Caudal-related (Cdx) genes are transcription factors that function as master homeobox regulators of HOX genes. Three Cdx genes (Cdx1, Cdx2 and Cdx4) have been identified in mammals, and function in part as mediators anterior-posterior patterning during development. However, accumulating data suggests a central role for Cdx genes in vertebrate hematopoiesis. This link was first established in the zebrafish model whereby Cdx4-deficient fish exhibited severe anaemia within the first day of development and fail to specify blood progenitors (Davidson et al, Nature 2003). Given the high degree of conservation of Cdx genes between species, we anticipated that the loss of Cdx4 in the mouse would result in a phenotype similar to the lethal bloodless phenotype observed in zebrafish. This initial hypothesis was supported by reports that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells or embryoid bodies (McKinney-Freeman et al, Blood 2008). To establish the function of the Cdx4 gene in mammalian hematopoiesis, we analyzed the hematopoietic system during development and in the adult hematopoietic compartment, using mice in which Cdx4 was either deleted in the germline, or conditionally deleted in the adult hematopoietic system using Mx-Cre mediated excision. Germline deletion resulted in fertile and viable animals born with expected Mendelian rations. Surprisingly, there were no apparent gross phenotypic manifestations of Cdx4 deletion in either context despite complete excision of the Cdx4 gene in the hematopoietic compartment. Peripheral blood counts were normal, as was flow cytometric analysis for the myeloid and lymphoid compartments as assessed using Mac1/Gr1 and various B- & T-cell markers. Using multi-parameter flow analysis and methylcellulose plating assays, we observed that loss of Cdx4 in both mouse models only caused subtle hematopoietic perturbations, such as an increase in thymus weight and a modest decrease in pre-B colony formation ability in vitro. However, there were no other apparent defects in the hematopoietic stem and progenitor compartments including GMP, CMP & MEP. There were also no marked differences in the expression of Hox genes in the hematopoietic compartment, even though striking dysregulation of various Hox genes was observed in Cdx4-deficient zebrafish model. We also assessed the effect of Cdx4 deletion on the development of leukemia mediated by MLL fusion genes. Using in vivo retroviral transduction/bone marrow transplantations and in vitro serial-replating assays, we observed that Cdx4 null mice remain susceptible to leukemic transformation by MLL-AF9, indicating that Cdx4 is not necessary for MLL-AF9 mediated leukemias. The only subtle differences observed were that Cdx4−/− progenitors transduced with MLLAF9 generated more differentiated transformed blast colony-forming units in vitro, and induced leukemias with longer disease latencies compared to their wild-type littermate controls. Thus, in contrast with the zebrafish system, we found that Cdx4 is dispensable for normal mammalian hematopoiesis, as well as for MLL-AF9 mediated leukemia. The striking differences we observed between the studies of the same gene in the two different vertebrate systems emphasizes the importance of not solely relying on a single model system to assess the role of transcription factors in development, and that the deficiency of even a highly conserved gene might perturb function to variable extents in different animal models.

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