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The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons. The removal of dying neurons by microglia plays a key role in both vertebrate nervous system development and several diseases. Here, the authors use a quantitative live imaging approach to investigate neuronal-microglial interactions at single-cell resolution and establish the functions of the phosphatidylserine receptors, TIM-4 and BAI1, in neuronal engulfment.

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.

Organisms are far greater than the sum of their differentiated cells, as the function of most cell types emerges from their organisation into three-dimensional tissues. Yet, the mechanisms underlying architectural diversity remain poorly understood, partly due to a lack of methods for directly comparing different tissue organisations. Here we establish nuQLOUD, an efficient imaging and computational framework that reduces complex tissues to clouds of nuclear positions, enabling the extraction of cell-type agnostic architectural features. Applying nuQLOUD to whole zebrafish embryos reveals that global tissue diversity can be efficiently reduced to two archetypes, termed ‘amorphous’ and ‘crystalline’. We investigate the role of cadherin cell adhesion molecules in controlling organisational diversity and demonstrate that their expression segregates along tissue-archetypal lines. Targeted perturbations identify N-cadherin as a general driver of the amorphous archetype. This organisation-centric approach provides a way to conceptualise tissue diversification and investigate the underlying mechanisms within a standardised, quantitative framework. Using whole-embryo imaging and point cloud analysis, Brambach et al. reveal that global tissue diversity can be reduced to two organisational archetypes, crystalline and amorphous, each requiring specific cadherin expression during development.

It is widely accepted that migrating cells and tissues navigate along pre-patterned chemoattractant gradients; here it is shown that migrating tissues can also determine their own direction by generating local gradients of chemokine activity, via polarized receptor-mediated internalization, that are sufficient to ensure robust collective migration. Cells that know their place The currently accepted view of how cells migrate directionally over long distances — an important driving force in embryogenesis — is that they navigate using pre-patterned chemoattractant guidance gradients. In this study Darren Gilmour and colleagues present the first in vivo evidence for a rather different mechanism: self-generated guidance gradient formation. Using zebrafish lateral line primordium as a model for collective cell migration, the authors show that migrating tissues can determine their own direction by generating local gradients in initially uniform extracellular guidance cues, producing a travelling wave. The atypical chemokine receptor Cxcr7 is the key regulator of the process, being both necessary and sufficient for self-directed migration. The finding that cells can autonomously determine their migration routes could have wider implications in processes such as cancer metastasis. The directed migration of cell collectives is a driving force of embryogenesis 1 , 2 , 3 . The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients 2 . 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 4 , 5 , 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 6 .

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.

Key Points Collective cell migration is defined as the coordinated movement of multiple cells that retain cell–cell contacts while coordinating their actin dynamics and intracellular signalling. Because the cells form a structural and functional unit, both active and passive cell translocation occur. The movement of connected cells contributes to morphogenesis, wound healing and cancer invasion and each process underlies homologous but distinct molecular mechanisms of cell–cell interaction and pro-migratory extracellular signalling. The concept of collective movement explains how the body forms and reshapes as well as how cancer cells destructively invade as a 'socially' organized mass. The collective migration of cells as cohesive groups is prevalent during embryogenesis, organ development, wound repair and tumour invasion. The mechanisms that underlie different forms of collective cell migration are not well understood, but some general principles are emerging. 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.

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.

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.

Groups of cells within a migrating collective assemble shared luminal cavities that trap and concentrate the signalling molecule fibroblast growth factor, providing a self-organising mechanism to focus and coordinate cell communication within tissues. How embryo cells get it together The developing embryo assembles complex tissues and organs through the coordinated differentiation of cell groups, a collective process that depends on highly efficient cell communication. How cells in growing tissues exert control on morphogens that direct their fate and are secreted into extracellular spaces has been unclear. Using live imaging Darren Gilmour and colleagues show that cells in the developing lateral line system of zebrafish arrange themselves in shared microenvironments or microlumina so that signals — such as fibroblast growth factor (FGF) — are more concentrated in some areas than in others. This allows a coordinated response from nearby cells according to their position, a feedback process that in turn influences the arrangement of the cells and propels further development. Morphogenesis is the process whereby cell collectives are shaped into differentiated tissues and organs 1 . 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 2 , optic-cups 3 and even mini-brains 4 . 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 5 . 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 6 and embryonic stem cells 7 , luminal signalling provides a potentially general mechanism to locally restrict, coordinate and enhance cell communication within tissues.

Microglia engulf dying neurons through efferocytosis, a critical function in both development and disease. How microglia process the engulfed neuronal material-especially lipids-remains poorly understood, despite its central role in neurodegeneration. Thus, we developed HuZIBRA, a scalable in vivo xenotransplantation model in which human iPSC-derived microglia-like cells (iMGLs) are introduced into the developing zebrafish brain (zf-hiMG), a system characterized by high levels of neuronal cell death and amenable to precise genetic and pharmacological manipulation. We show that human microglia-like cells recognize and engulf apoptotic zebrafish neurons, indicating conserved efferocytic mechanisms. In these cells, engulfed neuronal material accumulates into a distinct, lipid-rich intracellular compartment, the gastrosome, which we also observed in iMGLs placed in a human brain-like environment. The size of the human gastrosome dynamically reflects neuronal cell death levels and is regulated by key genes, including TREM2 and SLC37A2. Pharmacological inhibition of the cholesterol transporter NPC1 induces gastrosome expansion and lipid accumulation, recapitulating pathological features of Niemann-Pick disease type C. Thus, HuZIBRA provides a powerful in vivo platform to uncover cell-autonomous adaptive responses of human microglia to apoptotic and metabolic stress, with the gastrosome emerging as a key integrator of neuronal debris processing and disease-relevant lipid metabolism.