caudal


EVOLUTIONARY HOMOLOGS (part 2/2)

Mammalian Caudal homologs

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).

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).

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).

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).

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).

It was believed that Cdk2-cyclin E complexes are essential to drive cells through the G1-S phase transition. However, it was discovered recently that the mitotic kinase Cdk1 (Cdc2a) compensates for the loss of Cdk2. The present study tested whether Cdk2 can compensate for the loss of Cdk1. A knockin mouse was generated in which the Cdk2 cDNA was knocked into the Cdk1 locus (Cdk1Cdk2KI). Substitution of both copies of Cdk1 by Cdk2 led to early embryonic lethality, even though Cdk2 was expressed from the Cdk1 locus. In addition, Cdk2-/- Cdk1+/Cdk2KI mice were generated in which one copy of Cdk2 and one copy of Cdk1 were expressed from the Cdk1 locus and the Cdk2 gene was deleted from the endogenous Cdk2 locus. It was found that both male and female Cdk2-/- Cdk1+/Cdk2KI mice were sterile, similar to Cdk2-/- mice, even though they expressed the Cdk2 protein from the Cdk1 locus in testes. The translocational and cell cycle properties of knockin Cdk2 in Cdk2-/- Cdk1+/Cdk2KI cells were comparable to those of endogenous Cdk2, but premature transcriptional activation of Cdk1 was detected during liver regeneration in the absence of Cdk2. This study provides evidence of the molecular differences between Cdk2 and Cdk1 and highlights that the timing of transcriptional activation and the genetic locus play important roles in determining the function of Cdk proteins in vivo (Satyanarayana, 2008).

Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo

The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP (see Drosophila Caudal) expression as a readout of Hippo signaling activity, this study assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. TE specification and commitment was found to coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling (Posfai, 2017).

Cdx1 and Cdx2 and intestinal development

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).

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).

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).

Conditional ablation of the homeobox transcription factor Cdx2 from early endoderm results in the replacement of the posterior intestinal epithelium with keratinocytes, a dramatic cell fate conversion caused by ectopic activation of the foregut/esophageal differentiation program. This anterior homeotic transformation of the intestine was first apparent in the early embryonic Cdx2-deficient gut by a caudal extension of the expression domains of several key foregut endoderm regulators. While the intestinal transcriptome was severely affected, Cdx2 deficiency only transiently modified selected posterior Hox genes and the primary enteric Hox code was maintained. Further, Cdx2-directed intestinal cell fate adoption plays an important role in the establishment of normal epithelial-mesenchymal interactions, as multiple signaling pathways involved in this process were severely affected. It is concluded that Cdx2 controls important aspects of intestinal identity and development, and that this function is largely independent of the enteric Hox code (Gao, 2009).

Cdx1, whose gut expression pattern resembles that of Cdx2, has the capability to drive intestinal differentiation in a gain-of-function setting. Redundancy between all three Cdx proteins has been reported in a number of nonendoderm tissues. Therefore, it was surprising to see the near-complete homeotic transformation of the Cdx2-deficient intestine, as some compensation was anticipated. It has been established that Cdx1 activation is directly dependent on Cdx2. This transcriptional hierarchy between the two Cdx genes reflects their sequential expression pattern in the gut endoderm, where Cdx2 precedes Cdx1 by a few days. In fact, the expression of Cdx1 starts only when villus morphogenesis and epithelial maturation begin. The data provide further evidence for the evolutionary significance of the 'Parahox' cluster, where Cdx2, but not Cdx1 or Cdx4, is located. Thus, Cdx1 is controlled by the more ancient caudal ortholog Cdx2 in gut endoderm to facilitate the developmental and anatomical complexity of the organ (Gao, 2009).

Similar to Cdx1, Isx is another intestine-specific transcription factor whose expression initiates during epithelial differentiation, consistent with its dependency on Cdx2. In addition, the maintenance of HNF1α and HNF4α expression in the embryonic intestine is directly controlled by Cdx2. Single-gene ablation of Cdx1, Isx, Hnf1α, or Hnf4α in mice had no effect on the establishment of the intestinal epithelium, and Cdx1, Isx, HNF1α and HNF4α regulate the expression of numerous intestinal genes. The data support the notion that Cdx2 functions upstream of a group of pro-intestinal transcription factors, with which it synergizes to promote intestinal cell fate (Gao, 2009).

In contrast to significant understanding of signaling cascades that determine cell polarity in lower eukaryotic or immortalized cells, little is known about the transcriptional program that governs mammalian epithelial polarization in vivo. This study shows, using conditional gene ablation and three-dimensional tissue culture, that the homeobox transcription factor Cdx2 controls apical-basolateral polarity in mouse enterocytes and human colonic epithelial cells. Cdx2 regulates a comprehensive gene network involved in endo-lysosomal maturation and protein transport. In the absence of Cdx2, defective protein trafficking impairs apical-basal transport and induces ectopic lumen formation. These defects are partially recapitulated by suppression of key apical transport components, Rab11a and Kif3b, which are regulated by Cdx2. Furthermore, Cdx2 deficiency affects components that control the organization of microvillus actin cytoskeleton, leading to severe microvillus atrophy. These results demonstrate that Cdx2 regulates epithelial cell polarity and morphogenesis through control of apical protein transport and endo-lysosomal function (Gao, 2010).

Interactions between Cdx genes and retinoic acid modulate early cardiogenesis

Cdx transcription factors regulate embryonic positional identities and have crucial roles in anteroposterior patterning (AP) processes of all three germ layers. The zebrafish homologues cdx1a and cdx4 redundantly regulate posterior mesodermal derivatives inducing embryonic blood cell fate specification and patterning of the embryonic kidney. It is hypothesized that cdx factors restrict formation of anterior mesodermal derivatives such as cardiac cells by imposing posterior identity to developing mesodermal cells. Ectopic expression of Cdx1 or Cdx4 applied during the brief window of mesoderm patterning in differentiating murine embryonic stem cell (ESC) strongly suppresses cardiac development as assayed by expression of cardiac genes and formation of embryoid bodies (EB) containing 'beating' cell clusters. Conversely, in loss-of-function studies performed in cdx-deficient zebrafish embryos, a dose-dependent expansion was observed of tbx5a+ anterior-lateral plate mesoderm giving rise to cardiac progenitors. However, further cardiac development of these mesodermal cells required additional suppression of the retinoic acid (RA) pathway, possibly due to differential activity of inhibitory RA signals in cdx mutants. Together, these data suggest that cdx proteins affect cardiogenesis by regulating the formation of cardiogenic mesoderm and together with the RA pathway control the early development of cardiac precursor cells (Lengerke, 2011).

Caudal homologs and hindbrain/spinal cord development

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).

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 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).

Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst

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).

Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation

Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. This event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development (Niwa, 2005).

Cdx2 acts downstream of cell polarization to cell-autonomously promote trophectoderm fate in the early mouse embryo

The first lineage decision during mouse development is the establishment of trophectoderm and inner cell mass lineages, morphologically distinguishable at the blastocyst stage. The Caudal-like transcription factor Cdx2 is required for repression of inner cell mass genes Oct4 and Nanog in the trophectoderm. Expression of Cdx2 in the trophectoderm is thus one of the earliest known events in lineage determination. However, it is not clear whether the Cdx2 expression pattern is the cause or the consequence of this first lineage decision. This study shows that Cdx2 is initially ubiquitously expressed, and becomes progressively upregulated in outside, future trophectoderm cells prior to blastocyst formation. Ubiquitous Cdx2 expression begins around the time of cell polarization, but cell polarization is independent of zygotic Cdx2. Finally, it was shown functionally that Cdx2 is downstream of lineage allocation since Cdx2 mutant cells, which show cell-autonomous defects in expression of Oct4, Nanog, and the trophectoderm marker Eomesodermin, do not preferentially contribute to inner cell mass in chimeric blastocysts. Cdx2 therefore appears to act downstream of the first lineage decision, suggesting that processes influencing lineage allocation or morphogenesis may regulate Cdx2 expression along the inside/outside axis of the embryo (Ralston, 2008).

Stochastic patterning in the mouse pre-implantation embryo

Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, critical events leading to the divergence of TE, EPI and PE lineages were identified. Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, a model is proposed in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development (Dietrich, 2007).

Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos

During pre-implantation mouse development, embryos form blastocysts with establishment of the first two cell lineages: the trophectoderm (TE) which gives rise to the placenta, and the inner cell mass (ICM) which will form the embryo proper. Differentiation of TE is regulated by the transcription factor Caudal-related homeobox 2 (Cdx2), but the mechanisms which act upstream of Cdx2 expression remain unknown. This study shows that the TEA domain family transcription factor, Tead4, is required for TE development. Tead1, Tead2 and Tead4 were expressed in pre-implantation embryos, and at least Tead1 and Tead4 were expressed widely in both TE and ICM lineages. Tead4-/- embryos died at pre-implantation stages without forming the blastocoel. The mutant embryos continued cell proliferation, and adherens junction and cell polarity were not significantly affected. In Tead4-/- embryos, Cdx2 was weakly expressed at the morula stage but was not expressed in later stages. None of the TE specific genes, including Eomes and a Cdx2 independent gene, Fgfr2, was detected in Tead4-/- embryos. Instead, the ICM specific transcription factors, Oct3/4 and Nanog, were expressed in all the blastomeres. Tead4-/- embryos also failed to differentiate trophoblast giant cells when they were cultured in vitro. ES cells with normal in vitro differentiation abilities were established from Tead4-/- embryos. These results suggest that Tead4 has a distinct role from Tead1 and Tead2 in trophectoderm specification of pre-implantation embryos, and that Tead4 is an early transcription factor required for specification and development of the trophectoderm lineage, which includes expression of Cdx2 (Nishioka, 2008).

Responsiveness of Caudal homologs to retinoic acid

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).

Cdx2 regulation of posterior development through non-Hox targets

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, a conditional Cdx2 floxed allele was developed in mice and temporal inactivation was effected at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and data is presented suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. A model is proposed wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively (Savory, 2009).

It is notable that caudalizers, including RA, canonical Wnt and Fgf, are involved in both the development of the posterior embryo and in vertebral patterning. The latter function could be mediated, at least in part, through direct regulation of expression of Cdx family members. Based on these observations, the present findings suggest a model whereby Cdx2 functions directly upstream of factors involved both in axis elongation and in AP patterning, and therefore integrates aspects of retinoid, Fgf and Wnt signaling involved in these processes. In this regard, this model is consistent with the previously described Wnt3a-Cdx feedback loop in Xenopus. In addition, Cdx2 has also been shown to govern endoderm patterning and specification of the colon through Hox-independent means. Finally, it is notable that, in Drosophila, cad is required for specification of the posterior embryo through regulation of expression of gap and pair-rule genes and is subsequently needed for gastrulation and hindgut patterning. Three other genes, fkh, byn and wg, which are related to murine HNF-3 (Foxm1 — Mouse Genome Informatics), T and Wnt, are also required for Drosophila hindgut gastrulation. The overlapping expression patterns and cross-regulation of cad, fkh, byn and wg, certain aspects of which are conserved in the vertebrate homologues Cdx, T, HNF-3 and Wnt, have led to the hypothesis that these genes constitute an evolutionarily conserved 'cassette' that functions during gastrulation. The finding of a central role for Cdx2 within this cassette, aspects of which appear to be reflected across diverse vertebrate species, emphasizes a conserved role for Cdx/cad in AP pattering and elaboration of the posterior embryo (Savory, 2009).

Cdx mediates neural tube closure through transcriptional regulation of the planar cell polarity gene Ptk7

The vertebrate Cdx genes (Cdx1, Cdx2 and Cdx4) encode homeodomain transcription factors with well-established roles in anteroposterior patterning. To circumvent the peri-implantation lethality inherent to Cdx2 loss of function, the Cre-loxP system has been used to ablate Cdx2 at post-implantation stages, and a crucial role for Cdx2 function was confirmed in events related to axial extension. As considerable data suggest that the Cdx family members functionally overlap, this analysis was extended to assess the consequence of concomitant loss of both Cdx1 and Cdx2. This study report that Cdx1-Cdx2 double mutants exhibit a severely truncated anteroposterior axis. In addition, these double mutants exhibit fused somites, a widened mediolateral axis and craniorachischisis, a severe form of neural tube defect in which early neurulation fails and the neural tube remains open. These defects are typically associated with deficits in planar cell polarity (PCP) signaling in vertebrates. Consistent with this, it was found that expression of Ptk7 (Protein tyrosine kinase 7), which encodes a gene involved in PCP (a homolog of Drosophila Off-track), is markedly reduced in Cdx1-Cdx2 double mutants, and is a candidate Cdx target. Genetic interaction between Cdx mutants and a mutant allele of Scrib, a gene involved in PCP signaling, is suggestive of a role for Cdx signaling in the PCP pathway. These findings illustrate a novel and pivotal role for Cdx function upstream of Ptk7 and neural tube closure in vertebrates (Savory, 2011).

Cdx and Hox genes differentially regulate posterior axial growth in mammalian embryos

Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. This study shows that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant phenotypes. Conversely, posterior (paralog group 13) Hox genes can prematurely arrest posterior axial growth when precociously expressed. These data suggest that the transition from trunk to tail Hox gene expression successively regulates the construction and termination of axial structures in the mouse embryo. Thus, Hox genes seem to differentially orchestrate posterior expansion of embryonic tissues during axial morphogenesis as an integral part of their function in specifying head-to-tail identity. In addition, evidence is presented that Cdx and Hox transcription factors exert these effects by controlling Wnt signaling. Concomitant regulation of Cyp26a1 expression, restraining retinoic acid signaling away from the posterior growth zone, may likewise play a role in timing the trunk-tail transition (Young, 2009).

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. The phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead to a proposal that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects (van de Ven, 2011).

Transcriptional regulation of caudal homologs

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).

Cdx4 dysregulates Hox gene expression and generates acute myeloid leukemia alone and in cooperation with Meis1a in a murine model

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

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