N. Ray Dunn

The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse

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.

Teratoma formation by human embryonic stem cells: Evaluation of essential parameters for future safety studies

Transplantation of human embryonic stem cells (hESC) into immune-deficient mice leads to the formation of differentiated tumors comprising all three germ layers, resembling spontaneous human teratomas. Teratoma assays are considered the gold standard for demonstrating differentiation potential of pluripotent hESC and hold promise as a standard for assessing safety among hESC-derived cell populations intended for therapeutic applications. We tested the potency of teratoma formation in seven anatomical transplantation locations (kidney capsule, muscle, subcutaneous space, peritoneal cavity, testis, liver, epididymal fat pad) in SCID mice with and without addition of Matrigel, and found that intramuscular teratoma formation was the most experimentally convenient, reproducible, and quantifiable. In the same experimental setting, we compared undifferentiated hESC and differentiated populations enriched for either beating cardiomyocytes or definitive endoderm derivatives (insulin-secreting beta cells), and showed that all cell preparations rapidly formed teratomas with varying percentages of mesoderm, ectoderm, and endoderm. In limiting dilution experiments, we found that as little as two hESC colonies spiked into feeder fibroblasts produced a teratoma, while a more rigorous single-cell titration achieved a detection limit of 1/4000. In summary, we established core parameters essential for facilitating safety profiling of hESC-derived products for future therapeutic applications.

Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo

Genetic and biochemical data have identified Smad4 as a key intracellular effector of the transforming growth factor β (TGFβ superfamily of secreted ligands. In mouse, Smad4-null embryos do not gastrulate, a phenotype consistent with loss of other TGFβ-related signaling components. Chimeric analysis reveals a primary requirement for Smad4 in the extra-embryonic lineages; however, within the embryo proper, characterization of the specific roles of Smad4 during gastrulation and lineage specification remains limited. We have employed a Smad4 conditional allele to specifically inactivate the Smad4 gene in the early mouse epiblast. Loss of Smad4 in this tissue results in a profound failure to pattern derivatives of the anterior primitive streak, such as prechordal plate, node, notochord and definitive endoderm. In contrast to these focal defects, many well-characterized TGFβ- and Bmp-regulated processes involved in mesoderm formation and patterning are surprisingly unaffected. Mutant embryos form abundant extra-embryonic mesoderm, including allantois, a rudimentary heart and middle primitive streak derivatives such as somites and lateral plate mesoderm. Thus, loss of Smad4 in the epiblast results not in global developmental abnormalities but instead in restricted patterning defects. These results suggest that Smad4 potentiates a subset of TGFβ-related signals during early embryonic development, but is dispensable for others.

Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse

Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Rather, previous genetic analysis in the mouse has shown that bone morphogenetic protein 4 (Bmp4) is required for the formation of both PGCs and allantois. Bmp4 is expressed in both the trophoblast-derived extraembryonic ectoderm (ExE) and in the epiblast-derived extraembryonic mesoderm (ExM), in which the PGCs, allantois primordium, and angioblasts are first detected. We have shown that Bmp4 made in the ExE functions to induce precursors of PGCs and allantois in the adjacent epiblast, resulting in complete lack of both cell types in homozygous null mutants. However, the function of Bmp4 in the ExM is totally unknown. To address this question, we generated tetraploid (4N) chimeras by aggregating Bmp4 null ES cells with wild-type tetraploid embryos. In this combination, wild-type tetraploid cells contribute to the extraembryonic trophoblast and primitive endoderm lineages but are excluded from the epiblast and its derivatives, including the ExM. Our results clearly demonstrate that Bmp4 made in the ExM does not affect the establishment of either PGC or allantois lineages, but is required for PGC localization and survival and for the differentiation of the allantois. These findings suggest that Bmp4 expressed in epiblast-derived tissues plays vital roles in reproduction by regulating both the development of the germ line and the vascular connection between the embryo and the placenta.

Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo

TGFβ/activin/Nodal receptors activate both Smad2 and Smad3 intracellular effector proteins. The functional activities of these closely related molecules have been extensively studied in cell lines. We show both are expressed in the early mouse embryo from the blastocyst stage onwards and mediate Foxh1-dependent activation of the Nodal autoregulatory enhancer in vitro. Genetic manipulation of their expression ratios reveals that Smad3 contributes essential signals at early post-implantation stages. Thus, loss of Smad3 in the context of one wild-type copy of Smad2 results in impaired production of anterior axial mesendoderm, while selective removal of both Smad2 and Smad3 from the epiblast additionally disrupts specification of axial and paraxial mesodermal derivatives. Finally, we demonstrate that Smad2;Smad3 double homozygous mutants entirely lack mesoderm and fail to gastrulate. Collectively, these results demonstrate that dose-dependent Smad2 and Smad3 signals cooperatively mediate cell fate decisions in the early mouse embryo.

Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic β cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%–20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho‐SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage. STEM CELLS 2012; 30:631–642

p38MAPK Controls Expression of Multiple Cell Cycle Inhibitors and Islet Proliferation with Advancing Age

Aging is a complex organismal process that is controlled by genetic, environmental, and behavioral factors. Accumulating evidence supports a role for different cell cycle inhibitors in mammalian aging. Little is known, however, about the upstream signals that induce their expression. Here, we explore the role of p38MAPK by generating a dominant-negative allele (p38(AF)) in which activating phosphorylation sites Thr180 and Tyr182 are mutated. Heterozygous p38(AF) mice show a marked attenuation of p38-dependent signaling and age-induced expression of multiple cell cycle inhibitors in different organs, including pancreatic islets. As a result, aged p38(AF/+) mice show enhanced proliferation and regeneration of islets when compared to wild-type littermates. We further find an age-related reduction in expression of the p38-specific phosphatase Wip1. Wip1-deficient mice demonstrate decreased islet proliferation, while Wip1 overexpression rescues aging-related decline in proliferation and regenerative capacity. We propose that modulation of p38MAPK activity may provide new avenues for treating certain age-related degenerative diseases.

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFβ pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice

Modeling a pregnancy disorder Preeclampsia, a dangerous pregnancy disorder marked by high blood pressure, can lead to premature birth and be life-threatening to the mother and baby. Research leading to effective treatments has been hampered by a lack of informative animal models. Ho et al. identified ELABELA as a hormone produced by the placenta whose levels are lower in preeclampsia (see the Perspective by Wirka and Quertermous). ELABELA-deficient pregnant mice showed clinical signs of preeclampsia, including high blood pressure and elevated urine protein. A proportion of embryos lacking ELABELA displayed defective heart development, and full-term pups had low birth weights. Science, this issue p. 707; see also p. 643 ELABELA is a placental hormone that functions in preeclampsia and heart development during embryogenesis. Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

Repressed by a NuRD

Embryonic stem (ES) cells deficient in Mbd3, a component of the nucleosome remodelling complex (NuRD), exhibit LIF-independent self-renewal and a restricted potential to differentiate. As such, it is likely that NuRD is required for ES cell pluripotency, and represents a potential link between maintaining the undifferentiated state and the capacity to differentiate.