Carl-Philipp Heisenberg

Tensile forces govern germ-layer organization in zebrafish

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFβ-signalling (transforming growth factor β), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

Forces in Tissue Morphogenesis and Patterning

During development, mechanical forces cause changes in size, shape, number, position, and gene expression of cells. They are therefore integral to any morphogenetic processes. Force generation by actin-myosin networks and force transmission through adhesive complexes are two self-organizing phenomena driving tissue morphogenesis. Coordination and integration of forces by long-range force transmission and mechanosensing of cells within tissues produce large-scale tissue shape changes. Extrinsic mechanical forces also control tissue patterning by modulating cell fate specification and differentiation. Thus, the interplay between tissue mechanics and biochemical signaling orchestrates tissue morphogenesis and patterning in development.

Single-cell force spectroscopy

The controlled adhesion of cells to each other and to the extracellular matrix is crucial for tissue development and maintenance. Numerous assays have been developed to quantify cell adhesion. Among these, the use of atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) has recently been established. This assay permits the adhesion of living cells to be studied in near-physiological conditions. This implementation of AFM allows unrivaled spatial and temporal control of cells, as well as highly quantitative force actuation and force measurement that is sufficiently sensitive to characterize the interaction of single molecules. Therefore, not only overall cell adhesion but also the properties of single adhesion-receptor–ligand interactions can be studied. Here we describe current implementations and applications of SCFS, as well as potential pitfalls, and outline how developments will provide insight into the forces, energetics and kinetics of cell-adhesion processes.

Brain-derived Neurotrophic Factor is a Survival Factor for Cultured Rat Cerebellar Granule Neurons and Protects them Against Glutamate-induced Neurotoxicity

We have studied the effects of different neurotrophins on the survival and proliferation of rat cerebellar granule cells in culture. These neurons express trkB and trkC, the putative neuronal receptors for brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) respectively. Binding studies using iodinated BDNF and NT‐3 demonstrated that both BDNF and NT‐3 bind to the cerebellar granule neurons with a similar affinity of ˜ 2x10‐9 M. The number of receptors per granule cell was surprisingly high, ∼30x10‐4 and 2x 105 for BDNF and NT‐3, respectively. Both NT‐3 and BDNF elevated c‐fos mRNA in the granule neurons, but only BDNF up‐regulated the mRNA encoding the low‐affinity neurotrophin receptor (p75). In contrast to NT‐3, BDNF acted as a survival factor for the granule neurons. BDNF also induced sprouting of the granule neurons and significantly protected them against neurotoxicity induced by high (1 mM) glutamate concentrations. Cultured granule neurons also expressed low levels of BDNF mRNA which were increased by kainic acid, a glutamate receptor agonist. Thus, BDNF, but not NT‐3, is a survival factor for cultured cerebellar granule neurons and activation of glutamate receptor(s) up‐regulates BDNF expression in these cells.

The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation.

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.

Proteomics of early zebrafish embryos

BackgroundZebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed.ResultsAs a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.ConclusionThe provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.

Adhesion Functions in Cell Sorting by Mechanically Coupling the Cortices of Adhering Cells

Embryonic Cell Sorting and Movement Differential cell adhesion has long been thought to drive cell sorting. Maître et al. (p. 253, published online 23 August) show that cell sorting in zebrafish gastrulation is triggered by differences in the ability of cells to modulate cortex tension at cell-cell contacts, thereby controlling contact expansion. Cell adhesion functions in this process by mechanically coupling the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. In zebrafish epiboly the enveloping cell layer (EVL)—a surface epithelium formed at the animal pole of the gastrula—gradually spreads over the entire yolk cell to engulf it at the end of gastrulation. Behrndt et al. (p. 257) show that an actomyosin ring connected to the epithelial margin triggers EVL spreading both by contracting around its circumference and by generating a pulling force through resistance against retrograde actomyosin flow. Cell adhesion provides a mechanical scaffold for cell cortex tension to drive cell sorting during zebrafish gastrulation. Differential cell adhesion and cortex tension are thought to drive cell sorting by controlling cell-cell contact formation. Here, we show that cell adhesion and cortex tension have different mechanical functions in controlling progenitor cell-cell contact formation and sorting during zebrafish gastrulation. Cortex tension controls cell-cell contact expansion by modulating interfacial tension at the contact. By contrast, adhesion has little direct function in contact expansion, but instead is needed to mechanically couple the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. The coupling function of adhesion is mediated by E-cadherin and limited by the mechanical anchoring of E-cadherin to the cortex. Thus, cell adhesion provides the mechanical scaffold for cell cortex tension to drive cell sorting during gastrulation.

Non-canonical Wnt signalling and regulation of gastrulation movements.

Members of the Wnt family have been implicated in a variety of developmental processes including axis formation, patterning of the central nervous system and tissue morphogenesis. Recent studies have shown that a Wnt signalling pathway similar to that involved in the establishment of planar cell polarity in Drosophila regulates convergent extension movements during zebrafish and Xenopus gastrulation. This finding provides a good starting point to dissect the complex cell biology and genetic regulation of vertebrate gastrulation movements.

A role for Rho GTPases and cell–cell adhesion in single-cell motility in vivo

Cell migration is central to embryonic development, homeostasis and disease, processes in which cells move as part of a group or individually. Whereas the mechanisms controlling single-cell migration in vitro are relatively well understood, less is known about the mechanisms promoting the motility of individual cells in vivo. In particular, it is not clear how cells that form blebs in their migration use those protrusions to bring about movement in the context of the three-dimensional cellular environment. Here we show that the motility of chemokine-guided germ cells within the zebrafish embryo requires the function of the small Rho GTPases Rac1 and RhoA, as well as E-cadherin-mediated cell–cell adhesion. Using fluorescence resonance energy transfer we demonstrate that Rac1 and RhoA are activated in the cell front. At this location, Rac1 is responsible for the formation of actin-rich structures, and RhoA promotes retrograde actin flow. We propose that these actin-rich structures undergoing retrograde flow are essential for the generation of E-cadherin-mediated traction forces between the germ cells and the surrounding tissue and are therefore crucial for cell motility in vivo.

Forces driving epithelial spreading in zebrafish gastrulation

Embryonic Cell Sorting and Movement Differential cell adhesion has long been thought to drive cell sorting. Maître et al. (p. 253, published online 23 August) show that cell sorting in zebrafish gastrulation is triggered by differences in the ability of cells to modulate cortex tension at cell-cell contacts, thereby controlling contact expansion. Cell adhesion functions in this process by mechanically coupling the cortices of adhering cells at their contacts, allowing cortex tension to control contact expansion. In zebrafish epiboly the enveloping cell layer (EVL)—a surface epithelium formed at the animal pole of the gastrula—gradually spreads over the entire yolk cell to engulf it at the end of gastrulation. Behrndt et al. (p. 257) show that an actomyosin ring connected to the epithelial margin triggers EVL spreading both by contracting around its circumference and by generating a pulling force through resistance against retrograde actomyosin flow. Contraction of an actomyosin ring drives epithelial morphogenesis during embryonic development. Contractile actomyosin rings drive various fundamental morphogenetic processes ranging from cytokinesis to wound healing. Actomyosin rings are generally thought to function by circumferential contraction. Here, we show that the spreading of the enveloping cell layer (EVL) over the yolk cell during zebrafish gastrulation is driven by a contractile actomyosin ring. In contrast to previous suggestions, we find that this ring functions not only by circumferential contraction but also by a flow-friction mechanism. This generates a pulling force through resistance against retrograde actomyosin flow. EVL spreading proceeds normally in situations where circumferential contraction is unproductive, indicating that the flow-friction mechanism is sufficient. Thus, actomyosin rings can function in epithelial morphogenesis through a combination of cable-constriction and flow-friction mechanisms.