Transgenic major histocompatibility complex class I antigen expressed in mouse trophoblast affects maternal immature B cells.

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.

Maternal B lymphocytes specific for paternal histocompatibility antigens are partially deleted during pregnancy.

Although genetically different from its mother, a mammalian fetus bearing paternal alloantigens is normally not rejected. To investigate one of the many possible mechanisms involved in this important biologic phenomenon, we analyzed the consequences of fetal alloantigen recognition on maternal B lymphocytes. We used transgenic mice expressing a unique B cell receptor with a relatively high affinity for the MHC class I molecule H-2Kk on most B lymphocytes. We provide the first evidence for an alloantigen-specific B cell deletion in the spleens and bone marrow of transgenic mothers bearing H-2Kk-positive fetuses. This highly reproducible deletion affects < or =80% of Id-bearing B cells, starts at midpregnancy, and is only observed until term. Such a specific maternal B cell deletion could contribute to the success of the fetal allograft.

Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on microtubule dynamics.

To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Identification of a new exon of the brain microtubule-associated protein 2.

The 5'-region of the transcripts encoding the HMW-(MAP2b) and LMW-(MAP2c) microtubule associated proteins in the brain was amplified by RT-PCR. The sequencing of cloned PCR fragments allowed to identify a new variant of brain HMW-MAP2 which contained, compared to MAP2b, an insertion of 246 bp located downstream the 5'-junction between MAP2b and MAP2c and that does not alter the open reading frame of MAP2b. The number of amino acid residues encoded by this insertion increases the molecular weight by 8.5 kDa i.e. corresponds to the difference in apparent size between MAP2a and MAP2b. A LMW-MAP2 PCR amplification product containing this insertion has been also identified. Genomic Southern blot analysis confirmed that this region belongs to the MAP2 gene and is located on a single exon.

Tau and microtubule-associated protein 2c transfection and neurite outgrowth in ND 7/23 cells.

Neuronal hybrid ND 7/23 cells, which display sensorylike properties, develop neurites when cultured in the presence of either dibutyryl cyclic AMP plus nerve growth factor (DBcAMP + NGF) or retinoic acid or a phorbol ester derivative, although they express only trace amounts of the microtubule-associated tau proteins and low levels of microtubule-associated protein 2c (MAP2c). Nondifferentiated ND cells transfected with tau cDNAs did not develop neurites, whereas very short cell processes were formed in MAP2c-transfected cells. tau and MAP2 antibodies labeled microtubule bundles displayed in a ring array underneath the surface of the transfected cells and short microtubules starting from the cell center. After differentiation in the presence of DBcAMP + NGF, the same bundle organization was observed in the transfected cells. In addition, tau and MAP2 antibodies stained a short section of the formed neurites. These data demonstrate that the expression of tau protein is not sufficient to induce neurite extension and that other proteins induced by morphogens are more important to initiate morphological differentiation of this cell line.

Two novel HMW MAP2 variants with four microtubule-binding repeats and different projection domains.

The brain microtubule-associated protein MAP2 is composed of two high molecular (MAP2a and b) and one low molecular (MAP2c) weight isoforms. All these forms were thought to contain three repeated microtubule-binding domains in their C-terminal region but a MAP2c variant containing four repeats has recently been identified. We report here the existence of two high molecular weight MAP2 isoforms with four microtubule-binding domains in the sensory neuronal cell line ND 7/23. A stretch of 135 bp is missing in one of these forms suggesting that several HMW MAP2 variants can be produced by alternative splicing.

Microtubule-associated proteins 1A and LC2. Two proteins encoded in one messenger RNA.

The deduced amino acid sequence for the filamentous microtubule-associated protein (MAP) 1A, thought to be involved in stabilizing the mature neuronal cytoskeleton, has been determined from a series of overlapping cDNA clones. Though previously described as biochemically and immunologically distinct from MAP1B, we now demonstrate that MAP1A is structurally related to MAP1B, a protein associated with neurite outgrowth and process plasticity. The two MAPs exhibit regional amino acid sequence similarities spanning their potential microtubule binding domains placing both into a new MAP family. The cDNA sequence encoding MAP1A was also found to encode one of its associated light chains (LC) called LC2. Both proteins are found on a single mRNA in the same open reading frame and are translated as a pre-MAP1A/LC2-protein. The topological relationship between MAP1A and LC2 coding sequences is, therefore, identical to that previously shown for MAP1B and LC1 (Hammarback, J. A., Obar, R. A., Hughes, S. M., and Vallee, R. B. (1991) Neuron 7, 129-139). Based on these and earlier results, we conclude that LC1 and LC2 are structurally related polypeptides generated from distinct MAP polyprotein precursors but free to exchange between the two MAPs.