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How developing brains mechanically interact with the surrounding embryonic scalp layers (ie, epidermal and mesenchymal) in the preosteogenic head remains unknown. Between embryonic day (E) 11 and E13 in mice, before ossification starts in the skull vault, the angle between the pons and the medulla decreases, raising the possibility that when the elastic scalp is directly pushed outward by the growing brain and thus stretched, it recoils inward in response, thereby confining and folding the brain.Stress-release tests showed that the E11-13 scalp recoiled and that the in vivo prestretch prerequisite for this recoil was physically dependent on the brain (pressurization at 77-93 Pa) and on actomyosin and elastin within the scalp. In scalp-removed heads, brainstem folding was reduced, and the spreading of ink from the lateral ventricle to the spinal cord that occurred in scalp-intact embryos (with5 μL injection) was lost, suggesting roles of the embryonic scalp in brain morphogenesis and cerebrospinal fluid homeostasis. Under nonstretched conditions, scalp cell proliferation declined, while the restretching of the shrunken scalp rescued scalp cell proliferation.In the embryonic mouse head before ossification, a stretcher-compressor relationship elastically develops between the brain and the scalp, underlying their mechanically interdependent development.

Supplementary data contains additional methods and figures. The figures contain the following information: Supplementary Figure 1 extends Figure 1 providing evidence that Meflin is expressed in BM MSCs and PSCs in mice. Supplementary Figure 2 supports Figure 1 showing the development of the Meflin-ZDC mice. Supplementary Figure 3-5 extend Figure 1 providing additional evidence that Meflin is specifically expressed in PSCs and CAFs and its expression is regulated by calcipotriol. Supplementary Figure 6-9 support Figure 2 showing the detection of Meflin expression at the protein and mRNA levels in human and mouse PDAC. Supplementary Figure 10 and 11 support Figure 2 showing the data of single-cell RNA sequencing of CAFs isolated from PDAC and breast cancer mouse models. Supplementary Figure 12 supports Figure 3 showing the effects of Meflin depletion on the global gene expression profiles in mouse MSCs. Supplementary Figure 13 supports Figure 3 showing the analysis of tumors developed in WT and Meflin-KO KPC mice. Supplementary Figure 14 supports Figure 6 showing the development of the Meflin-CreERT2 mice. Supplementary Figure 15 and 16 support Figure 7 showing the effect of exogenous Meflin expression on MSC gene expression and the growth of xenograft tumors. Supplementary Figure 17 support Figure 7 showing the role of Meflin in collagen remodeling in mouse and human PDAC. Supplementary Table 1 and 2 show the description of the cohort of PDAC patients included in the analysis and univariate and multivariate Cox regression analysis of human PDAC cases.

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression.Significance:Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects.

In our previous study, fatty acid-binding protein 5 (FABP5) was expressed in septoclasts with long processes which are considered to resorb uncalcified matrix of the growth plate (GP) cartilage, and no apparent abnormalities were detected in the histo-architecture of the GP of FABP5-deficient (FABP5−/−) mice. Those finding lead us to hypothesize that another FABP can compensate the deletion of FABP5 in septoclasts of its gene-mutant mice. Based on the hypothesis, the present study examined the expression levels of several other FABPs in septoclasts and their morphology in FABP5−/− mouse tibiae. Processes of FABP5−/− septoclasts tend to be shorter than wild septoclasts. FABP4-positive septoclasts in FABP5−/− mice were more numerous than those cells in wild mice.Peroxisome proliferator-activated receptor (PPAR) γ was expressed in FABP4-positive septoclasts of FABP5−/− mice as well as mice administered with GW1929, a PPARγ agonist, suggesting that the occurrence of PPARγ induces an increase of FABP4-positive septoclasts. The present finding suggests that the functional exertion of FABP5 in septoclasts is supplemented by FABP4 in normal and FABP5−/− mice, and that the expression of FABP4 is up-regulated in accompany with PPARγ in FABP5−/− for maintenance of resorptive activity in the GP.

Long-chain fatty acids (LCFAs) and retinoic acid (RA) are abundant in the growth plates (GPs) of long bones; however, their roles have not been elucidated. We observed that epidermal fatty acid-binding protein (E-FABP/FABP5) with a high affinity for both LCFAs and RA is exclusively expressed in the septoclasts located at the chondro-osseous junction (COJ) of the GP.E-FABP expressed in septoclasts is involved in both LCFA metabolism and RA signaling as an intracellular transporter of both LCFAs and RA. Septoclasts with shortened cytoplasmic processes are associated with cartilage resorptive activity downregulation because of E-FABP deficiency or excess or deficiency of RA. In ontogeny, the septoclasts are differentiated from the pericytes and involved in the resorption of the uncalcified matrix of the cartilage templates in endochondral ossification.Septoclasts originate from pericytes and express E-FABP to play crucial roles in uncalcified matrix resorption by LCFA metabolism and RA signaling during endochondral ossification.

The inner/apical surface of the embryonic brain wall is important as a major site for cell production by neural progenitor cells (NPCs). We compared the mechanical properties of the apical surfaces of two neighboring but morphologically distinct cerebral wall regions in mice from embryonic day (E) E12–E14. Through indentation measurement using atomic force microscopy (AFM), we first found that Young’s modulus was higher at a concave-shaped apical surface of the pallium than at a convex-shaped apical surface of the ganglionic eminence (GE). Further AFM analysis suggested that contribution of actomyosin as revealed with apical surface softening by blebbistatin and stiffness of dissociated NPCs were both comparable between pallium and GE, not accounting for the differential apical surface stiffness. We then found that the density of apices of NPCs was greater, with denser F-actin meshwork, in the apically stiffer pallium than in GE. A similar correlation was found between the decreasing density between E12 and E14 of NPC apices and the declining apical surface stiffness in the same period in both the pallium and the GE. Thus, one plausible explanation for the observed difference (pallium > GE) in apical surface stiffness may be differential densification of NPC apices. In laser ablation onto the apical surface, the convex-shaped GE apical surface showed quicker recoils of edges than the pallial apical surface did, with a milder inhibition of recoiling by blebbistatin than in pallium. This greater pre-stress in GE may provide an indication of how the initially apically concave wall then becomes an apically convex “eminence.”

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression., ファイル公開：2020/10/01

In the developing central nervous system, cell departure from the apical surface is the initial and fundamental step to form the 3D, organized architecture. Both delamination of differentiating cells and repositioning of progenitors to generate outer radial glial cells (oRGs) contribute to mammalian neocortical expansion; however, a comprehensive understanding of their mechanisms is lacking. Here, we demonstrate that Lzts1, a molecule associated with microtubule components, promotes both cell departure events. In neuronally committed cells, Lzts1 functions in apical delamination by altering apical junctional organization. In apical RGs (aRGs), Lzts1 expression is variable, depending on Hes1 expression levels. According to its differential levels, Lzts1 induces diverse RG behaviors: planar division, oblique divisions of aRGs that generate oRGs, and their mitotic somal translocation. Loss-of-function of lzts1 impairs all these cell departure processes. Thus, Lzts1 functions as a master modulator of cellular dynamics, contributing to increasing complexity of the cerebral architecture during evolution., Outer radial glial cells (oRGs) are undifferentiated cells that divide in the subventricular zone during neurodevelopment, but the underlying mechanisms are not fully understood. Here the authors show that Lzts1 positively controls both neuronal delamination and generation of oRG-like cell types.

During brain enlargement between embryonic day (E) 11 and E13, within the limited mouse cranial space enclosed by the scalp consisting of epidermal and preosteogenic mesenchymal layers, the angle between the pons and the medulla decreases. This raises the possibility that the scalp, upon being pushed outwardly by the brain and stretched, in return inwardly recoils to confine and fold the brain. Our stress-releasing tests showed that the scalp recoiled to shrink more extensively at E12~13 than at E15~16 and that the in vivo pre-stretch prerequisite for this recoil response depended physically on the brain (pressurization at 77~93 Pa) and on actomyosin and elastin within the scalp layers. Under non-stretched conditions, scalp cell proliferation declined, while re-stretching of the shrunken scalp rescued proliferation. In scalp-removed heads, pons–medulla folding was reduced, and the spreading of ink from the lateral ventricle to the spinal cord that occurred in scalp-intact embryos (with >5 μl injection) was lost, suggesting that the scalp plays roles in brain morphogenesis and cerebrospinal fluid homeostasis. Thus, the brain and the scalp mechanically interact and collaborate.

The mammalian secondary palate is formed through complex developmental processes: growth, elevation, and fusion. Although it is known that the palatal elevation pattern changes along the anterior–posterior axis, it is unclear what molecules are expressed and whether their locations change before and after elevation. We examined the expression regions of molecules associated with palatal shelf elevation (Pax9, Osr2, and Tgfβ3) and tissue deformation (F-actin, E-cadherin, and Ki67) using immunohistochemistry and RT–PCR in mouse embryos at E13.5 (before elevation) and E14.5 (after elevation). Pax9 was expressed at significantly higher levels in the lingual/nasal region in the anterior and middle parts, as well as in the buccal/oral region in the posterior part at E13.5. At E14.5, Pax9 was expressed at significantly higher levels in both the lingual/nasal and buccal/oral regions in the anterior and middle parts and the buccal/oral regions in the posterior part. Osr2 was expressed at significantly higher levels in the buccal/oral region in all parts at E13.5 and was more strongly expressed at E13.5 than at E14.5 in all regions. No spatiotemporal changes were found in the other molecules. These results suggested that Pax9 and Osr2 are critical molecules leading to differences in the elevation pattern in palatogenesis.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized β-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

Rationale: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. Objective: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. Methods and Results: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-β, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-β and augments its intracellular signaling. Conclusions: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

SummaryMammalian neocortex exhibits a disproportionally “luxurious” representation of somatotopies in its lateral region, which depends on dorsal-to-ventral expansion of the pallium during development. Despite recent studies elucidating the molecular mechanisms underlying the cortical arealization/patterning, we know very little about how the cortex expands ventrally and the nature of the underlying force-generating events. We found that neurons born earliest (at embryonic day 10 [E10]) in the mouse pallium migrated ventrally and then extended corticofugal axons, which together formed a morphogenetic flow of the preplate that persists until E13. These neurons exerted pulling and pushing forces at the process and the soma, respectively. Ablation of these E10-born neurons attenuated both deflection of radial glial fibers (by E13) and extension of the cortical plate (by E14), which should occur ventrally, and subsequently shrank the postnatal neocortical map dorsally. This previously unrecognized preplate stream physically primes neocortical expansion and somatotopic map formation.

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle–dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 μm from the surface, which is compressed and elastic because of the apical surface’s contractility, laterally pushes the densely neighboring processes of non–M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor’s daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development., Author summary The development of large brain structures, such as the mammalian cerebral cortex, depends on the continuous and efficient production of cells by neural progenitor cells. Neural progenitor cells are elongated and span the developing brain wall. The nuclei and bodies of these cells move cyclically between the apical and basal surfaces, and they divide every time they reach the apical surface. While we understand how individual cells achieve this cycle, how the movements of several progenitor cells are coordinated with one another remains elusive. By using a combination of live imaging and mechanical experiments, coupled with mathematical simulations, we show that cell crowding at the apical surface, where progenitor cells divide, creates a subapical microzone that is compressed and elastic. We then show that when each mother cell rounds up, preparing for division, it pushes this elastic microzone laterally, thereby storing mechanical energy. After cell division, this mechanical energy is transferred to the daughter cells, propelling them along the axis of movement in the direction of the basal surface, in an energy-saving manner. Our mathematical simulations show that timely departure of newly generated daughter cells is critical for the overall tissue structure of the cerebral proliferative zone.

The thick outer subventricular zone (OSVZ) is characteristic of the development of human neocortex. How this region originates from the ventricular zone (VZ) is largely unknown. Recently, we showed that over-proliferation-induced acute nuclear densification and thickening of the VZ in neocortical walls of mice, which lack an OSVZ, causes reactive delamination of undifferentiated progenitors and invasion by these cells of basal areas outside the VZ. In this study, we sought to determine how VZ cells behave in non-rodent animals that have an OSVZ. A comparison of mid-embryonic mice and ferrets revealed: (1) the VZ is thicker and more pseudostratified in ferrets. (2) The soma and nuclei of VZ cells were horizontally and apicobasally denser in ferrets. (3) Individual endfeet were also denser on the apical (ventricular) surface in ferrets. (4) In ferrets, apicalward nucleokinesis was less directional, whereas basalward nucleokinesis was more directional; consequently, the nuclear density in the periventricular space (within 16μm of the apical surface) was smaller in ferrets than in mice, despite the nuclear densification seen basally in ferrets. These results suggest that species-specific differences in nucleokinesis strategies may have evolved in close association with the magnitudes and patterns of nuclear stratification in the VZ.