Differential Hox expression in murine embryonic stem cell models of normal and malignant hematopoiesis.

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFRß. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFRß) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis.

Patterns of Wnt/Fzd/LRP gene expression during embryonic hematopoiesis.

Wnt signaling plays several roles in hematopoiesis, promoting hemopoietic stem cell (HSC) self-renewal, providing proliferative signals for immature progenitors and regulating lineage commitment. To ascertain which Wnt proteins and receptors are important during hematopoietic development, we used two systems; in vitro hematopoietic differentiation of embryonic stem (ES) cells and tissues isolated from sites specific for hematopoiesis during mouse embryogenesis. Initially genes involved in hematopoiesis were profiled and indicate differentiating ES cells undergo a wave of primitive hematopoiesis (Day 3.75) similar to the mouse yolk sac, followed by a wave of more definitive hematopoiesis (Day 7.75) comparable to the aorta-gonad-mesonephros (AGM) and E15.5 liver with lineage commitment by Day 15. A similar biphasic expression pattern occurred for Wnt/Fzd/LRP genes with Wnt 3, 5a, 8a, Fzd4, and LRP5 becoming upregulated during primitive hematopoiesis, followed by Wnt3a, 6, 7b, 10b, and 16 during more definitive hematopoiesis. High expression of Wnt5a, Fzd4, and LRP5 during the first phase of hematopoiesis suggests these genes are involved in early hematopoietic regulation. Wnt3a and 16 were also expressed at specific stages, with Wnt16 detected when the earliest lymphoid progenitors are formed (AGM and 2 degrees BC of ES differentiation). Wnt3a expression corresponded with the induction of definitive hematopoiesis a period, which involves rapid expansion of HSC (Day 7.75 of ES differentiation, AGM and E15.5 liver). Supplementation with Wnt3a during ES hematopoietic differentiation increased proliferation and appeared to promote stem cell expansion. Overall this study provides valuable information on the Wnt/Fzd/LRP involved in supporting embryonic hematopoiesis.

Tel/PDGFRbeta induces stem cell differentiation via the Ras/ERK and STAT5 signaling pathways.

OBJECTIVE: Fusion genes involving the platelet-derived growth factor receptor-beta (PDGFRbeta) are found in a subgroup of myeloproliferative neoplasms, with one such fusion, Tel/PDGFRbeta found in a subset of chronic myelomonocytic leukemia patients. Tel/PDGFRbeta results in constitutive activation of several signaling pathways and induces a myeloproliferative disease in mice, with signals via tyrosines 579/581 identified as being important for this phenotype. In this study, we have used a tetracycline-regulated system to express wild-type and the mutated F2 Tel/PDGFRbeta to identify the key signaling pathways, which drive Tel/PDGFRbeta-induced differentiation of embryonic stem (ES) cells. MATERIALS AND METHODS: The leukemic oncogene Tel/PDGFRbeta and Tel/PDGFRbeta-F2 were inducibly expressed in ES cells and their effects on self-renewal, signal transduction, and gene expression patterns analyzed. RESULTS: Tel/PDGFRbeta activated several major signal transduction pathways (signal transducers and activators of transcription [STAT] 3, STAT5, mitogen-activated protein kinases, phosphatidylinositol-3 kinase) in ES cells, but only specific inhibition of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) or STAT5 pathways was able to significantly prevent Tel/PDGFRbeta-induced differentiation and restore ES-cell self-renewal. Inhibiting the tyrosine kinase activity of the oncogene using Gleevec or PDGFRbeta inhibitor III also substantially prevented Tel/PDGFRbeta-induced differentiation and its ability to upregulate key genes involved in myelopoiesis. Tyrosines 579/581 played a critical role in mediating signals via the Ras/ERK and STAT5 pathways, with dual targeting of the tyrosine kinase activity of Tel/PDGFRbeta and the MEK/ERK pathway completely preventing Tel/PDGFRbeta-induced differentiation. CONCLUSION: These findings suggest that targeted disruption of key signaling pathways in combination with the tyrosine kinase activity of leukemic oncogenes, such as Tel/PDGFRbeta, may result in more efficacious therapies for suppressing leukemic progression in the clinical setting.