ECM Mechanics in Central Nervous System Morphogenesis Open Access

The shape of a tissue is often linked to its function, the chambers of the heart, or alveoli of the lungs, for example. Ensuring these tissues develop into the correct shape and structure (a process called morphogenesis) is an important part of development, and understanding how this morphogenesis occurs is a key question in developmental biology. This has been explored from many different angles, with a major focus on understanding the gene expression changes that drive proliferation, differentiation, migration, and apoptosis of cells.

More recently, the field has started to investigate the mechanical factors that drive the emergence of three-dimensional structures, including collective cellular behaviours and active and passive mechanical forces [1, 2]. The mechanics of morphogenesis have been well studied in some areas of developmental biology but remain poorly understood in others. One such area is the morphogenesis of the central nervous system (CNS). CNS development is characterized by several mechanical events, for example, the closure of the neural tube [3], cortical folding patterns on the surface of the brain [4, 5], and the formation of neural circuits [6, 7].

The mechanical forces at play during these morphogenic events can be found at a cellular, tissue, and extracellular level and can be transmitted across the developing tissue in a variety of ways (Fig. 1). For example, forces generated by the actomyosin cytoskeleton within a cell can be transmitted to other cells or to the extracellular matrix (ECM), and vice versa, mechanical signals from the extracellular space can be transmitted to the actomyosin cytoskeleton via the ECM. These signals can also be relayed via membrane-bound receptors and intracellular pathways to the nucleus in a process termed mechanotransduction. This transduction of mechanical forces into intracellular biochemical signals is often triggered by a “molecular clutch” mechanism. This is when external mechanical forces are sensed via the binding of ECM to the membrane receptor integrin. Integrins can then transduce these forces to the cytoskeleton and transmit molecular signals to the nucleus [8, 9].

Although more research has focused on cell-generated forces, it is important to study how cells generate, transmit, and sense forces through the cell membrane and out to their environment. How cells collectively sense and alter mechanical properties to help shape a developing tissue is still being explored, and the role of the ECM in this process is one area yet to be understood. It is becoming more evident that there is a need to bridge the gap between our current knowledge of neural tissue morphogenesis and how this process is regulated by ECM dynamics and extracellular forces.

In the last few decades, many advances have been made in understanding ECM composition and dynamics across different tissues, thanks to new technologies and multidisciplinary approaches that have allowed researchers to overcome several experimental limitations [10, 11]. The latest advances in mass spectrometry and proteomic technologies are revealing the diversity of the matrisome, which is defined as the collection of genes encoding ECM and ECM-associated proteins. The MatrisomeDB database provides important insight on the tissue-specific variation of ECM components in multicellular organisms [12, 13]. However, the composition of the ECM in the developing CNS has not yet been extensively studied. In this review, we will summarize what is known and focus on the major ECM components, their mechanical properties, and new advances in the technologies used to study their properties and regulation during CNS morphogenesis.

CNS morphogenesis starts when specialized cells in the ectoderm form the neuroepithelium. These cells will delaminate and give rise to neuroblasts, the precursors of glia and neurons [14, 15], and they are surrounded by ECM that provides the right microenvironment to instruct their behaviour and fate [16, 17]. This ECM is organized into three major types, each with a different composition: the interstitial matrix, the basement membrane, and perineuronal nets [18].

The interstitial matrix is secreted by both neuronal and glial cells and has a soft, flexible, and fibrous texture. This property is a result of the structural components it contains, like hyaluronan, proteoglycans, linker proteins, glycoproteins (tenascins, laminins, fibronectin), and fibrous proteins (elastin, collagens) [19, 20]. In addition to their role in signalling pathways, the network of proteoglycans and glycoproteins that is embedded in the interstitial matrix also contribute to the ability of the ECM to take up fluid. This is particularly important for morphogenesis as changes in tissue shape and structure can be buffered by changing the volume of the interstitial extracellular space, or by preserving the volume and effectively resisting the mechanical strain being generated [21, 22].

The basement membrane is a thin, specialized layer of the ECM, composed primarily of laminins and collagens [23]. In the CNS, the main basement membranes are found (i) on the outer, pial surface [24] (therefore called the pial basement membrane) and (ii) surrounding blood vessels [25] (the vascular basement membrane). Both of these basement membranes act as gatekeepers, regulating the flow of fluids and molecules between the vessels/meningeal space and the CNS parenchyma, while also providing structural support [18, 22]. The pial basement membrane surrounds the entire CNS and is mostly secreted by meningeal fibroblasts [24]. It separates the cells of the CNS from the meninges and cerebrospinal fluid (CSF) on the surface of the brain. The vascular basement membrane is crucial for the structure, function, and integrity of the blood-brain barrier. Both the pial and vascular basement membranes directly interact with the neuroepithelium and cells of the developing brain, and they are involved in transmitting external signals that can impact cell behaviour [26, 27]. This includes promoting proliferation and the neurogenic potential of radial glial progenitors early in development [28, 29].

Perineuronal nets are specialized ECM structures that surround the cell bodies, dendrites, and initial axon segments of inhibitory neurons in the CNS [30]. They form a dense network composed of proteoglycans and glycoproteins, alongside other molecules. These nets emerge postnatally and gradually mature over time [31] and play a vital role in memory modulation by supporting synaptic plasticity, maintaining homeostasis, and regulating inhibitory signals near neurons [32, 33]. As perineuronal nets appear later in development, this review will largely focus on the interstitial matrix and basement membrane.

The role that these three distinct types of ECM play in the morphogenesis of the developing CNS remains an open question. Understanding the functional and architectural diversity of the ECM in the CNS will enable researchers to learn more about the impact it has on tissue dynamics and morphogenesis across the scales, including its role in tissue mechanics. In recent years, it has become evident that the ECM is not only a passive scaffold but also an important source of forces. Based on its composition, the ECM exhibits several mechanical properties, such as viscoelasticity, tensile and compressive strength, topology, and stiffness. All of these properties can influence morphogenesis, for example, via altering neural cell movements and morphology [34].

There is growing evidence for the role of mechanical forces in neural development. The overall stiffness of neural tissue changes over the course of development; the adult human brain has a stiffness in the range of 1 kPa [35], whereas the foetal brain is much less stiff, in the range of 100 Pa [36]. In addition, it has been shown that ECM-generated forces, for example, the pressure generated from the swelling of hyaluronic acid, play a crucial role in the formation of cortical folds [36], similar to its role in the morphogenesis of other organs [37]. These studies highlight the need for a comprehensive understanding of how the ECM and mechanical forces act in unison to orchestrate aspects of CNS morphogenesis (Fig. 2).

In this review, we will focus on the role of different ECM components in CNS morphogenesis, in particular those that contribute to the mechanical properties of the ECM (collagens, elastin, glycosaminoglycans [GAGs], and proteoglycans), and how the ECM is regulated during this morphogenesis. We will also highlight the key areas where the field currently has limited information and where novel insights could help us fill this gap in our knowledge.

Rigidity and resistance to stretching are properties required in tissues and organs that undergo mechanical overloading, such as skin, bones, muscles, or tendons. These properties are provided by the most abundant ECM protein in the human body: collagens. The mechanical properties of collagens have been measured in vitro, including stiffness, tensile modulus, and strain. These versatile properties of collagens are crucial to both determine the stiffness of a tissue [38] and to respond to it. Collagens play a crucial role in mechanotransduction, being one of the first proteins to sense mechanical stress [39]. They can remodel under strain, a process that involves cross-linking and reorientation of fibres as well as degradation and synthesis of new fibres. This makes collagen fibres crucial players in connective tissue homeostasis and morphogenesis. It has been reported that the half-life of collagen varies across different organs and throughout the lifespan [40], suggesting that collagen turnover may be tailored in a tissue-specific manner and according to the mechanical fatigue present [39]. The role of collagens in tissues with limited mechanical fatigue, such as the CNS, has been less well explored.

Collagens are still the most represented ECM component in tissues that are less stiff, such as the CNS [41]. One reason for this is that collagens are both part of the basement membrane (collagen IV, VI) and the interstitial matrix (collagen I, collagen VI, XVII). This enables collagens to have many structural and mechanical roles across many different aspects of development, such as neuronal migration, maturation, and synaptogenesis [42]. Where collagen is produced in the CNS has been extensively reviewed elsewhere [41], and it has been reported that many of the CNS progenitors and neuronal and glial cells secrete it. Collagens are also secreted into CNS tissues from the meningeal layer of cells that surround the brain and the spinal cord in vertebrates. In particular, these cells express and secrete high amount of fibrous collagens [43, 44] throughout development. However, the exact spatial and temporal dynamics of this collagen expression are still poorly understood.

The spatial and temporal abundance of collagens has also been hypothesized to correlate with morphogenic events during neural crest emergence in avian embryos [45]. Specifically, it has been suggested that collagen I binding with fibronectins in the interstitial matrix may alter the 3D structure and functionality of the ECM as neural crest cells migrate along it. In addition, the distribution of collagen IV and laminin, vital components of the basement membrane, aligns with the tissue remodelling that takes place during neural crest cells migration and organization into ganglia [45]. More recently, it has been shown that the assembly of collagen IV initiates ventral nerve cord morphogenesis in Drosophila in a cellular-independent manner [46], suggesting a potential role for mechanical forces.

To modulate the mechanical properties of a tissue, collagens require direct interactions with other ECM components such as fibronectin, the first protein sensing mechanical stress [47, 48], or elastin. These interactions are important for the construction of the ECM network; for example, fibroblasts will first deposit relaxed fibronectin fibres to act as a template for collagen I assembly. However, once collagen I assembly is complete, fibronectin is no longer able to sense mechanical stress, suggesting a reciprocal mechanoregulation occurs [49]. Investigating these interactions during morphogenesis could uncover more about the mechanical forces at play and how the cells receive them, especially given the high presence of collagen and fibronectin in many different organs during development.

Elastin is the ECM protein that confers biological tissues the ability to withstand a deformation and be able to return to its original shape, the property of elasticity. It has been shown that elastin is present in the interstitial matrix of the adult brain, with high levels of elastin-derived peptides in the CSF during ageing [50], after ischemic stroke [51], and increased levels in neurodegenerative disorders, such as Alzheimer’s disease [52, 53]. However, there is currently little evidence of the presence of elastin in the developing CNS. As discussed above, the human foetal brain appears to be less stiff than the adult brain, suggesting that there may be a change in the composition of the ECM, one of which could be elastin levels.

Alternatively, we may not have collected data from the correct time point in CNS development to detect elastin abundance. Understanding when elastin becomes abundant in the CNS will help unravel its function in morphogenesis. It is known that elastic fibres confer structural stability to tissue, and there is evidence from other embryonic organs in different model systems that elastin and the proteins related to elastin remodelling play a crucial role during morphogenesis. For example, during gut looping in the developing chick embryo there is upregulation of elastic fibre-related genes [54], and in the developing zebrafish heart elastin is detected in the valve where more mechanical strength is required [55]. How the spatial and temporal abundance of elastin is regulated alongside these morphogenic events is an interesting question to explore and could help inform the timepoints of CNS development to examine further.

Elasticity is an important component of biological tissues, but the composition of the ECM confers other properties that prevents these tissues from being considered completely elastic. This is largely due to the interactions between collagens and elastin with glycoproteins, proteoglycans, and their chains, the GAGs, which are present in the interstitial matrix as well as in the basement membrane. Proteoglycans provide a scaffold for the attachment of collagen and elastin fibres, influencing tissue elasticity [56]. They also have an active role in protecting collagen fibres from overstretching [57]. However, proteoglycans can also attract and retain large amounts of water, due to the presence of negatively charged GAG chains, forming a hydrated gel that provides mechanical support and hydration to the tissue [58]. This interaction with water helps to maintain structural integrity and compressibility and makes these tissues viscoelastic.

Examples of these proteoglycan interactions can be found in decorin, which can regulate the organization and diameter of collagen fibres [59], and versican, which interacts with hyaluronan and/or hyaluronic acid [60]. The above-mentioned examples have been mostly studied in skin or blood vessels; however, the same proteins can be found in abundance in the CNS, with the chondroitin sulphate proteoglycans being the most abundant [61]. These glycans are essential to maintain the functional and structural integrity of the CNS tissue. They have known roles in cell differentiation, migration, proliferation, cell adhesion, synaptic plasticity, and axonal growth and have been increasingly studied over the past years [62‒64]. In particular, chondroitin sulphate proteoglycans restrict cell migration and limit axonal growth [65], whereas heparan sulphate proteoglycans promote neuronal precursor cell proliferation [66] and contribute to axon pathfinding [67].

Later in development, chondroitin sulphate proteoglycans are the main components of perineuronal nets, playing an important role in synaptogenesis and synaptic plasticity [68]. In addition, proteoglycans are also involved in glial scar formation [69] after CNS lesions. Although scar formation can initiate the healing process, it has been shown that after CNS injury, small leucine-rich proteoglycans are enriched in the areas of injury in both human and rodent tissue but not in the highly regenerative zebrafish. Their presence is thought to impede axon regeneration by altering tissue mechanics [70], and their inhibition could potentially provide a therapeutic target.

Taken together, all these studies highlight a gap in our knowledge of the physical and mechanical attributes of glycans in the CNS, aside from providing simple structural support. Understanding how they contribute to the mechanical properties of the tissue could uncover novel roles of these families of ECM components in the morphogenesis of the CNS, as well as other tissues during development.

To enable the large-scale cell and tissue movements required for morphogenesis, the ECM must be able to remodel quickly. This remodelling has been implicated in organ morphogenesis during development, for example, in the intestine [71], ear canal [37], and brain [36]. Within the brain, it has been reported that the ECM composition and distribution does indeed change over time; sulphated GAGs represent up to 60% of brain mass during embryogenesis but only 20% in the adult [72]. It has therefore been suggested that ECM remodelling is important to ensure tissue homeostasis, including balancing of the mechanical properties of the tissue, and that this may play an important role in tissue growth and morphogenesis during the development of the CNS.

ECM dynamics are tightly regulated by enzymes, such as matrix metalloproteinases (MMPs), disintegrin and metalloproteinase with thrompospondin type I motifs (ADAMTs), hyaluronidase/chondroitinases, and their tissue-specific inhibitors (TIMPs or tissue inhibitors of metalloproteinases). Changing the ECM composition will most likely influence mechanical forces, and interestingly vice versa, mechanical forces also appear to influence ECM dynamics. In vitro studies have shown that ECM dynamics are indeed regulated by mechanical forces in some systems. For example, osteoblastic cells upregulate the expression of MMPs and TIMPs when exposed to mechanical strain [73]. In a more recent study in zebrafish, it has been shown that mechanical forces control the remodelling of cardiac ECM, in part through TIMP downregulation [74].

In the context of CNS morphogenesis, Small and Crawford have extensively described the role of MMPs in different model organisms in their review [75]. Notably, the level of expression of MMPs and ADAMs in the CNS is different during development compared to the adult, highlighting how highly dynamic the ECM is in this region. In the adult human brain, most of the MMPs are absent and only upregulated in pathological conditions [76]. Although ADAMs are still present in the adult brain, the lack of MMPs suggests ECM remodelling may play a more prominent role in development, a critical period for morphogenesis. This raises the question of how the degree of ECM remodelling may be linked to the differences in mechanical properties reported in developing CNS tissues compared to the adult. We currently know relatively little about the mechanical properties of the CNS, or how they impact ECM remodelling. Further experiments, and possible novel methods, are required to unravel the role mechanical forces play in regulating ECM dynamics in these complex CNS tissues.

The biochemical and mechanical properties of ECM have been well studied in vitro and in 2D, and these data have resulted in the development of new materials and have uncovered key aspects of ECM in development. There is now a growing movement to investigate the same biochemical and mechanical properties in 3D and complex tissues to better understand their role in tissue morphogenesis. This is often not an easy experiment to design, especially in vivo, with many technical difficulties limiting success. For this reason, computational models have been used to help understand how mechanical properties could impact CNS tissue morphogenesis (Fig. 2). In the last few decades, several mathematical and computational models of CNS morphogenesis have been developed, which have been extensively reviewed elsewhere [77, 78].

A common conundrum for many computational models is that they require some level of input data, ideally real-life measurements of the tissue and forces being modelled, but these data are not readily available, hence the need for computational models to investigate the same question. Technological advances over the last few decades have helped to bridge this gap between real-life data and modelling of CNS development. Recent advances in movement correction algorithms have enabled high-resolution images of foetal development to be taken using magnetic resonance imaging (MRI), capturing fundamental morphometric information of the developing human brain [79]. Much of these data are collected for clinical reasons, including changes in morphogenesis, with a focus on tissue structure. For example, in utero MRI is used to quantify cortical overgrowth in foetuses with isolated ventriculomegaly or to quantify and map local tissue growth patterns underlying gyrification during healthy cortical development [80‒82]. These findings and foetal MRI data are being combined with computational models to help map the developing brain but also to start to make predictions on typical versus atypical development [83]. This could be further enhanced by incorporating mechanical measurements from ex vivo and/or in vitro studies, to better model tissue morphogenesis, such as cortical folding.

Another mechanical force in the developing CNS that is often understudied is fluid flow and pressure. The ventricles of the early CNS are filled with a specialized fluid, the CSF, which is a critical player in CNS development and is one of the major physical contributors of flow and pressure. Following the closure of the neural tube, the ventricular system and the spinal cord are formed and quickly fill with CSF. The flow rate and pressure of this CSF are driven by differences in the pressure gradient created by the secretion of the CSF and the exchange with the interstitial fluid, together with cardiac, respiratory, and body movements. The CSF is composed of different molecules, such as enzymes, growth factors, cytokines, and ECM proteins. Interestingly, embryonic CSF is rich in chondroitin sulphate proteoglycans, which facilitate the water retention in the ventricles, thereby generating the hydrostatic pressure that drives the expansion of the brain [84, 85]. At a cellular level, motile cilia line the ventricular and spinal cord inner surface and partially contribute to the fluid flow.

The importance of fluid flow during development has been extensively reviewed by Daems et al. [86]. Given that several factors influence CSF flow, decoupling the individual contribution to it remains significantly challenging. However, it would be very interesting to individually model how each of these changes, such as mechanical pressure and viscosity, could affect neural development (Fig. 2). In addition to this, changes in cardiac pulse affected by various conditions and/or inflammation during gestation that ultimately influence CSF flow could also be quantified in humans but also modelled in other ex vivo and/or in vitro systems. Taking advantage of the principles underlying MRI, the mechanical properties of tissue stiffness and viscoelasticity of organs can be measured in real time using magnetic resonance elastography (MRE) [87]. This is currently not possible in foetal MRI, but with further advances in the field we may be able to measure such properties in utero, gaining invaluable data not only for modelling but for our understanding of the mechanical factors at play in CNS development.

In this review, we have focused on the role of the mechanical properties of the ECM during CNS morphogenesis (Fig. 1). In the last few decades, it has been evident that the ECM is not simply a passive, supportive substrate but an important and instructive player during morphogenesis. A clear gap in our knowledge of CNS morphogenesis is understanding what mechanical factors are at play and how these factors influence ECM dynamics and vice versa how ECM dynamics alter the mechanical forces. More specifically, this should be investigated in all three different ECM types as most of the past research has been focused on the basement membrane. Bridging the gap in our knowledge is particularly needed for morphogenesis in 3D and complex tissues/embryos. In this context, further advancement in imaging techniques and methods to measure mechanical forces, together with computational models (Fig. 2), will be needed to shed light on the currently poorly accessible timepoints of CNS development. Understanding more about how the CNS is sculpted during development will not only help further our understanding of the factors influencing this development, but also those leading to developmental malformations.

The authors declare no conflict of interest.

The authors would like to thank the following funding sources: the Medical Research Council MR/S025065/1 to K.R.L., Heart of Racing to K.R.L. and the European Molecular Biology Organization (EMBO) Postdoctoral Fellowship ALTF 609-2022 to A.G.

A.G., K.S., and K.R.L. wrote and edited the manuscript. A.G. and K.S. made the illustrations.