram syndrome (Fan, 2003).

The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ (Arnold, 2008).

A maternal T-box gene controls primary germ layer specification in Xenopus embryos

A putative T-box transcription factor (Xombi) has the ability to induce sites of invagination that resemble the blastopore lip in the ectoderm of Xenopus embryos. Maternal Xombi transcript is first localized to the oocyte's vegetal cortex and cytoplasm, early sources of mesoderm and endoderm-inducing signals. Soon after zygotic transcription begins, there is a wave of Xombi expression (beginning in dorsal mesoderm and then extending to lateral and ventral mesoderm), that precedes and parallels blastopore lip formation at the border between the mesoderm and endoderm. Transcripts encoding brachyury, Xwnt8 and goosecoid colocalize with Xombi transcripts within the marginal zone; ectopic expression of Xombi induces expression of all three mesodermal genes. Xombi expression is induced by the secreted mesoderm inducers ActivinA, ActivinB and Xnr1, and eFGF, and by brachyury, another Xenopus T-box containing gene. The time course and location of Xombi expression, its biological activities and the partial dependence of Xombi expression and blastopore lip formation on fibroblast growth factor (FGF) signaling suggest that Xombi contributes to a traveling wave of morphogenesis and differentiation during Xenopus gastrulation. Xombi is most closely related to mouse Tbx2 (57% identity) , Drosophila Optomotor blind, (54% identity) and Xenopus Brachyury. Outside its putative DNA-binding domain, Xombi shows no obvious homology to other members of the gene family (Lustig, 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).

The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, affinity purified polyclonal antibodies to Apod were prepared and compared to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus: one referred to as VegT, and a smaller molecular weight isoform referred to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. It is concluded that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and responses to growth factors (Stennard, 1999).

In Xenopus eggs, localized molecules have been identified; some of these (such as Vg1 and Xwnt-11) can specify cell fates by functioning as inducers or patterning agents. A new member of the T-box family of transcription factors, named Brat, is expressed maternally and its transcripts are localized to the vegetal hemisphere of the egg. Brat homology to Drosophila Optomotor blind is 51%, while homology to T-related gene is only 45%. Homology to Xenopus Brachyury is 47%. During early embryonic cleavage, Brat mRNA becomes partitioned primarily within vegetal cells that are fated to form the endoderm. Zygotic expression of Brat begins at the onset of gastrulation within the presumptive mesoderm of the marginal zone. Consistent with its zygotic expression pattern, Brat induces, in a dose-dependent manner, a full spectrum of mesodermal genes that are expressed in tissues throughout the dorsal-ventral axis. Brat also induces endoderm, consistent with its vegetal localization, making Brat a good candidate for a maternal determinant of the endoderm. Endogenous Brat is required for mesoderm formation. Brat might directly activate transcription of the Xbra gene, or it might function indirectly to promote Xbra transcription. Brat plays a dual role in the formation of endodermal and mesodermal tissues. The first phase of its expression is maternal when Brat transcripts become localized to the vegetal pole of oocytes early in oogenesis. In full-grown oocytes, the transcripts reside mostly in the vegetal cortex. In a second phase, Brat can respond to mesoderm-inducing factors, and Brat can be activated by basic FGF or activin B proteins (Horb, 1997).

The maternal transcription factor VegT (T-box protein Brat or Antipodean) is important for establishing the primary germ layers in Xenopus. The vegetal masses of embryos lacking maternal VegT do not produce mesoderm-inducing signals and mesoderm formation in these embryos occurs ectopically -- from the vegetal area, rather than the equatorial zone of the blastula. The efficiency of the depletion of maternal VegT mRNA have been increased and the effects on mesoderm formation has been studied. Maternal VegT is required for the formation of 90% of mesodermal tissue, as measured by the expression of mesodermal markers MyoD, cardiac actin, Xbra, Xwnt8 and alphaT4 globin. Furthermore, the transcription of FGFs and TGFbetas, Xnr1, Xnr2, Xnr4 and derriere (TGFbeta family member Vg1) does not occur in VegT-depleted embryos. A test was performed to see whether these growth factors may be endogenous factors in mesoderm induction. Their ability to rescue the phenotype of VegT-depleted embryos has been studied under conditions where their expression has been restricted to the vegetal mass. Xnr1, Xnr2, Xnr4 and derriere mRNA all rescue mesoderm formation, as well as the formation of blastopores and the wild-type body axis. Derriere rescues trunk and tail while nr1, nr2 and nr4 rescue head, trunk and tail. It is concluded that mesoderm induction in Xenopus depends on a maternal transcription factor regulating these zygotic growth factors (Kofron, 1999).

mRNA encoding the T-box transcription factor VegT is located throughout the vegetal pole of the Xenopus egg and is believed to play an important part in endoderm and mesoderm formation. VegT has been found to generate endoderm both by cell-autonomous action and by generating TGF-beta family signals, the latter being entirely responsible for its mesoderm-inducing activity. Signaling molecules induced cell-autonomously by VegT include derrière, Xnr4 and activin B. Xnr1 and Xnr2 are also induced, but primarily in a non-autonomous manner. All of these signaling molecules are found in the blastula and gastrula vegetal pole and induce both endoderm and mesoderm in the animal cap assay, and hence are good candidates both for the endogenous zygotic mesoderm-inducing signal and for reinforcing the vegetal expression of endoderm markers (Clements, 1999).

The Xenopus nodal related-1 (Xnr1) gene has a complex expression pattern in embryos, with two temporal phases. In the first phase, transcripts are first detected in perinuclear sites in the vegetal region of the blastula. During gastrulation, this expression disappears and transcripts become localized to the dorsal marginal zone. Expression stops and then restarts in a second phase at neurula and tailbud stages, first in two symmetric patches near the posterior end of the notochord, and then asymmetrically in a large domain in the left lateral plate mesoderm. The regulation of the early phase of expression of Xnr1 has been investigated. The T-box transcription factor VegT can induce Xnr1. It had previously been shown that Xnr1 can induce VegT in ectoderm cells and it is shown that the early expression of Xnr1 is regulated by an autoregulatory loop. By inspection of the Xnr1 promoter sequence, two non-palindromic T-box-binding sites, which are 10 bp apart, has been investigated. Using mutational analysis, these elements are shown to be required for the VegT induction of Xnr1. The Xnr1 promoter shows striking homologies with the Xnr3 promoter. In particular, two elements that are required for Wnt signaling are conserved between these two promoters, but the two T-box sites are not conserved, and Xnr3 is not induced by VegT. A region of the promoter containing the T-box sites and the Wnt sites is sufficient to drive expression of a reporter gene in a dorsal domain in transgenic Xenopus at the gastrula stage. This pattern of expression of the transgene in gastrulae is not dependent on the T-box sites (Hyde, 2000).

During cleavage stages, maternal VegT mRNA and protein are localized to the Xenopus embryo’s vegetal region from which the endoderm will arise and where several zygotic gene transcripts will be localized. Previous loss-of-function experiments on this T-box transcription factor have suggested a role for VegT in Xenopus endoderm formation. Whether VegT is required to initiate endoderm formation was investigated using a loss of function approach. The endodermal genes Bix1, Bix3, Bix4, Milk (Bix2), Mix.1, Mix.2, Mixer, Xsox17 a, Gata4, Gata5, Gata6 and endodermin, as well as the anterior endodermal genes Xhex and cerberus, and the organizer specific gene, Xlim1, are all downstream of maternal Veg T. The TGFbetas, Xnr1, Xnr2, Xnr4 and derrière rescue expression of these aformentioned genes, supporting the idea that cell interactions are critical for proper endoderm formation. Additionally, inhibitory forms of Xnr2 and Derrière block the ability of VegT mRNA injection to rescue VegT-depleted embryos. Furthermore, a subset of endodermal genes was rescued in VegT-depleted vegetal masses by induction from an uninjected vegetal mass. Finally, a gene hierarchy downstream of VegT has been established by testing the ability of Mixer and Gata5 to rescue the expression of other endodermal genes. These results identify VegT as the maternal regulator of endoderm initiation and illustrate the complexity of zygotic pathways activated by VegT in the embryo's vegetal region (Xanthos, 2001).

A gene hierarchy for endodermal formation is proposed. Maternal VegT directly activates Xnr1 expression. Nodal signaling then initiates the expression of the early endodermal genes such as Mixer and Gata5 in gastrulae. It is also possible that VegT directly activates expression of these genes, although the severe reduction of expression observed with cmXnr2 in this study indicates that TGFbeta signaling plays an important role in their expression. Although Mixer and Gata5 are both considered to be general endodermal markers, the results suggest they act in separable pathways -- Mixer maintaining Xsox17alpha expression and Gata5 initiating Xlim1 expression in the early gastrula. Furthermore, the rescue experiments suggest that Mixer and Gata5 cooperate to initiate Xhex and Gata4 expression in the early gastrula. Later endodermal markers were examined in Mixer and Gata5 mRNA-injected, VegT-depleted embryos at the taibud stage. Several genes including endodermin, Gata6 and Xsox17alpha were not only rescued but even overexpressed at this stage. It is difficult to interpret these results since Mixer is not normally expressed in wild-type embryos at this time. However, it is likely that endodermal genes may be overexpressed because VegT-depleted embryos lack mesoderm, which may normally act as an inhibitory influence on endoderm formation (Xanthos, 2001).

Because activin remains expressed in VegT-depleted embryos and endoderm does not form, it is unlikely to be important in the initiation of endoderm formation. Maternal VegT directly initiates transcription of Bix1, Bix4 and possibly other homeobox genes, as well as the nodals. Zygotic TGFbs indirectly or directly activate expression of early endodermal transcription factors: this is consistent with previous work showing one of these genes, Mix.2, to be downstream of TGFbeta signaling. The zygotic TGFbetas also cause Smad activation, which then acts in concert with other vegetally expressed transcription factors such as Mixer to activate gene expression. Xsox17a mRNA does not rescue endodermal gene expression, and Mixer mRNA only rescues Xsox17alpha, suggesting that they may require partners such as the Smads or beta-catenin. Recently, it has been shown that Xsox17alpha/beta and Xsox3 physically interact with beta-catenin, and that Mixer interacts with Smad2. In contrast, ectopic expression of Xsox17alpha or Mixer in animal caps induces an array of endodermal markers. This could be explained by inherent differences in the embryo's animal and vegetal regions (Xanthos, 2001).

Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-ß factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localized throughout the vegetal pole, and ß-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, a minimal promoter has been defined that mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional ß-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that ß-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a ß-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. A model is proposed in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of ß-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of ß-catenin/XTcf-3 (Hilton, 2003).

RNA localization is a key mechanism for generating cell and developmental polarity in a wide variety of organisms. A role has been investigated for the Xenopus homolog of the double-stranded RNA-binding protein Staufen in RNA localization during oogenesis. Xenopus Staufen (XStau) is present in a ribonucleoprotein complex, and associates with both a kinesin motor protein and vegetally localized RNAs Vg1 and VegT. A functional role for XStau was revealed through expression of a dominant-negative version that blocks localization of Vg1 RNA in vivo. These results suggest a central role for XStau in RNA localization in Xenopus oocytes, and provide evidence that Staufen is a conserved link between specific mRNAs and the RNA localization machinery (Yoon, 2004).

One cause for the range of RNAs recognized by Staufen probably lies in the nature of the interaction between dsRBDs and dsRNA, which is generally non-sequence specific. Vg1 and VegT contain potentially double-stranded regions, but they are specifically bound by XStau in vivo. So the question remains as to how Staufen could interact specifically with disparate RNA targets. It is proposed that there are two classes of RNA-binding factors involved in RNA localization. One class recognizes and binds to RNA localization elements in a sequence-specific manner. Examples of such factors in Xenopus include Vg1 RNA-binding proteins hnRNP I and Vg1RBP/vera. This class of factors may be cell-type specific and act to establish a core ribonucleoprotein complex for transport. The other class of factors, such as XStau, may act not at the level of sequence-specific RNA recognition, but rather, recognize the core RNP complex and mediate the interaction with the localization machinery. In such a model, some dsRBDs would interact in a non-sequence specific manner with double-stranded regions of RNA presented on the RNP, while other dsRBDs could interact with protein components of the core RNP. Consistent with this idea, dsRBD2 and dsRBD5 of Drosophila Staufen do not bind RNA in vitro, whereas dsRBD1, dsRBD3 and dsRBD4 bind dsRNA sequence nonspecifically. Dominant-negative XStau234 is defective in interaction with hnRNP I, suggesting that XStau dsRBD1 or dsRBD5 could potentially facilitate interaction between XStau and hnRNP I. It is suggested that this interaction is in the context of an RNP, and hnRNP I and Vg1RBP/vera have been shown to associate with Vg1 and VegT RNAs in the nucleus, prior to recruitment of XStau to the cytoplasmic RNP. The observed biochemical interaction between XStau and kinesin could further suggest a role for XStau in motor recruitment, although this remains an issue for future investigation. Thus, Staufen may represent a central component of the RNA localization machinery, perhaps linking the localized RNP cargoes with the motors that move them (Yoon, 2004).

The neuron-specific transcription factor T-box brain 1 (TBR1) regulates brain development. Disruptive mutations in the TBR1 gene have been repeatedly identified in patients with autism spectrum disorders (ASDs). This study shows that Tbr1 haploinsufficiency results in defective axonal projections of amygdalar neurons and the impairment of social interaction, ultrasonic vocalization, associative memory and cognitive flexibility in mice. Loss of a copy of the Tbr1 gene altered the expression of Ntng1, Cntn2 and Cdh8 and reduced both inter- and intra-amygdalar connections. These developmental defects likely impair neuronal activation upon behavioral stimulation, which is indicated by fewer c-FOS-positive neurons and lack of GRIN2B induction in Tbr1+/- amygdalae. Upregulation of amygdalar neuronal activity by local infusion of a partial NMDA receptor agonist, d-cycloserine, ameliorates the behavioral defects of Tbr1+/- mice. This study suggests that TBR1 is important in the regulation of amygdalar axonal connections and cognition.

T-box proteins regulate posterior body morphogenesis in zebrafish

The vertebrate posterior body is formed by a combination of the gastrulation movements that shape the head and anterior trunk and posterior specific cell behaviors. This study investigates whether genes that regulate cell movements during gastrulation [no tail (ntl)/brachyury, knypek (kny; encoding a glypican) and pipetail (ppt)/wnt5] interact to regulate posterior body morphogenesis. Both kny;ntl and ppt;ntl double mutant embryos exhibit synergistic trunk and tail shortening by early segmentation. Gene expression analysis in the compound mutants indicates that anteroposterior germ-layer patterning is largely normal and that the tail elongation defects are not due to failure to specify or maintain posterior tissues. Moreover, ntl interacts with ppt and kny to synergistically regulate the posterior expression of the gene encoding bone morphogenetic protein 4 (bmp4) but not of other known T-box genes, fibroblast growth factor genes or caudal genes. Examination of mitotic and apoptotic cells indicates that impaired tail elongation is not simply due to decreased cell proliferation or increased cell death. Cell tracing in ppt;ntl and kny;ntl mutants demonstrates that the ventral derived posterior tailbud progenitors move into the tailbud. However, gastrulation-like convergence and extension movements and cell movements within the posterior tailbud are impaired. Furthermore, subduction movements of cells into the mesendoderm are reduced in kny;ntl and ppt;ntl mutants. It is proposed that Ntl and the non-canonical Wnt pathway components Ppt and Kny function in parallel, partially redundant pathways to regulate posterior body development. This work initiates the genetic dissection of posterior body morphogenesis and links genes to specific tail-forming movements. Moreover, genetic evidence is provided for the notion that tail development entails a continuation of mechanisms regulating gastrulation together with mechanisms unique to the posterior body (Marlow, 2004).

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, tbx6 was identified as a posterior mesodermal gene activated by both of these signaling pathways. A genomic fragment was isolated from the tbx6 gene that recapitulates the endogenous tbx6 expression, and this was used to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. The tbx6 promoter was found to utilize distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, overexpression of either signaling pathway was found to activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. A model is presented in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures (Szeto, 2004).

T/TBX6, controls Notch signaling

Notch signaling in the presomitic mesoderm (psm) is critical for somite formation and patterning. WNT signals regulate transcription of the Notch ligand Dll1 in the tailbud and psm. LEF/TCF factors cooperate with TBX6 to activate transcription from the Dll1 promoter in vitro. Mutating either T or LEF/TCF sites in the Dll1 promoter abolishes reporter gene expression in vitro as well as in the tail bud and psm of transgenic embryos. These results indicate that WNT activity, in synergy with TBX6, regulates Dll1 transcription and thereby controls Notch activity, somite formation, and patterning (Hofmann, 2004).

Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. The molecular basis for this presumed hierarchy has been examined and it has been shown that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-ß-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1 (Galceran, 2004).

T-box family, notochord and the midline

Continued Optomotor blind Evolutionary homologs part 2/3 | part 3/3

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

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