Key Points An international panel established the first ever diagnostic criteria for iMCD based on review of 244 clinical cases and 88 tissue samples. The criteria require multicentric lymphadenopathy with defined histopathology, ≥2 clinical/laboratory changes, and exclusion of iMCD mimics.

Animal somatic cell DNA is characterized by a bimodal pattern of methylation: tissue-specific genes are methylated in most cell types whereas housekeeping genes have 5' CpG islands which are constitutively unmethylated. Because methyl moieties derived from the gametes are erased in the morula and early blastula, this profile must be re-established in every generation; this is apparently accomplished by a wave of non-CpG island de novo methylation that occurs at implantation. Using transfection into embryonic stem cells and transgenic mice as a model system, we now show that Sp1 elements play a key role in protecting a CpG island in the adenine phosphoribosyltransferase (APRT) gene from de novo methylation. This recognition mechanism represents a critical step in embryogenesis, as it is responsible for setting up the correct genome methylation pattern which, in turn, is involved in regulating basal gene expression in the organism.

The Ipl (Tssc3) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the Igf2 gene. Expression of Ipl is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele. Ipl encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (Ipl neo and Ipl loxP ) and another line deleted for the similar but nonimprinted gene Tih1. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the Ipl neo allele and was decreased slightly in nulls with the Ipl loxP allele. When bred into an Igf2 mutant background, the Ipl deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of Tih1. These results show a nonredundant function for Ipl in restraining placental growth. The data further indicate that Ipl can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size. The Ipl gene, also known as Tssc3, lies on distal chromosome 7 of the mouse and human chromosome 11p15.5 (1, 2). This region contains multiple imprinted genes clustered in 1 Mb of DNA. Two of these, p57 Kip2 (Cdkn1c) and Igf2, control fetal and placental growth in mice (3-7) and humans (8). Similar to these genes, Ipl is highly expressed in the extraembryonic tissues (1), but in contrast to these genes, Ipl is expressed only weakly in the embryo proper (1, 9). Ipl encodes a cytoplasmic protein with a pleckstrin-homology (PH) domain (9), thus by analogy with other PH-domain proteins it may modulate cell signaling, intracellular trafficking, or other processes that depend on phosphatidylinositol lipid second messengers. Ipl has two close relatives: TDAG51 and Tih1. Of these genes, Tih1 is most similar to Ipl (9). Tih1 is located in a nonimprinted region of mouse chromosome 1, and it is expressed biallelically (9). To determine the function of Ipl and to accumulate data relevant to the biological rationale for imprinting, we created mice with germ-line deletions of Ipl and Tih1. Materials and MethodsGene Deletions, Genotyping, and Gene Expression. To make the Ipl neo targeting vector, a 5-kb 5Ј genomic NotI restriction fragment and a 6-kb 3Ј EcoRI͞XbaI fragment flanking Ipl were ligated upstream and downstream of Pgk-Neo in pPNT1, yielding pPNT-Ipl. To make the Ipl loxP targeting vector, the Pgk-Neo cassette of pPNT-⌬Ipl was replaced by that from pLNL, which includes flanking loxP sites. To verify homologous recombination, we used genomic PCR products upstream and downstream of the 5Ј and 3Ј cassettes. Recombination of the loxP sites was induced by crossing to HSP-Cre transgenic mice (10). The Ipl deletions (encompassing nucleotides 77,444-78,990 of GenBank a...

Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is caused by donor T cells that mediate host tissue injury through multiple inflammatory mechanisms. Blockade of individual effector molecules has limited efficacy in controlling GVHD. Here, we report that Notch signaling is a potent regulator of T-cell activation, differentiation, and function during acute GVHD. Inhibition of canonical Notch signaling in donor T cells markedly reduced GVHD severity and mortality in mouse models of allogeneic HSCT. Although Notch-deprived T cells proliferated and expanded in response to alloantigens in vivo, their ability to produce interleukin-2 and inflammatory cytokines was defective, and both CD4+ and CD8+ T cells failed to up-regulate selected effector molecules. Notch inhibition decreased the accumulation of alloreactive T cells in the intestine, a key GVHD target organ. However, Notch-deprived alloreactive CD4+ T cells retained significant cytotoxic potential and antileukemic activity, leading to improved overall survival of the recipients. These results identify Notch as a novel essential regulator of pathogenic CD4+ T-cell responses during acute GVHD and suggest that Notch signaling in T cells should be investigated as a therapeutic target after allogeneic HSCT.

The maternally expressed/paternally silenced genes Phlda2 (a.k.a. Ipl/Tssc3), Slc22a1l, Cdkn1c, Kcnq1, and Ascl2 are clustered in an imprinted domain on mouse chromosome 7. Paternal deletion of a cis-acting differentially methylated DNA element, Kvdmr1, causes coordinate loss of imprinting and over-expression of all of these genes and the resulting conceptuses show intrauterine growth restriction (IUGR). To test the specific contribution of Phlda2 to IUGR in the Kvdmr1-knockout, we crossed Kvdmr1(+/-) males with Phlda2(+/-) females. Conceptuses with the (Phlda2(+/+); Kvdmr1(+/-)) genotype showed fetal and placental growth retardation. Restoration of Phlda2 dosage to normal, as occurred in the conceptuses with the (Phlda2(-/+); Kvdmr1(+/-)) genotype, had a marginally positive effect on fetal weights and no effect on post-natal weights, but significantly rescued the placental weights. As we previously reported, loss of Phlda2 expression in the wild-type background (Phlda2(-/+); Kvdmr1(+/+) genotype) caused placentomegaly. Thus Phlda2 acts as a true rheostat for placental growth, with overgrowth after gene deletion and growth retardation after loss of imprinting. Consistent with this conclusion, we observed significant placental stunting in BAC-transgenic mice that over-expressed Phlda2 and one flanking gene, Slc22a1l, but did not over-express Cdkn1c.

We searched for novel imprinted genes in a region of human chromosome 11p15.5, which contains several known imprinted genes. Here we describe the cloning and characterization of the IPL ( I mprinted in P lacenta and L iver) gene, which shows tissue-specific expression and functional imprinting, with the maternal allele active and the paternal allele relatively inactive, in many human and mouse tissues. Human IPL is highly expressed in placenta and shows low but detectable expression in fetal and adult liver and lung. Mouse Ipl maps to the region of chromosome 7 which is syntenic with human 11p15.5 and this gene is expressed in placenta and at higher levels in extraembryonic membranes (yolk sac), fetal liver and adult kidney. Mouse and human IPL show sequence similarity to TDAG51 , a gene which was shown to be essential for Fas expression and susceptibility to apoptosis in a T lymphocyte cell line. Like several other imprinted genes, mouse and human IPL genes are small and contain small introns. These data expand the repertoire of known imprinted genes and will be helpful in testing the mechanism of genomic imprinting and the role of imprinted genes in growth regulation.

SUMMARYIn vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-l transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra,