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Retinoic acid (RA) plays a critical role in normal development, growth, and maintenance of certain tissues. The action of RA is thought to be mediated in part by the three nuclear receptors (RARa, -13, and -y), each of which is expressed as multiple isoforms. To investigate the function ofthe

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, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation 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 we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hoxindependent and -dependent pathways, respectively. DEVELOPMENT 4100 system to derive a conditional null allele. In agreement with previous studies (Chawengsaksophak et al., 2004), loss of Cdx2 in the postimplantation embryo resulted in axial truncation posterior to the forelimb. This axial truncation, together with the nature of many of the genes impacted by Cdx2 loss, suggested that precocious cessation of the generation of presomitic mesoderm (PSM) is the primary basis for this phenotype. To further define the role of Cdx2 and to elucidate the mechanistic basis for this phenotype, we sought to determine whether any of the affected genes were direct Cdx2 targets. Chromatin immunoprecipitation (ChIP) demonstrated occupancy of the Wnt3a, Cyp26a1 and T promoters by Cdx2 in vivo. Moreover, all of these promoters harbor functional Cdx response elements (CDREs) as determined by transfection analysis or transgenic assays. Thus, Cdx2 directly regulates the expression of multiple players essential for the development of the posterior embryo. Taken together with previous work, these findings suggest that Cdx2 is required to couple the generation of paraxial mesoderm through multiple Hox-independent mechanisms with Hoxdependent AP vertebral patterning. MATERIALS AND METHODS Gene targeting and the generation of Cdx2 -/-mutantsA 5 kb fragment of genomic Cdx2 sequence encompassing the first intron through to the 3Ј UTR was subcloned into pBluescript II KS + . A floxed thymidine kinase/neomycin resistance cassette (loxPGK-TK-Neolox) (Iulianella and Lohnes, 2002) was cloned into the BglII site in intron 1, and a single loxP site was inserted into the NruI site in intron 2, generating the targeting vector (see Fig. S1A in the supplementary material). R1 embryonic stem cells were electroporated with...

Cdx1 encodes a mammalian homeobox gene involved in vertebral patterning. Retinoic acid (RA) is likewise implicated in vertebral patterning. We have previously shown that 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, we confirm prior results which demonstrated that Cdx1 can be directly regulated by Wnt signaling, and identify functional LEF/TCF response motifs essential for this response. We also find that Cdx1 expression is markedly attenuated in a stage- and tissue-specific fashion in the Wnt3a hypomorph vestigial tail, and present data demonstrating that Wnt3a and RA synergize strongly to activate Cdx1. Finally, we show that 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.

Cdx1, Cdx2 and Cdx4 encode homeodomain transcription factors that are involved in vertebral anterior-posterior (AP) patterning. Cdx1 and Cdx2 are also expressed in the intestinal epithelium during development, suggesting a role in this tissue. Intestinal defects have not been reported in Cdx1 null mutants, while Cdx2 null mutants die at embryonic day 3.5 (E3.5), thus precluding assessment of the null phenotype at later stages. To circumvent this latter shortcoming, we have used a conditional Cre-lox strategy to inactivate Cdx2 in the intestinal epithelium. Using this approach, we found that ablation of Cdx2 at E13.5 led to a transformation of the small intestine to a pyloric stomach-like identity, although the molecular nature of the underlying mesenchyme remained unchanged. Further analysis of Cdx1-Cdx2 double mutants suggests that Cdx1 does not play a critical role in the development of the small intestine, at least after E13.5.

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Clonality is, at present, the only means by which the self-renewal potential of a given stem cell can be determined. To assess the clonality of human embryonic stem cells (hESC), a protocol involving seeding wells at low cell densities is commonly used to surmount poor cloning efficiencies. However, factors influencing the accuracy of such an assay have not been fully elucidated. Using clonogenic assays together with time-lapse microscopy, numerical analyses, and regulated gene expression strategies, we found that individual and collective cell movements are inherent properties of hESCs and that they markedly impact the accuracy of clonogenic assays. Analyses of cell motility using mean-square displacement and paired migration correlation indicated that cell movements become more straight-line or ballistic and less random-walk as separation distance decreases. Such motility-induced reaggregation (rather than a true clone) occurs approximately 70% of the time if the distance between two hESCs is <6.4 mum, and is not observed if the distance is >150 mum. Furthermore, newly formed small hESC colonies have a predisposition toward the formation of larger colonies through asymmetric colony expansion and movement, which would not accurately reflect self-renewal and proliferative activity of a true hESC clone. Notably, inhibition of Rho-associated kinase markedly upregulated hESC migration and reaggregation, producing considerable numbers of false-positive colonies. Conversely, E-cadherin upregulation significantly augmented hESC clonogenicity via improved survival of single hESCs without influencing cell motility. This work reveals that individual cell movement, asymmetric colony expansion, Rho-associated kinase, and E-cadherin all work together to influence hESC clonogenicity, and provides additional guidance for improvement of clonogenic assays in the analysis of hESC self-renewal.

The vertebrate Cdx genes (Cdx1 Cdx2 and Cdx4 in the mouse) encode homeodomain transcription factors related to the Drosophila caudal gene. The vertebrate Cdx gene products have been implicated in the development of the posterior embryo. In particular, loss- and gain-of-function experiments suggest that Cdx members are direct regulators of Hox genes and likely impart posterior information, in part, through this mechanism. Several signaling molecules, notably retinoic acid (RA*) and members of the Wnt (wingless) and fibroblast growth factor (FGF) families, are also implicated in patterning of the posterior vertebrate embryo. Interestingly, recent work indicates that members of the Cdx family are targets of Wnt, RA and FGF signaling, suggesting that Cdx factors act to convey the activity of these signaling molecules to Hox genes. This article will briefly review Cdx expression and function, with particular emphasis on vertebrate model systems.