Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation.
The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.
Lako
M
M
Department of Biological Sciences, South Road, University of Durham, Durham DH1 3LE, UK. majlinda.lako@durham.ac.uk
Lindsay
S
S
Lincoln
J
J
Cairns
P M
PM
Armstrong
L
L
Hole
N
N
D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology*; D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000096:biosynthesis; D013997:Time Factors; D014162:Transfection; D014170:Transformation, Genetic; D014212:Tretinoin / Q000494:pharmacology; D015854:Up-Regulation; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D000098908:Brachyury Protein
D004121:Dimethyl Sulfoxide; D005326:Fetal Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D020825:T-Box Domain Proteins; D014212:Tretinoin; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D000098908:Brachyury Protein