Tissue and cell interactions in mammalian PGC development

ABSTRACT Primordial germ cells (PGCs) form early in embryo development and are crucial precursors to functioning gamete cells. Considerable research has focussed on identifying the transcriptional characteristics and signalling pathway requirements that confer PGC specification and development, enabling the derivation of PGC-like cells (PGCLCs) in vitro using specific signalling cocktails. However, full maturation to germ cells still relies on co-culture with supporting cell types, implicating an additional requirement for cellular- and tissue-level regulation. Here, we discuss the experimental evidence that highlights the nature of intercellular interactions between PGCs and neighbouring cell populations during mouse PGC development. We posit that the role that tissue interactions play on PGCs is not limited solely to signalling-based induction but extends to coordination of development by robust regulation of the proportions and position of the cells and tissues within the embryo, which is crucial for functional germ cell maturation. Such tissue co-development provides a dynamic, contextual niche for PGC development. We argue that there is evidence for a clear role for inter-tissue dependence of mouse PGCs, with potential implications for generating mammalian PGCLCs in vitro.

Polar auxin transport dynamics of primary and secondary vein patterning in dicot leaves

The growth regulator auxin plays a central role in the phyllotaxy, shape, and venation patterns of leaves. The auxin spatial localization underlying these phenomena involves polar auxin transport (PAT) at the cellular level, particularly the preferential allocation of PIN efflux proteins to certain areas of the plasma membrane. Two general mechanisms have been studied: an up-the-gradient (UTG) allocation dependent on neighbouring-cell auxin concentrations, and a with-the-flux (WTF) allocation dependent on the flow of auxin across walls. We have developed a combined UTG+WTF model to quantify the observed auxin flows both towards (UTG) and away from (WTF) auxin maxima during primary and secondary vein patterning in leaves. The model simulates intracellular and membrane kinetics and intercellular transport, and is solved for a 2D leaf of several hundred cells. In addition to normal development, modelling of increasing PAT inhibition generates, as observed experimentally: a switch from several distinct vein initiation sites to many less-distinct sites; a delay in vein canalization; inhibited connection of new veins to old; and finally loss of patterning in the margin, loss of vein extension, and confinement of auxin to the margin. The model generates the observed formation of discrete auxin maxima at leaf vein sources and shows the dependence of secondary vein patterning on the efficacy of auxin flux through cells. Simulations of vein patterning and leaf growth further indicate that growth itself may bridge the spatial scale from the cell-cell resolution of the PIN-auxin dynamics to vein patterns on the whole-leaf scale.

Force networks, torque balance and Airy stress in the planar vertex model of a confluent epithelium

The vertex model is a popular framework for modelling tightly packed biological cells, such as confluent epithelia. Cells are described by convex polygons tiling the plane and their equilibrium is found by minimizing a global mechanical energy, with vertex locations treated as degrees of freedom. Drawing on analogies with granular materials, we describe the force network for a localized monolayer and derive the corresponding discrete Airy stress function, expressed for each N -sided cell as N scalars defined over kites covering the cell. We show how a torque balance (commonly overlooked in implementations of the vertex model) requires each internal vertex to lie at the orthocentre of the triangle formed by neighbouring edge centroids. Torque balance also places a geometric constraint on the stress in the neighbourhood of cellular trijunctions, and requires cell edges to be orthogonal to the links of a dual network that connect neighbouring cell centres and thereby triangulate the monolayer. We show how the Airy stress function depends on cell shape when a standard energy functional is adopted, and discuss implications for computational implementations of the model.

Cell-based simulations of Notch-dependent cell differentiation on growing domains

Notch signalling controls cell differentiation and proliferation in many tissues. The Notch signal is generated by the interaction between the Notch receptor of one cell with the Notch ligand (Delta or Jagged) of a neighbouring cell. Therefore, the pathway requires cell-cell contact in order to be active. During organ development, cell differentiation occurs concurrently with tissue growth and changes in cell morphology. How growth impacts on Notch signalling and cell differentiation remains poorly understood. Here, we developed a modelling environment to simulate Notch signalling in a growing tissue. We use our model to simulate the differentiation process of pancreatic progenitor cells. Our results suggest that Notch-mediated differentiation in the developing pancreas is first mediated by geometric effects that result in loss of Notch signalling on the tissue boundary, leading to the differentiation of tip versus trunk cells. A second wave of differentiation further happens in the trunk cells due to a reduction in the expression of the ligand Jagged, which has been shown to be controlled by signalling factors secreted from the surrounding mesenchyme. Our results bring new insights into how cells coordinate tissue growth with cell fate specification during organ development.

Force chains in cell–cell mechanical communication

Force chains (FCs) are a key determinant of the micromechanical properties and behaviour of heterogeneous materials, such as granular systems. However, less is known about FCs in fibrous materials, such as the networks composing the extracellular matrix (ECM) of biological systems. Using a finite-element computational model, we simulated the contraction of a single cell and two nearby cells embedded in two-dimensional fibrous elastic networks and analysed the tensile FCs that developed in the ECM. The role of ECM nonlinear elasticity on FC formation was evaluated by considering linear and nonlinear, i.e. exhibiting ‘buckling’ and/or ‘strain-stiffening’, stress–strain curves. The effect of the degree of cell contraction and network coordination value was assessed. We found that nonlinear elasticity of the ECM fibres influenced the structure of the FCs, facilitating the transition towards more distinct chains that were less branched and more radially oriented than the chains formed in linear elastic networks. When two neighbouring cells contract, a larger number of FCs bridged between the cells in nonlinear networks, and these chains had a larger effective rigidity than the chains that did not reach a neighbouring cell. These results suggest that FCs function as a route for mechanical communication between distant cells and highlight the contribution of ECM fibre nonlinear elasticity to the formation of FCs.

Substrate deformations induce directed keratinocyte migration

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.

Tricellular junction proteins promote disentanglement of daughter and neighbour cells during epithelial cytokinesis

In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, we explored in Drosophila epithelium, de novo formation of tricellular septate junctions (TCJs). We found that upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughter-neighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells and their neighbours during cytokinesis.

Experimental study on in-plane compressive response of irregular honeycombs

Compared with regular honeycombs, irregular honeycombs are more representative of real foams, and thus more suitable for the study of foam mechanics. In this paper, the deformation and failure progression in the irregular honeycombs are investigated by analysing the images captured in order to gain an improved understanding on foam failure. Irregular honeycombs with varying cell wall thickness, cell size and cell shape were manufactured using a three-dimensional printer and tested under compression. The behaviour of irregular honeycombs is found to be different from that of regular honeycombs. In irregular honeycombs, cell walls start to fracture at some point, initially at a low speed from multiple locations. The global stress reaches its maximum value shortly after the first fracture of cell walls. Only a few cell walls buckle in the specimens with cells of irregular shape. Fracture is more likely to occur to thin and long cell walls aligned within a medium angle (around 30 to 60°) to the compressive load. However, the susceptibility of a cell wall is to fracture is also affected by its neighbouring cell walls. Strong and stiff neighbouring cell walls could shield load away and protect it from breaking. Because of this, it is better to think of a weak spot as a region, rather than an individual cell or cell wall. Overall, the more uniform cell wall size and thickness are, the better the mechanical performance of cellular solids is.