From morphogen to morphogenesis and back.

A long-term aim of the life sciences is to understand how organismal shape is encoded by the genome. An important challenge is to identify mechanistic links between the genes that control cell-fate decisions and the cellular machines that generate shape, therefore closing the gap between genotype and phenotype. The logic and mechanisms that integrate these different levels of shape control are beginning to be described, and recently discovered mechanisms of cross-talk and feedback are beginning to explain the remarkable robustness of organ assembly. The 'full-circle' understanding of morphogenesis that is emerging, besides solving a key puzzle in biology, provides a mechanistic framework for future approaches to tissue engineering.

Collective cell migration in morphogenesis, regeneration and cancer.

The collective migration of cells as a cohesive group is a hallmark of the tissue remodelling events that underlie embryonic morphogenesis, wound repair and cancer invasion. In such migration, cells move as sheets, strands, clusters or ducts rather than individually, and use similar actin- and myosin-mediated protrusions and guidance by extrinsic chemotactic and mechanical cues as used by single migratory cells. However, cadherin-based junctions between cells additionally maintain 'supracellular' properties, such as collective polarization, force generation, decision making and, eventually, complex tissue organization. Comparing different types of collective migration at the molecular and cellular level reveals a common mechanistic theme between developmental and cancer research.

Luminal signalling links cell communication to tissue architecture during organogenesis.

Morphogenesis is the process whereby cell collectives are shaped into differentiated tissues and organs. The self-organizing nature of morphogenesis has been recently demonstrated by studies showing that stem cells in three-dimensional culture can generate complex organoids, such as mini-guts, optic-cups and even mini-brains. To achieve this, cell collectives must regulate the activity of secreted signalling molecules that control cell differentiation, presumably through the self-assembly of microenvironments or niches. However, mechanisms that allow changes in tissue architecture to feedback directly on the activity of extracellular signals have not been described. Here we investigate how the process of tissue assembly controls signalling activity during organogenesis in vivo, using the migrating zebrafish lateral line primordium. We show that fibroblast growth factor (FGF) activity within the tissue controls the frequency at which it deposits rosette-like mechanosensory organs. Live imaging reveals that FGF becomes specifically concentrated in microluminal structures that assemble at the centre of these organs and spatially constrain its signalling activity. Genetic inhibition of microlumen assembly and laser micropuncture experiments demonstrate that microlumina increase signalling responses in participating cells, thus allowing FGF to coordinate the migratory behaviour of cell groups at the tissue rear. As the formation of a central lumen is a self-organizing property of many cell types, such as epithelia and embryonic stem cells, luminal signalling provides a potentially general mechanism to locally restrict, coordinate and enhance cell communication within tissues.

TimerQuant: a modelling approach to tandem fluorescent timer design and data interpretation for measuring protein turnover in embryos.

Studies on signalling dynamics in living embryos have been limited by a scarcity of in vivo reporters. Tandem fluorescent protein timers provide a generic method for detecting changes in protein population age and thus provide readouts for signalling events that lead to changes in protein stability or location. When imaged with quantitative dual-colour fluorescence microscopy, tandem timers offer detailed 'snapshot' readouts of signalling activity from subcellular to organismal scales, and therefore have the potential to revolutionise studies in developing embryos. Here we use computer modelling and embryo experiments to explore the behaviour of tandem timers in developing systems. We present a mathematical model of timer kinetics and provide software tools that will allow experimentalists to select the most appropriate timer designs for their biological question, and guide interpretation of the obtained readouts. Through the generation of a series of novel zebrafish reporter lines, we confirm experimentally that our quantitative model can accurately predict different timer responses in developing embryos and explain some less expected findings. For example, increasing the FRET efficiency of a tandem timer actually increases the ability of the timer to detect differences in protein half-life. Finally, while previous studies have used timers to monitor changes in protein turnover, our model shows that timers can also be used to facilitate the monitoring of gene expression kinetics in vivo.

Directional tissue migration through a self-generated chemokine gradient.

The directed migration of cell collectives is a driving force of embryogenesis. The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients. One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them, a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion.

Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.

The directed migration of cell collectives drives the formation of complex organ systems. A characteristic feature of many migrating collectives is a 'tissue-scale' polarity, whereby 'leader' cells at the edge of the tissue guide trailing 'followers' that become assembled into polarised epithelial tissues en route. Here, we combine quantitative imaging and perturbation approaches to investigate epithelial cell state transitions during collective migration and organogenesis, using the zebrafish lateral line primordium as an in vivo model. A readout of three-dimensional cell polarity, based on centrosomal-nucleus axes, allows the transition from migrating leaders to assembled followers to be quantitatively resolved for the first time in vivo. Using live reporters and a novel fluorescent protein timer approach, we investigate changes in cell-cell adhesion underlying this transition by monitoring cadherin receptor localisation and stability. This reveals that while cadherin 2 is expressed across the entire tissue, functional apical junctions are first assembled in the transition zone and become progressively more stable across the leader-follower axis of the tissue. Perturbation experiments demonstrate that the formation of these apical adherens junctions requires dynamic microtubules. However, once stabilised, adherens junction maintenance is microtubule independent. Combined, these data identify a mechanism for regulating leader-to-follower transitions within migrating collectives, based on the relocation and stabilisation of cadherins, and reveal a key role for dynamic microtubules in this process.

Organizing moving groups during morphogenesis.

The directed migration of cells drives the formation of many complex organ systems. Although in this morphogenetic context cells display a strong preference for migrating in organized, cohesive groups, little is known about the mechanisms that coordinate their movements. Recent studies on several model systems have begun to dissect the organization of these migrating tissues in vivo and have shown that cell guidance is mediated by a combination of chemical and mechanical cues.

A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo.

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterized by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live-imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarized vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterized by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia.

Conserved exchange of paralog proteins during neuronal differentiation.

Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas ∼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line.

The shape of most complex organ systems arises from the directed migration of cohesive groups of cells. Here, we dissect the role of the chemokine guidance receptor Cxcr4b in regulating the collective migration of one such cohesive tissue, the zebrafish lateral line primordium. Using in vivo imaging, we show that the shape and organization of the primordium is surprisingly labile, and that internal cell movements are uncoordinated in embryos with reduced Cxcr4b signaling. Genetic mosaic experiments reveal that single cxcr4b mutant cells can migrate in a directional manner when placed in wild-type primordia, but that they are specifically excluded from the leading edge. Moreover, a remarkably small number of SDF1a-responsive cells are able to organize an entire cxcr4b mutant primordium to restore migration and organogenesis in the lateral line. These results reveal a role for chemokine signaling in mediating the self-organizing migration of tissues during morphogenesis.

Collective cell migration guided by dynamically maintained gradients.

How cell collectives move and deposit subunits within a developing embryo is a question of outstanding interest. In many cases, a chemotactic mechanism is employed, where cells move up or down a previously generated attractive or repulsive gradient of signalling molecules. Recent studies revealed the existence of systems with isotropic chemoattractant expression in the lateral line primordium of zebrafish. Here we propose a mechanism for a cell collective, which actively modulates an isotropically expressed ligand and encodes an initial symmetry breaking in its velocity. We derive a closed solution for the velocity and identify an optimal length that maximizes the tissues' velocity. A length dependent polar gradient is identified, its use for pro-neuromast deposition is shown by simulations and a critical time for cell deposition is derived. Experiments to verify this model are suggested.

Going your own way: Self-guidance mechanisms in cell migration.

How cells and tissues migrate from one location to another is a question of significant biological and medical relevance. Migration is generally thought to be controlled by external hardwired guidance cues, which cells follow by polarizing their internal locomotory machinery in the imposed direction. However, a number of recently discovered 'self-guidance' mechanisms have revealed that migrating cells have more control over the path they follow than previously thought. Here, directional information is generated by the migrating cells themselves via a dynamic interplay of cell-intrinsic and -extrinsic regulators. In this review, we discuss how self-guidance can emerge from mechanisms acting at different levels of scale and how these enable cells to rapidly adapt to environmental challenges.

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis.

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe the function of Smoothelin-like 2 (SMTNL2), a member of the smooth-muscle-related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during development in multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of coronin-1B. Although coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular cortex.

ErbB2 and ErbB3 regulate amputation-induced proliferation and migration during vertebrate regeneration.

Epimorphic regeneration is a unique and complex instance of postembryonic growth observed in certain metazoans that is usually triggered by severe injury [Akimenko et al., 2003; Alvarado and Tsonis, 2006; Brockes, 1997; Endo et al., 2004]. Cell division and migration are two fundamental biological processes required for supplying replacement cells during regeneration [Endo et al., 2004; Slack, 2007]. However, the connection between the early stimuli generated after injury and the signals regulating proliferation and migration during regeneration remain largely unknown. Here we show that the oncogenes ErbB2 and ErbB3, two members of the EGFR family, are essential for mounting a successful regeneration response in vertebrates. Importantly, amputation-induced progenitor proliferation and migration are significantly reduced upon genetic and/or chemical modulation of ErbB function. Moreover, we also found that NRG1 and PI3K functionally interact with ErbB2 and ErbB3 during regeneration and interfering with their function also abrogates the capacity of progenitor cells to regenerate lost structures upon amputation. Our findings suggest that ErbB, PI3K and NRG1 are components of a permissive switch for migration and proliferation continuously acting across the amputated fin from early stages of vertebrate regeneration onwards that regulate the expression of the transcription factors lef1 and msxB.

The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and Cxcr4b.

Tissue migration is a collective behavior that plays a key role in the formation of many organ systems. Although tissue movements are guided by extrinsic cues, in many contexts, their receptors need to be active only at the leading edge to ensure morphogenesis. This has led to the prevalent view that extrinsic signals exert their influence by controlling a small number of leader cells. The zebrafish lateral-line primordium is a cohesive cohort of over 100 cells that is guided through CXCR4-SDF1 signaling. Recent work has shown that Cxcr4b activity is only required in cells at the very tip, raising the question of what controls cell behavior within trailing regions. Here, we present the first mutant in zebrafish SDF1a/CXCL12a and show, surprisingly, that the resultant phenotype is stronger than a null mutation in its cognate receptor, Cxcr4b, indicating the involvement of other SDF1a receptors. A candidate approach identified Cxcr7/RDC1, whose expression is restricted to cells behind the leading edge. Morpholino knockdown of Cxcr7 leads to a novel phenotype in which the migration of trailing cells is specifically affected, causing tissue stretching, a defect rescued by the reintroduction of wild-type cells specifically at the back of the primordium. Finally, we present evidence that Cxcr4b and Cxcr7 act independently to regulate group migration. We provide the first example where a single extrinsic guidance cue, SDF1a, directly controls the migration of both leading and trailing edges of a tissue through the activation of two independent receptors, CXCR4b and CXCR7.

Getting back on track: exploiting canalization to uncover the mechanisms of developmental robustness.

Developing embryos can adapt dynamically to noise and variation to generate organs of incredible precision, a process termed 'canalization'; however, the underlying robustness mechanisms are poorly understood. Technological developments, both in quantitative imaging and high precision perturbation, are now enabling targeted investigation into developmental robustness in vivo. Here, we will first distil the common design features of studies that have exploited the canalization behaviour of specific systems to interrogate developmental adaptation, to provide a general experimental framework for future investigations in other contexts. We will then highlight, using a selection of recent case studies, how this approach is revealing that tissues and embryos can fix themselves in unexpected ways.

An image-based data-driven analysis of cellular architecture in a developing tissue.

Quantitative microscopy is becoming increasingly crucial in efforts to disentangle the complexity of organogenesis, yet adoption of the potent new toolbox provided by modern data science has been slow, primarily because it is often not directly applicable to developmental imaging data. We tackle this issue with a newly developed algorithm that uses point cloud-based morphometry to unpack the rich information encoded in 3D image data into a straightforward numerical representation. This enabled us to employ data science tools, including machine learning, to analyze and integrate cell morphology, intracellular organization, gene expression and annotated contextual knowledge. We apply these techniques to construct and explore a quantitative atlas of cellular architecture for the zebrafish posterior lateral line primordium, an experimentally tractable model of complex self-organized organogenesis. In doing so, we are able to retrieve both previously established and novel biologically relevant patterns, demonstrating the potential of our data-driven approach.

Dynamic Buffering of Extracellular Chemokine by a Dedicated Scavenger Pathway Enables Robust Adaptation during Directed Tissue Migration.

How tissues migrate robustly through changing guidance landscapes is poorly understood. Here, quantitative imaging is combined with inducible perturbation experiments to investigate the mechanisms that ensure robust tissue migration in vivo. We show that tissues exposed to acute "chemokine floods" halt transiently before they perfectly adapt, i.e., return to the baseline migration behavior in the continued presence of elevated chemokine levels. A chemokine-triggered phosphorylation of the atypical chemokine receptor Cxcr7b reroutes it from constitutive ubiquitination-regulated degradation to plasma membrane recycling, thus coupling scavenging capacity to extracellular chemokine levels. Finally, tissues expressing phosphorylation-deficient Cxcr7b migrate normally in the presence of physiological chemokine levels but show delayed recovery when challenged with elevated chemokine concentrations. This work establishes that adaptation to chemokine fluctuations can be "outsourced" from canonical GPCR signaling to an autonomously acting scavenger receptor that both senses and dynamically buffers chemokine levels to increase the robustness of tissue migration.

Migration and function of a glial subtype in the vertebrate peripheral nervous system.

Glia-axon interactions are essential for the development and function of the nervous system. We combine in vivo imaging and genetics to address the mechanism by which the migration of these cells is coordinated during embryonic development. Using stable transgenic lines, we have followed the migration of one subset of glial cells and their target axons in living zebrafish embryos. These cells coalesce at an early stage and remain coupled throughout migration, with axons apparently pathfinding for glia. Mutant analysis demonstrates that axons provide instructive cues that are sufficient to control glial guidance. Furthermore, mutations in the transcription factor Sox10/cls uncouple the migration of axons and glia. Finally, genetic ablation of this glial subtype reveals an essential role in nerve fasciculation.

Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.