FunGenES: UED-LF2: HOXB4 can enhance the differentiation of embryonic stem cells by modulating the haematopoietic niche

ABSTRACT: Haematopoietic differentiation of embryonic stem (ES) cells in vitro has been used as a model to study early haematopoietic development and it is well documented that haematopoietic differentiation can be enhanced by over-expression of HOXB4. HOXB4 is expressed in haematopoietic progenitor cells (HPC) where it promotes self-renewal, but it is also expressed in the primitive streak of the gastrulating embryo. This led us to hypothesise that HOXB4 might modulate gene expression in pre- haematopoietic mesoderm and that this property might contribute to its pro-haematopoietic effect in differentiating ES cells. To test our hypothesis we developed a conditionally activated HOXB4 expression system using the mutant oestrogen receptor (ERT2) and showed that a pulse of HOXB4 prior to HPC emergence in differentiating ES cells led to an increase in haematopoietic differentiation. Expression profiling revealed an increase in the expression of genes associated with paraxial mesoderm that gives rise to the haematopoietic niche. Cell mixing experiments suggest that HOXB4 activation can indeed have a paracrine, as well as a cell autonomous, effect on haematopoietic differentiation. We provide evidence that this may, in part, be mediated by modulation of the Wnt pathway via the HOXB4 target gene, Frzb. Constructs: HOXB4-ERT2 constructs were generated that utilised the CAG promoter to drive expression of the Hoxb4ERT2 fusion cDNA followed by an IRES element and the puromycin resistance gene (pCAG Hoxb4ERT2IP); CGR8.5 cells bearing Hoxb4VP16 fusion construct. Fusion of HOXB4-ERT2 with the transactivation domain of the Herpes simplex VP16 (Brickman et al. 2000) (Zamparini et al. 2006) was generated by PCR as were 2 mutants: Hoxb4 (N266P)VP16/Pcs2+ and Hoxb4 (d266)VP16/Pcs2+ which had mutations in an asparagine (residue 266) in the third helix of the homeodomain which functions in DNA recognition (Hanes and Brent 1989).

ORGANISM(S): Mus musculus

SUBMITTER: Oliver Hummel 

PROVIDER: E-MTAB-547 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress