Abstract
We report here the identification and characterization of a novel paired-like homeobox-containing gene (Ehox). This gene, identified in embryonic stem (ES) cells, is differentially expressed during in vitro ES cell differentiation. We have assessed Ehox function using the ES cell in vitro differentiation system. This has involved molecular and biological analyses of the effects of sense or antisense Ehox expression (using episomal vectors) on ES cell differentiation. Analysis of antisense Ehox-expressing ES cells indicates that they are unable to express marker genes associated with hematopoietic, endothelial, or cardiac differentiation following removal of leukemia inhibitory factor. In contrast, overexpression of Ehox using the sense construct accelerated the appearance of these differentiation markers. ES cell self-renewal and differentiation assays reveal that inhibition of Ehox activity results in the maintenance of a stem cell phenotype in limiting concentrations of leukemia inhibitory factor and the almost complete impairment of the cardiomyocyte differentiation capacity of these cells. We therefore conclude that Ehox is a novel homeobox-containing gene that is essential for the earliest stages of murine ES cell differentiation.
Highlights
Murine embryonic stem (ES)1 cells are derived from the inner cell mass of the day 3.5 post coitus blastocyst and can be maintained in culture as a self-renewing totipotent population in the presence of the growth factor, leukemia inhibitory factor (LIF) (1, 2)
The full-length cDNA for JB46 was isolated from a day 5 embryoid bodies (EBs) full-length cDNA library and sequenced (Fig. 1a)
Genomic sequence data base searches with Ehox cDNA sequences has revealed a number of regions that are identical to sequences from the murine X chromosome, confirming this as the chromosomal location of Ehox
Summary
Cloning and Sequencing of Full-length Ehox—To derive full-length Ehox cDNA clones, a day 5 EB cDNA library was generated using the Zap-Express kit (Stratagene, Amsterdam, The Netherlands) and all procedures were performed according to the instructions of the manufacturer. Transfected E14/T cells were selected in 2 g/ml puromycin for 6 days, and clones were pooled and maintained under selection throughout subsequent culture. Northern Blotting—20 g of total RNA isolated from undifferentiated E14/T ES cells supertransfected with episomal plasmids to express Ehox sense, antisense, or empty vector was loaded per lane of a 2% formaldehyde, 1% agarose gel. Day each culture was co-transfected with the Ehox expression vectors used in ES cell supertransfections. Alkaline Phosphatase Assay—In initial experiments, 1 ϫ 104 transfected E14/T cells were plated in duplicate onto 35-mm gelatin-coated wells in standard ES cell medium containing 100, 10, 1, or 0 units/ml LIF and 2 g/ml puromycin. In Vitro Differentiation of Cardiomyocytes—EBs were generated in 10-l hanging drop cultures containing 300 cells in GMEM, 10% FCS, 100 units/ml LIF, and 2 g/ml puromycin for 2 days. EBs with beating cardiomyocytes were scored daily for the 10 days
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
