Integrins are key plasma membrane proteins that mediate cell-ECM adhesion and communication, and they rely on a conformational change for their activation and bidirectional signaling. However, there are few in vivo studies of integrin activation. Here, we identify Integrin α5 (Itgα5)-associated proteins in the physiological setting of zebrafish somite morphogenesis. Using label-free mass spectrometry, we compared Itgα5-associated proteins in different integrin activation states. As expected, we found active Itgα5 enriched extracellular matrix (ECM) proteins. Surprisingly, inactive Itgα5 incapable of binding ligand recruits actin cytoskeletal proteins as efficiently as the active integrin. We validated Itgα5's linking to actin adaptors using Parallel Reaction Monitoring (PRM). We then focused on α-actinin 4 (Actn4), an actin cross-linker, which we find preferentially associates with inactive Itgα5. Along zebrafish somite boundaries, Itgα5 and Actn4 displayed on and off co-localization, and Actn4 showed a stronger correlation with wild-type and inactive Itgα5 compared with the active Itgα5. We also found that deleting the actin-binding domain (Actn4ABDdel) resulted in cytoplasmic retention and loss of colocalization with Itgα5. These findings suggest that Itgα5 and Actn4 cooperate during somite boundary formation and that actin cytoskeleton reorganization facilitates their colocalization. Furthermore, we showed ligand-binding-deficient Itgα5 associated with Paxillin a (Pxna), a scaffold protein highly enriched at somite boundaries and strongly correlated with activated Itgα5. This study provides novel insights into in vivo integrin activation and integrin-actin interactions and broadens our understanding of integrin's role in tissue morphogenesis. Data are available via ProteomeXchange with identifiers PXD024942, PXD065495, PXD058516, PXD058550, and PXD058747.

Phenotypic variation is often observed in individuals with the same mutation. However, the mechanisms that contribute to this variation remain largely unknown. Fibronectin mutants in both mouse and zebrafish fail to form a functional cardiovascular system, although the penetrance and expressivity of this phenotype vary depending on the genetic background. Here we investigate the variation of the zebrafish natter phenotype, which is caused by a nonsense mutation in fibronectin 1a (fn1a). natter/fn1a mutants exhibit incompletely penetrant cardia bifida, a phenotype caused by the failure of cardiac progenitors to migrate to the midline. To examine whether this variation is related to the nonsense mutation, we first generated a large deletion in fn1a that removes the proximal promoter and first 17 exons. Characterisation of this allele found that mutants display variable cardiac phenotypes indistinguishable from those observed in natter/fn1a mutants. As phenotypic variation is often associated with changes in paralogous gene expression, we next examined the expression of the fn1a paralogue, fn1b, and observed its upregulation specifically in the natter/fn1a mutants that exhibit a severe phenotype. However, overexpression and double mutant analyses suggest that fn1b expression levels do not modulate the natter/fn1a mutant phenotype. During these studies, we observed a small proportion of natter/fn1a mutants with a wild-type (WT)-like phenotype. Selectively raising WT looking mutant larvae increased the proportion of natter/fn1a mutants displaying the WT-like phenotype from 1.7% to 38.6% in just three generations, indicating the selection of a genetic modifier of the mutant phenotype. We mapped this modifier to the integrin alpha 5 (itgα5) locus through whole-genome sequencing. Furthermore, we found that manipulating itgα5 expression influenced the severity of the fn1a mutant phenotype, and that the variance in itgα5 expression was increased in fn1a mutants exhibiting a severe phenotype. Taken together, these results indicate that itgα5 modifies the fn1a mutant phenotype.

Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimer's disease (AD), particularly in individuals carrying the APOE-ε4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (Aβ42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-ε4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 ( FN1 ) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-ε4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-ε4 carriers. Here, we demonstrate that APOE-ε4 , Aβ42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-ε4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract: Accessibility text:This image illustrates the effects of different APOE isoforms (ApoE-ε3 and ApoE-ε4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-ε4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-ε3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-ε4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. Highlights:APOE-ε4 drives fibronectin deposition in Alzheimer's, disrupting astrocyte-endothelia interactions. APOE-ε4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-ε4 pathology.

Animal tissues exist within a continuum of fluid to solid states, and transitions between states are important for embryonic development, wound healing and cancer metastasis. Fluid-to-solid transitions are governed by the ratio of adhesive energy to kinetic energy. Here, we find that presomitic mesoderm solidification is driven by an intrinsic decline in cell speed along with an increase in adhesion mediated by Cadherin 2 in parallel with fibronectin and its receptor Integrin α5. A computational model of cell-cell adhesion in the central tissue mesenchyme and cell-ECM adhesion on the tissue surface explains the observed phenotypes. Further, we identify negative feedback within the ECM as fibronectin supports the formation of a separate layer of Fibrillin 2b matrix that inhibits solidification. These data reveal a tissue fluidity code in which solidification is promoted by cadherins in parallel with Integrin α5 and fibronectin, whereas negative feedback through Fibrillin 2b promotes fluidization.

APOEε4 significantly increases the risk of developing Alzheimer’s disease (AD). Cognitively healthy APOEε4 -carriers exist, suggesting potential protective mechanisms against APOEε4 . We hypothesized that some APOEε4- carriers may have genetic variations protecting them from developing APOEε4 -mediated AD pathology. We aim to identify these protective genetic variants. Whole genome sequencing (WGS) and cerebrospinal fluid (CSF) proteomics were performed from human cohorts to identify potential protective variants segregating exclusively among APOEε4 carriers. Bioinformatic analyses were performed to select candidate target genes. Immunohistochemistry on postmortem human brain tissues and mouse models expressing human APOE variants were performed along with in-vivo functional studies in adult zebrafish AD model. WGS analyses revealed 510 potential gene variants segregating exclusively among APOEε4 carriers, which included rare and loss-of-function (LOF) variants. Pathway analysis of these genes showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of LOF in ECM proteins. This was further validated by CSF proteome profiling and subsequent analyses in APOEε4 carriers and non-carriers. Fibronectin-1 (FN1) and Collagen-6A2 ( COL6A2 ) were prioritized as candidate target genes for postmortem validation and in-vivo functional studies. FN1 protein was increased in APOEε4 carriers resulting in thickened ECM at the basement membrane around the blood vessels, potentially impairing pathology-induced responses such as clearance and immune system activity. This observation is validated in human brains, mouse models and zebrafish model; therefore, the pathological association of FN1 to AD is evolutionarily conserved. Supporting this hypothesis, in-vivo functional study in zebrafish model with LOF mutations in fn1b revealed that fibronectin LOF enhanced gliovascular remodeling and microglial activation while reducing astrogliosis, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. The vascular deposition of the ECM components FN1 and COL6A2 are increased in APOEε4 carriers. Rare variant in FN1 protect against APOEε4- mediated pathogenesis in AD. We propose a new disease mechanisms and potential therapeutic intervention targets for vascular contribution to dementia to mitigate the risk of developing AD.

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

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 β1a; β1b 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.

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.

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.

Dysregulated transcription factors (TFs) that rewire gene expression circuitry are frequently identified as key players in disease. Although several TFs have been drugged with small molecules, the majority of oncogenic TFs are not currently pharmaceutically tractable due to their paucity of ligandable pockets. The first generation of transcription factor targeting chimeras (TRAFTACs) was developed to target TFs for proteasomal degradation by exploiting their DNA binding ability. In the current study, we have developed the second generation TRAFTACs ("oligoTRAFTACs") composed of a TF-binding oligonucleotide and an E3 ligase-recruiting ligand. Herein, we demonstrate the development of oligoTRAFTACs to induce the degradation of two oncogenic TFs, c-Myc and brachyury. In addition, we show that brachyury can be successfully degraded by oligoTRAFTACs in chordoma cell lines. Furthermore, zebrafish experiments demonstrate in vivo oligoTRAFTAC activity. Overall, our data demonstrate oligoTRAFTACs as a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway.

The study of how neighboring tissues physically interact with each other, inter-tissue adhesion, is an emerging field at the interface of cell biology, biophysics and developmental biology. Inter-tissue adhesion can be mediated by either cell-extracellular matrix adhesion or cell-cell adhesion, and both the mechanisms and consequences of inter-tissue adhesion have been studied in vivo in numerous vertebrate and invertebrate species. In this Review, we discuss recent progress in understanding the many functions of inter-tissue adhesion in development and evolution. Inter-tissue adhesion can couple the motion of adjacent tissues, be the source of mechanical resistance that constrains morphogenesis, and transmit tension required for normal development. Tissue-tissue adhesion can also create mechanical instability that leads to tissue folding or looping. Transient inter-tissue adhesion can facilitate tissue invasion, and weak tissue adhesion can generate friction that shapes and positions tissues within the embryo. Lastly, we review studies that reveal how inter-tissue adhesion contributes to the diversification of animal morphologies.

Many diseases, including cancer, stem from aberrant activation and overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity are able to be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription-factor degradation. Herein, we show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription-factor of interest (TOI) and to HaloTag fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins with minor changes to the chimeric oligonucleotide.

The reproducibility of embryonic development is a remarkable feat of biological organization, but the underlying mechanisms are poorly understood. Clearly, gene regulatory networks are central to the orderly progression of development, but noisy molecular and cellular processes should reduce reproducibility. Here, we identify ergodicity, a type of dynamical stability, as underlying the reproducibility of development. In ergodic systems, a single timepoint measurement equals a time average. Focusing on the zebrafish tailbud, we define gene expression and cell motion states using a parallel statistical analyses of single cell RNA sequencing data and in vivo timelapse cell tracking data and a change point detection algorithm. Strikingly, the cell motion state transitions in each embryo exhibit the same patterns for both a single timepoint and a 2-3 hour time average. Both the cell motion and gene expression cell states exhibit balanced influx and outflux rates reflecting a spatiotemporal stability. Stated simply, these data indicate the pattern of changes in the tailbud doesn’t change. This ergodic pattern of cell state transitions may represent an emergent meta-state that links gene networks to the reproducible progression of embryogenesis.

Embryonic development is a complex process in which cells divide, migrate, and differentiate in a precise spatiotemporal pattern. Cell-cell communication among neighboring cells plays a central role in specifying cell fate and in coordinating development. Embryonic development also relies on physical interaction between cells and coordinated changes in cell shape. A more recently investigated phenomenon is the coupling of development of adjacent tissues via inter-tissue adhesion. In this issue of EMBO Reports, Monnot and colleagues identify a role for inter-tissue adhesion in the development of adjacent sensory organs in the zebrafish. Specifically, eye morphogenesis influences the organ shape and retrograde axon growth in the adjacent olfactory placode via a shared extracellular matrix.

Integrins are heterodimeric cell surface receptors composed of an alpha and beta subunit that mediate cell adhesion to extracellular matrix proteins such as fibronectin. We previously studied integrin alpha 5 beta 1 activation during zebrafish somitogenesis, and in the present study, we characterize the integrin alpha V fibronectin receptors. Integrins are activated via a conformational change, and we perform single-molecule biophysical measurements of both integrin activation via fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) and integrin intra-heterodimer stability via fluorescence cross-correlation spectroscopy (FCCS) in living embryos. We find that integrin heterodimers that exhibit robust cell surface expression, including alpha V beta 3, alpha V beta 5, and alpha V beta 6, are never activated in this in vivo context, even in the presence of fibronectin matrix. In contrast, activatable integrins, such as integrin alpha V beta 1, and alleles of alpha V beta 3, alpha V beta 5, alpha V beta 6 that are biased to the active conformation exhibit poor cell surface expression and have a higher intra-heterodimer dissociation constant (K-D). These observations suggest that a weak integrin intra-heterodimer affinity decreases integrin cell surface stability and increases integrin activatability.

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.

New research demonstrates that mechanics can serve as a means of information propagation in developing embryos. Historically, the study of embryonic development has had a dichotomy between morphogens and pattern formation on the one hand and morphogenesis and mechanics on the other. Secreted signals are the preeminent means of information propagation between cells and used to control cell fate, while physical forces act downstream or in parallel to shape tissue morphogenesis. However, recent work has blurred this division of function by demonstrating that mechanics can serve as a means of information propagation. Adhesive or repulsive interactions can propagate through a tissue as a wave. These waves are rapid and directional and can be used to control the flux of cells through a developmental trajectory. Here, two examples are reviewed in which mechanics both guides and mediates morphogenesis and two examples in which mechanics intertwines with morphogens to regulate cell fate.

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.