Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus finds novel myomeres with odd shapes and unusually prominent sclerocoels.

Serial block-face scanning electron microscopy of the tail tip of post-metamorphic amphioxus (Branchiostoma floridae) revealed some terminal myomeres never been seen before with other techniques. The morphology of these myomeres differed markedly from the chevron shapes of their more anterior counterparts. Histologically, these odd-shaped myomeres ranged from empty vesicles bordered by undifferentiated cells to ventral sacs composed of well-developed myotome, dermatome, and sclerotome. Strikingly, several of these ventral sacs gave rise to a nipple-like dorsal projection composed either entirely of sclerotome or a mixture of sclerotome and myotome. Considered as a whole, from posterior to anterior, these odd-shaped posterior myomeres suggested that their more substantial ventral part may represent the ventral limb of a chevron, while the delicate projection represents a nascent dorsal limb. This scenario contrasts with formation of chevron-shaped myomeres along most of the antero-posterior axis. Although typical chevron formation in amphioxus is surprisingly poorly studied, it seems to be attained by a dorso-ventral extension of the myomere accompanied by the assumption of a V-shape; this is similar to what happens (at least superficially) in developing fishes. Another unusual feature of the odd-shaped posterior myomeres of amphioxus is their especially distended sclerocoels. One possible function for these might be to protect the posterior end of the central nervous system from trauma when the animals burrow into the substratum.

Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages.

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.

Live Imaging of Epithelial-Mesenchymal Transition in Mesoderm Cells of Gastrulating Drosophila Embryos.

Epithelial-mesenchymal transitions (EMTs) drive the generation of cell diversity during both evolution and development. More and more evidence has pointed to a model where EMT is not a binary switch but a reversible process that can be stabilized at intermediate states. Despite our vast knowledge on the signaling pathways that trigger EMT, we know very little about how EMT happens in a step-wise manner. Live imaging of cells that are undergoing EMT in intact, living, animals will provide us valuable insights into how EMT is executed at both the cellular and molecular levels and help us identify and understand the intermediate states. Here, we describe how to image early stages of EMT in the mesoderm cells of live Drosophila melanogaster embryos and how to image contractile myosin that suspends EMT progression.

Right, left and cilia: How asymmetry is established.

The initial breaking of left-right (L-R) symmetry in the embryo is controlled by a motile-cilia-driven leftward fluid flow in the left-right organiser (LRO), resulting in L-R asymmetric gene expression flanking the LRO. Ultimately this results in left- but not right-sided activation of the Nodal-Pitx2 pathway in more lateral tissues. While aspects of the initial breaking event clearly vary between vertebrates, events in the Lateral Plate Mesoderm (LPM) are conserved through the vertebrate lineage. Evidence from model systems and humans highlights the role of cilia both in the initial symmetry breaking and in the ability of more lateral tissues to exhibit asymmetric gene expression. In this review we concentrate on the process of L-R determination in mouse and humans.

Distinct interactions between epithelial and mesenchymal cells control cell morphology and collective migration during sponge epithelial to mesenchymal transition.

Epithelial and mesenchymal cell types are basic for animal multicellularity and they have complementary functions coordinated by cellular interactions. Sponges are especially important model organisms to address the evolutionary basis of morphogenetic programs for epithelial and mesenchymal organization in animals. Evolutionary studies in sponges can contribute to the understanding of the mechanisms that control tissue maintenance and tumor progression in humans. In the present study, sponge mesenchymal and epithelial cells were isolated from the demosponge Hymeniacidon heliophila, and aggregate formation was observed by video microscopy. Epithelial-mesenchymal interaction, epithelial transition, and cell migration led to sponge cell aggregation after drastic stress. Based on their different morphologies, adhesion specificities, and motilities, we suggest a role for different sponge cell types as well as complementary functions in cell aggregation. Micromanipulation under the microscope and cell tracking were also used to promote specific grafting-host interaction, to further test the effects of cell type interaction. The loss of cell polarity and flattened shape during the epithelial to mesenchymal cell transition generated small immobile aggregates of round/amoeboid cells. The motility of these transited epithelial-cell aggregates was observed by cell tracking using fluorescent dye, but only after interaction with streams of migratory mesenchymal cells. Cell motility occurred independently of morphological changes, indicating a progressive step in the transition toward a migratory mesenchymal state. Our data suggest a two-step signaling process: (a) the lack of interaction between mesenchymal and epithelial cells triggers morphological changes; and (b) migratory mesenchymal cells instruct epithelial cells for directional cell motility. These results could have an impact on the understanding of evolutionary aspects of metastatic cancer cells. HIGHLIGHTS: Morphogenetic movements observed in modern sponges could have a common evolutionary origin with collective cell migration of human metastatic cells. A sponge regenerative model was used here to characterize epithelial and mesenchymal cells, and for the promotion of grafting/host interactions with subsequent cell tracking. The transition from epithelial to mesenchymal cell type can be observed in sponges in two steps: (a) withdrawal of epithelial/mesenchymal cell interactions to trigger morphological changes; (b) migratory mesenchymal cells to induce epithelial cells to a collective migratory state.

Chronic otitis media is initiated by a bulla cavitation defect in the FBXO11 mouse model.

Auditory bulla cavitation defects are a cause of otitis media, but the normal cellular pattern of bulla mesenchyme regression and its failure are not well understood. In mice, neural-crest-derived mesenchyme occupies the bulla from embryonic day 17.5 (E17.5) to postnatal day 11 (P11) and then regresses to form the adult air-filled bulla cavity. We report that bulla mesenchyme is bordered by a single layer of non-ciliated epithelium characterized by interdigitating cells with desmosome cell junctions and a basal lamina, and by Bpifa1 gene expression and laminin staining of the basal lamina. At P11-P12, the mesenchyme shrinks: mesenchyme-associated epithelium shortens, and mesenchymal cells and extracellular matrix collagen fibrils condense, culminating in the formation of cochlea promontory mucosa bordered by compact non-ciliated epithelial cells. FBXO11 is a candidate disease gene in human chronic otitis media with effusion and we report that a bulla cavitation defect initiates the pathogenesis of otitis media in the established mouse model Jeff (Fbxo11Jf/+ ). Persistent mesenchyme in Fbxo11Jf/+ bullae has limited mesenchymal cell condensation, fibrosis and hyperplasia of the mesenchyme-associated epithelium. Subsequent modification forms fibrous adhesions that link the mucosa and the tympanic membrane, and this is accompanied by dystrophic mineralization and accumulation of serous effusion in the bulla cavity. Mouse models of bulla cavitation defects are important because their study in humans is limited to post-mortem samples. This work indicates new diagnostic criteria for this otitis media aetiology in humans, and the prospects of studying the molecular mechanisms of murine bulla cavitation in organ culture.

Androgen Regulates Dimorphic F-Actin Assemblies in the Genital Organogenesis.

Impaired androgen activity induces defective sexual differentiation of the male reproductive tract, including hypospadias, an abnormal formation of the penile urethra. Androgen signaling in the urethral mesenchyme cells (UMCs) plays essential roles in driving dimorphic urethral development. However, cellular events for sexual differentiation remain virtually unknown. In this study, histological analyses, fluorescent staining, and transmission electron microscopy (TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin dynamics and migratory behaviors of UMCs were further analyzed by time-lapse imaging. We observed a prominent accumulation of F-actin with poorly assembled extracellular matrix (ECM) in female UMCs. In contrast, thin fibrils of F-actin co-aligning with the ECM through membrane receptors were identified in male UMCs. Processes for dimorphic F-actin assemblies were temporally identified during an androgen-regulated masculinization programming window and spatially distributed in several embryonic reproductive tissues. Stage-dependent modulation of the F-actin sexual patterns by androgen in UMCs was also demonstrated by time-lapse analysis. Moreover, androgen regulates coordinated migration of UMCs. These results suggest that androgen signaling regulates the assembly of F-actin from cytoplasmic accumulation to membranous fibrils. Such alteration appears to promote the ECM assembly and the mobility of UMCs, contributing to male type genital organogenesis.

Paraxial Nodal Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species.

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel 'parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.

Light and electron microscopic studies of the intestinal epithelium in Notoplana humilis (Platyhelminthes, Polycladida): the contribution of mesodermal/gastrodermal neoblasts to intestinal regeneration.

Some free-living flatworms in the phylum Platyhelminthes possess strong regenerative capability that depends on putative pluripotent stem cells known as neoblasts. These neoblasts are defined based on several criteria, including their proliferative capacity and the presence of cellular components known as chromatoid bodies. Polyclads, which are marine flatworms, have the potential to be a good model system for stem cell research, yet little information is available regarding neoblasts and regeneration. In this study, transmission electron microscopy and immunostaining analyses, using antibodies against phospho-histone H3 and BrdU, were used to identify two populations of neoblasts in the polyclad Notoplana humilis: mesodermal neoblasts (located in the mesenchymal space) and gastrodermal neoblasts (located within the intestine, where granular club cells and phagocytic cells are also located). Light and electron microscopic analyses also suggested that phagocytic cells and mesodermal/gastrodermal neoblasts, but not granular club cells, migrated into blastemas and remodeled the intestine during regeneration. Therefore, we suggest that, in polyclads, intestinal regeneration is accomplished by mechanisms underlying both morphallaxis (remodeling of pre-existing tissues) and epimorphosis (de novo tissue formation derived from mesodermal/gastrodermal neoblasts). Based on the assumption that gastrodermal neoblasts, which are derived from mesodermal neoblasts, are intestinal stem cells, we propose a model to study intestinal regeneration.

The distribution and ultrastructure of the forming blood capillaries and the effect of apoptosis on vascularization in mouse embryonic molar mesenchyme.

Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.

[Interrelation of nuclear envelope with cell organelles in peritoneal cells during ontogenesist and in experimental pathology].

Visceral and parietal peritoneum was studied by electron microscopy in albino mice both in the process of ontogenesis and after its injury induced by the the intraperitoneal injection of 0.5% novocaine solution. It was shown that during the early stages of intrauterine development (Day 13) most of the mesotheliocytes and mesenchymal cells contained predominantly free ribosomes (polysomes) in their cytoplasm while other organelles were rare and were located near the nuclear envelope. Subsequently, the number of membranous organelles increased while that of polysomes decreased. One day after the injury of the mesothelium, undifferentiated mesotheliocytes containing numerous polysomes in their cytoplasm appeared at the margin of wound surface. In these cells the protrusion 9f membranes of nuclear envelope and their association with the membranous organelles (endoplasmic reticulum, Golgi complex, mitochondria) were detected. The observed interrelations between the nuclear envelope and the membranous cytoplasmic organelles is considered to be a possible way of their formation in the undifferentiated cells. Rare occurrence of this phenomenon in adults animals under the pathological condition and its absence during the physiological regeneration is considered as a manifestation of the law of histogenetic recapitulation.

Scanning electron microscopic evidence for physical segmental boundaries in the anterior presomitic mesoderm.

The metameric pattern of the axial skeleton is established during embryogenesis by somite formation from the unsegmented paraxial mesoderm (presomitic mesoderm). Here, we have investigated the morphology of the anterior presomitic mesoderm of chick embryos using scanning electron microscopy. We found periodically arranged transverse clefts in the anterior region of the presomitic mesoderm. These gaps can be regarded as physical boundaries between prospective somites in the determined zone of the presomitic mesoderm. This study provides additional evidence suggesting that prospective somite boundaries are not only marked by defined zones of gene expression, but are also accompanied by changes in cellular morphology that give rise to identifiable morphological segments.

From blood islands to blood vessels: morphologic observations and expression of key molecules during hyaloid vascular system development.

PURPOSE: The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. METHODS: Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. RESULTS: Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. CONCLUSIONS: Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.

The pine-cone body: an intermediate structure between the cap mesenchyme and the renal vesicle in the developing nod mouse kidney revealed by an ultrastructural study.

Nephrogenesis is mainly characterized by the interaction of two distinct renal constituents, the ureteric bud and the metanephric mesenchyme. In this paper we describe by means of light and electron microscopic techniques the morphological events that take place during the early stages of cap mesenchymal formation. Samples of normal renal tissue were excised from newborn NOD mice and processed by standard light and electron microscopy techniques. In all samples examined we detected the presence of several cap mesenchymal aggregates in different stages of differentiation. They varied from small solid nodules with few ovoid cells to bigger pine-cone-like aggregates, characterized by a peculiar distribution and morphology of their cellular constituents. Our data highlight, for the first time, the presence of a specific cap mesenchymal structure, the pine-cone body and show, at ultrastructural level, how each cap aggregate epithelializes proceeding in stages from a condensed mesenchymal aggregate to the renal vesicle, through the intermediate "pine-cone body" stage.

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture.

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.

Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses.

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response.

Mesenchymal-epithelial differentiation of adamantinoma of long bones: an immunohistochemical and ultrastructural study.

Three cases of adamantinoma were studied by electron microscopy and immunohistochemistry. In the tubular pattern, well-differentiated epithelial cells and glandular structures were present, in addition to ill-defined glands. In the basaloid pattern, less differentiated epithelial cells with discohesion were seen in the central epithelial masses. This study established the epithelial nature of some tubular structures with slit-like lumina, easily misinterpreted as capillaries by light microscopy. Results also showed that the irregular spaces observed within the basaloid pattern probably result from cell discohesion. Moreover, this investigation demonstrates the epithelial nature of a subset of spindle cells within the stroma of adamantinoma and offers ultrastructural evidence for a probable mesenchymal-epithelial transformation as its histogenesis.

Influence of the mesenchymal cell source on oral epithelial development.

The extent of the influence of mesenchymal tissue on epithelial development is still debated, and elucidation of epithelial-mesenchymal interactions should be of relevance for controlling normal as well as pathological growth and development. The aim of the present study was to elucidate the influence of the mesenchymal cell type on oral mucosa epithelial development in vitro, using tissue-engineering principles, by including three different sources for mesenchymal cell type, viz. oral mucosa, skin and cornea, each of them presenting a distinct type of epithelium in situ. We investigated epithelial-mesenchymal interactions, considering both morphological criteria and protein expression (filaggrin, keratin 10, keratin 12, keratin 13 and laminin 5). The results of the histology, immunohistochemistry and transmission electron microscopy of the three types of tissue-engineered constructs composed of mesenchymal cells of different sources (oral, dermal and corneal fibroblasts) and of the same oral epithelial cells showed that the mesenchymal cell source had a significant influence on the thickness and ultrastructure of the epithelium, but not on the differentiation of oral epithelial cells, which might be an intrinsic property of these cells due to their genetic programming.

Deficiency in Crumbs homolog 2 (Crb2) affects gastrulation and results in embryonic lethality in mice.

The Crumbs family of transmembrane proteins has an important role in the differentiation of the apical membrane domain in various cell types, regulating such processes as epithelial cell polarization. The mammalian Crumbs protein family is composed of three members. Here, we inactivated the mouse Crb2 gene with gene-targeting techniques and found that the protein is crucial for early embryonic development with severe abnormalities appearing in Crb2-deficient embryos at late-gastrulation. Our findings indicate that the primary defect in the mutant embryos is disturbed polarity of the epiblast cells at the primitive streak, which affects epithelial to mesenchymal transition (EMT) during gastrulation, resulting in impaired mesoderm and endoderm formation, and embryonic lethality by embryonic day 12.5. These findings therefore indicate a novel role for the Crumbs family of proteins.

Atomic force microscopy and high-content analysis: two innovative technologies for dissecting the relationship between epithelial-mesenchymal transition-related morphological and structural alterations and cell mechanical properties.

Epithelial-mesenchymal transition (EMT) is a complex series of cellular reprogramming events culminating in striking alterations in morphology towards an invasive mesenchymal phenotype. Increasingly, evidence suggests that EMT exerts a pivotal role in pathophysiological situations including fibrosis and cancer. Core to these dynamical changes in cellular polarity and plasticity is discrete modifications in cytoskeletal structure. In particular, newly established actin-stress fibres supplant a preceding system of highly organised cortical actin. Although cumulative studies have contributed to elucidation of the detailed signalling pathways that underpin this elaborate molecular process, there remains a deficiency regarding its precise contribution to cellular biomechanics. The advent of atomic force microscopy (AFM) and high-content analysis (HCA) provides two innovative technologies for dissecting the relationship between EMT-related morphological and structural alterations and cell mechanical properties. AFM permits acquisition of high resolution topographical images and detailed analysis of cellular viscoelasticity while HCA facilitates a comprehensive and perspicacious assessment of morphological changes. In combination, they offer the possibility of novel insights into the dynamic traits of transitioning cells. Herein, a detailed protocol describing AFM and HCA techniques for evaluation of transforming growth factor-ß1-induced EMT of alveolar epithelial cells is provided.