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.

Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids.

Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.

Constructing cellular niche properties by localized presentation of Wnt proteins on synthetic surfaces.

Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires ∼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/ß-catenin-dependent transcription. Immobilization efficiency can be measured 12-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12-18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.

Immobilized WNT Proteins Act as a Stem Cell Niche for Tissue Engineering.

The timing, location, and level of WNT signaling are highly regulated during embryonic development and for the maintenance of adult tissues. Consequently the ability to provide a defined and directed source of WNT proteins is crucial to fully understand its role in tissue development and to mimic its activity in vitro. Here we describe a one-step immobilization technique to covalently bind WNT3A proteins as a basal surface with easy storage and long-lasting activity. We show that this platform is able to maintain adult and embryonic stem cells while also being adaptable for 3D systems. Therefore, this platform could be used for recapitulating specific stem cell niches with the goal of improving tissue engineering.

Different roles of cadherins in the assembly and structural integrity of the desmosome complex.

Adhesion between cells is established by the formation of specialized intercellular junctional complexes, such as desmosomes. Desmosomes contain isoforms of two members of the cadherin superfamily of cell adhesion proteins, desmocollins (Dsc) and desmogleins (Dsg), but their combinatorial roles in desmosome assembly are not understood. To uncouple desmosome assembly from other cell-cell adhesion complexes, we used micro-patterned substrates of Dsc2aFc and/or Dsg2Fc and collagen IV; we show that Dsc2aFc, but not Dsg2Fc, was necessary and sufficient to recruit desmosome-specific desmoplakin into desmosome puncta and produce strong adhesive binding. Single-molecule force spectroscopy showed that monomeric Dsc2a, but not Dsg2, formed Ca(2+)-dependent homophilic bonds, and that Dsg2 formed Ca(2+)-independent heterophilic bonds with Dsc2a. A W2A mutation in Dsc2a inhibited Ca(2+)-dependent homophilic binding, similar to classical cadherins, and Dsc2aW2A, but not Dsg2W2A, was excluded from desmosomes in MDCK cells. These results indicate that Dsc2a, but not Dsg2, is required for desmosome assembly through homophilic Ca(2+)- and W2-dependent binding, and that Dsg2 might be involved later in regulating a switch to Ca(2+)-independent adhesion in mature desmosomes.

Fabricating surfaces with distinct geometries and different combinations of cell adhesion proteins.

A step-by-step procedure is described for functionalizing the surface of a glass coverslip so that a single cell contacts distinct patterns of extracellular matrix and cell-cell adhesion proteins. This dual-micropatterned substratum is accomplished through a two-step process. First, extracellular matrix (ECM) is microcontact-printed onto a silanized glass surface using electron beam lithography, etched resist-coated wafers, and Polydimethylsiloxane (PDMS) stamps of differing geometries. Then, non-ECM-coated surfaces are incubated sequentially with biotin, NeutrAvidin, and biotinylated Protein A to attach Fc-cadherin fusion proteins, Fc, or PEG. Cells are seeded at low density onto the functionalized surface for single-cell analysis of protein recruitment/turnover and cellular motility.

Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions.

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of alphaE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin- and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.