Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency.

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.

A graphical and computational modeling platform for biological pathways.

A major endeavor of systems biology is the construction of graphical and computational models of biological pathways as a means to better understand their structure and function. Here, we present a protocol for a biologist-friendly graphical modeling scheme that facilitates the construction of detailed network diagrams, summarizing the components of a biological pathway (such as proteins and biochemicals) and illustrating how they interact. These diagrams can then be used to simulate activity flow through a pathway, thereby modeling its dynamic behavior. The protocol is divided into four sections: (i) assembly of network diagrams using the modified Edinburgh Pathway Notation (mEPN) scheme and yEd network editing software with pathway information obtained from published literature and databases of molecular interaction data; (ii) parameterization of the pathway model within yEd through the placement of 'tokens' on the basis of the known or imputed amount or activity of a component; (iii) model testing through visualization and quantitative analysis of the movement of tokens through the pathway, using the network analysis tool Graphia Professional and (iv) optimization of model parameterization and experimentation. This is the first modeling approach that combines a sophisticated notation scheme for depicting biological events at the molecular level with a Petri net-based flow simulation algorithm and a powerful visualization engine with which to observe the dynamics of the system being modeled. Unlike many mathematical approaches to modeling pathways, it does not require the construction of a series of equations or rate constants for model parameterization. Depending on a model's complexity and the availability of information, its construction can take days to months, and, with refinement, possibly years. However, once assembled and parameterized, a simulation run, even on a large model, typically takes only seconds. Models constructed using this approach provide a means of knowledge management, information exchange and, through the computation simulation of their dynamic activity, generation and testing of hypotheses, as well as prediction of a system's behavior when perturbed.

Modelling the Structure and Dynamics of Biological Pathways.

There is a need for formalised diagrams that both summarise current biological pathway knowledge and support modelling approaches that explain and predict their behaviour. Here, we present a new, freely available modelling framework that includes a biologist-friendly pathway modelling language (mEPN), a simple but sophisticated method to support model parameterisation using available biological information; a stochastic flow algorithm that simulates the dynamics of pathway activity; and a 3-D visualisation engine that aids understanding of the complexities of a system's dynamics. We present example pathway models that illustrate of the power of approach to depict a diverse range of systems.

Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors.

Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut derivatives.

A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance.

BACKGROUND: The class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development. RESULTS: Here we examine the function of Oct4 homologs in Xenopus embryos and compare this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, we defined a set of evolutionary-conserved Oct4/POUV targets. Most of these targets were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos. CONCLUSIONS: Currently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, we identified a core set of functionally relevant factors that linked the maintenance of adhesion to Oct4/POUV. We found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency.

Oct4: the final frontier, differentiation defining pluripotency.

The transcription factor OCT4 is a cornerstone of pluripotency, and yet OCT4 has also been associated with differentiation in a number of contexts. Reporting in this issue of Developmental Cell, Frum et al. (2013) show that OCT4's major early activity in the blastocyst is to support primitive endoderm differentiation.

Transcriptional activation by Oct4 is sufficient for the maintenance and induction of pluripotency.

Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes.

Investigation of microsphere-mediated cellular delivery by chemical, microscopic and gene expression analysis.

Amino functionalized cross-linked polystyrene microspheres of well defined sizes (0.2-2 mum) have been prepared and shown to be efficient and controllable delivery devices, capable of transporting anything from small dye molecules to bulky proteins into cells. However, the specific mechanism of cellular entry is largely unknown and widely variant from study to study. As such, chemical, biological and microscopic methods are used to elucidate the mechanism of cellular uptake for polystyrene microspheres of 0.2, 0.5 and 2 mum in mouse melanoma cells. Uptake is found to be wholly unreliant upon energetic processes, while lysosomal and endosomal tracking agents failed to show co-localisation with lysosomes/endosomes, suggesting a non-endocytic uptake pathway. To further explore the consequences of microsphere uptake, gene expression profiling is used to determine if there is a transcriptional response to "beadfection" in both murine and human cells. None of the common transcriptional responses to enhanced endocytosis are observed in beadfected cells, further supporting a non-endocytic uptake mechanism. Furthermore, the microspheres are noted to have a limited interaction with cells at a transcriptional level, supporting them as a non-toxic delivery vehicle.

Microsphere-based tracing and molecular delivery in embryonic stem cells.

Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.

Differentiation of embryonic stem cells into anterior definitive endoderm.

Anterior definitive endoderm (ADE) is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other visceral organs. Here we describe a method for the differentiation of mouse embryonic stem (ES) cells to endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (aggregation or embryoid body) culture and in a serum-free adherent monolayer culture. Purified ES cell-derived ADE cells appear committed to endodermal fates and can undergo further differentiation in vitro towards liver and pancreas with enhanced efficiency.

Anterior definitive endoderm from ESCs reveals a role for FGF signaling.

The use of embryonic stem cell (ESC) differentiation to generate functional hepatic or pancreatic progenitors and as a tool for developmental biology is limited by an inability to isolate in vitro equivalents of regionally specified anterior definitive endoderm (ADE). To address this, we devised a strategy using a fluorescent reporter gene under the transcriptional control of the anterior endoderm marker Hex alongside the definitive mesendoderm marker Cxcr4. Isolation of Hex(+)Cxcr4(+) differentiating ESCs yielded a population expressing ADE markers that both can be expanded and is competent to undergo differentiation toward liver and pancreatic fates. Hex reporter ESCs were also used to define conditions for ADE specification in serum-free adherent culture and revealed an unexpected role for FGF signaling in the generation of ADE. Our findings in defined monolayer differentiation suggest FGF signaling is an important regulator of early anterior mesendoderm differentiation rather than merely a mediator of morphogenetic movement.

Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabolism.

AKAP121 focuses distinct signaling events from membrane to mitochondria by binding and targeting cAMP-dependent protein kinase (PKA), protein tyrosine phosphatase (PTPD1), and mRNA. We find that AKAP121 also targets src tyrosine kinase to mitochondria via PTPD1. AKAP121 increased src-dependent phosphorylation of mitochondrial substrates and enhanced the activity of cytochrome c oxidase, a component of the mitochondrial respiratory chain. Mitochondrial membrane potential and ATP oxidative synthesis were enhanced by AKAP121 in an src- and PKA-dependent manner. Finally, siRNA-mediated silencing of endogenous AKAP121 drastically impaired synthesis and accumulation of mitochondrial ATP. These findings indicate that AKAP121, through its role in enhancing cAMP and tyrosine kinase signaling to distal organelles, is an important regulator in mitochondrial metabolism.

A synthetic peptide reproducing the mitochondrial targeting motif of AKAP121: a conformational study.

The conformational features of a peptide derived by the 10-30 sequence of the mitochondrial domain of AKAP121 [Ac-1XKKPLALPGMLALLGWWWFFSRKKX25-NH2 (X=beta-Ala)] in water and in a water/trifluoroethanol (TFE) mixture at 298 K have been determined by NMR and CD spectroscopy. Backbone clustering analysis of NMR-derived structures led to the identification of a single representative structure in water/TFE. The structure of the peptide consists mainly of an alpha-helix, whose core is the region 7-23, with a less ordered N-terminal part. These data are confirmed by CD analysis. It is noteworthy that the high hydrophobic Trp16-Phe20 segment, that might also mediate interaction with tubulin, is organized in an alpha-helical wheel. Our conformational data can be the starting point for the development of highly selective peptides that interfere with the biological function of the Protein Kinase A scaffold protein AKAP121.

Mitochondrial AKAP121 binds and targets protein tyrosine phosphatase D1, a novel positive regulator of src signaling.

A-kinase anchor protein 121 (AKAP121) and its spliced isoform AKAP84 anchor protein kinase A (PKA) to the outer membrane of mitochondria, focusing and enhancing cyclic AMP signal transduction to the organelle. We find that AKAP121/84 also binds PTPD1, a src-associated protein tyrosine phosphatase. A signaling complex containing AKAP121, PKA, PTPD1, and src is assembled in vivo. PTPD1 activates src tyrosine kinase and increases the magnitude and duration of epidermal growth factor (EGF) signaling. EGF receptor phosphorylation and downstream activation of ERK 1/2 and Elk1-dependent gene transcription are enhanced by PTPD1. Expression of a PTPD1 mutant lacking catalytic activity inhibits src and downregulates ERK 1/2 but does not affect the activity of c-Jun N-terminal kinase 1/2 and p38alpha mitogen-activated protein kinase. AKAP121 binds to and redistributes PTPD1 from the cytoplasm to mitochondria and inhibits EGF signaling. Our findings indicate that PTPD1 is a novel positive regulator of src signaling and a key component of the EGF transduction pathway. By binding and/or targeting the phosphatase on mitochondria, AKAP121 modulates the amplitude and persistence of src-dependent EGF transduction pathway. This represents the first example of physical and functional interaction between AKAPs and a protein tyrosine phosphatase.

Role of the ERK-mediated signaling pathway in mesenchyme formation and differentiation in the sea urchin embryo.

Mesoderm and mesodermal structures in the sea urchin embryo are entirely generated by two embryologically distinct populations of mesenchyme cells: the primary (PMC) and the secondary (SMC) mesenchyme cells. We have identified the extracellular signal-regulated kinase (ERK) as a key component of the regulatory machinery that controls the formation of both these cell types. ERK is activated in a spatial-temporal manner, which coincides with the epithelial-mesenchyme transition (EMT) of the prospective PMCs and SMCs. Here, we show that ERK controls EMT of both primary and secondary mesenchyme cells. Loss and gain of function experiments demonstrate that ERK signaling is not required for the early specification of either PMCs or SMCs, but controls the maintenance and/or the enhancement of expression levels of regulatory genes which participate in the process of specification of these cell types. In addition, ERK-mediated signaling is essential for the transcription of terminal differentiation genes encoding proteins that define the final structures generated by PMCs and SMCs. Our findings suggest that ERK has a central pan-mesodermal role in coupling EMT and terminal differentiation of all mesenchymal cell types in the sea urchin embryo.