bifid
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).
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).
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
derriËre, 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 embryoís 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 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).
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).
Continued Optomotor blind Evolutionary homologs part 2/3 | part 3/3
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