The maternal gene spn-4 encodes a predicted RRM protein required for mitotic spindle orientation and cell fate patterning in early C. elegans embryos.

C. elegans embryogenesis begins with a stereotyped sequence of asymmetric cell divisions that are largely responsible for establishing the nematode body plan. These early asymmetries are specified after fertilization by the widely conserved, cortically enriched PAR and PKC-3 proteins, which include three kinases and two PDZ domain proteins. During asymmetric cell divisions in the early embryo, centrosome pairs initially are positioned on transverse axes but then rotate to align with the anteroposterior embryonic axis. We show that rotation of the centrosomal/nuclear complex in an embryonic cell called P(1) requires a maternally expressed gene we name spn-4. The predicted SPN-4 protein contains a single RNA recognition motif (RRM), and belongs to a small subfamily of RRM proteins that includes one Drosophila and two human family members. Remarkably, in mutant embryos lacking spn-4 function the transversely oriented 'P(1)' mitotic spindle appears to re-specify the axis of cell polarity, and the division remains asymmetric. spn-4 also is required for other developmental processes, including the specification of mesendoderm, the restriction of mesectoderm fate to P(1) descendants, and germline quiescence during embryogenesis. We suggest that SPN-4 post-transcriptionally regulates the expression of multiple developmental regulators. Such SPN-4 targets might then act more specifically to generate a subset of the anterior-posterior asymmetries initially specified after fertilization by the more generally required PAR and PKC-3 proteins.

DNA replication defects delay cell division and disrupt cell polarity in early Caenorhabditis elegans embryos.

In early Caenorhabditis elegans embryos, asymmetric cell divisions produce descendants with asynchronous cell cycle times. To investigate the relationship between cell cycle regulation and pattern formation, we have identified a collection of embryonic-lethal mutants in which cell divisions are delayed and cell fate patterns are abnormal. In div (for division delayed) mutant embryos, embryonic cell divisions are delayed but remain asynchronous. Some div mutants produce well-differentiated cell types, but they frequently lack the endodermal and mesodermal cell fates normally specified by a transcriptional activator called SKN-1. We show that mislocalization of PIE-1, a negative regulator of SKN-1, prevents the specification of endoderm and mesoderm in div-1 mutant embryos. In addition to defects in the normally asymmetric distribution of PIE-1, div mutants also exhibit other losses of asymmetry during early embryonic cleavages. The daughters of normally asymmetric divisions are nearly equal in size, and cytoplasmic P-granules are not properly localized to germline precursors in div mutant embryos. Thus the proper timing of cell division appears to be important for multiple aspects of asymmetric cell division. One div gene, div-1, encodes the B subunit of the DNA polymerase alpha-primase complex. Reducing the function of other DNA replication genes also results in a delayed division phenotype and embryonic lethality. Thus the other div genes we have identified are likely to encode additional components of the DNA replication machinery in C. elegans.

cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis.

A maternally expressed Caenorhabditis elegans gene called cyk-1 is required for polar body extrusion during meiosis and for a late step in cytokinesis during embryonic mitosis. Other microfilament- and microtubule-dependent processes appear normal in cyk-1 mutant embryos, indicating that cyk-1 regulates a specific subset of cytoskeletal functions. Because cytokinesis initiates normally and cleavage furrows ingress extensively in cyk-1 mutant embryos, we propose that the wild-type cyk-1 gene is required for a late step in cytokinesis. Cleavage furrows regress after completion of mitosis in cyk-1 mutants, leaving multiple nuclei in a single cell. Positional cloning and sequence analysis of the cyk-1 gene reveal that it encodes an FH protein, a newly defined family of proteins that appear to interact with the cytoskeleton during cytokinesis and in the regulation of cell polarity. Consistent with cyk-1 function being required for a late step in embryonic cytokinesis, we show that the CYK-1 protein co-localizes with actin microfilaments as a ring at the leading edge of the cleavage furrow, but only after extensive furrow ingression. We discuss our findings in the context of other studies suggesting that FH genes in yeast and insects function early in cytokinesis to assemble a cleavage furrow.

Positive and negative selection invoke distinct signaling pathways.

During T cell development, interaction of the T cell receptor (TCR) with cognate ligands in the thymus may result in either maturation (positive selection) or death (negative selection). The intracellular pathways that control these opposed outcomes are not well characterized. We have generated mice expressing dominant-negative Ras (dnRas) and Mek-1 (dMek) transgenes simultaneously, either in otherwise normal animals, or in animals expressing a transgenic TCR, thereby permitting a comprehensive analysis of peptide-specific selection. In this system, thymocyte maturation beyond the CD4+8+ stage is blocked almost completely, whereas negative selection, assessed using an in vitro deletion protocol, is quantitatively intact. This suggests that activation of the mitogen-activated protein kinase (MAPK) cascade is necessary for positive selection, but irrelevant for negative selection. Generation of gamma/delta and of CD4-8- alpha/beta T cells proceeds normally despite blockade of the MAPK cascade. Hence, only cells that mature via conventional, TCR-mediated repertoire selection require activation of the MAPK pathway to complete their maturation.

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.