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Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ layers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The peptide spanning residues 48-61 of hen egg white lysozyme (HEL) presented by I-A(k) gives rise to two T cell populations, referred to as type A and B, that distinguish the complex generated intracellularly upon processing of HEL from that formed with exogenous peptide. Here, we ascribe this difference to recognition of distinct conformers of the complex and show that formation of the two complexes results from antigen processing in different intracellular compartments and is dependent upon H2-DM. While the type A complex preferentially formed in a lysosome-like late vesicle, the type B complex failed to form in this compartment; this distinction was abolished in antigen-presenting cells lacking DM. Experiments in vitro indicated that H2-DM acts directly on the complex to eliminate the type B conformation. We conclude that different antigen-processing pathways generate distinct MHC-peptide conformers, priming T cells with distinct specificity that may play unique roles in immunity.

Smad proteins transmit TGFbeta signals from the cell surface to the nucleus. Here we analyze Smad2 mutant embryos created using ES cell technology. Smad2 function is not required for mesoderm production per se, but, rather unexpectedly, in the absence of Smad2 the entire epiblast adopts a mesodermal fate giving rise to a normal yolk sac and fetal blood cells. In contrast, Smad2 mutants entirely lack tissues of the embryonic germ layers. Smad2 signals serve to restrict the site of primitive streak formation and establish anterior-posterior identity within the epiblast. Chimera experiments demonstrate these essential activities are contributed by the extraembryonic tissues. Thus, the extraembryonic tissues play critical roles in establishing the body plan during early mouse development.

We used a "hit and run" gene targeting strategy to generate mice expressing only the p31 isoform of the conserved invariant (Ii) chain associated with major histocompatibility complex (MHC) class II molecules. Spleen cells from these mice appear indistinguishable from wild type with respect to class II subunit assembly, transport, peptide acquisition, surface expression, and the ability to present intact protein antigens. Moreover, these mutant mice have normal numbers of thymic and peripheral CD4+ T cells, and intact CD4+ T-dependent proliferative responses towards a soluble antigen. In short, MHC class II expression and function are surprisingly unaffected in mice lacking p41 invariant chain, implying that the p31 and p41 isoforms may be functionally redundant in the intact animal.

The conserved invariant chain associates with highly polymorphic alpha and beta subunits guiding class II transport through the secretory pathway. Early associations of these three polypeptides inside antigen-presenting cells are poorly understood. The present experiments provide a detailed picture of the structure and fate of class II alpha and beta subunits in invariant chain mutants possessing different MHC haplotypes. In the absence of invariant chain, A alpha bA beta b is predominantly expressed as free A alpha b and A beta b chains by both splenocytes and activated LPS/IL-4 blasts, confirming that A alpha bA beta b assembly is strongly dependent on invariant chain coexpression. A quite different situation exists with respect to other allelic products. In the absence of invariant chain, A alpha kA beta k, E alpha kE beta k, and A alpha dA beta d molecules assemble efficiently and are conformationally similar to mature wild-type heterodimers. The contribution of invariant chain to subunit assembly thus differs for allelic variants, suggesting that sequential associations of alpha, beta, and invariant chain may be affected by polymorphic differences.