The pioneer transcription factor Zelda facilitates the exit from regeneration and restoration of patterning in Drosophila.

For a damaged tissue to regenerate, the injured site must repair the wound, proliferate, and restore the correct patterning and cell types. We found that Zelda, a pioneer transcription factor largely known for its role in embryonic zygotic genome activation, is dispensable for normal wing development but crucial for wing disc patterning during regeneration. Impairing Zelda function during disc regeneration resulted in adult wings with a plethora of cell fate errors, affecting the veins, margins, and posterior compartment identity. Using CUT&RUN, we identified and validated targets of Zelda including the cell fate genes cut, Delta and achaete, which failed to return to their normal expression patterns upon loss of Zelda. In addition, Zelda controls expression of factors previously established to preserve cell fate during regeneration like taranis and osa, which stabilizes engrailed expression during regeneration, thereby preserving posterior identity. Finally, Zelda ensures proper expression of the integrins encoded by multiple edematous wings and myospheroid during regeneration to prevent blisters in the resuting adult wing. Thus, Zelda is crucial for maintaining cell fate and structural architecture of the regenerating tissue.

Mechanical stress driven by rigidity sensing governs epithelial stability.

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find that spontaneous half-integer defect formation underpinning large isotropic stress fluctuations initiate hole opening events. Our data show that monolayer rupture due to high tensile stress is promoted by the weakening of cell-cell junctions that could be induced by cell division events or local cellular stretching. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity during immune response and morphogenesis.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability.

Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level.

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

Direct measurement of near-nano-Newton forces developed by self-organizing actomyosin fibers bound α-catenin.

BACKGROUND INFORMATION: Actin cytoskeleton contractility plays a critical role in morphogenetic processes by generating forces that are then transmitted to cell-cell and cell-ECM adhesion complexes. In turn, mechanical properties of the environment are sensed and transmitted to the cytoskeleton at cell adhesion sites, influencing cellular processes such as cell migration, differentiation and survival. Anchoring of the actomyosin cytoskeleton to adhesion sites is mediated by adaptor proteins such as talin or α-catenin that link F-actin to transmembrane cell adhesion receptors, thereby allowing mechanical coupling between the intracellular and extracellular compartments. Thus, a key issue is to be able to measure the forces generated by actomyosin and transmitted to the adhesion complexes. Approaches developed in cells and those probing single molecule mechanical properties of α-catenin molecules allowed to identify α-catenin, an F-actin binding protein which binds to the cadherin complexes as a major player in cadherin-based mechanotransduction. However, it is still very difficult to bridge intercellular forces measured at cellular levels and those measured at the single-molecule level. RESULTS: Here, we applied an intermediate approach allowing reconstruction of the actomyosin-α-catenin complex in acellular conditions to probe directly the transmitted forces. For this, we combined micropatterning of purified α-catenin and spontaneous actomyosin network assembly in the presence of G-actin and Myosin II with microforce sensor arrays used so far to measure cell-generated forces. CONCLUSIONS: Using this method, we show that self-organizing actomyosin bundles bound to micrometric α-catenin patches can apply near-nano-Newton forces. SIGNIFICANCE: Our results pave the way for future studies on molecular/cellular mechanotransduction and mechanosensing.

Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers.

Actomyosin machinery endows cells with contractility at a single-cell level. However, within a monolayer, cells can be contractile or extensile based on the direction of pushing or pulling forces exerted by their neighbours or on the substrate. It has been shown that a monolayer of fibroblasts behaves as a contractile system while epithelial or neural progentior monolayers behave as an extensile system. Through a combination of cell culture experiments and in silico modelling, we reveal the mechanism behind this switch in extensile to contractile as the weakening of intercellular contacts. This switch promotes the build-up of tension at the cell-substrate interface through an increase in actin stress fibres and traction forces. This is accompanied by mechanotransductive changes in vinculin and YAP activation. We further show that contractile and extensile differences in cell activity sort cells in mixtures, uncovering a generic mechanism for pattern formation during cell competition, and morphogenesis.

Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus.

Limbal Stem Cell Deficiency (LSCD) is a painful and debilitating disease that results from damage or loss of the Corneal Epithelial Stem Cells (CESCs). Therapies have been developed to treat LSCD by utilizing epithelial stem cell transplants. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients. A major hurdle has been the lack of vertebrate models to study CESCs. Here we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea stem cell deficiency, such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and can stimulate mitosis in the remaining cells in the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one half of the cornea. These cases initially develop localized stem cell deficiency characteristics on the treated side. The localized PUV treatment is also capable of stimulating some mitosis in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored. Our study provides insight into the response of cornea cells following stem cell depletion, and establishes Xenopus as a suitable model for studying CESCs, stem cell deficiency, and other cornea diseases. This model will also be valuable for understanding the nature of transplanted CESCs, which will lead to progress in the development of therapeutics for LSCD.

Molecular markers for corneal epithelial cells in larval vs. adult Xenopus frogs.

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for maintaining the integrity and transparency of the cornea. These stem cells (SCs) are widely used in corneal transplants and ocular surface reconstruction. Molecular markers are essential to identify, isolate and enrich for these cells, yet no definitive CESC marker has been established. An extensive literature survey shows variability in the expression of putative CESC markers among vertebrates; being attributed to species-specific variations, or other differences in developmental stages of these animals, approaches used in these studies and marker specificity. Here, we expanded the search for CESC markers using the amphibian model Xenopus laevis. In previous studies we found that long-term label retaining cells (suggestive of CESCs and TACs) are present throughout the larval basal corneal epithelium. In adult frogs, these cells become concentrated in the peripheral cornea (limbal region). Here, we used immunofluorescence to characterize the expression of nine proteins in the corneas of both Xenopus larvae and adults (post-metamorphic). We found that localization of some markers change between larval and adult stages. Markers such as p63, Keratin 19, and ß1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog corneal epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detected in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, we identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none is unique to CESCs or TACs. Our results confirm that there is no single conserved CESC marker in vertebrates. This molecular characterization of the Xenopus cornea facilitates its use as a vertebrate model to understand the functions of key proteins in corneal homeostasis and wound repair.

The role of sensory innervation in cornea-lens regeneration.

BACKGROUND: Numerous sensory nerves in the cornea contribute to normal tissue homeostasis. Interestingly, cells within the basal corneal epithelium can regenerate new lenses in the frog, Xenopus. In this study, we investigated whether cornea sensory nerves or their neuropeptides are important for supporting cornea-lens regeneration. RESULTS: Attempts to sever the trigeminal nerve trunk, which provides sensory nerve branches to the cornea, did not inhibit lens regeneration. However, using this approach we found that it was not possible to completely disrupt sensory innervation, as these nerves are able to quickly regenerate back to the cornea. On the other hand, attenuation of neuropeptide levels with capsaicin was found to significantly inhibit lens regeneration, as visualized by a reduction of Substance P. These treatments also led to a reduction of cornea sensory innervation. Interestingly, inhibition of the Substance P-preferred receptor NK-1 with Spantide II did not affect lens-regeneration rates. CONCLUSIONS: This study provides evidence that cornea nerves support cornea-lens regeneration, which could occur through the release of various neurotrophic factors. Substance P, however, does not appear to be the critical component of this signaling pathway. Further studies are needed to investigate what role other known neurotrophic factors may play in this process.

The yeast stress inducible Ssa Hsp70 reduces α-synuclein toxicity by promoting its degradation through autophagy.

The mechanism underlying the role of Hsp70s in toxicity associated with intracellular accumulation of toxic protein inclusions is under intense investigation. In current study, we examined the roles of all different isoforms of yeast cytosolic Ssa Hsp70 on α-synuclein mediated cellular toxicity. The study showed that yeast cells expressing stress-inducible Ssa3 or Ssa4 as sole Ssa Hsp70 isoforms, reduced α-synuclein toxicity better than those expressing a constitutive counterpart. The protective effect of stress-inducible Ssa Hsp70s was not α-syn specific, but more general to other inclusion forming proteins such as polyQ. We show that the protective effect is not by induction of a general stress response in Ssa3 cells rather by promoting α-synuclein degradation through autophagy. The present study revealed that effect of Hsp70s was isoform dependent, and that autophagy protects Ssa3 cells from the deleterious effects of toxic protein inclusions.

Tubular microscaffolds for studying collective cell migration.

Epithelial cells demonstrate different collective migratory modes when encountering two (2D) and three dimensional (3D) microenvironment. While planar micropatterns and constraint have been shown to strongly impact collective cell migration (CCM), how out-of-plane curvature and 3D confinement will affect epithelial organization and dynamics remains largely unknown. This is likely due to lack of proper 3D microscaffolds for studying CCM. In this chapter, we briefly review the latest achievement in microengineering approaches to control 3D microenvironment of epithelial development. Then, we introduce convenient and simple methods of fabricating elastomeric tubular biocompatible microchannels as 3D cell culture scaffolds. Afterwards, we describe in detail the experimental set-up for observing 3D coordinated cell migration on curved surfaces and under spatial constraint. Finally, we provide an approach to analyze 3D dynamics using available techniques for 2D images.

Emergent patterns of collective cell migration under tubular confinement.

Collective epithelial behaviors are essential for the development of lumens in organs. However, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and confined surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1-10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical-basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular confinement and/or curvature. In contrast to flat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our findings provide insights into the emerging migratory modes for epithelial migration and growth under tubular confinement, which are reminiscent of the in vivo scenario.

Soft tubular microfluidics for 2D and 3D applications.

Microfluidics has been the key component for many applications, including biomedical devices, chemical processors, microactuators, and even wearable devices. This technology relies on soft lithography fabrication which requires cleanroom facilities. Although popular, this method is expensive and labor-intensive. Furthermore, current conventional microfluidic chips precludes reconfiguration, making reiterations in design very time-consuming and costly. To address these intrinsic drawbacks of microfabrication, we present an alternative solution for the rapid prototyping of microfluidic elements such as microtubes, valves, and pumps. In addition, we demonstrate how microtubes with channels of various lengths and cross-sections can be attached modularly into 2D and 3D microfluidic systems for functional applications. We introduce a facile method of fabricating elastomeric microtubes as the basic building blocks for microfluidic devices. These microtubes are transparent, biocompatible, highly deformable, and customizable to various sizes and cross-sectional geometries. By configuring the microtubes into deterministic geometry, we enable rapid, low-cost formation of microfluidic assemblies without compromising their precision and functionality. We demonstrate configurable 2D and 3D microfluidic systems for applications in different domains. These include microparticle sorting, microdroplet generation, biocatalytic micromotor, triboelectric sensor, and even wearable sensing. Our approach, termed soft tubular microfluidics, provides a simple, cheaper, and faster solution for users lacking proficiency and access to cleanroom facilities to design and rapidly construct microfluidic devices for their various applications and needs.

Cell contractility arising from topography and shear flow determines human mesenchymal stem cell fate.

Extracellular matrix (ECM) of the human Mesenchymal Stem Cells (MSCs) influences intracellular tension and is known to regulate stem cell fate. However, little is known about the physiological conditions in the bone marrow, where external forces such as fluid shear stress, apart from the physical characteristics of the ECM, influence stem cell response. Here, we hypothesize that substrate topography and fluid shear stress alter the cellular contractile forces, influence the genetic expression of the stem cells and hence alter their lineage. When fluid shear stress was applied, human MSCs with higher contractility (seeded on 1 µm wells) underwent osteogenesis, whereas those with lower contractility (seeded on 2 µm gratings) remained multipotent. Compared to human MSCs seeded on gratings, those seeded on wells exhibited altered alignment and an increase in the area and number of focal adhesions. When actomyosin contractility was inhibited, human MSCs did not exhibit differentiation, regardless of the topographical feature they were being cultured on. We conclude that the stresses generated by the applied fluid flow impinge on cell contractility to drive the stem cell differentiation via the contractility of the stem cells.

Regeneration of Leydig cells in ectopically autografted adult mouse testes.

Ectopic autografting of testis tissue is a promising approach for studying testicular development, male germline preservation and restoration of male fertility. In this study, we examined the fate of various testicular cells in adult mouse testes following ectopic autografting at 1, 2, 4 and 8 weeks post grafting. Histological examination showed no evidence of re-establishment of spermatogenesis in autografts, and progressive degeneration of seminiferous tubules was detected. Expression of germ cell-specific proteins such as POU5F1, DAZL, TNP1, TNP2, PRM1 and PRM2 revealed that, although proliferating and differentiating spermatogenic germ cells such as spermatogonia, spermatocytes and spermatids could survive in autografts until 4 weeks, only terminally differentiated germ cells such as sperm persisted in autografts until 8 weeks. The presence of Sertoli and peritubular myoid cells, as indicated by expression of WT1 and ACTA2 proteins, respectively, was evident in the autografts until 8 weeks. Interestingly, seminal vesicle weight and serum testosterone level were restored in autografted mice by 8 weeks post grafting. The expression of Leydig cell-specific proteins such as CYP11A1, HSD3B2 and LHCGR showed revival of Leydig cell (LC) populations in autografts over time since grafting. Elevated expression of PDGFRA, LIF, DHH and NEFH in autografts indicated de novo regeneration of LC populations. Autografted adult testis can be used as a model for investigating Leydig cell regeneration, steroidogenesis and regulation of the intrinsic factors involved in Leydig cell development. The success of this rodent model can have therapeutic applications for adult human males undergoing sterilizing cancer therapy.

SMMRNA: a database of small molecule modulators of RNA.

We have developed SMMRNA, an interactive database, available at http://www.smmrna.org, with special focus on small molecule ligands targeting RNA. Currently, SMMRNA consists of ∼770 unique ligands along with structural images of RNA molecules. Each ligand in the SMMRNA contains information such as Kd, Ki, IC50, ΔTm, molecular weight (MW), hydrogen donor and acceptor count, XlogP, number of rotatable bonds, number of aromatic rings and 2D and 3D structures. These parameters can be explored using text search, advanced search, substructure and similarity-based analysis tools that are embedded in SMMRNA. A structure editor is provided for 3D visualization of ligands. Advance analysis can be performed using substructure and OpenBabel-based chemical similarity fingerprints. Upload facility for both RNA and ligands is also provided. The physicochemical properties of the ligands were further examined using OpenBabel descriptors, hierarchical clustering, binning partition and multidimensional scaling. We have also generated a 3D conformation database of ligands to support the structure and ligand-based screening. SMMRNA provides comprehensive resource for further design, development and refinement of small molecule modulators for selective targeting of RNA molecules.