KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation.

BACKGROUND: Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development. METHODS: KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers. RESULTS: The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression. CONCLUSIONS: From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

The soybean Eu3 gene encodes an Ni-binding protein necessary for urease activity.

Mutation in Eu3 eliminates activity of both soybean ureases, the embryo-specific (encoded by Eu1) and the tissue-ubiquitous (encoded by Eu4). eu3-e1 is a completely recessive null allele. Eu3-e3 is a semi-dominant specifying 0.1% wild-type urease activity in the homozygous state and 5-10% as a heterozygote (Meyer-Bothling et al. 1987). Antibodies to plant UreG, a homologue of the bacterial urease accessory protein, revealed a 32 kDa protein (p32) in embryos of the Eu3/Eu3 precursor genotype. p32 is identical to UreG by the criteria of size, antigenicity, and its ability to bind Ni2+, a trait expected from the deduced histidine-rich N-terminus of UreG. UreG was absent in eu3-e1/eu3-e1, and lack of UreG co-segregated with eu3-e1. Eu3-e3 specified a UreG transcript which coded valine in place of alanine at residue 142 (A142V) confirming thatEu3 encodes UreG, which is renamed Eu3. Eu3 (A142V) retained Ni-binding ability. Eu3 is directly involved in urease activation, since anti-Eu3 (UreG) antibodies inhibited the in vitro activation of urease. Eu1 (embryo urease) and Eu3 accumulated in parallel in the developing embryo. The presence of Eu1 was not necessary for the high embryonic level of Eu3. However, the presence of Eu3 appeared to be important for accumulation of Eu1, perhaps by stabilizing it by Ni insertion. At the level of sensitivity employed Eu3 was detected in crude extracts of embryos but not non-embryonic tissues which have 1/500th the embryo urease activity. Functional Eu3, however, is necessary for activation of the ubiquitous urease in non-embryonic tissues.

Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean.

Plant orthologs of the bacterial urease accessory genes ureD and ureF, which are required for the insertion of the nickel ion at the active site, have been isolated from soybean ( Glycine max L. Merr.), tomato ( Lycopersicon esculentum) and Arabidopsis thaliana. The functionality of soybean UreD and UreF was tested by measuring their ability to complement urease-negative mutants of Schizosaccharomyces pombe, a eukaryote which produces a "plant-like" urease of ~90 kDa. The S. pombe ure4 mutant was complemented by a 12-kb fragment of S. pombe genomic DNA, which was shown by PCR to contain a putative ureD gene. However, ure4 was not complemented by a UreD cDNA soybean, expressed under the control of a strong promoter. In contrast, an S. pombe ure3 mutation was complemented by both a 10-kb fragment of S. pombe DNA containing ureF and the UreF cDNA from soybean. Soybean Eu2 is a candidate urease accessory gene; its product cooperates with the Eu3 protein in activating apourease in vitro. However, the sequences of UreD and UreF transcripts from two eu2/eu2 mutants, recovered as RT-PCR products, revealed no mutational alteration, suggesting that Eu2 encodes neither UreD nor UreF.