The developmental mechanics of divergent buckling patterns in the chick gut.

Tissue buckling is an increasingly appreciated mode of morphogenesis in the embryo, but it is often unclear how geometric and material parameters are molecularly determined in native developmental contexts to generate diverse functional patterns. Here, we study the link between differential mechanical properties and the morphogenesis of distinct anteroposterior compartments in the intestinal tract-the esophagus, small intestine, and large intestine. These regions originate from a simple, common tube but adopt unique forms. Using measured data from the developing chick gut coupled with a minimal theory and simulations of differential growth, we investigate divergent lumen morphologies along the entire early gut and demonstrate that spatiotemporal geometries, moduli, and growth rates control the segment-specific patterns of mucosal buckling. Primary buckling into wrinkles, folds, and creases along the gut, as well as secondary buckling phenomena, including period-doubling in the foregut and multiscale creasing-wrinkling in the hindgut, are captured and well explained by mechanical models. This study advances our existing knowledge of how identity leads to form in these regions, laying the foundation for future work uncovering the relationship between molecules and mechanics in gut morphological regionalization.

Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis.

The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein ß" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.

Mechanical forces remodel the cardiac extracellular matrix during zebrafish development.

The cardiac extracellular matrix (cECM) is fundamental for organ morphogenesis and maturation, during which time it undergoes remodeling, yet little is known about whether mechanical forces generated by the heartbeat regulate this remodeling process. Using zebrafish as a model and focusing on stages when cardiac valves and trabeculae form, we found that altering cardiac contraction impairs cECM remodeling. Longitudinal volumetric quantifications in wild-type animals revealed region-specific dynamics: cECM volume decreases in the atrium but not in the ventricle or atrioventricular canal. Reducing cardiac contraction resulted in opposite effects on the ventricular and atrial ECM, whereas increasing the heart rate affected the ventricular ECM but had no effect on the atrial ECM, together indicating that mechanical forces regulate the cECM in a chamber-specific manner. Among the ECM remodelers highly expressed during cardiac morphogenesis, we found one that was upregulated in non-contractile hearts, namely tissue inhibitor of matrix metalloproteinase 2 (timp2). Loss- and gain-of-function analyses of timp2 revealed its crucial role in cECM remodeling. Altogether, our results indicate that mechanical forces control cECM remodeling in part through timp2 downregulation.

Cardiac Development and Factors Influencing the Development of Congenital Heart Defects (CHDs): Part I.

The traditional description of cardiac development involves progression from a cardiac crescent to a linear heart tube, which in the phase of transformation into a mature heart forms a cardiac loop and is divided with the septa into individual cavities. Cardiac morphogenesis involves numerous types of cells originating outside the initial cardiac crescent, including neural crest cells, cells of the second heart field origin, and epicardial progenitor cells. The development of the fetal heart and circulatory system is subject to regulatation by both genetic and environmental processes. The etiology for cases with congenital heart defects (CHDs) is largely unknown, but several genetic anomalies, some maternal illnesses, and prenatal exposures to specific therapeutic and non-therapeutic drugs are generally accepted as risk factors. New techniques for studying heart development have revealed many aspects of cardiac morphogenesis that are important in the development of CHDs, in particular transposition of the great arteries.

Bioprinting Cell-Laden Hydrogels for Studies of Epithelial Tissue Morphogenesis.

The extracellular matrix (ECM) provides dynamic structural and molecular signals that affect the form and function of developing tissues. In order to parse how the individual features of the ECM impact cell- and tissue-level behavior during development, engineered culture models should reproduce key structural and molecular features of native ECM. Here, we describe a protocol for bioprinting epithelial cell aggregates embedded within a collagen-Matrigel ink in order to study the dynamic interplay between epithelial tissues and aligned networks of type I collagen fibers. Collagen fiber alignment and geometry can be spatially controlled by modulating the printing speed, nozzle geometry, surface chemistry, and degree of molecular crowding in the printing ink. We provide detailed procedures for generating epithelial cell aggregates, microextrusion printing collagen-Matrigel bioinks, culturing the three-dimensional (3D)-printed tissues, and imaging 3D-printed collagen-Matrigel constructs.

Arabidopsis Root Microbiome Microfluidic (ARMM) Device for Imaging Bacterial Colonization and Morphogenesis of Arabidopsis Roots.

Imaging the spatiotemporal dynamics of host-microbiota interactions is of particular interest for augmenting our understanding of these complex systems. This is especially true of plant-microbe interactions happening around, on, and inside plant roots where relatively little is understood about the dynamics of these systems. Over the past decade, a number of microfluidic devices have been developed to grow plants hydroponically in gnotobiotic conditions and image morphogenesis of the root and/or dynamics with fluorescently labeled bacteria from the plant root microbiome. Here we describe the construction and use of our Arabidopsis Root Microbiome Microfluidic (ARMM) device for imaging fluorescent protein expressing bacteria and their colonization of Arabidopsis roots. In contrast to other plant root imaging devices, we designed this device to have a larger chamber for observing Arabidopsis root elongation and plant-microbe interactions with older seedlings (between 1.5 and 4 weeks after germination) and a 200 µm chamber depth to specifically maintain thin Arabidopsis roots within the focal distance of the confocal microscope. Our device incorporates a new approach to growing Arabidopsis seedlings in screw-top tube caps for simplified germination and transfer to the device. We present representative images from the ARMM device including high resolution cross section images of bacterial colonization at the root surface.

PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system.

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis.

Lumenogenesis within the epiblast represents a critical step in early human development, priming the embryo for future specification and patterning events. However, little is known about the specific mechanisms that drive this process due to the inability to study the early embryo in vivo. While human pluripotent stem cell (hPSC)-based models recapitulate many aspects of the human epiblast, most approaches for generating these 3D structures rely on ill-defined, reconstituted basement membrane matrices. Here, we designed synthetic, nonadhesive polyethylene glycol (PEG) hydrogel matrices to better understand the role of matrix mechanical cues in iPSC morphogenesis, specifically elastic modulus. First, we identified a narrow range of hydrogel moduli that were conducive to the hPSC viability, pluripotency, and differentiation. We then used this platform to investigate the effects of the hydrogel modulus on lumenogenesis, finding that matrices of intermediate stiffness yielded the most epiblast-like aggregates. Conversely, stiffer matrices impeded lumen formation and apico-basal polarization, while the softest matrices yielded polarized but aberrant structures. Our approach offers a simple, modular platform for modeling the human epiblast and investigating the role of matrix cues in its morphogenesis.

Cyclic muscle contractions reinforce the actomyosin motors and mediate the full elongation of C. elegans embryo.

The paramount importance of mechanical forces in morphogenesis and embryogenesis is widely recognized, but understanding the mechanism at the cellular and molecular level remains challenging. Because of its simple internal organization, Caenorhabditis elegans is a rewarding system of study. As demonstrated experimentally, after an initial period of steady elongation driven by the actomyosin network, muscle contractions operate a quasi-periodic sequence of bending, rotation, and torsion, that leads to the final fourfold size of the embryos before hatching. How actomyosin and muscles contribute to embryonic elongation is investigated here theoretically. A filamentary elastic model that converts stimuli generated by biochemical signals in the tissue into driving forces, explains embryonic deformation under actin bundles and muscle activity, and dictates mechanisms of late elongation based on the effects of energy conversion and dissipation. We quantify this dynamic transformation by stretches applied to a cylindrical structure that mimics the body shape in finite elasticity, obtaining good agreement and understanding of both wild-type and mutant embryos at all stages.

Zn2+-dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses.

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.

Differential proliferation regulates multi-tissue morphogenesis during embryonic axial extension: integrating viscous modeling and experimental approaches.

A major challenge in biology is to understand how mechanical interactions and cellular behavior affect the shapes of tissues and embryo morphology. The extension of the neural tube and paraxial mesoderm, which form the spinal cord and musculoskeletal system, respectively, results in the elongated shape of the vertebrate embryonic body. Despite our understanding of how each of these tissues elongates independently of the others, the morphogenetic consequences of their simultaneous growth and mechanical interactions are still unclear. Our study investigates how differential growth, tissue biophysical properties and mechanical interactions affect embryonic morphogenesis during axial extension using a 2D multi-tissue continuum-based mathematical model. Our model captures the dynamics observed in vivo by time-lapse imaging of bird embryos, and reveals the underestimated influence of differential tissue proliferation rates. We confirmed this prediction in quail embryos by showing that decreasing the rate of cell proliferation in the paraxial mesoderm affects long-term tissue dynamics, and shaping of both the paraxial mesoderm and the neighboring neural tube. Overall, our work provides a new theoretical platform upon which to consider the long-term consequences of tissue differential growth and mechanical interactions on morphogenesis.

Salivary gland developmental mechanics.

The salivary gland undergoes branching morphogenesis to elaborate into a tree-like structure with numerous saliva-secreting acinar units, all joined by a hierarchical ductal system. The expansive epithelial surface generated by branching morphogenesis serves as the structural basis for the efficient production and delivery of saliva. Here, we elucidate the process of salivary gland morphogenesis, emphasizing the role of mechanics. Structurally, the developing salivary gland is characterized by a stratified epithelium tightly encased by the basement membrane, which is in turn surrounded by a mesenchyme consisting of a dense network of interstitial matrix and mesenchymal cells. Diverse cell types and extracellular matrices bestow this developing organ with organized, yet spatially varied mechanical properties. For instance, the surface epithelial sheet of the bud is highly fluidic due to its high cell motility and weak cell-cell adhesion, rendering it highly pliable. In contrast, the inner core of the bud is more rigid, characterized by reduced cell motility and strong cell-cell adhesion, which likely provide structural support for the tissue. The interactions between the surface epithelial sheet and the inner core give rise to budding morphogenesis. Furthermore, the basement membrane and the mesenchyme offer mechanical constraints that could play a pivotal role in determining the higher-order architecture of a fully mature salivary gland.

Biophysics of morphogenesis in the vertebrate lung.

Morphogenesis is a physical process that sculpts the final functional forms of tissues and organs. Remarkably, the lungs of terrestrial vertebrates vary dramatically in form across species, despite providing the same function of transporting oxygen and carbon dioxide. These divergent forms arise from distinct physical processes through which the epithelium of the embryonic lung responds to the mechanical properties of its surrounding mesenchymal microenvironment. Here we compare the physical processes that guide folding of the lung epithelium in mammals, birds, and reptiles, and suggest a conceptual framework that reconciles how conserved molecular signaling generates divergent mechanical forces across these species.

Gears of life: A primer on the simple machines that shape the embryo.

A simple machine is a basic of device that takes mechanical advantage to apply force. Animals and plants self-assemble through the operation of a wide variety of simple machines. Embryos of different species actuate these simple machines to drive the geometric transformations that convert a disordered mass of cells into organized structures with discrete identities and function. These transformations are intrinsically coupled to sequential and overlapping steps of self-organization and self-assembly. The processes of self-organization have been explored through the molecular composition of cells and tissues and their information networks. By contrast, efforts to understand the simple machines underlying self-assembly must integrate molecular composition with the physical principles of mechanics. This primer is concerned with effort to elucidate the operation of these machines, focusing on the "problem" of morphogenesis. Advances in understanding self-assembly will ultimately connect molecular-, subcellular-, cellular- and meso-scale functions of plants and animals and their ability to interact with larger ecologies and environmental influences.

A Lifeact-EGFP quail for studying actin dynamics in vivo.

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.

Deficiency of CAMSAP2 impairs olfaction and the morphogenesis of mitral cells.

In developing olfactory bulb (OB), mitral cells (MCs) remodel their dendrites to establish the precise olfactory circuit, and these circuits are critical for individuals to sense odors and elicit behaviors for survival. However, how microtubules (MTs) participate in the process of dendritic remodeling remains elusive. Here, we reveal that calmodulin-regulated spectrin-associated proteins (CAMSAPs), a family of proteins that bind to the minus-end of the noncentrosomal MTs, play a crucial part in the development of MC dendrites. We observed that Camsap2 knockout (KO) males are infertile while the reproductive tract is normal. Further study showed that the infertility was due to the severe defects of mating behavior in male mice. Besides, mice with loss-of-function displayed defects in the sense of smell. Furthermore, we found that the deficiency of CAMSAP2 impairs the classical morphology of MCs, and the CAMSAP2-dependent dendritic remodeling process is responsible for this defect. Thus, our findings demonstrate that CAMSAP2 plays a vital role in regulating the development of MCs.

The contest between artificial management and natural environment determines the adaptive strategies of leaf morphogenesis in Sabina chinensis.

Sabina chinensis is a typically heteromorphic leaf evergreen tree worldwide with both ornamental and ecological value. However, the shaping mechanism of heteromorphic leaves of S. chinensis and its adaptability to environment are important factors determining its morphology. The morphological change of S. chinensis under different habitats (tree around) and treatments (light, pruning and nutrients) was investigated. Our findings suggested that the prickle leaves proportion was associated with low light intensity and soil nutrient scarcity. Stems and leaves are pruned together to form clusters of large prickle leaves, while only pruning leaves often form alternately growing small prickle leaves and scale leaves, and the length of the prickle leaves is between 0.5 cm and 1 cm. The gene expression of prickle leaves is higher than that of scale leaves under adverse environmental conditions, and the gene expression correlations between small prickle leaf and scale leaf were the highest. Homologous and heterologous mutants of gene structure in prickle leaves were larger than those in scale leaves. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed that phenylpropanone and flavonoid biosynthesis were common enrichment pathways, and that the enrichment genes were mainly related to metabolism, genetic information processing and organismal systems. Therefore, we concluded that the occurrence of the heteromorphic leaf phenomenon was related to the changes in photosynthesis, mechanical damage and nutrient supplementation. The organic matter in the S. chinensis prickle leaves was reduced under environmental stresses, and it will be allocated to the expression of prickle leaf or protective cuticles formation.

Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Evaluating predictions of the patterning cascade model of crown morphogenesis in the human lower mixed and permanent dentition.

OBJECTIVE: The patterning cascade model of crown morphogenesis has been studied extensively in a variety of organisms to elucidate the evolutionary history surrounding postcanine tooth form. The current research is the first to use a large modern human sample to examine whether the crown configuration of lower deciduous and permanent molars aligns with expectations derived from the model. This study has two main goals: 1) to determine if metameric and antimeric pairs significantly differ in size, accessory trait expression, and relative intercusp spacing, and 2) assess whether the relative distance among early-forming cusps accounts for observed variation in accessory cusp expression. METHODS: Tooth size, intercusp distance, and morphological trait expression data were collected from 3D scans of mandibular dental casts representing participants of the Harvard Solomon Islands Project. Paired tests were utilized to compare tooth size, accessory trait expression, and relative intercusp distance between diphyodont metameres and permanent antimeres. Proportional odds logistic regression was implemented to investigate how the odds of greater accessory cusp expression vary as a function of the distance between early-developing cusps. RESULTS/SIGNIFICANCE: Comparing paired molars, significant differences were identified for tooth size and cusp 5 expression. Several relative intercusp distances emerged as important predictors of cusp 6 expression, however, results for cusp 5 and cusp 7 did not match expected patterns. These findings support previous quantitative genetic results and suggest the development of neighboring crown structures represents a zero-sum partitioning of cellular territory and resources. As such, this study contributes to a better understanding of the foundations of deciduous and permanent molar crown variation in humans.

The first embryo, the origin of cancer and animal phylogeny. III. The totipotency as revealed by morphogenesis and the neoplasia controlled by cellular differentiation.

We have extensively described that the neoplastic process (NP) has deep evolutionary roots and we have made specific predictions about the connection between cancer and the formation of the first embryo, which allowed for the evolutionary radiation of metazoans. My main hypothesis is that the NP is at the heart of cellular mechanisms responsible for animal morphogenesis, and given its embryological basis, also at the center of cell differentiation-one of the most interesting and relevant aspects of embryogenesis. In this article, I take forward the idea of the role of physics in the modeling of the neoplastic functional module (NFM) and its contribution to morphogenesis to reveal the totipotency of the zygote. In my consideration of these arguments, I examine mechanical and biophysical clues and their intimate connection with cellular differentiation. I expound on how cancer biology is perfectly intertwined with embryonic differentiation and why it is considered a disease of cell differentiation. The neoplasia is controlled by textural gradients that lead to cell differentiation within the embryo. Thus, the embryo would be a benign tumor. Finally, inspired by evolutionary history and by what the nervous system represents for current biology and based on the impressive nervous system of ctenophores as seen in fossil records, I propose a hypothesis with physical foundations (mechanical morphogenesis) for the formation of a preneural pattern of the nervous system of the first animal embryo.