Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

Changes in S1P1 and S1P2 expression during embryonal development and primitive endoderm differentiation of F9 cells

Sphingosine 1-phosphate (S1P) is a ligand for S1P family receptors (S1P(1)-S1P(5)). Of these receptors, S1P(1), S1P(2), and S1P(3) are ubiquitously expressed in adult mice, while S1P(4) and S1P(5) are tissue specific. However, little is known of their expression during embryonal development. We performed Northern blot analyses in mouse embryonal tissue and found that such expression is developmentally regulated. We also examined the expression of these receptors during primitive endoderm (PrE) differentiation of mouse F9 embryonal carcinoma (EC) cells, a well-known in vitro endoderm differentiation system. S1P(2) mRNA was abundantly expressed in F9 EC cells, but little S1P(1) and no S1P(3), S1P(4), or S1P(5) mRNA was detectable. However, S1P(1) mRNA expression was induced during EC-to-PrE differentiation. Studies using small interference RNA of S1P(1) indicated that increased S1P(1) expression is required for PrE differentiation. Thus, S1P(1) may play an important function in PrE differentiation that is not substituted for by S1P(2).     

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible.We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells.Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.     

Genomics of TGF-β1 signaling in stem cell commitment and dendritic cell development

Dendritic cells are professional antigen presenting cells and central for establishing and maintaining immunity and immunological tolerance. They develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Dendritic cell development and function are regulated by specific cytokines, including transforming growth factor type β1 (TGF-β1). Our previous work demonstrated the importance of TGF-β1 signaling for dendritic cell development and subset specification. Here, we used genome-wide gene expression profiling with DNA microarrays to investigate the activity of TGF-β1 on gene expression in dendritic cell development. This study identified specific gene categories induced by TGF-β1 with an impact on dendritic cell biology.