SUMMARY Exomeres are a recently discovered type of extracellular nanoparticle with no known biological function. Herein, we describe a simple ultracentrifugation-based method for separation of exomeres from exosomes. Exomeres are enriched in Argonaute 1–3 and amyloid precursor protein. We identify distinct functions of exomeres mediated by two of their cargo, the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N-glycans, and the EGFR ligand, amphiregulin (AREG). Functional ST6Gal-I in exomeres can be transferred to cells, resulting in hypersialylation of recipient cell-surface proteins including β1-integrin. AREG-containing exomeres elicit prolonged EGFR and downstream signaling in recipient cells, modulate EGFR trafficking in normal intestinal organoids, and dramatically enhance the growth of colonic tumor organoids. This study provides a simplified method of exomere isolation and demonstrates that exomeres contain and can transfer functional cargo. These findings underscore the heterogeneity of nanoparticles and should accelerate advances in determining the composition and biological functions of exomeres.

In the mouse embryo, the splanchnic mesodermal cells of the anterior heart field (AHF) migrate from the pharynx to contribute to the early myocardium of the outflow tract (OT) and right ventricle (RV). Recent studies have attempted to distinguish the AHF from other precardiac populations, and to determine the genetic and molecular mechanisms that regulate its development. Here, we have used an Fgf8 lacZ allele to demonstrate that Fgf8 is expressed within the developing AHF. In addition, we use both a hypomorphic Fgf8 allele (Fgf8 neo ) and Cre-mediated gene ablation to show that Fgf8 is essential for the survival and proliferation of the AHF. Nkx2.5Cre is expressed in the AHF, primary heart tube and pharyngeal endoderm, while TnT-Cre is expressed only within the specified heart tube myocardium. Deletion of Fgf8 by Nkx2.5Cre results in a significant loss of the Nkx2.5 Cre lineage and severe OT and RV truncations by E9.5, while the remaining heart chambers (left ventricle and atria) are grossly normal. These defects result from significant decreases in cell proliferation and aberrant cell death in both the pharyngeal endoderm and splanchnic mesoderm. By contrast, ablation of Fgf8 in the TnT-Cre domain does not result in OT or RV defects, providing strong evidence that Fgf8 expression is crucial in the pharyngeal endoderm and/or overlying splanchnic mesoderm of the AHF at a stage prior to heart tube elongation. Analysis of downstream signaling components, such as phosphorylated-Erk and Pea3, identifies the AHF splanchnic mesoderm itself as a target for Fgf8 signaling.

Using single transcription factors to reprogram cells could produce important insights into the epigenetic mechanisms that direct normal differentiation, or counter inappropriate plasticity, or even provide new ways of manipulating normal ontogeny in vitro to control lineage diversification and differentiation. We enforced Pdx1 expression from the Neurogenin-3-expressing endocrine commitment point onward and found during the embryonic period a minor increased b-cell allocation with accompanying reduced a-cell numbers. More surprisingly, almost all remaining Pdx1-containing glucagon/Arx-producing cells underwent a fairly rapid conversion at postnatal stages, through glucagon-insulin double positivity, to a state indistinguishable from normal b cells, resulting in complete a-cell absence. This a-to-b conversion was not caused by activating Pdx1 in the later glucagon-expressing state. Our findings reveal that Pdx1 can work singlehandedly as a potent context-dependent autonomous reprogramming agent, and suggest a postnatal differentiation evaluation stage involved in normal endocrine maturation.Supplemental material is available for this article.Received April 27, 2011; revised version accepted July 5, 2011.A major hurdle for cell replacement-based diabetes therapy is the difficulty of supplying vast numbers of functioning insulin-producing b cells. One method could be through the reprogramming of alternative cell types. While this process might be easier with closely lineagerelated cells, even substantially different cells may be susceptible (e.g., Zhou et al. 2008).Recent studies reveal significant plasticity between pancreatic a and b cells under certain induced conditions, implying a potential route to b cells through a cells. In a near-total b-cell destruction and regeneration model in adult mice, a proportion of new b cells were produced from a cells via a bihormonal glucagon (Thorel et al. 2010). The interconversion presumably occurs in response to a combination of the physiological need to replenish b cells and regenerationinduced stress, raising questions as to the local or systemic signals triggered by such lesions. Direct superimposition of a pro-b-lineage condition was reported when Pax4 expression was forced in pancreatic or endocrine progenitors or in embryonic a cells to redirect endocrine differentiation or coax pre-existing a cells into b cells. The converted cells seemed similar to normal b cells and temporarily improved glycemia under induced diabetes, although the effect was superseded by uncontrolled a-cell neogenesis and fatality caused by extreme hyperglycemia (Collombat et al. 2009). These studies on the ability of a single lineage-allocating transcription factor to sustain complete cell fate conversion suggest that similar analyses for other transcription factors could be insightful. Determining which factors induce specific types of lineage reprogramming, as well as the repertoire of cellular competence states amenable to fate switching, could lead to pharmacological intervention to activat...

+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9 + and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9 + Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3 HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9 + Neurog3 TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9 + Neurog3 TA.LO endocrine-biased progenitors feed production of Neurog3 HI endocrine-committed cells during pancreas organogenesis.[Keywords: Neurog3; progenitor; endocrine-biased; mitotic] Supplemental material is available for this article. During mammalian organogenesis, lineage specification and commitment involve passage through distinct progenitor/precursor states that rely on different combinations and levels of transcription factors (Wilkinson et al. 2013;Cano et al. 2014). In the current model of pancreatic endocrine cell formation, Neurogenin3 (Neurog3) expression in the epithelium rapidly progresses to a high-level production of protein (Neurog3 HI ) that leads to endocrine fate commitment, cell cycle exit, and delamination toward proto-islet clusters. Meta-analysis of published literature (see below), however, is suggestive of a broader pattern of lower-level Neurog3 expression across the epithelium that is substantially more prevalent than the actively delaminating endocrine-committed Neurog3 HI population. The present study is focused on determining whether this low-Neurog3-expressing subpopulation represents the early-phase expression in post-mitotic cells on their way to becoming Neurog3 HI cells or endocrine fate-biased but uncommitted mitotic cells that have self-maintaining progenitor characteristics.Pancreas organogenesis is divided into a primary transition and secondary transition (Pan and Wright 2011). During the primary transition (embryonic day 9.5 [E9.5] to E12.5), multipotent progenitor cells undergo apical polarization, forming microlumens that then coalesce to generate an epithelial plexus. Within this epithelial plexus, progenitor cells (around E12.5) segregate into "tip" and "trunk" domains. In the short-term, tip domains

Petal growth is central to floral morphogenesis, but the underlying genetic basis of petal growth regulation is yet to be elucidated. In this study, we found that the basal region of the ray floret petals of Gerbera hybrida was the most sensitive to treatment with the phytohormones gibberellin (GA) and abscisic acid (ABA), which regulate cell expansion during petal growth in an antagonistic manner. To screen for differentially expressed genes (DEGs) and key regulators with potentially important roles in petal growth regulation by GA or/and ABA, the RNA-seq technique was employed. Differences in global transcription in petals were observed in response to GA and ABA and target genes antagonistically regulated by the two hormones were identified. Moreover, we also identified the pathways associated with the regulation of petal growth after application of either GA or ABA. Genes relating to the antagonistic GA and ABA regulation of petal growth showed distinct patterns, with genes encoding transcription factors (TFs) being active during the early stage (2 h) of treatment, while genes from the “apoptosis” and “cell wall organization” categories were expressed at later stages (12 h). In summary, we present the first study of global expression patterns of hormone-regulated transcripts in G. hybrida petals; this dataset will be instrumental in revealing the genetic networks that govern petal morphogenesis and provides a new theoretical basis and novel gene resources for ornamental plant breeding.

Background and Purpose-Notch signaling activity regulates arteriogenesis. Presenilin 1 (PS1) mediates Notch signaling activity via cleavage of Notch, liberating Notch intracellular domain (NICD). We tested the hypothesis that simvastatin enhances arteriogenesis after stroke by increasing PS1 activation of the Notch signaling pathway. Methods-Rats were subjected to middle cerebral artery occlusion (MCAo) and treated with or without simvastatin (1 mg/kg) starting 24 hours after stroke and daily for 7 days; they were euthanized 14 days after stroke. Immunostaining, Western blot, and real-time polymerase chain reaction assays were performed. Results-Simvastatin significantly increased arterial diameter, density, and vascular smooth muscle cell proliferation, and upregulated PS1, Notch1, and NICD expression in the ischemic border tissue and in the cerebral arteries compared with MCAo control rats, respectively. However, simvastatin did not increase arteriogenesis, PS1, and NICD expression in sham control animals. To investigate the mechanisms of simvastatin-induced arteriogenesis, primary cerebral artery cultures were used. Rats were subjected to MCAo and treated with or without simvastatin daily for 7 days. The cerebral arteries derived from these stroke rats were cultured in matrigel and treated with or without a ␥40-secretase inhibitor II, which blocks Notch signaling activity, inhibiting NICD production. Arterial cell migration was measured. simvastatin treatment significantly increased arterial cell migration compared to control MCAo artery, whereas inhibition of Notch signaling activity by the ␥40-secretase inhibitor II significantly attenuated simvastatin-induced arterial cell migration. Conclusions-These data indicate that simvastatin increases arteriogenesis after stroke, and that simvastatin upregulation of PS1 expression and Notch signaling activity may facilitate an increase in arteriogenesis.

Anthocyanins and flavonols have vital roles in flower coloration, plant development, and defense. Because anthocyanins and flavonols share the same subcellular localization and common biosynthetic substrates, these pathways may compete for substrates. However, the mechanism regulating this potential competition remains unclear. Here, we identified GhMYB1a, an R2R3-MYB transcription factor involved in the regulation of anthocyanin and flavonol accumulation in gerbera (Gerberahybrida). GhMYB1a shares high sequence similarity with that of other characterized regulators of flavonol biosynthesis. In addition, GhMYB1a is also phylogenetically grouped with these proteins. The overexpression of GhMYB1a in gerbera and tobacco (Nicotianatabacum) resulted in decreased anthocyanin accumulation and increased accumulation of flavonols by upregulating the structural genes involved in flavonol biosynthesis. We further found that GhMYB1a functions as a homodimer instead of interacting with basic helix-loop-helix cofactors. These results suggest that GhMYB1a is involved in regulating the anthocyanin and flavonol metabolic pathways through precise regulation of gene expression. The functional characterization of GhMYB1a provides insight into the biosynthesis and regulation of flavonols and anthocyanins.

Circulating tumor cells (CTCs) are shed from the primary tumor into the circulatory system and act as seeds that initiate cancer metastasis to distant sites. CTC enumeration has been shown to have a significant prognostic value as a surrogate marker in various cancers. The widespread clinical utility of CTC tests, however, is still limited due to the inherent rarity and heterogeneity of CTCs, which necessitate robust techniques for their efficient enrichment and detection. Significant recent advances have resulted in technologies with the ability to improve yield and purity of CTC enrichment as well as detection sensitivity. Current efforts are largely focused on the translation and standardization of assays to fully realize the clinical utility of CTCs. In this review, we aim to provide a comprehensive overview of CTC enrichment and detection techniques with an emphasis on novel approaches for rapid quantification of CTCs.