A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix.

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young's modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

Comparative Analysis of Brain Stiffness Among Amniotes Using Glyoxal Fixation and Atomic Force Microscopy.

Brain structures are diverse among species despite the essential molecular machinery of neurogenesis being common. Recent studies have indicated that differences in the mechanical properties of tissue may result in the dynamic deformation of brain structure, such as folding. However, little is known about the correlation between mechanical properties and species-specific brain structures. To address this point, a comparative analysis of mechanical properties using several animals is required. For a systematic measurement of the brain stiffness of remotely maintained animals, we developed a novel strategy of tissue-stiffness measurement using glyoxal as a fixative combined with atomic force microscopy. A comparison of embryonic and juvenile mouse and songbird brain tissue revealed that glyoxal fixation can maintain brain structure as well as paraformaldehyde (PFA) fixation. Notably, brain tissue fixed by glyoxal remained much softer than PFA-fixed brains, and it can maintain the relative stiffness profiles of various brain regions. Based on this method, we found that the homologous brain regions between mice and songbirds exhibited different stiffness patterns. We also measured brain stiffness in other amniotes (chick, turtle, and ferret) following glyoxal fixation. We found stage-dependent and species-specific stiffness in pallia among amniotes. The embryonic chick and matured turtle pallia showed gradually increasing stiffness along the apico-basal tissue axis, the lowest region at the most apical region, while the ferret pallium exhibited a catenary pattern, that is, higher in the ventricular zone, the inner subventricular zone, and the cortical plate and the lowest in the outer subventricular zone. These results indicate that species-specific microenvironments with distinct mechanical properties emerging during development might contribute to the formation of brain structures with unique morphology.

Brain-stiffness-mimicking tilapia collagen gel promotes the induction of dorsal cortical neurons from human pluripotent stem cells.

The mechanical properties of the extracellular microenvironment, including its stiffness, play a crucial role in stem cell fate determination. Although previous studies have demonstrated that the developing brain exhibits spatiotemporal diversity in stiffness, it remains unclear how stiffness regulates stem cell fate towards specific neural lineages. Here, we established a culture substrate that reproduces the stiffness of brain tissue using tilapia collagen for in vitro reconstitution assays. By adding crosslinkers, we obtained gels that are similar in stiffness to living brain tissue (150-1500 Pa). We further examined the capability of the gels serving as a substrate for stem cell culture and the effect of stiffness on neural lineage differentiation using human iPS cells. Surprisingly, exposure to gels with a stiffness of approximately 1500 Pa during the early period of neural induction promoted the production of dorsal cortical neurons. These findings suggest that brain-stiffness-mimicking gel has the potential to determine the terminal neural subtype. Taken together, the crosslinked tilapia collagen gel is expected to be useful in various reconstitution assays that can be used to explore the role of stiffness in neurogenesis and neural functions. The enhanced production of dorsal cortical neurons may also provide considerable advantages for neural regenerative applications.

Systematic profiling of spatiotemporal tissue and cellular stiffness in the developing brain.

Accumulating evidence implicates the significance of the physical properties of the niche in influencing the behavior, growth and differentiation of stem cells. Among the physical properties, extracellular stiffness has been shown to have direct effects on fate determination in several cell types in vitro. However, little evidence exists concerning whether shifts in stiffness occur in vivo during tissue development. To address this question, we present a systematic strategy to evaluate the shift in stiffness in a developing tissue using the mouse embryonic cerebral cortex as an experimental model. We combined atomic force microscopy measurements of tissue and cellular stiffness with immunostaining of specific markers of neural differentiation to correlate the value of stiffness with the characteristic features of tissues and cells in the developing brain. We found that the stiffness of the ventricular and subventricular zones increases gradually during development. Furthermore, a peak in tissue stiffness appeared in the intermediate zone at E16.5. The stiffness of the cortical plate showed an initial increase but decreased at E18.5, although the cellular stiffness of neurons monotonically increased in association with the maturation of the microtubule cytoskeleton. These results indicate that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. Taken together, our method profiles the stiffness of living tissue and cells with defined characteristics and can therefore be utilized to further understand the role of stiffness as a physical factor that determines cell fate during the formation of the cerebral cortex and other tissues.

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells.

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

The mammalian DM domain transcription factor Dmrta2 is required for early embryonic development of the cerebral cortex.

Development of the mammalian telencephalon is precisely organized by a combination of extracellular signaling events derived from signaling centers and transcription factor networks. Using gene expression profiling of the developing mouse dorsal telencephalon, we found that the DM domain transcription factor Dmrta2 (doublesex and mab-3-related transcription factor a2) is involved in the development of the dorsal telencephalon. Consistent with its medial-high/lateral-low expression pattern in the dorsal telencephalon, Dmrta2 null mutants demonstrated a dramatic reduction in medial cortical structures such as the cortical hem and the choroid plexus, and a complete loss of the hippocampus. In this mutant, the dorsal telencephalon also showed a remarkable size reduction, in addition to abnormal cell cycle kinetics and defective patterning. In contrast, a conditional Dmrta2 deletion in the telencephalon, which was accomplished after entry into the neurogenic phase, resulted in only a slight reduction in telencephalon size and normal patterning. We also found that Dmrta2 expression was decreased by a dominant-negative Tcf and was increased by a stabilized ß-catenin form. These data suggest that Dmrta2 plays pivotal roles in the early development of the telencephalon via the formation of the cortical hem, a source of Wnts, and also in the maintenance of neural progenitors as a downstream of the Wnt pathway.

IgSF molecule MDGA1 is involved in radial migration and positioning of a subset of cortical upper-layer neurons.

Mdga1, encoding a GPI-anchored immunoglobulin superfamily molecule containing an MAM domain, is expressed by a specific subset of neurons, including layer II/III projection neurons, in the mouse neocortex. To investigate the function of Mdga1 in corticogenesis, we generated Mdga1-deficient mice and backcrossed them to obtain a congenic background. Gross anatomy of the Mdga1-deficient brain at postnatal day (P) 14 showed no obvious phenotype. However, the migration of Mdga1-mutant neurons to the superficial cortical plate was clearly delayed. Most Mdga1-mutant neurons reached the lower portion of the upper cortical layer by embryonic day 18.5 and stayed there through P0. By P7, the location of the mutant cells was the same as wild-type. The location of Cux2-expressing upper-layer neurons in the cortical plate was largely unaffected. These observations indicated that Mdga1 is involved in the migration and positioning of a subset of cortical neurons and suggested that the radial migration of upper-layer neurons might be differentially regulated.

Paths, elongation, and projections of ascending chick embryonic spinal commissural neurons after crossing the floor plate.

The paths of embryonic chick spinal commissural neurons originating from the lumbosacral (LS) 2 spinal segment and born around Hamburger-Hamilton stage (HH) 18 were observed by labeling the axons with an in ovo electroporation method designed to limit the electroporated area to approximately one somite length. After crossing the floor plate, these axons followed two major paths, one ventral and one dorsal, and a minor path running between the major ones. These axons reached the brachial region by HH28, passed through the cervical region at HH29, and entered the medullary area by HH30. The dorsal axons entered the developing cerebellum by HH33, crossed the midline again, and spread into the rostral-ipsilateral area of the developing cerebellum so that most of them were confined to lobules II-III by HH39. A small population of ventrally running axons turned to enter the cerebellum, and the rest entered the superior medullary velum between the cerebellum and the midbrain. The LS2-originating axons that ascended ipsilaterally into the cerebellum followed a single path, and their extension was delayed compared with that of the commissural axons. Some of the ipsilateral axons innervated the cerebellum; the rest entered the superior medullary velum. These direct observations of the formation of part of the spinocerebellar projections in chick will be a useful reference for future analyses of the underlying mechanisms.

MDGA1, an IgSF molecule containing a MAM domain, heterophilically associates with axon- and muscle-associated binding partners through distinct structural domains.

Molecules belonging to the immunoglobulin superfamily (IgSF) are reported to be involved in intercellular communication in the developing nervous system. We have identified a novel GPI-anchored IgSF molecule containing a MAM (meprin, A5 protein, PTPmu) domain, named MDGA1, by screening for genes that are expressed by subpopulations of cells in the embryonic chick spinal cord. MDGA1 is selectively expressed by brachial LMCm motor neurons, some populations of DRG neurons, and interneurons. We found that MDGA1 interacts heterophilically with axon-rich regions, mainly through its MAM domain. Interestingly, MDGA1 also interacts with differentiating muscle through its N-terminal region, which contains Ig domains. These results suggest that MDGA1 functions in MDGA1-expressing nerves en route to and at their target site.