Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^

The role of Lmx1b in the control of dorsoventral compartmentalization in the distal mesenchyme of the developing mouse limb

Lmx1b encodes a LIM-homeodomain transcription factor required for dorso-ventral (D-V) patterning in the mesenchyme of the vertebrate limb. In the absence of Lmx1b function, limbs exhibit a bi-ventral pattern indicating that Lmx1b is required for cells to adopt a dorsal cell fate. However, how Lmx1b specifies dorsal cell fates in the mesenchyme of the distal limb is unknown. Lmx1b is initially expressed throughout the dorsal and ventral limb bud mesenchyme, then becomes dorsally restricted around E10.5. At this stage, there is a sharp boundary between Lmx1b expressing and Lmx1b non-expressing cells. How the dorso-ventral Lmx1b expression boundary is formed and maintained is currently unknown. One mechanism that may contribute to establishing and/or maintaining the Lmx1b expression boundary is if the dorsal mesenchyme is a lineage-based compartment, where different groups of non-mingling cells are separated. Compartment formation has been proposed to rely on compartment-specific selector gene activity which functions to restrict cells to a compartment and specifies the identity of cells within that compartment. Based on the evidence that the dorsal limb identity relies on the expression of Lmx1b in the dorsal half of the limb mesenchyme, we hypothesized that Lmx1b might function as a dorsal limb bud mesenchyme selector gene to set up a dorsal compartment. To test this hypothesis, we developed an inducible CreERT2/ loxP based fate mapping approach that permanently marks Lmx1b wild-type and mutant cells and examined the distribution of their descendents in the developing limb. Our data is the first to show that dorso-ventral lineage compartments exist in the limb bud mesenchyme. Furthermore, Lmx1b is required for maintenance of the dorso-ventral compartment lineage boundary. The behavior of Lmx1b mutant cells that cross into the ventral mesenchyme, as well as previous chimera analysis in which mutant cells spread evenly in the ventral limb and form patches in the dorsal side, suggest that cell affinity differences prevent intermingling of dorsal and ventral cells. ^