caudal
A mouse gene, Cdx-1, was isolated from an embryonic cDNA library using a Drosophila caudal gene probe. The deduced amino acid sequence of Cdx-1 contains conserved sequence domains along the entire gene, as well as a highly conserved caudal-type homeo box. A structural comparison suggests a common ancestral origin of mouse Cdx-1 and Drosophila caudal. The expression of Cdx-1 during embryogenesis was analyzed by Northern blotting and in situ hybridization. Cdx-1-specific transcripts are localized in the epithelial lining of the intestines beginning at day 14 of gestation. The expression of Cdx-1 in the intestine continues into adulthood, but cannot be detected in any other tissues. The Cdx-1 gene is the first homeo-box-containing gene expressed in cells derived from the embryonic endoderm (Duprey, 1988).
Expression of mouse Cdx-1 gene begins with the onset of the head process formation (day 7.5) in ectodermal and mesodermal cells of the primitive streak. Expression extends initially to the middle of the prospective hindbrain and subsequently regresses caudad to the spinal cord level by day 9.5. The mesoderm-specific expression is detected in the first somites and can be followed during their differentiation to the myotome of the dorsal somitic edge by day 12. The developing limb buds and the mesonephros exhibit expression up to day 12. No signal is detected in notochordal cells and cells of the definitive endoderm. Thus, Cdx-1 is expressed during gastrulation when anterior-posterior positional values are established along the embryonic axes. Furthermore, the expression correlates with the formation of segmented tissue in the posterior hindbrain, the spinal cord and structures like the mesonephros (Meyer, 1993).
Cdx-4 is expressed transiently from 7 until 10 days, starting at the beginning of gastrulation in the allantois and
posterior tip of the primitive streak. At the mid-streak stage, Cdx-4 expression moves rostrally,
and protein and mRNA are detected in all cells over the posterior half of the primitive streak. As
development proceeds, Cdx-4 gene products continue to be restricted to the posterior of the
embryo, including the remnants of the primitive streak. Cdx-4 is expressed in neurectoderm,
presomitic and lateral plate mesoderm, and hindgut endoderm (Gamer, 1993).
The holy grail of developmental biology is the search for a link between homeotic genes and the expression of downstream targets. Intestine-specific transcription of mouse sucrase-isomaltase,
a gene that is expressed exclusively in differentiated enterocytes, is dependent on binding of a
tissue-specific homeodomain protein (mouse Cdx-2) to an evolutionarily conserved promoter
element in the sucrase-isomaltase gene. Mouse Cdx-2 binds as a dimer to its regulatory element
and that dimerization in vitro is dependent on the electrochemical balance of the cell. These findings suggest that members of the Cdx gene
family play a fundamental role in the establishment of the intestinal phenotype (Suh, 1994).
Cdx1 is expressed along the embryonic axis from day 7.5 postcoitum until day 12, by which time the anterior limit of expression has regressed from the hindbrain level to the forelimb bud region. To assign a functional role for Cdx1 in murine embryonic development, the gene was inactivated via homologous recombination. Viable fertile homozygous mutant mice were obtained that show anterior homeotic transformations of vertebrae. These abnormalities were concomitant with posterior shifts of Hox gene expression domains in the somitic mesoderm. The presence of putative Cdx1-binding sites in Hox gene control regions as well as in vitro transactivation of Hoxa-7 indicates a direct regulation (Subramanian, 1995).
Three mouse homologs of Drosophila Caudal (Cdx-1, Cdx-2, and Cdx-4) have been investigated. Cdx-1 and 2 are both strongly
expressed in the adult mid- and hindgut, while Cdx-1 and 4 have been shown to be activated in the
embryonic primitive streak. Cdx-2 expression
begins at 3.5 days and is confined to the trophectoderm, being absent from the inner cell mass.
From 8.5 days, Cdx-2 begins to be expressed in embryonic tissues,
principally (unlike Cdx-1) in the posterior part of the gut from its earliest formation, as well as in the
tail bud and in the caudal part of the neural tube. Cdx-2 is, therefore, transcribed well before any
other member of the Cad homolog group (Beck, 1995).
Classical embryological experiments suggest that a posterior signal is required for
patterning the developing anteroposterior axis. In this paper, a potential role in Xenopus is investigated
for FGF signaling during this process. During normal development, embryonic fibroblast growth
factor (eFGF) (See Drosophila Branchless) is expressed in the dorsal mesoderm, specifically, in the notochord and in the posterior mesoderm around the closing blastopore. Overexpression of eFGF from the start of gastrulation results in a posteriorised
phenotype of reduced head and enlarged proctodeum. The overexpression of eFGF causes the up-regulation of a
number of posteriorly expressed genes, and prominent among these are Xcad3, a caudal
homolog, and the Hox genes, in particular HoxA7. There is both an increase of expression within the normal domains and an extension of expression towards the anterior. Application of eFGF-loaded beads to specific regions of gastrulae reveals that anterior truncations arise
from an effect on the developing dorsal axis. Similar anterior truncations are caused by the dorsal overexpression of Xcad3 or HoxA7. This suggests that this aspect of the eFGF
overexpression phenotype is caused by the ectopic activation of posterior genes in anterior regions. Further results using the dominant negative FGF receptor show that the normal expression of posterior Hox genes is dependent on FGF signaling and that this regulation is likely
mediated by the activation of Xcad3. It has been demonstrated that the eFGF regulates the transcription of Xbra (Drosophila homolog: T-related gene) and that Xbra can in turn activate eFGF expression. Xbra does not directly activate Hox gene expression. However, at the very least, Xbra clearly plays an indirect role in anteroposterior specification through its regulation of eFGF expression in the notochord and the posterior of the embryo. The biological activity of eFGF, together with its expression in the posterior of the embryo, make it a good candidate to fulfil the role of the 'transforming' activity proposed by Nieuwkoop in his 'activation and transformation' model for neural patterning (Pownall, 1996).
A detailed and systematic study of the expression of the Cdx1 protein was carried out during embryonic intestinal development, postnatal cytodifferentiation and in the regenerating (after radiation-induced damage) intestine of the mouse. Using antibodies directed against Cdx1, it is shown that the Cdx1 protein is localised in the proliferating immature epithelium during intestinal development. It becomes restricted to the proliferative crypt compartment during postnatal differentiation, as well as in the adult intestine. The mesenchymal layer is completely negative both during embryonic development and in the postnatal intestine. The expression of the protein is first clearly detected throughout the simple columnar epithelium at day 15 of development. This expression progressively becomes restricted to the regions of epithelial proliferation in the crypts of the adult mouse by day 40 of post-natal development. There were occasional cells that were Cdx1 positive in the villi. During regeneration of the epithelium after radiation-induced damage, Cdx1 expression diminishes during the initial phase of cellular regression. The expression is then very strong in the regenerating epithelial foci, but not in the quiescent sterilised crypts between day 4 and 6. The normal pattern is restored between day 6 and 7. The Paneth cells are negative. The physical segregation of Cdx1 with the proliferative compartment and the hierarchy of cell renewal in the intestinal epithelium is an important example of how regulatory genes function in the maintenance and in the dysfunction of renewing tissues (Subramanian, 1998).
Mouse Cdx and Hox genes presumably evolved from genes on a common ancestor cluster involved in anteroposterior patterning. Drosophila caudal (cad) is involved in specifying the posterior end of the early embryo, and is essential for patterning tissues derived from the most caudal segment, the analia. Two of the three mouse Cdx paralogs, Cdx 1 and Cdx2, are expressed early in a Hox-like manner in the three germ layers. In the nascent paraxial mesoderm, both genes are expressed in cells contributing first to the most rostral, and then to progressively more caudal parts of the vertebral column. Later, expression regresses from the anterior sclerotomes, and is only maintained for Cdx1 in the dorsal part of the somites, and for both genes in the tail bud. Cdx1 null mutants show anterior homeosis of upper cervical and thoracic vertebrae. Cdx2-null embryos die before gastrulation, and Cdx2 heterozygotes display anterior transformations of lower cervical and thoracic vertebrae. The genetic interactions between Cdx1 and Cdx2 are analyzed in compound mutants. Combining mutant alleles for both genes gives rise to anterior homeotic transformations along a more extensive length of the vertebral column than do single mutations. The most severely affected Cdx1 null/Cdx2 heterozygous mice display a posterior shift of their cranio-cervical, cervico-thoracic, thoraco-lumbar, lumbo-sacral and sacro-caudal transitions. The effects of the mutations in Cdx1 and Cdx2 were co-operative in severity, and a more extensive posterior shift of the expression of three Hox genes was observed in double mutants. The alteration in Hox expression boundaries occurred early. It is concluded that both Cdx genes cooperate at early stages in instructing the vertebral progenitors all along the axis, at least in part by setting the rostral expression boundaries of Hox genes. In addition, Cdx mutants transiently exhibit alterations in the extent of Hox expression domains in the spinal cord, reminiscent of the strong effects of overexpressing Cdx genes on Hox gene expression in the neurectoderm. Phenotypical alterations in the peripheral nervous system were observed at mid-gestation stages. Strikingly, the altered phenotype at caudal levels included a posterior truncation of the tail, mildly affecting Cdx2 heterozygotes, but more severely affecting Cdx1/Cdx2 double heterozygotes and Cdx1 null/Cdx2 heterozygotes. Mutations in Cdx1 and Cdx2 therefore also interfere with axis elongation in a cooperative way. The function of Cdx genes in morphogenetic processes during gastrulation and tail bud extension, and their relationship with the Hox genes are discussed in the light of available data in Amphioxus, C. elegans, Drosophila and mice (van den Akker, 2002).
The patterning effect of Cdx genes at rostral levels is more likely to result from the regulatory action of Cdx on 3' Hox genes than from a 'posterior' homeotic role of the Cdx gene products. Work in Drosophila and C. elegans has strongly suggested that Cdx gene products positively regulate several genes of the Hox cluster in the ancestral situation: cad regulates ftz in the fly, and pal-1 regulates mab-5 and vab-7 in worms. Cdx target sequences probably already existed in the ancestral Hox cluster, as witnessed by the direct transcriptional activation of mab-5 by pal-1 in the V6 cells of C. elegans. In the mouse, Hox genes with rostral expression boundaries at the level of cervical to sacral contain potential Cdx-binding sites in their regulatory regions. The existence of this molecular crosstalk would have given Cdx gene products the possibility to regulate the 5' Hox genes and posterior development, as well as 3' Hox genes and more anterior patterning. Direct Cdx/Hox regulatory interactions have been observed in vertebrates. Loss of expression of a Hoxb8/lacZ transgene in mesoderm and neurectoderm upon inactivation of the Cdx-binding sites may indicate a fundamental requirement of Cdx gene products in aiding trunk Hox genes to achieve their correct expression patterns. Whether the Cdx genes directly contribute positional information to paraxial mesoderm cells, or whether they transduce this information via the Hox genes is not easy to establish at this point, in the absence of total Hox disruption, or without inactivating all Cdx binding sites in the Hox clusters (van den Akker, 2002).
The early, maximally extending expression domain of Cdx1 corresponds to that of the most 3' Hox genes, with a rostral expression boundary at the level of the preotic sulcus, the limit between rhombomeres 2 and 3. Cdx1 and Cdx2 are initially and transiently expressed as early as Hoxb1 in the posterior part of the primitive streak at the late streak stage. These Cdx genes therefore display features of 3'-most Hox genes, in spite of the fact that they are later involved in generating and patterning posteriormost structures. cad in the ParaHox cluster might be paralogous to the 5' neighbour of AbdB in the Hox cluster, eve. evx2 has in fact been shown to function as a posterior Hoxd gene in distal structures of the mouse limbs. Nevertheless, comparative analysis of the amino acid sequence of the homeodomains reveals that Cdx1 and Cdx2 are closer to Hox paralogy groups 8 and 9, and even to Hox paralogy group 1 and 2 than to the most posterior paralogy group 13 and to Evx proteins. In addition, the Cdx gene products possess a Pbx recognition motif, which is absent in 5'-most Abdb Hox proteins, such as paralogy group 13, and in Evx gene products. This motif in Cdx1 shares four of the five consensus residues with that of Hoxb4. It therefore seems that mammalian Cdx genes are relatively closely related to 3' Hox genes, although to a lesser extent than their 3' neighbours on the ParaHox cluster, Gsh1 and Pdx1. This could possibly explain the existence of similarities in their regulation (van den Akker, 2002).
Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is
generated in the embryo by signaling gradients of an as yet unknown nature. Transcription factors were sought that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, an in vivo assay was devised to detect responsiveness of cis-acting sequences to such
differentially active factors. This assay was used to analyze a Hoxb8 regulatory element, and the most pronounced response was detected in a short stretch of DNA containing a cluster of potential CDX binding sites. Differentially expressed
DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro (included among these proteins are cdx gene products). Binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory
element to drive regionally restricted expression in the embryo. Ectopic expression of cdx gene products
anteriorizes expression of reporter transgenes driven by the CDX binding regulatory element, as well as that of the endogenous Hoxb8 gene,
in a manner that is consistent with CDX genes being essential transducers of positional information. These data suggest that, in
contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting
through CDX binding sites in their enhancers. This may represent the ancestral mode of action for caudal homologs, which
are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development (Charite, 1998).
There are three mouse homologs of Drosophila Caudal: CDX1, CDX2 and CDX4. Cdx2 null mutants die between 3.5 and 5.5 days post coitum. Cdx2 heterozygotes exhibit a variable phenotype, with many showing tail abnormalities or stunted growth. Skeletal analysis demonstrates a homeotic shift of vertebrae and compatible malformations of the ribs. Within the first three months of life, 90% of Cdx2 heterozygotes develop multiple intestinal adenomatous polyps, particularly in the proximal colon. These polyps occasionally contain areas of true metaplasia. In contrast to the surrounding intestinal epithelium, the neoplastic cells do not express Cdx2 from the remaining allele. These results suggest that Cdx2 mutation is the primary event in the genesis of some intestinal tumors (Chawengsaksophak, 1997).
To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line
Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and
antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the
cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two
typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the
endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum.
Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and
cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit,
laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly,
most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes, rather
than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression,
a homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a
decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice,
Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like
structures. Laminin-1 (see Drosophila Laminin A)is known to stimulate intestinal cell differentiation in vitro. The differentiating effect of laminin-1 coatings on Caco2-TC7 cells is
accompanied by an upregulation of Cdx2. To further document this observation, a series
of Caco2 clones was analyzed in which the production of laminin-alpha1 chain was differentially inhibited by antisense
RNA. A positive correlation exists between the level of Cdx2 expression, that of endogenous
laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken
together, these results suggest (1) that Cdx1 and Cdx2 homeobox genes play distinct roles in the
intestinal epithelium; (2) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype
of differentiated villus enterocytes, and (3) that Cdx2 expression is modulated by basement membrane
components. It is concluded that Cdx2 plays a key role in the extracellular matrix-mediated
intestinal cell differentiation (Lorentz, 1997).
Using a xenograft model of fetal intestinal anlagen implanted under the
skin of nude mice, the expression of five homeobox genes
(HoxA-4, HoxA-9, HoxC-8, Cdx-1 and Cdx-2) was examined. In homotypic associations of fetal endoderm and mesenchyme that
recapitulate normal development, the overall pattern of homeobox gene expression is
maintained: HoxA-9 (homologous to Drosophila Abd-B) and HoxC-8 (homologous to Drosophila abd-A) were the highest in the colon and ileum, respectively, and
HoxA-4 (homologous to Drosophila Deformed) is expressed all along the intestine. Cdx-1 and Cdx-2 (Both homologs of Drosophila caudal) exhibit an increasing
gradient of expression from small intestine to colon. Grafting per se causes a faint
upregulation of HoxA-9 and HoxC-8 in small intestinal regions where these genes are not
normally expressed, while the endoderm-mesenchyme dissociation-association step
provokes a decay of Cdx-1 in the colon. In heterotopic associations of colonic endoderm
with small intestinal mesenchyme, the colonic epithelium exhibits heterodifferentiation into a
small intestinal-like phenotype. In this case, a decay of HoxA-9 expression and
an upregulation of HoxC-8 is observed. Heterodifferentiation of the colonic epithelium is
accompanied by a downregulation of Cdx-1 and Cdx-2 to a level similar to that found in the
normal small intestine. To demonstrate that mesenchyme-derived cells can influence Cdx-1
and Cdx-2 expression in the bowel epithelium, fetal jejunal endoderm was associated with
intestinal fibroblastic cell lines that either support small intestinal-like or colonic-like
morphogenesis. A lower expression of both homeobox genes occurs in grafts presenting
the small intestinal phenotype than in those showing glandular colonic-like differentiation.
Taken together, these results suggest that homeobox genes participate in the control of the
positional information and/or cell differentiation in the intestinal epithelium. They also
indicate that the level of Cdx-1 and Cdx-2 homeobox gene expression is influenced by
epithelial-mesenchymal cell interactions in the intestinal mucosa (Duluc, 1997).
In vertebrates, each vertebra along the anteroposterior axis has a characteristic structure. Several
transcription factors and cell signaling molecules expressed in the primitive streak ectoderm and/or the tailbud play essential roles in
establishing the correct anteroposterior specification of vertebrae during mouse development. Anteroposterior specification of the somitic mesodermal cells is established before they form the somite, likely during gastrulation.
Grafting experiments in the chick embryo have demonstrated that presumptive vertebral cells acquire specificity
along their anteroposterior axis before somite formation. In addition, it appears that several transcription factors, including Cdx-1, and cell signaling
molecules, including Gdf-11, FGF receptor-1 (FGFR1), and activin receptor IIB (ActRIIB), expressed in the primitive streak ectoderm and/or the tail-bud play essential roles in establishing the correct antero-posterior specification of the vertebrae during mouse development. Wnt-3a mutants exhibit
homeotic transformations in the vertebrae along their entire body axis.
Mutation of cdx-1 results in an anterior transformation, as occurs in Wnt-3a mutants. Reduced expression of cdx-1 is observed in the primitive streak and tail bud region of Wnt-3a mutant
embryos. These results indicate that Wnt-3a is necessary for correct anteroposterior patterning of vertebra, and that cdx-1 may be one of the
mediator genes of Wnt-3a signaling in this process (Ikeya, 2001).
Inactivation of Cdx2 by homologous recombination results in the development of forestomach epithelium at ectopic sites in pericaecal
areas of the midgut of heterozygote mice. Local factors subsequently result in the secondary induction of tissues exhibiting an orderly
sequence of tissue types between the ectopic forestomach tissue and the surrounding colon. Clonal analysis of this secondarily generated
tissue using Y chromosome painting in chimaeric mice indicates that once differentiated to express Cdx2, host colonic epithelium can
form only small intestinal-type epithelium, while Cdx2 mutant cells give rise to a succession of gastric-type tissue but never to a small intestine
morphology. These results indicate a difference in potency between forestomach and midgut precursor endodermal cells (Beck, 2003).
The vertebrate caudal proteins, being upstream regulators of the Hox genes, play a role in establishment of the body plan. Analysis is described of two orthologous caudal genes (chick cdx-A and mouse cdx-1) by use of lacZ reporters expressed in transgenic mouse embryos. The expression patterns show many similarities to the expression of endogenous mouse cdx-1. At 8.7 days, cdx/lacZ activity within neurectoderm and mesoderm forms posterior-to-anterior gradients, and the possibility is discussed that similar gradients of cdx gene expression may function as morphogen gradients for the establishment of Hox gene expression boundaries. The observations suggest that gradients form by decay of cdx/lacZ activity in cells that have moved anterior to the vicinity of the node. The cdx-A/lacZ expression pattern requires an intron enhancer that includes two functional control elements: a DR2-type retinoic acid response element and a Tcf/ß-catenin binding motif. These motifs are structurally conserved in mouse cdx-1 (Gaunt, 2003).
The chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and their anterior expansion
is also limited to the level of the otic vesicle. Using a dominant negative
form of a Xenopus Cdx gene (XcadEnR) it has been found that the
effect of FGF treatment on 5' HoxB genes is mediated in part
through the activation and function of CDX activity. Conversely, the 3'
HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to
RA but not FGF treatments at these stages. In ovo
electroporation of a dominant negative retinoid receptor construct
(dnRAR) shows that retinoid signaling is required to initiate expression.
Elevating CDX activity by ectopic expression of an activated form of a
Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically
activates and anteriorly expands Hoxb4 expression. In a similar
manner, when ectopic expression of XcadVP16 is combined with FGF
treatment, it was found that Hoxb9 expression expands anteriorly into the
hindbrain region. These findings suggest a model whereby, over the window of
early development examined, all HoxB genes are actually competent
to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms
that axially restrict the Cdx domains of expression, serve to prevent
3' genes from responding to FGF signaling in the hindbrain. FGF may have
a dual role in both modulating the accessibility of the HoxB complex
along the axis and in activating the expression of Cdx genes. The
position of the shift in RA or FGF responsiveness of Hox genes may be time
dependent. Hence, the specific Hox genes in each of these complementary groups
may vary in later stages of development or other tissues. These results
highlight the key role of Cdx genes in integrating the input of
multiple signaling pathways, such as FGFs and RA, in controlling initiation of
Hox expression during development and the importance of understanding
regulatory events/mechanisms that modulate Cdx expression (Bel-Vialar, 2002).
The spinal cord is a unique vertebrate feature that originates, together with the hindbrain, from the caudal neural plate. Whereas the hindbrain subdivides into rhombomeres, the spinal cord remains unsegmented. Cdx transcription factors have been identified as key determinants of the spinal cord region in zebrafish. Loss of Cdx1a and Cdx4 functions causes posterior expansion of the hindbrain at the expense of the unsegmented spinal cord. By contrast, cdx4 overexpression in the hindbrain impairs rhombomere segmentation and patterning and induces the expression of spinal cord-specific genes. Using cell transplantation, Cdx factors function has been identfied directly within the neural ectoderm to specify spinal cord. Overexpression of 5' Hox genes fails to rescue hindbrain and spinal cord defects associated with cdx1a/cdx4 loss-of-function, suggesting a Hox-independent mechanism of spinal cord specification. In the absence of Cdx function, the caudal neural plate retains hindbrain characteristics and remains responsive to surrounding signals, particularly retinoic acid, in a manner similar to the native hindbrain. It is proposed that by preventing the posterior-most region of the neural plate from following a hindbrain developmental program, Cdx factors help determine the size of the prospective hindbrain and spinal cord territories (Skromne, 2007).
Blastocyst formation marks the segregation of the first two cell lineages in the mammalian preimplantation embryo: the inner cell mass (ICM) that will form the embryo proper and the trophectoderm (TE) that gives rise to the trophoblast lineage. Commitment to ICM lineage is attributed to the function of the two transcription factors, Oct4 (encoded by Pou5f1) and Nanog. However, a positive regulator of TE cell fate has not been described. The T-box protein eomesodermin (Eomes) and the caudal-type homeodomain protein Cdx2 are expressed in the TE, and both Eomes and Cdx2 homozygous mutant embryos die around the time of implantation. A block in early TE differentiation occurs in Eomes mutant blastocysts. However, Eomes mutant blastocysts implant, and Cdx2 and Oct4 expression is correctly restricted to the ICM TE. Blastocoel formation initiates in Cdx2 mutants but epithelial integrity is not maintained and embryos fail to implant. Loss of Cdx2 results in failure to downregulate Oct4 and Nanog in outer cells of the blastocyst and subsequent death of those cells. Thus, Cdx2 is essential for segregation of the ICM and TE lineages at the blastocyst stage by ensuring repression of Oct4 and Nanog in the TE (Strumpf, 2005).
Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. Cdx1 is a direct retinoid target gene, suggesting that Cdx1 may convey some of the effects of retinoid signaling. However, RA appears to be essential for only early stages of Cdx1 expression, and therefore other factors must be involved in maintaining later stages of expression. Based on function and pattern of expression, Wnt family members, in particular Wnt3a, are candidates for regulation of expression of Cdx1. Consistent with this, Cdx1 can be directly regulated by Wnt signaling, and functional LEF/TCF response motifs essential for this response have been identified. Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and Wnt3a and RA synergize strongly to activate Cdx1. Cdx1 positively regulates its own expression. These data prompt a model whereby retinoid and Wnt signaling function directly and synergistically to initiate Cdx1 expression in the caudal embryo. Expression is then maintained, at least in part, by an autoregulatory mechanism at later stages (Prinos, 2001).
Exogenous retinoic acid (RA) can evoke vertebral homeosis when administered during late gastrulation. These vertebral transformations correlate with alterations of the rostral limit of Hox gene expression in the prevertebrae, suggesting that retinoid signaling regulates the combinatorial expression of Hox genes dictating vertebral identity. Conversely, loss of certain RA receptors (RARs) results in anterior homeotic transformations principally affecting the cervical region. Despite these observations, the relationship between retinoid signaling, somitic Hox expression, and vertebral patterning is poorly understood. Cdx1 homozygous null mutants exhibit anterior homeotic transformations, some of which are reminiscent of those in RARgamma null offspring. In Cdx1 mutants, these transformations occur concomitant with posteriorized prevertebral expression of certain Hox genes. Cdx1 is a direct RA target, suggesting an indirect means by which retinoid signaling may impact vertebral patterning. To further investigate this relationship, a complete allelic series of Cdx1-RARgamma mutants was generated and the skeletal phenotype assessed either following normal gestation or after administration of RA. Synergistic interactions between these null alleles were observed in compound mutants, and the full effects of exogenous RA on vertebral morphogenesis requires Cdx1. These findings are consistent with a role for RA upstream of Cdx1 as regards axial patterning. However, exogenous RA attenuates several defects inherent to Cdx1 null mutants. This finding, together with the increased phenotypic severity of RARgamma-Cdx1 double null mutants relative to single nulls, suggests that these pathways also function in parallel, likely by converging on common targets (Allan, 2001).
Initiation of Hox genes requires interactions between numerous factors and
signaling pathways in order to establish their precise domain boundaries in
the developing nervous system. There are distinct differences in the
expression and regulation of members of the Hox gene family within a complex, suggesting
that multiple competing mechanisms are used to initiate Hox gene expression
domains in early embryogenesis. In this study, by analyzing the response of
HoxB genes to both RA and FGF signaling in neural tissue during early
chick embryogenesis (HH stages 7-15), two distinct groups of
Hox genes have been defined based on their reciprocal sensitivity to RA or FGF during this developmental period. The sharp reciprocal transition from RA to FGF responsiveness in moving from the 3' (Hoxb1 to Hoxb5) to the 5' (Hoxb6-Hoxb9) Hox genes is surprising. In mouse the 3' Hox genes do not respond
uniformly to RA treatment, since there is a progressive temporal shift in their competence or ability to respond to RA during gastrulation, such that
successively more 5' genes respond in later time windows. Hence, it had been suggested that the most posterior 5' Hox genes might also be progressively sensitive to RA in later stages at the end of or after gastrulation. The expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and these same genes are refractory to RA treatment at these stages (Bel-Vialar, 2002).
Hox gene products are key players in establishing positional identity along the anteroposterior (AP) axis. In vertebrates, gain or loss of Hox expression along the AP axis often leads to inappropriate morphogenesis, typically manifesting as homeotic transformations that affect the vertebrae and/or hindbrain. Various signalling pathways are known to impact on Hox expression, including the retinoid signalling pathway. Exogenous retinoic acid (RA), disruption of enzymes involved in maintaining normal embryonic RA distribution or mutation of the retinoid receptors (RARs and RXRs) can all impact on Hox expression with concomitant effects on AP patterning. Several Hox loci have well characterized RA response elements (RAREs), which have been shown to regulate functionally relevant Hox expression in the neurectoderm. A similar crucial function for any RARE in mesodermal Hox expression has, however, not been documented. The means by which RA regulates mesodermal Hox expression could therefore be either through an undocumented direct mechanism or through an intermediary; these mechanisms are not necessarily exclusive. In this regard, it has been found that Cdx1 may serve as such an intermediary. Cdx1 encodes a homeobox transcription factor that is crucial for normal somitic expression of several Hox genes, and is regulated by retinoid signalling in vivo and in vitro likely through an atypical RARE in the proximal promoter. In order to more fully understand the relationship between retinoid signalling, Cdx1 expression and AP patterning, mice have been derived in which the RARE has been functionally inactivated. These RARE-null mutants exhibit reduced expression of Cdx1 at all stages examined, vertebral homeotic transformations and altered Hox gene expression which correlates with certain of the defects seen in Cdx1-null offspring. These findings are consistent with a pivotal role for retinoid signalling in governing a subset of expression of Cdx1 crucial for normal vertebral patterning (Houle, 2003).
Anteroposterior (AP) patterning of the developing neural tube is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. The interactions between FGF and retinoic signaling pathways have been studied. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and
HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, it was found that posterior expression of FGFR1 and FGFR4 is dependent on the expression of RARalpha2. Anterior expression is also altered with FGFR1 expression being lost, whereas FGFR4 expression is expanded beyond its normal expression domain. RARalpha2 is required for the
expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. It is concluded that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP
neural patterning depends on a series of mutually interactive feedback loops
among FGFs, RARs and HOX genes (Shiotsugu, 2004).
During mammalian development, the Cdx1 homeobox gene
exhibits an early period of expression when the embryonic
body axis is established, and a later period where
expression is restricted to the embryonic intestinal
endoderm. Cdx1 expression is maintained throughout
adulthood in the proliferative cell compartment of the
continuously renewed intestinal epithelium, the crypts. In
this study, evidence in vitro and in vivo is provided that
Cdx1 is a direct transcriptional target of the Wnt/beta-catenin
signaling pathway. Upon Wnt stimulation, expression of
Cdx1 can be induced in mouse embryonic stem (ES) cells
as well as in undifferentiated rat embryonic endoderm.
Tcf4-deficient mouse embryos show abrogation of Cdx1
protein in the small intestinal epithelium, making Tcf4
the likely candidate to transduce Wnt signal in this part
of gut. The promoter region of the Cdx1 gene contains
several Tcf-binding motifs, and these bind Tcf/Lef1/beta-catenin
complexes and mediate beta-catenin-dependent
transactivation. The transcriptional regulation of the
homeobox gene Cdx1 in the intestinal epithelium by Wnt/beta-catenin
signaling underlines the importance of this
signaling pathway in mammalian endoderm development (Lickert, 2000).
Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. The nature of Fgf response element (FRE) has been investigated in a posterior neural gene, Xcad3 (Xenopus caudal homolog), which plays a crucial role of posterior neural development. Evidence suggests that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s) (Haremaki, 2003).
The reporter constructs containing the FREs exhibit high dose dependence on Fgf similar to that shown for endogenous Xcad3, when examined in the embryonic cell culture assay. Sequence and mutagenesis analyses reveal that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs (EBMs and TLBMs respectively) that lie in juxtaposition. The EBM
is known to serve as the binding site for Ets family transcription factors
that are nuclear effectors of the Fgf/Ras/Mapk pathway.
Indeed, functional and physical analyses show that Ets proteins are involved
in the Fgf response of Xcad3 as transcriptional activators, and that
Xcad3 is directly targeted by the Fgf signaling pathway. This
conclusion is consistent with the previous observation that Fgf can induce
Xcad3 expression in the animal cap assay within 2 hours of its
addition and even in the presence of the protein synthesis inhibitor
cycloheximide, which indicates that Xcad3 is an immediate early
target of Fgf signaling (Haremaki, 2003 and references therein).
TLBMs could serve as the binding sites for Tcf/Lef family transcription
factors that are nuclear effectors of the Wnt/ß-catenin pathway. It was anticipated that XTcf3 would functioned as a co-activator of Ets proteins, since Wnt signaling has been suggested as being involved in activation of posterior neural genes. Surprisingly, however, functional analysis reveals that
XTcf3 acts as a repressor of Xcad3. The data suggest that the
endogenous pool of ß-catenin in ectoderm cells is considerably smaller
compared with that of XTcf3 co-repressors such as XCtBP and Groucho. This in
turn implies that Wnt signaling could activate Xcad3 expression in
embryonic cells, when they are provided with a larger pool of ß-catenin.
Marginal zone cells of the early gastrula embryo, where Xcad3 is
initially expressed, are among such candidate cells, since a relatively large
amount of ß-catenin is translocated into the nucleus in these cells.
Recently, a mutant function of Tcf3 as a repressor has revealed in the
zebrafish headless mutant that carries a mutation in Tcf3. In this mutant, expression of midbrain-hindbrain boundary genes such as En2 and Pax2 is de-repressed in more anterior neural region, leading to severe head
defects. It would be interesting to know whether similar anterior expansion is seen in Cdx gene expression in this mutant (Haremaki, 2003 and references therein).
Sox2 is de-repressed by Bmp antagonists in the neurogenic region of
ectoderm during neural induction. Sox2, which shares a cognate DNA
bindings motif with Tcf/Lef family members, is required as a co-activator for
the Fgf response of Xcad3. Sox2 is likely to compete with XTcf3 for
TLBMs in the composite FREs to cooperate with Ets proteins that bind to
adjacent EBMs. Physical analysis supports this idea. Both Sox and Ets family
transcription factors interact with specific partner factors to direct signals to target genes, but direct partnership between them has not been reported. Collectively, these results indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on the FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox proteins and negatively acting Tcf protein (Haremaki, 2003).
In the mouse, Cdx1 is essential for normal anteroposterior vertebral
patterning through regulation of a subset of Hox genes. Retinoic acid (RA) and
certain Wnts have also been implicated in vertebral patterning, although the
relationship between these signaling pathways and the regulation of mesodermal
Hox gene expression is not fully understood. Prior work has shown that
Cdx1 is a direct target of both Wnt and retinoid signaling pathways,
and might therefore act to relay these signals to the Hox genes. Wnt and RA
are believed to impact on Cdx1 through an atypical RA-response
element (RARE) and Lef/Tcf-response elements (LRE), respectively, in the
proximal promoter. To address the roles of these regulatory motifs and
pathways, mice mutated for the LRE or the LRE plus the RARE were produced. In
contrast to RARE-null mutants, which exhibit limited vertebral defects,
LRE-null and LRE+RARE-null mutants exhibited vertebral malformations affecting
the entire cervical region that closely phenocopied the malformations seen in
Cdx1-null mutants. Mutation of the LRE also greatly reduced induction
of Cdx1 by RA, demonstrating a requirement for Wnt signaling in the
regulation of this gene by retinoids. LRE and LRE+RARE mutants also exhibited
vertebral fusions, suggesting a defect in somitogenesis. As Wnt signaling is
implicated in somitogenesis upstream of the Notch pathway, it is conceivable
that Cdx1 might play a role in this process. However, none of the Notch
pathway genes assessed was overtly affected (Pilon, 2007).
HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, CDX2, and CDX4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. CDX4 is expressed normally in early hematopoietic progenitors and is expressed aberrantly in ~25% of acute myeloid leukemia (AML) patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system and dysregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture, and generate a partially penetrant, long-latency AML in bone marrow transplant recipients. Coexpression of the Hox cofactor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure-function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains but not the putative Pbx cofactor interaction motif. Together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia (Bansal, 2006).
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