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Summary The mammalian brain contains few niches for neural stem cells (NSCs) capable of generating new neurons, whereas other regions are primarily gliogenic. Here we leverage the spatial separation of the sub-ependymal zone NSC niche and the olfactory bulb, the region to which newly generated neurons from the sub-ependymal zone migrate and integrate, and present a comprehensive proteomic characterization of these regions in comparison to the cerebral cortex, which is not conducive to neurogenesis and integration of new neurons. We find differing compositions of regulatory extracellular matrix (ECM) components in the neurogenic niche. We further show that quiescent NSCs are the main source of their local ECM, including the multi-functional enzyme transglutaminase 2, which we show is crucial for neurogenesis. Atomic force microscopy corroborated indications from the proteomic analyses that neurogenic niches are significantly stiffer than non-neurogenic parenchyma. Together these findings provide a powerful resource for unraveling unique compositions of neurogenic niches., Graphical Abstract, Highlights • Proteomics define the NSC niche-specific extracellular matrix • Detergent-solubility profiling reveals extracellular matrix architecture • Transglutaminase 2 regulates neurogenesis • Stiffness is increased in the neurogenic niches of the brain, The physical properties of stem cell niches are thought to mediate important regulatory functions. Here we provide a proteomic resource of the neural stem cell niche in comparison to gliogenic brain parenchyma, highlighting stiffness and the enzyme transglutaminase 2 as key regulators of neurogenesis.

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements.

Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements., eLife digest Neurons in the brain form an intricate network that follows a precise template. For example, in a young frog embryo, the neurons from the eyes send out thin structures, called axons, which navigate along a well-defined path and eventually connect with the visual centres of the brain. This journey requires the axons to take a sharp turn so they can wire with the right brain structures. Axons find their paths not only by following chemical signals but also by reacting to the stiffness of their environment. In an older frog embryo for instance, the brain is stiffer at the front, and softer at the back. As neurons from the eyes make their way through the brain, they turn to follow this gradient, moving away from stiffer areas towards the softer regions. Here, Thompson, Pillai et al. investigate when and how this stiffness gradient is established in frogs. To do so, a new technique was developed. Called time-lapse in vivo atomic force microscopy, the method measures how brain stiffness changes over time in a live embryo, while also taking images of the growing axons. The experiments show that the stiffness gradient arose within tens of minutes, just as the first ‘pioneering’ axons from the eyes began to grow across the brain. These axons then responded to the gradient, turning towards the softer tissue. Changes in the number of cells in the underlying brain tissue governed the formation of the gradient, with rapidly stiffening areas containing more cells than those that remained soft. In fact, using drugs that stop cells from dividing reduced both the mechanical gradient and the turning response of the axons. The technique developed by Thompson, Pillai et al. is a useful tool that can help elucidate how variations in stiffness control the brain wiring process. It could also be used to look into how other developmental or regenerative processes, such as the way neurons reconnect after injuries to the brain or spinal cord, may be regulated by mechanical tissue properties.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.

During development, normal functioning, as well as in certain pathological conditions, cells are influenced not only by biochemical but also by mechanical signals. Over the past two decades, atomic force microscopy (AFM) has become one of the key tools to investigate the mechanical properties and interactions of biological samples. AFM studies have provided important insights into the role of mechanical signaling in different biological processes. In this chapter, we introduce different applications of AFM-based force measurements, from experimental setup and sample preparation to data acquisition and analysis, with a special focus on nervous system mechanics. Combined with other microscopy techniques, AFM is a powerful tool to reveal novel information about molecular, cell, and tissue mechanics.

Development of the anterior-posterior axis is a self-organizing process in the absence of maternal cues in the mouse embryo

SummaryThe ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.

Ageing causes a decline in tissue regeneration owing to a loss of function of adult stem cell and progenitor cell populations1. One example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source of this loss of function is the stem cell ‘niche’—a set of cell-extrinsic cues that include chemical and mechanical signals3,4. Here we show that the OPC microenvironment stiffens with age, and that this mechanical change is sufficient to cause age-related loss of function of OPCs. Using biological and synthetic scaffolds to mimic the stiffness of young brains, we find that isolated aged OPCs cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, the proliferation and differentiation rates of OPCs are increased. We identify the mechanoresponsive ion channel PIEZO1 as a key mediator of OPC mechanical signalling. Inhibiting PIEZO1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the ageing CNS. We also show that PIEZO1 is important in regulating cell number during CNS development. Thus we show that tissue stiffness is a crucial regulator of ageing in OPCs, and provide insights into how the function of adult stem and progenitor cells changes with age. Our findings could be important not only for the development of regenerative therapies, but also for understanding the ageing process itself. Aged progenitor cells in the rat central nervous system can be made to behave as young cells by reducing the stiffness of the tissue microenvironment, or by inhibiting the mechanosensitive protein PIEZO1.