The switch from membrane-bound to secreted-form IgM that occurs during differentiation of B lymphocytes has long been known to involve regulated processing of the heavy chain pre-mRNA. Here, we show that accumulation of one subunit of an essential polyadenylation factor (CstF-64) is specifically repressed in mouse primary B cells and that overexpression of CstF-64 is sufficient to switch heavy chain expression from membrane-bound (microm) to secreted form (micros). We further show that CstF-64 is limiting for formation of intact CstF, that CstF has a higher affinity for the microm poly(A) site than for the micros site, and that the microm site is stronger in a reconstituted in vitro processing reaction. Our results indicate that CstF-64 plays a key role in regulating IgM heavy chain expression during B cell differentiation.

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessels can lead to myriad diseases that affect one in four people worldwide. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessels has not yet been described. We report the existence of a secreted, splice variant of vascular endothelial growth factor receptor-2 (sVegfr-2) that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without accompanying changes in blood vasculature. sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival, and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 is a molecular uncoupler of blood and lymphatic vessels whose modulation might have a therapeutic role in lymphatic vascular malformations, transplantation, and potentially in tumor lymphangiogenesis and lymphedema.

The relative abundance of the mRNAs encoding the membrane (tLm) and secreted (p,u) forms of immunoglobulin ,u heavy chain is regulated during B-cell maturation by a change in the mode of RNA processing. Current models to explain this regulation involve either competition between cleavage-polyadenylation at the proximal (IL.) poly(A) site and cleavage-polyadenylation at the distal (i'm) poly(A) site [poly(A) we noted that in a wide variety of organisms, the sequence at the 5' splice junction of the C t4-to-M1 intron is significantly different from the consensus 5' splice junction sequence and is therefore suboptimal with respect to its complementary base pairing with Ul small nuclear RNA. When we mutated this suboptimal sequence into the consensus sequence, the ,u mRNA production in plasmacytoma cells was shifted from predominately p,s to exclusively p.m This result unequivocally demonstrated that splicing of the C,u4-to-Ml exon is in competition with usage of the , poly(A) site. A key feature of this regulatory phenomenon appears to be the appropriately balanced efficiencies of these two processing reactions. Consistent with predictions of the p,u site-splice model, B cells were found to contain .m precursor RNA that had undergone the C,u4-to-M1 splice but had not yet been polyadenylated at the pm site.The mRNAs encoding the membrane-bound (m) and secreted (s) forms of IgM heavy chain (p) are produced from a single primary transcript which is alternatively processed at its 3' end. If the primary transcript is cleaved and polyadenylated at the proximal (p,j) poly(A) site, R, mRNA is produced. If instead it undergoes splicing of the C,u4 and Ml exons, which removes the proximal poly(A) site, and is cleaved and polyadenylated at a distal (Rm) poly(A) site, it gives rise to Am mRNA. The relative abundance of these two mRNAs is regulated during B-cell maturation, the p.m mRNA being predominant in early stages and the Rs mRNA being heavily favored in mature plasma cells (2, 15).The differential expression of ,um and ps mRNAs could potentially be regulated at several levels, including (i) transcriptional termination, if polymerase terminates before reaching the p.m site, (ii) mRNA stability, if either of the mRNAs were preferentially destabilized in a stage-specific manner, and (iii) RNA processing, if any of the processing events were in direct competition with each other. Although transcriptional termination contributes to the P-s >>». phenotype in some plasmacytomas, it clearly does not do so in all cells exhibiting this phenotype (13,18,22,23,27,40,57), and therefore cannot be a primary determinant of the regulatory mechanism. The relative stability of Rs

The ␣-fetoprotein (AFP) and H19 genes are transcribed at high levels in the mammalian fetal liver but are rapidly repressed postnatally. This repression in the liver is controlled, at least in part, by the Afr1 gene. Afr1 was defined >25 years ago when BALB͞cJ mice were found to have 5-to 20-fold higher adult serum AFP levels compared with all other mouse strains; subsequent studies showed that this elevation was due to higher Afp expression in the liver. H19, which has become a model for genomic imprinting, was identified initially in a screen for Afr1-regulated genes. The BALB͞cJ allele (Afr1 b ) is recessive to the wild-type allele (Afr1 a ), consistent with the idea that Afr1 functions as a repressor. By high-resolution mapping, we identified a gene that maps to the Afr1 interval on chromosome 15 and encodes a putative zinc fingers and homeoboxes (ZHX) protein. In BALB͞cJ mice, this gene contains a murine endogenous retrovirus within its first intron and produces predominantly an aberrant transcript that no longer encodes a functional protein. Liver-specific overexpression of a Zhx2 transgene restores wild-type H19 repression on a BALB͞cJ background, confirming that this gene is responsible for hereditary persistence of Afp and H19 in the livers of BALB͞cJ mice. Thus we have identified a genetically defined transcription factor that is involved in developmental gene silencing in mammals. We present a model to explain the liver-specific phenotype in BALB͞cJ mice, even though Afr1 is a ubiquitously expressed gene.development ͉ genetics ͉ positional cloning