BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.     

Efficient Cloning of cDNAs of Retinoic Acid-Responsive Genes in P19 Embryonal Carcinoma Cells and Characterization of a Novel Mouse Gene, Stra1 (Mouse LERK-2/Eplg2)

Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stral, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.     

Antigen challenge inhibits thymic emigration

T cell development in the thymus involves a series of TCR-mediated control points including TCR-beta selection and positive and negative selection. Approximately half of the thymic sojourn is spent in the medulla, where thymocytes undergo final maturation before emigrating to the periphery. Although it is acknowledged that thymic emigration is an active process, relatively little is known about how this is regulated, why it takes so long, and whether TCR-mediated signaling can influence this step. Using wild-type and TCR transgenic mice, we found that Ag injected i.v. or intrathymically led to a striking reduction in the number of recent thymic emigrants (RTE) in the periphery. This was caused by inhibition of T cell export rather than peripheral deletion, because a cohort of RTE that was already released before in vivo Ag challenge was not depleted, and similar results were observed in Bim-deficient mice, which have impaired T cell deletion. Within the thymus, the loss of RTE was associated with retention of medullary thymocytes rather than increased negative selection. In addition to Ag-specific inhibition of export, some TCR-independent suppression of emigration was also observed that appeared to be partly the result of the inflammatory cytokine TNF. Thus, in addition to its accepted role in intrathymic selection events, TCR signaling can also play an important role in the regulation of thymic emigration.     

Age-related changes in litter inputs explain annual trends in soil CO2 effluxes over a full Eucalyptus rotation after afforestation of a tropical savannah

Land use changes such as savannah afforestation with eucalypts impact the soil carbon (C) balance, therefore affecting soil CO₂ efflux (F _s ), a major flux in the global C cycle. We tested the hypothesis that F _s increases with stand age after afforestation, due to an increasing input of fresh organic matter to the forest floor. In a Eucalyptus plantation established on coastal savannahs in Congo, bimonthly measurements of F _s were carried out for 1 year on three adjacent stands aged 0.9, 4.4 and 13.7 years and presenting similar growth patterns. Litterfall and litter accumulation on the forest floor were quantified over a chronosequence. Equations were derived to estimate the contribution of litter decomposition to F _s throughout the rotation. Litterfall increased with stand age after savannah afforestation. F _s , that was strongly correlated on a seasonal basis with soil water content (SWC) in all stands, decreased between ages 0.9 year and 4.4 years due to savannah residue depletion, and increased between ages 4.4 years and 13.7 years, mainly because of an increasing amount of decomposing eucalypt litter. The aboveground litter layer therefore appeared as a major source of CO₂, whose contribution to F _s in old stands was estimated to be about four times higher than that of the eucalypt-derived soil organic C pool. The high litter contribution to F _s in older stands might explain why 13.7 years-old stand F _s was limited by moisture all year round whereas SWC did not limit F _s for large parts of the year in the youngest stands.     

Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2^−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2^−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2^−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2^−/−Bim^+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2^+/−Bim^+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2^−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.     

Degenerative disorders caused by Bcl-2 deficiency prevented by loss of its BH3-only antagonist Bim.

Apoptosis is triggered when proapoptotic members of the Bcl-2 protein family bearing only the BH3 association domain bind to Bcl-2 or its homologs and block their antiapoptotic activity. To test whether loss of the BH3-only protein Bim could prevent the cellular attrition caused by Bcl-2 deficiency, we generated mice lacking both genes. Mice without Bcl-2 have a fragile lymphoid system, become runted, turn gray, and succumb to polycystic kidney disease. Concomitant absence of Bim prevented all these disorders. Indeed, loss of even one bim allele restored normal kidney development, growth, and health. These results demonstrate that Bim levels set the threshold for initiation of apoptosis in several tissues and suggest that degenerative diseases might be alleviated by blocking BH3-only proteins.     

Dynamics of Nutrient Translocation in Stemwood across an Age Series of a Eucalyptus Hybrid

Despite the continuous nature of growth of eucalyptus hybridsin Congo, taper functions fitted to stem profiles of one clonethroughout stand development, combined with annual tree measurements,made it possible to locate accurately the position of annualrings in stems. Annual rings were identified on discs of woodsampled every 4 m in four trees cut from 1-, 2-, 3-, 4-, 5-,6- and 7-year-old stands. Chemical analysis, performed individuallyfor each ring per level and per tree sampled, made it possibleto quantify the changes in nutrient content of the rings duringstand development. Nutrient translocation in stemwood was thuscalculated in a stepwise manner between trees of two successiveages. The cumulated nutrient translocations in stemwood fromthe 1-year-old stage to the 6-year-old stage amounted to 18·5kg ha^-1N, 4·2 kg ha^-1P, 38·8 kg ha^-1K, 1·5kg ha^-1Ca and 3·2 kg ha^-1Mg. They represented 11, 18,121, 6, and 15% of the amounts of N, P, K, Ca and Mg accumulatedin stemwood, respectively, at the 7-year-old stage (loggingage). Negative translocations of N, P, Ca and Mg in stemwoodbetween 6 and 7 years might indicate an improvement in the nutritivestatus of the stand at the end of the rotation. Much translocationof K in stemwood suggests that this process might be involvedin the high use efficiency of this element. Copyright 2001 Annalsof Botany Company Translocation, stemwood, ring, nutrient, Eucalyptus, age series