Rare genetic variation in fibronectin 1 (FN1) protects against APOEε4 in Alzheimer's disease.

The risk of developing Alzheimer's disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4-mediated AD pathology. To test this, we leveraged whole-genome sequencing (WGS) data in the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain (COL6A2) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. An independent analysis in a large cohort of 7185 APOEε4 homozygous carriers found that rs140926439 variant in FN1 was protective of AD (OR = 0.29; 95% CI [0.11, 0.78], P = 0.014) and delayed age at onset of disease by 3.37 years (95% CI [0.42, 6.32], P = 0.025). The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4-mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b-the ortholog for human FN1. We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling, and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests that vascular deposition of FN1 is related to the pathogenicity of APOEε4, and LOF variants in FN1 may reduce APOEε4-related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.

Segmental assembly of fibronectin matrix requires rap1b and integrin α5.

BACKGROUND: During segmentation of the zebrafish embryo, inside-out signaling activates Integrin α5, which is necessary for somite border morphogenesis. The direct activator of Integrin α5 during this process is unknown. One candidate is Rap1b, a small monomeric GTPase implicated in Integrin activation in the immune system. RESULTS: Knockdown of rap1b, or overexpression of a dominant negative rap1b, causes a mild axis elongation defect in zebrafish. However, disruption of rap1b function in integrin α5(-/-) mutants results in a strong reduction in Fibronectin (FN) matrix assembly in the paraxial mesoderm and a failure in somite border morphogenesis along the entire anterior-posterior axis. Somite patterning appears unaffected, as her1 oscillations are maintained in single and double morphants/mutants, but somite polarity is gradually lost in itgα5(-/-) ; rap1b MO embryos. CONCLUSIONS: In itgα5(-) (/) (-) mutants, rap1b is required for proper somite border morphogenesis in zebrafish. The loss of somite borders is not a result of aberrant segmental patterning. Rather, somite boundary formation initiates but is not completed, due to the failure to assemble FN matrix along the nascent boundary. We propose a model in which Rap1b activates Integrin/Fibronectin receptors as part of an "inside-out" signaling pathway that promotes Integrin binding to FN, FN matrix assembly, and subsequent stabilization of morphological somite boundaries.

Live imaging of Fibronectin 1a-mNeonGreen and Fibronectin 1b-mCherry knock-in alleles during early zebrafish development.

Within the developing embryo, cells assemble and remodel their surrounding extracellular matrix during morphogenesis. Fibronectin is an extracellular matrix glycoprotein and is a ligand for several members of the Integrin adhesion receptor family. Here, we compare the expression pattern and loss of function phenotypes of the two zebrafish fibronectin paralogs fn1a and fn1b. We engineered two fluorescently tagged knock-in alleles to facilitate live in vivo imaging of the Fibronectin matrix. Genetic complementation experiments indicate that the knock-in alleles are fully functional. Fn1a-mNeonGreen and Fn1b-mCherry are co-localized in ECM fibers on the surface of the paraxial mesoderm and myotendinous junction. In 5-days old zebrafish larvae, Fn1a-mNeonGreen predominantly localizes to the branchial arches, heart ventricle, olfactory placode and within the otic capsule while Fn1b-mCherry is deposited at the pericardium, proximal convoluted tubule, posterior hindgut and at the ventral mesoderm/cardinal vein. We examined Fn1a-mNeonGreen and Fn1b-mCherry in maternal zygotic integrin α5 mutants and integrin ß1a; ß1b double mutants and find distinct requirements for these Integrins in assembling the two Fibronectins into ECM fibers in different tissues. Rescue experiments via mRNA injection indicate that the two fibronectins are not fully inter-changeable. Lastly, we examined cross-regulation between the two Fibronectins and find fn1a is necessary for normal Fn1b fibrillogenesis in the presomitic mesoderm, but fn1b is dispensable for the normal pattern of Fn1a deposition.

Rare genetic variation in Fibronectin 1 ( FN1 ) protects against APOEe4 in Alzheimer's disease.

The risk of developing Alzheimer's disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4 ; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4- mediated AD pathology. To test this, we leveraged whole genome sequencing (WGS) data in National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain ( COL6A2 ) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4 -mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b - the ortholog for human FN1 . We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests vascular deposition of FN1 is related to the pathogenicity of APOEε4 , LOF variants in FN1 may reduce APOEε4 -related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.

Automated time-lapse data segmentation reveals in vivo cell state dynamics.

Embryonic development proceeds as a series of orderly cell state transitions built upon noisy molecular processes. We defined gene expression and cell motion states using single-cell RNA sequencing data and in vivo time-lapse cell tracking data of the zebrafish tailbud. We performed a parallel identification of these states using dimensional reduction methods and a change point detection algorithm. Both types of cell states were quantitatively mapped onto embryos, and we used the cell motion states to study the dynamics of biological state transitions over time. The time average pattern of cell motion states is reproducible among embryos. However, individual embryos exhibit transient deviations from the time average forming left-right asymmetries in collective cell motion. Thus, the reproducible pattern of cell states and bilateral symmetry arise from temporal averaging. In addition, collective cell behavior can be a source of asymmetry rather than a buffer against noisy individual cell behavior.

Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses. Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction. However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging. Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity. We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1-dependent stimuli from the single-cell level to that of the entire organism. GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo. GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.

Essential roles of fibronectin in the development of the left-right embryonic body plan.

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left-right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5ß1, is also required for the development of the left-right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left-right axis of asymmetry in vertebrates.

Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling.

Extracellular matrixes (ECMs) coat and subdivide animal tissues, but it is unclear how ECM formation is restricted to tissue surfaces and specific cell interfaces. During zebrafish somite morphogenesis, segmental assembly of an ECM composed of Fibronectin (FN) depends on the FN receptor Integrin alpha5beta1. Using in vivo imaging and genetic mosaics, our studies suggest that incipient Itgalpha5 clustering along the nascent border precedes matrix formation and is independent of FN binding. Integrin clustering can be initiated by Eph/Ephrin signaling, with Ephrin reverse signaling being sufficient for clustering. Prior to activation, Itgalpha5 expressed on adjacent cells reciprocally and non-cell-autonomously inhibits spontaneous Integrin clustering and assembly of an ECM. Surface derepression of this inhibition provides a self-organizing mechanism for the formation and maintenance of ECM along the tissue surface. Within the tissue, interplay between Eph/Ephrin signaling, ligand-independent Integrin clustering and reciprocal Integrin inhibition restricts de novo ECM production to somite boundaries.

Integrinalpha5 and delta/notch signaling have complementary spatiotemporal requirements during zebrafish somitogenesis.

Somitogenesis is the process by which the segmented precursors of the skeletal muscle and vertebral column are generated during vertebrate embryogenesis. While somitogenesis appears to be a serially homologous, reiterative process, we find that there are differences between the genetic control of early/anterior and late/posterior somitogenesis. We demonstrate that point mutations can cause segmentation defects in either the anterior, middle, or posterior somites in the zebrafish. We find that mutations in zebrafish integrinalpha5 disrupt anterior somite formation, giving a phenotype complementary to the posterior defects seen in the notch pathway mutants after eight/deltaD and deadly seven/notch1a. Double mutants between the notch pathway and integrinalpha5 display somite defects along the entire body axis, with a complete loss of the mesenchymal-to-epithelial transition and Fibronectin matrix assembly in the posterior. Our data suggest that notch- and integrinalpha5-dependent cell polarization and Fibronectin matrix assembly occur concomitantly and interdependently during border morphogenesis.

Fibronectin is a smart adhesive that both influences and responds to the mechanics of early spinal column development.

An extracellular matrix of Fibronectin adheres the neural tube to the two flanking columns of paraxial mesoderm and is required for normal vertebrate development. Here, we find that the bilaterally symmetric interfaces between the zebrafish neural tube and paraxial mesoderm function as optimally engineered adhesive lap joints with rounded edges, graded Fibronectin 'adhesive' and an arced adhesive spew filet. Fibronectin is a 'smart adhesive' that remodels to the lateral edges of the neural tube-paraxial mesoderm interfaces where shear stress is highest. Fibronectin remodeling is mechanically responsive to contralateral variation morphogenesis, and Fibronectin-mediated inter-tissue adhesion is required for bilaterally symmetric morphogenesis of the paraxial mesoderm. Strikingly, however, perturbation of the Fibronectin matrix rescues the neural tube convergence defect of cadherin 2 mutants. Therefore, Fibronectin-mediated inter-tissue adhesion dynamically coordinates bilaterally symmetric morphogenesis of the vertebrate trunk but predisposes the neural tube to convergence defects that lead to spina bifida.

In embryos, the spinal cord starts out as a flat sheet of cells that curls up to form a closed cylinder called the neural tube. The folding tube is attached to the surrounding tissues through an extracellular matrix of proteins and sugars. Overlapping strands of a protein from the extracellular matrix called Fibronectin connect the neural tube to adjacent tissues, like a kind of biological glue. However, it remained unclear what effect this attachment had on the embryonic development of the spinal cord. Connecting two overlapping objects with glue to form what is known as an 'adhesive lap joint' is common in fields such as woodworking and aeronautical engineering. The glue in these joints comes under shearing stress whenever the two objects it connects try to pull apart. But, thanks to work in engineering, it is possible to predict how different joints will perform under tension. Now, Guillon et al. have deployed these engineering principles to shed light on neural tube development. Using zebrafish embryos and computational models, Guillon et al. investigated what happens when the strength of the adhesive lap joints in the developing spine changes. This revealed that Fibronectin works like a smart adhesive: rather than staying in one place like a conventional glue, it moves around. As the neural tube closes, cells remodel the Fibronectin, concentrating it on the areas under the highest stress. This seemed to both help and hinder neural tube development. On the one hand, by anchoring the tube equally to the left and right sides of the embryo, the Fibronectin glue helped the spine to develop symmetrically. On the other hand, the strength of the adhesive lap joints made it harder for the neural tube to curl up and close. If the neural tube fails to close properly, it can lead to birth defects like spina bifida. One of the best-known causes of these birth defects in humans is a lack of a vitamin known as folic acid. Cell culture experiments suggest that this might have something to do with the mechanics of the cells during development. It may be that faulty neural tubes could close more easily if they were able to unglue themselves from the surrounding tissues. Further use of engineering principles could shed more light on this idea in the future.

Organization of Embryonic Morphogenesis via Mechanical Information.

Embryonic organizers establish gradients of diffusible signaling molecules to pattern the surrounding cells. Here, we elucidate an additional mechanism of embryonic organizers that is a secondary consequence of morphogen signaling. Using pharmacological and localized transgenic perturbations, 4D imaging of the zebrafish embryo, systematic analysis of cell motion, and computational modeling, we find that the vertebrate tail organizer orchestrates morphogenesis over distances beyond the range of morphogen signaling. The organizer regulates the rate and coherence of cell motion in the elongating embryo using mechanical information that is transmitted via relay between neighboring cells. This mechanism is similar to a pressure front in granular media and other jammed systems, but in the embryo the mechanical information emerges from self-propelled cell movement and not force transfer between cells. The propagation likely relies upon local biochemical signaling that affects cell contractility, cell adhesion, and/or cell polarity but is independent of transcription and translation.

A Sawtooth Pattern of Cadherin 2 Stability Mechanically Regulates Somite Morphogenesis.

Differential cadherin (Cdh) expression is a classical mechanism for in vitro cell sorting. Studies have explored the roles of differential Cdh levels in cell aggregates and during vertebrate gastrulation, but the role of differential Cdh activity in forming in vivo tissue boundaries and boundary extracellular matrix (ECM) is unclear. Here, we examine the interactions between cell-cell and cell-ECM adhesion during somitogenesis, the formation of the segmented embryonic precursors of the vertebral column and musculature. We identify a sawtooth pattern of stable Cdh2 adhesions in which there is a posterior-to-anterior gradient of stable Cdh2 within each somite, while there is a step-like drop in stable Cdh2 along the somite boundary. Moreover, we find that the posterior somite boundary cells with high levels of stable Cdh2 have the most columnar morphology. Cdh2 is required for maximal cell aspect ratio and thus full epithelialization of the posterior somite. Loss-of-function analysis also indicates that Cdh2 acts with the fibronectin (FN) receptor integrin α5 (Itgα5) to promote somite boundary formation. Using genetic mosaics, we demonstrate that differential Cdh2 levels are sufficient to induce boundary formation, Itgα5 activation, and FN matrix assembly in the paraxial mesoderm. Elevated cytoskeletal contractility is sufficient to replace differential Cdh2 levels in genetic mosaics, suggesting that Cdh2 promotes ECM assembly by increasing cytoskeletal and tissue stiffness along the posterior somite boundary. Throughout somitogenesis, Cdh2 promotes ECM assembly along tissue boundaries and inhibits ECM assembly in the tissue mesenchyme.

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

Cell-fibronectin interactions propel vertebrate trunk elongation via tissue mechanics.

During embryonic development and tissue homeostasis, cells produce and remodel the extracellular matrix (ECM). The ECM maintains tissue integrity and can serve as a substrate for cell migration. Integrin α5 (Itgα5) and αV (ItgαV) are the α subunits of the integrins most responsible for both cell adhesion to the ECM protein fibronectin (FN) and FN matrix fibrillogenesis. We perform a systems-level analysis of cell motion in the zebrafish tail bud during trunk elongation in the presence and absence of normal cell-FN interactions. Itgα5 and ItgαV have well-described roles in cell migration in vitro. However, we find that concomitant loss of itgα5 and itgαV leads to a trunk elongation defect without substantive alteration of cell migration. Tissue-specific transgenic rescue experiments suggest that the FN matrix on the surface of the paraxial mesoderm is required for body elongation via its role in defining tissue mechanics and intertissue adhesion.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function.

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1(-/-). The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.

beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.

The Tübingen large-scale zebrafish genetic screen completed in 1996 identified a set of five genes required for orderly somite segmentation. Four of them have been molecularly identified and three were found to code for components of the Notch pathway, which are required for the coordinated oscillation of gene expression, known as the segmentation clock, in the presomitic mesoderm (PSM). Here, we show that the final member of the group, beamter (bea), codes for the Notch ligand DeltaC, and we present and characterize two new alleles, including one allele encoding for a protein truncated in the 7th EGF repeat and an allele deleting only the DSL domain which was previously shown to be necessary for ligand function. Interestingly however, when we over-express any of the mutant deltaC mRNAs, we observe antimorphic effects on both hindbrain neurogenesis and hypochord formation. Expression of bea/deltaC oscillates in the PSM, and a triple fluorescent in situ analysis of its oscillation in relation to that of other oscillating genes in the PSM reveals differences in subcellular localization of the oscillating mRNAs in individual cells in different oscillation phases. Mutations in aei/deltaD and bea/deltaC differ in the way they disrupt the oscillating expression of her1 and deltaC. Furthermore, we find that the double mutants have significantly stronger defects in hypochord formation but not in somitogenesis or hindbrain neurogenesis, indicating genetically that the two delta's may function either semi-redundantly or distinctly, depending upon context.

her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis.

Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. Our study focuses on how the oscillations in the expression of these two genes is affected in the mutants aei/deltaD and des/notch1, in 'morpholino knockdowns' of deltaC and her1 and in double 'mutant' combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. We also show that a later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression.