Frizzled gene expression and negative regulation of canonical WNT-β-catenin signaling in mouse F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-β-catenin signaling, thereby allowing PE cells to differentiate

Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells

Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs) derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells

A Model for Equity-Oriented PreK-12 Engineering

In this paper, we address the societally important issue of developing a more equitable approach to preK-12 engineering education. Our primary emphasis is on K-8 grades—a time when first impressions of engineering may be developed. Calls for increased participation by all students, including those who have been historically marginalized, motivate the need for theoretically grounded ways of developing and assessing educational programs. This paper draws from sociocultural learning theory and applies four theoretical and empirical analyses to derive design principles for equity that can inform curriculum, instruction, and assessment of preK-12 engineering education programs. We present a model for equity-oriented preK-12 engineering learning and delineate its dimensions and principles, which include socially engaged engineering, authentic engineering practices, asset-oriented pedagogies, and student engineering identity. We illustrate each with examples and discuss ways of implementing equity-oriented engineering curricula

Retinoic acid and the development of the endoderm

Retinoic acid (RA) is an important signaling molecule in the development of the endoderm and an important molecule in protocols used to generate endodermal cell types from stem cells. In this review, we describe the RA signaling pathway and its role in the patterning and specification of the extra embryonic endoderm and different endodermal organs. The formation of endoderm is an ancient evolutionary feature and RA signaling appears to have coevolved with the vertebrate lineage. Towards that end, we describe how RA participates in many regulatory networks required for the formation of extraembryonic structures as well as the organs of the embryo proper

Knocking out a negative regulator of Hedgehog signaling blocks differentiation of cells into neurons

Hedgehog (Hh) signaling, one of many different protein signaling pathways found in mammals, is vital in many stage of neural development. A major negative regulator of Hh signaling is a protein known as Suppressor of Fused (SUFU), which acts to sequester the full length Gli transcription factors, proteins that can turn genes on and off, in the cytoplasm or facilitates its conversion to a repressive form. The P19 embryonal carcinoma cell line is a model of hind-brain neuronal differentiation and the involvement of Hh signaling, in particular the role of SUFU in this process has yet to be explored. We hypothesize that SUFU is required for P19 neuronal differentiation to occur, and that altering the expression of this protein through CRISPR/Cas9 knockout will result in a loss of differentiation potential. Throughout normal, retinoic acid (RA) induced, differentiation of P19 cells the abundance of SUFU mRNA levels show modest changes, although protein abundance does not change. Knocking out Sufu blocked neuronal differentiation, without changing the stem-ness of the cells or their ability to grow normally. During RA-induced neuron formation, Hh signaling is inhibited in the later stages of the differentiation process, as seen by increased Gli repressor levels over those days. It is likely, then, that knocking out the negative regulator SUFU does not facilitate the conversion of Gli proteins from their full-length active state to their smaller repressive state in those final stages of neuron formation. Together these results show the dynamic nature of Hh signaling during neuron development and showcase the critical role of SUFU in regulating this developmental event

Reproductive Biology of Middle Mississippi River Shovelnose Sturgeon: Insights from Seasonal and Age Variation in Plasma Sex Steroid and Calcium Concentrations

Shovelnose sturgeon Scaphirhynchus platorynchus are endemic to the Mississippi River drainage and are commercially harvested for roe in several states. Status of shovelnose sturgeon populations throughout much of its range is unknown or declining; Mississippi River stocks are experiencing recruitment overfishing and may be at risk of collapse. Restoration of shovelnose sturgeon populations will require additional information on their reproductive biology, including age at maturity and reproductive status and cycles. The objectives of this study were to determine the effects of reproductive stage, fish age, and season on plasma 17-β estradiol (E2) and calcium (Ca2+) concentrations in female shovelnose sturgeon and plasma testosterone (T) concentrations in male shovelnose sturgeon from the middle Mississippi River (MMR). We also assessed the relationship between plasma vitellogenin (VTG) and Ca2+ concentrations for female shovelnose sturgeon. Plasma E2 and Ca2+ concentrations in females and T concentrations in males differed among reproductive stages, consistent with results of prior research on shovelnose sturgeon and other sturgeon species. VTG and Ca2+ concentrations were strongly correlated in female shovelnose sturgeon, indicating that Ca2+ can be used as a surrogate for VTG assays for identification of vitellogenic females, as has been shown for other sturgeon species. Age at maturity was estimated at 10 years for males and 9 years for females based on T and E2 profiles, consistent with other recent age at maturity estimates in the MMR and Wabash River determined by gonadal examination. Peaks in plasma sex steroid and Ca2+ concentrations during April and October possibly reflected the spring spawning season and provide additional evidence in support of recent documentation of fall spawning by shovelnose sturgeon in the MMR. Additional research is needed to elucidate fall spawning by shovelnose sturgeon in the MMR

Socially Engaged Engineering: A Framework for K-8 Education

Socially engaged engineering provides for student learning of the design, analysis, and practices of engineering as well as the ways that engineering is situated in sociocultural contexts. This paper provides a conceptual framework regarding socially engaged engineering for K-8 educators, researchers, and curriculum designers. The framework identifies ways to support youth learning of engineering and considerations of technical, social, environmental, and ethical dimensions of engineering. As engineering enters K-8 educational settings, it is important to introduce the discipline in equity-oriented ways. We draw from the field of engineering for social justice to build this framework for examining engineering at the macro-, meso-, and microscales. Situating engineering in sociocultural contexts can be motivating to learners and provide perspectives on the nature of engineering. Our framework was concurrently designed with Youth Engineering Solutions (YES; https://YouthEngineeringSolutions.org) curricular units. To test the applicability of our principles, we applied them to K-8 YES curricula for school and out-of-school environments. Through the coevolution of principles and curricular materials, we developed age-appropriate learning objectives for three levels—lower elementary, upper elementary, and middle school. This paper shares the principles and progression, showing worked examples from curricula to demonstrate how the principles translate into curricular resources. We discuss constraints to the implementation of socially engaged engineering curricula, including those imposed in educational settings and the ideological assumptions about science, engineering, and STEM disciplines

NOX1 and NOX4 are required for the differentiation of mouse F9 cells into extraembryonic endoderm

Mouse F9 cells differentiate to primitive endoderm (PrE) when treated with retinoic acid (RA). Differentiation is accompanied by increased reactive oxygen species (ROS) levels, and while treating F9 cells with antioxidants attenuates differentiation, H2O2 treatment alone is sufficient to induce PrE. We identified the NADPH oxidase (NOX) complexes as candidates for the source of this endogenous ROS, and within this gene family, and over the course of differentiation, Nox1 and Nox 4 show the greatest upregulation induced by RA. Gata6, encoding a master regulator of extraembryonic endoderm is also up-regulated by RA and we provide evidence that NOX1 and NOX4 protein levels increase in F9 cells overexpressing Gata6. Pan-NOX and NOX1-specific inhibitors significantly reduced the ability of RA to induce PrE, and this was recapitulated using a genetic approach to knockdown Nox1 and/or Nox4 transcripts. Interestingly, overexpressing either gene in untreated F9 cells did not induce differentiation, even though each elevated ROS levels. Thus, the data suggests that ROS produced during PrE differentiation is dependent in part on increased NOX1 and NOX4 levels, which is under the control of GATA6. Furthermore, these results suggest that the combined activity of multiple NOX proteins is necessary for the differentiation of F9 cells to primitive endoderm

Response to Comment on “Using Nested Average Electricity Allocation Protocols …”

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134265/1/jiec12476.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134265/2/jiec12476_am.pd

Evolutionary characterization of pig interferon-inducible transmembrane gene family and member expression dynamics in tracheobronchial lymph nodes of pigs infected with swine respiratory disease viruses

Studies have found that a cluster of duplicated gene loci encoding the interferon-inducible transmembrane proteins (IFITMs) family have antiviral activity against several viruses, including influenza A virus. The gene family has 5 and 7 members in humans and mice, respectively. Here, we confirm the current annotation of pig IFITM1, IFITM2, IFITM3, IFITM5, IFITM1L1 and IFITM1L4, manually annotated IFITM1L2, IFITM1L3, IFITM5L, IFITM3L1 and IFITM3L2, and provide expressed sequence tag (EST) and/or mRNA evidence, not contained with the NCBI Reference Sequence database (RefSeq), for the existence of IFITM6, IFITM7 and a new IFITM1-like (IFITM1LN) gene in pigs. Phylogenic analyses showed seven porcine IFITM genes with highly conserved human/mouse orthologs known to have anti-viral activity. Digital Gene Expression Tag Profiling (DGETP) of swine tracheobronchial lymph nodes (TBLN) of pigs infected with swine influenza virus (SIV), porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus or porcine circovirus type 2 over 14 days post-inoculation (dpi) showed that gene expression abundance differs dramatically among pig IFITM family members, ranging from 0 to over 3,000 tags per million. In particular, SIV upregulated IFITM1 by 5.9 fold at 3 dpi. Bayesian framework further identified pig IFITM1 and IFITM3 as differentially expressed genes in the overall transcriptome analysis. In addition to being a component of protein complexes involved in homotypic adhesion, the IFITM1 is also associated with pathways related to regulation of cell proliferation and IFITM3 is involved in immune responses