Genes directing flower development in Arabidopsis.

We describe the effects of four recessive homeotic mutations that specifically disrupt the development of flowers in Arabidopsis thaliana. Each of the recessive mutations affects the outcome of organ development, but not the location of organ primordia. Homeotic transformations observed are as follows. In agamous-1, stamens to petals; in apetala2-1, sepals to leaves and petals to staminoid petals; in apetala3-1, petals to sepals and stamens to carpels; in pistillata-1, petals to sepals. In addition, two of these mutations (ap2-1 and pi-1) result in loss of organs, and ag-1 causes the cells that would ordinarily form the gynoecium to differentiate as a flower. Two of the mutations are temperature-sensitive. Temperature shift experiments indicate that the wild-type AP2 gene product acts at the time of primordium initiation; the AP3 product is active later. It seems that the wild-type alleles of these four genes allow cells to determine their place in the developing flower and thus to differentiate appropriately. We propose that these genes may be involved in setting up or responding to concentric, overlapping fields within the flower primordium.     

B-1 B cell development

CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.     

Analysis of VH gene replacement events in a B cell lymphoma

We have analyzed a series of recombinational events at the IgH chain locus of the B cell lymphoma, NFS-5. Each of these recombinational events results in the replacement of the VH gene segment of the rearranged H chain gene (VhDJh) with that of an upstream germline gene segment. Replacements on the productive and nonproductive alleles have been observed. In each case, the recombination occurs in close proximity to a highly conserved heptameric sequence (5'TACTGTG3') which is located at the 3' end of the VH coding region. In the two examples of recombination on the productive allele that have been analyzed, the initial VHQ52 gene is replaced by different VH7183 genes. On the non-productive allele, sequential replacement events have been analyzed: the initial VHQ52 rearrangement is first replaced by a closely related VHQ52 gene, followed by a second replacement using a VHQ52 pseudogene. Southern blot analysis using VH probes indicates that these recombinations may be accompanied by the deletion of germline VH genes belonging to both the VHQ52 and VH7183 families, suggesting that these gene families are interspersed in the NFS/N mouse.     

The chemokine network in cancer--much more than directing cell movement

Cytokine and chemokine gradients are central to the directed movement of cells in both homeostatic and pathological processes. Most cancers have a complex chemokine network which can influence immune responses to the tumor, direct the extent and cellular composition of the leukocyte infiltrate and also play a role in angiogenesis. Tumor cells can also hijack the chemokine system and gain expression of certain chemokine receptors and respond to specific chemokine gradients. Chemokine receptor expression and activation on malignant cells may be central to the growth, survival and migration of cancer cells from the primary tumor. Chemokine receptors, both CC and CXC have been detected on malignant cells and the relevant ligands are sometimes expressed at the tumor site and at sites of tumor spread, suggesting a role for the chemokine family in malignant growth and metastasis.     

Redundancy in B cell developmental pathways: c-Cbl inactivation rescues early B cell development through a B cell linker protein-independent pathway

Deficiency in the adaptor protein B cell linker protein (BLNK) results in a substantial but incomplete block in B cell development, suggesting that alternative pathways exist for B lineage differentiation. Another adaptor protein, c-Cbl, plays a negative regulatory role in several BCR-signaling pathways. We therefore investigated the role of c-Cbl during B cell development and addressed the possibility that redundancies in pathways for B cell differentiation could be further revealed by eliminating negative effects mediated by c-Cbl. Strikingly, c-Cbl inactivation reversed a number of the critical defects in early B cell differentiation that are seen in BLNK-deficient mice. c-Cbl(-/-)BLNK(-/-) mice exhibited normalized down-regulation of pre-BCR and CD43, up-regulation of MHC class II, and augmented L chain rearrangement, resulting in a successful transition from pre-B cells to immature B cells. c-Cbl inactivation also reversed the potentially tumor-predisposing hyperproliferative response of BLNK(-/-) pre-B cells to IL-7. Pre-BCR cross-linking induced enhanced and prolonged tyrosine phosphorylation in c-Cbl(-/-)BLNK(-/-) pre-BCR(+) pre-B cells compared with c-Cbl(+/-)BLNK(-/-) cells, including elevated phosphorylation of Lyn, Syk, Btk, and phospholipase C-gamma2. Our studies suggest that some, but not all, pre-BCR-triggered developmental events can be mediated by BLNK-independent pathways that are negatively regulated by c-Cbl, and further suggest that different events during early B cell development require different strength or duration of pre-BCR signaling.     

B cell antigen receptor signaling and internalization are mutually exclusive events

Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.     

Role of beta-catenin in B cell development and function

beta-Catenin is a central mediator of Wnt signaling pathway, components of which have been implicated in B cell development and function. B cell progenitors and bone marrow stromal cells express Wnt ligands, Frizzled receptors and Wnt antagonists, suggesting fine tuned regulation of this pathway in B cell development. In particular, deletion of Frizzled 9 gene results in developmental defects at the pre-B stage of development and an accumulation of plasma cells. Furthermore, Wnt signals regulate B cell proliferation through lymphocyte enhancer-binding factor-1. However, it is not known whether Wnt signaling in B cell development is mediated by beta-catenin and whether beta-catenin plays a role in mature B cell function. In this report, we show that mice bearing B cell-specific deletion of beta-catenin have normal B cell development in bone marrow and periphery. A modest defect in plasma cell generation in vitro was documented, which correlated with a defective expression of IRF-4 and Blimp-1. However, B cell response to T-dependent and T-independent Ags in vivo was found to be normal. Thus, beta-catenin expression was found to be dispensable for normal B cell development and function.     

Genetic isolation of transport signals directing cell surface expression

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.     

Aberrant recombination events in B cell lines derived from a kappa-deficient human.

We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus.     

Finding their niche: chemokines directing cell migration in the thymus

T lymphocytes are generated throughout life, arising from bone marrow-derived progenitors that complete an essential developmental process in the thymus. Thymic T cell education leads to the generation of a self-restricted and largely self-tolerant peripheral T-cell pool and is facilitated by interactions with thymic stromal cells residing in distinct supportive niches. The signals governing thymocyte precursor migration into the thymus, directing thymocyte navigation through thymic microenvironments and mature T-cell egress into circulation were, until recently, largely unknown, but presumed to be mediated to a large extent by chemokine signalling. Recent studies have now uncovered various specific functions for members of the chemokine superfamily in the thymus. These studies have not only revealed distinct but also in some cases overlapping roles for several chemokine family members in various thymocyte migration events and have also shown that homing and positioning of other cells in the thymus, such as dendritic cells and natural killer T cells is also chemokine-dependent. Here, we discuss current understanding of the role of chemokines in the thymus and highlight key future avenues for investigation in this field.     

Late events in B cell activation. Expression of membrane alkaline phosphatase activity

Alkaline phosphatase (APase) has been previously described as a membrane marker correlating with B cell proliferation after stimulation by selected B cell mitogens. We have found, however, that the appearance of B cell membrane APase correlates more closely with differentiation than with proliferation. This conclusion has been drawn from the following observations: 1) APase activity appears well after peak B cell thymidine uptake, 2) mitogens which stimulate only B cell proliferation (Salmonella typhimurium mitogen) fail to induce expression of the enzyme, and 3) when proliferation of mitogen-activated B cells is inhibited, APase activity is not suppressed and may even be augmented. In addition to membrane expression, APase is also spontaneously shed into the surrounding milieu, perhaps as a result of endogenous phospholipase activity. By using a group of well-characterized inhibitors, the APase activity was shown to belong to class I (similar to the bone/liver/kidney class). Because APase always appears in differentiating but not proliferating cells, we would propose that the enzyme appearance is a late marker of B cell activation, associated with cell progression to differentiation and consequent IgM synthesis.     

The maize mutant polymitotic affects cell cycle events during microspore development

The maize (Zea mays L.) male-sterile mutant polymitotic (po) was analyzed utilizing in-vitro cell culture and immunocytochemistry methods to better understand the relationship between the mutant phenotype and cell cycle events during microspore development. Using a live meiocyte culture system, initiation and progression through abnormal post-meiotic cell cycles at the end of meiosis II was documented in the po mutant with a CCD camera and a computer image analysis system. Our results showed that premature chromosomal condensation precedes abnormal post-meiotic cell cycle progression in the po mutant at the end of meiosis II. Temporal analysis of the po mutant in-vitro revealed that unsynchronized post-meiotic divisions occurred immediately following the end of meiosis II and did not require interaction with the surrounding somatic tissue. Furthermore, the altered distribution of p34^cdc2 protein kinase from a nuclear to a cytoplasmic location was identified in tetrads during onset of the unsynchronized divisions in the po mutant. In contrast, a predominantly nuclear location of p34^cdc2 was observed during interphase of the wild-type tetrads at the same stage. Received: 25 March 1999 / Accepted: 6 May 1999     

Expression of adhesion structures during B cell development in man

We have used three-color flow cytometry to study the expression of adhesion structures during B cell development in man. The results indicate that the cell-surface molecule(s) recognized by 515, a mAb which defines a broadly expressed family of cell-surface glycoproteins that includes lymphocyte homing receptors, exhibit a clear bimodal distribution (515lo and 515hi); 515hi cells were found exclusively on more mature B cells which already expressed high levels of CD20. Earlier, less mature B cells, identified by their expression of CD10, were uniformly 515lo. In contrast, the CD11a/LFA-1 Ag was acquired gradually over the course of B cell development. B cells which expressed high levels of CD20 expressed three to six times as much CD11a/LFA-1 as cells which expressed CD10. Interestingly, expression of the 515hi phenotype was tightly correlated with that of Leu-8, a marker previously shown to define maturational and functions subsets of B cells. These data document the coordinated regulation of multiple cell surface structures during B cell ontogeny, and demonstrate that adhesion structures necessary for proper B cell function are precisely up-regulated during B cell differentiation in man.