The C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/β-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the misspecification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis.

The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35(-) embryonic arrest phenotype was variable. We report here that the NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and that CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. Our data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, our results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm.

Abstract1α,25(OH) 2 -vitamin D 3 (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca 2+ channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca 2+ concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca 2+ -dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca 2+ -dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.