During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7⁺ cells with satellite cell characteristics, including the ability to form dystrophin⁺ fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

It is well known that cell fate decisions in the mouse organizer region during gastrulation ultimately govern gut formation and patterning, left–right axis determination, and development of the central nervous system. Previous studies suggest that signaling pathways activated by Nodal, bone morphogenetic protein (BMP), and Wnt ligands coordinately regulate patterning of the streak and the formation of midline organizing tissues, but the specific contributions of these molecules within discrete cell lineages are poorly defined. Here we removedSmad2activity in the epiblast, using a conditional inactivation strategy. Abrogation ofSmad2does not compromise primitive streak (PS) formation or gastrulation movements, but rather results in a failure to correctly specify the anterior definitive endoderm (ADE) and prechordal plate (PCP) progenitors. To selectively lowerNodalactivity in the posterior epiblast, we generated a novel allele lacking the proximal epiblast enhancer (PEE) governingNodalexpression in the PS. As for conditional inactivation ofSmad2, germ-line deletion of the PEE selectively disrupts development of the anterior streak. In striking contrast, the node and its midline derivatives, the notochord and floor plate, develop normally in both categories of mutant embryos. Finally, we show that removal of one copy ofSmad3in the context of aSmad2-deficient epiblast results in a failure to specify all axial midline tissues. These findings conclusively demonstrate that gradedNodal/Smad2signals govern allocation of the axial mesendoderm precursors that selectively give rise to the ADE and PCP mesoderm.

Blimp1, a zinc-finger containing DNA-binding transcriptional repressor, functions as a master regulator of B cell terminal differentiation. Considerable evidence suggests that Blimp1 is required for the establishment of anteroposterior axis formation and the formation of head structures during early vertebrate development. In mouse embryos, Blimp1 is strongly expressed in axial mesendoderm, the tissue known to provide anterior patterning signals during gastrulation. Here, we describe for the first time the defects caused by loss of Blimp1 function in the mouse. Blimp1 deficient embryos die at mid-gestation, but surprisingly early axis formation, anterior patterning and neural crest formation proceed normally. Rather, loss of Blimp1 expression disrupts morphogenesis of the caudal branchial arches and leads to a failure to correctly elaborate the labyrinthine layer of the placenta. Blimp1 mutant embryos also show widespread blood leakage and tissue apoptosis, and, strikingly, Blimp1 homozygous mutants entirely lack PGCs. At the time of PGC allocation around 7.25 days post coitum, Blimp1 heterozygous embryos exhibit decreased numbers of PCGs. Thus Blimp1 probably acts to turn off the default pathway that allows epiblast cells to adopt a somatic cell fate, and shifts the transcriptional program so that they become exclusively allocated into the germ cell lineage.

The SAGA (Spt–Ada–Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.