Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis

Imaging Approaches and the Quantitative Analysis of Heart Development.

Heart morphogenesis is a complex and dynamic process that has captivated researchers for almost a century. This process involves three main stages, during which the heart undergoes growth and folding on itself to form its common chambered shape. However, imaging heart development presents significant challenges due to the rapid and dynamic changes in heart morphology. Researchers have used different model organisms and developed various imaging techniques to obtain high-resolution images of heart development. Advanced imaging techniques have allowed the integration of multiscale live imaging approaches with genetic labeling, enabling the quantitative analysis of cardiac morphogenesis. Here, we discuss the various imaging techniques used to obtain high-resolution images of whole-heart development. We also review the mathematical approaches used to quantify cardiac morphogenesis from 3D and 3D+time images and to model its dynamics at the tissue and cellular levels.Grant support PGC2018-096486-B-I00 from the Spanish Ministerio de Ciencia e Innovación and Grant H2020-MSCA-ITN-2016-722427 from the EU Horizon 2020 program to M.T. M.S. was supported by a La Caixa Foudation PhD fellowship (LCF/BQ/DE18/11670014) and The Company of Biologists travelling fellowship (DEVTF181145). The CNIC is supported by the Spanish Ministery of Science and the ProCNIC Foundation.S

On growth and force: mechanical forces in development.

The EMBO/EMBL Symposium 'Mechanical Forces in Development' was held in Heidelberg, Germany, on 3-6 July 2019. This interdisciplinary symposium brought together an impressive and diverse line-up of speakers seeking to address the origin and role of mechanical forces in development. Emphasising the importance of integrative approaches and theoretical simulations to obtain comprehensive mechanistic insights into complex morphogenetic processes, the meeting provided an ideal platform to discuss the concepts and methods of developmental mechanobiology in an era of fast technical and conceptual progress. Here, we summarise the concepts and findings discussed during the meeting, as well as the agenda it sets for the future of developmental mechanobiology

Zebrafish Eye Development: Rac and the Creepy Crawlers of the Eye

During vertebrate eye development the optic vesicles protrude from either side of the brain and form the optic cups. As an optic cup starts to surround the lens a groove on the ventral side of the eye forms, known as the choroid fissure (CF). Normally, the CF will close around the optic nerve and hyaloid vasculature. If this process does not occur properly it results in a keyhole opening in the eye known as coloboma. This results in blindness and affects nearly 1 in 4-5,000 births. Zebrafish were utilized as a model for eye development to study CF closure (CFC) as they utilize similar gene expression and cellular signaling. Previously, a transient β-catenin/actin fusion seam within the fusing CF was observed indicating the formation of cell-to-cell contacts. Rac, a small G-protein, regulates actin cytoskeleton reorganization and formation of lamellipodia required for cell-to-cell adhesion. These lamellipodia increase interactions between cells increasing contacts that could form adherens junctions. I hypothesized Rac would be expressed prior to CFC and dissipate upon CFC completion, similar to adhesion proteins. To determine Rac’s localization, embryos were cryosectioned at 47 and 49-hours post-fertilization (hpf) and Rac immunofluorescence was observed. These data demonstrated Rac is present within the CF edges at 47 hpf and dissipates the CF fusion seam as CFC progresses in wildtypes embryos around 49 hpf. Quantification of these data further demonstrated a progressive fusion event that initiates in the central section of the CF and moves bidirectionally towards the proximal and distal edges, emulating a zipper-like fashion. in vivo analysis of Rx3:GFP embryos (neuroretina labeled) identified a subpopulation of cells that are present within the CF at 24 hpf. This population of cells appear highly protrusive and “reach” in multiple directions. Further analysis of Rac embryos identified these “reaching cells” as Rac positive. in vivo analysis of this cell population revealed that seven identified categories of reaching cells can be divided into three stages of CFC. Rac is also required for reaching cells. When observing the Rac-DN embryo no reaching cells were ever observed, regardless of heat-shocking time. The Rac-DN embryos showed an abnormal optic cup angle and unusual cuboidal cells shapes (early heat-shock). In later heat-shocked times the abnormal angle and unusual cell shapes were resolved, however, there were unusual division patterns that were observed. Further investigation is ongoing to identify the role of Rac in this cell population and the role of “reaching cells” during zebrafish eye development

Modeling Human Intestinal Disease: Ontogeny Of Postembryonic Zebrafish Intestinal Morphology

The goal of this study was to establish zebrafish as a model organism for intestinal motility disorders in humans. Zebrafish provide a high throughput model that allows for the examination of the intestine throughout the life of an organism. While the zebrafish intestine shares a high degree of morphological homology with the human intestine, little is known about the maturation process. To begin to understand the maturation of the intestine, I characterized its appearance throughout the larval period and into metamorphosis. I found that the onset of metamorphosis coincides with a minimum standard length that ranges from 4.4 to 5.2 mm. Using a stage range of larval and metamorphic specimens, I used histological methods to follow the distribution of goblet cells, the mucin-producing secretory cell type of the intestinal epithelium. I found that goblet cells differentiate in a step-wise manner over the larval and metamorphic periods. I also determined the timing of gut looping for the intestine, a key morphological difference in the larval and adult intestine. I found that gut loops appear a short time after metamorphosis begins. In future studies, we seek to establish an assay for gut motility for studying mutant lines that have altered intestinal profiles. The zebrafish intestine may provide a robust model for understanding human intestinal physiology and disease

Regulation of Early Zebrafish Embryogenesis by Calcium Signaling and Dachsous1b Cadherin

Early animal embryogenesis entails a dynamic combination of embryonic cleavages, axial patterning, and gastrulation movements to shape a basic body plan. The underlying molecular signaling responsible for regulating this process remains poorly understood. In this thesis work, I first review recent progress in understanding of gastrulation movements in various model organisms brought by advances in imaging techniques. The externally developing and optically translucent zebrafish embryo is an ideal model organism to study vertebrate embryonic development by in vivo imaging. The objective of my thesis research is to leverage experimental advantages in the zebrafish model to uncover novel regulators and elucidate the molecular mechanisms involved in early vertebrate embryogenesis. Calcium signaling has been implicated in the control of many aspects of embryonic development. However, the spatiotemporal dynamics of calcium signaling during embryogenesis are not well characterized. By generating stable transgenic zebrafish lines ubiquitously expressing GCaMP6s, a genetically encoded calcium indicator, I demonstrated higher activities of calcium signaling during cleavage and blastula stages compared to previous reports. In addition, I showed that superficial dorsal-biased calcium signaling during blastula and gastrula stages was strongly correlated with and dependent on the dorsal organizer establishment. In the developing gastrulae, I directly visualized calcium activity in the dorsal forerunner cells and showed it was modulated by Nodal signaling in a cell non-autonomous manner. The GCaMP6s transgenic lines revealed with unprecedented spatiotemporal resolution the dynamic calcium signaling during early zebrafish embryogenesis and provide a superior tool for future studies. In zebrafish, mutations in atypical cadherin dachsous1b/dchs1b cause pleiotropic embryonic defects, including abnormal cleavages. Using the GCaMP6s transgenic reporter to examine the furrow-associated calcium activity in zebrafish dchs1b mutants, I showed that abnormal cleavages in dchs1b mutants were due to furrow progression defects during cytokinesis. These defects were likely caused by misregulated microtubules, as in vivo imaging of fluorescently marked microtubules during cleavage stages revealed reduced microtubule dynamics and impaired midzone microtubule assembly in dchs1b mutants. I further identified Ttc28 cytoplasmic protein as a molecular link between Dchs1b and microtubule dynamics. My biochemical experiments revealed that Dchs1b physically interacts via its intracellular domain with the tetratricopeptide repeat domain of Ttc28, and controls its subcellular distribution. Moreover, genetic inactivation of ttc28 resulted in increased microtubule dynamics and suppressed the microtubule defects in dchs1b mutants, suggesting a mechanism through which Dchs1b controls embryonic cleavages. In the last part of my thesis, I aimed to determine whether the chemokine ligand Ccl19.a1, a potential upstream regulator of calcium signaling, is required for axial patterning in zebrafish. I demonstrated that TALEN-generated ccl19a.1 mutations produce mildly dorsalized phenotypes and partially suppress the ventralized ichabod/ctnnb2 mutant phenotypes to influence axis formation, providing a genetic evidence for Ccl19.1 acting as a negative regulator of β-catenin and axis formation. Together, my work make several advances in understanding early vertebrate embryogenesis: it characterizes dynamic calcium signaling during zebrafish embryogenesis with a superior spatiotemporal resolution, reveals that Dchs1b regulates microtubule dynamics and embryonic cleavages by interacting with Ttc28 and regulating its subcellular distribution, and provides genetic evidence that Ccl19a.1 is necessary to limit β-catenin activity and consequently axis formation in zebrafish

Forces Generated by Cell Intercalculation Tow Epidermal Sheets in Mammalian Tissue Morphogenesis

Underlying the dramatic tissue movements of development—the bending, folding, squeezing, pushing, pulling, mass movements, and individual movements—are processes of cell migration. Throughout our lives, cell migration plays a role in the maintenance and regeneration of our tissues, and even in the development and progression of disease. To carry out the complex tasks of development and tissue morphogenesis, cells must coordinate their behaviors and migrate collectively. Insights into these collective behaviors have come from elegant studies of gastrulation movements in model organisms such as flies, frogs, and fish, uncovering conserved cellular and molecular mechanisms. However, the extent to which these mechanisms are refined, reiterated, and combined in the complex tissue environments of late development and adulthood is not well understood, highlighting the need for new models. Here, I develop a model of collective cell movements in late mammalian development by studying embryonic eyelid closure in mice, a process in which an epithelium locally reshapes, expands, and moves over another epithelium. Using a combination of lineage tracing and genetic ablation facilitated by ultrasound-guided lentiviral injection, I establish that the migratory cells of the eyelid front are derived from the epidermis rather than the periderm, and that the periderm is not required in the process as previously hypothesized. Quantitative analyses of cell proliferation, including inhibition of cell divisions in vivo, reveal that closure is primarily driven by cell motility rather than by proliferation. Optimizing conditions for the culture and live imaging of skin explants ex vivo, I reveal that cells of the eyelid front elongate perpendicularly to the axis of closure, extend mediolateral protrusions, and intercalate along this axis. Laser ablation and quantitative analyses of tissue deformation reveal that it is this intercalation, and not assembly and constriction of a supracellular actin cable, that drives eyelid closure. This mechanism is a novel mode of epithelial fusion in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. Functional analyses in vivo show that this mechanism requires alpha- 5 beta -1 integrin/fibronectin and myosin II-dependent cell motility, is potentially organized by non-canonical Wnts/PCP, and is supported by a concomitant reduction in cadherins through localized Wnt/beta -catenin signaling. These studies establish eyelid closure as a model in which well-described mechanisms of collective cell movement are integrated into a complex morphogenetic process, set the stage for future study of the interplay between canonical and non-canonical Wnts/PCP in regulating cell motility and intercalation, and present an opportunity to uncover novel regulators of collective migration

IDENTIFICATION OF THE MOLECULAR MECHANISMS OF ZEBRAFISH INNER EAR HAIR CELL REGENERATION USING HIGHTHROUGHPUT GENE EXPRESSION PROFILING

All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors, i.e. hair cells, but mammals only possess a very limited capacity for regeneration after birth. As a result, mammals suffer from permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates, we chose to study the hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique, Digital Gene Expression (DGE), which leverages the power of next-generation sequencing. We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological up-regulation of stat3 signaling accelerated hair cell regeneration without over-producing cells. Taking other published datasets into account, we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target not only for hair cell regeneration, but also for a much broader application in tissue repair and injury healing. The dissertation contains four supplemental files. Supplemental file 1 contains raw data of five expression profiles generated by DGE. It is a tab-delimited text file with six columns. The first column contains the sequences of the tags and the second to sixth columns contain the count of the corresponding tags in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 2 contains UniGene clusters identified from unambiguously mapped tags. It is a tab-delimited text file with six columns. The first column contains the UniGene IDs. The second to sixth columns contain the count of the corresponding UniGene clusters in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 3 contains candidate genes identified by comparison of the expression profiles during regeneration to the control profiles. It is a tab-delimited text file with 19 columns. The contents in each column are specified in the header. Supplemental file 4 contains a list of the candidate genes known to be expressed in the inner ear and/or the lateral line system during development. It is a tab-delimited text file with four columns which contain UniGene IDs, ZFIN IDs, Entrez Gene IDs, and gene symbols respectively

Collective Cell Migration Drives Morphogenesis of the Kidney Nephron

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis

Rigidity percolation uncovers a structural basis for embryonic tissue phase transitions

Embryo morphogenesis is impacted by dynamic changes in tissue material properties, which have been proposed to occur via processes akin to phase transitions (PTs). Here, we show that rigidity percolation provides a simple and robust theoretical framework to predict material/structural PTs of embryonic tissues from local cell connectivity. By using percolation theory, combined with directly monitoring dynamic changes in tissue rheology and cell contact mechanics, we demonstrate that the zebrafish blastoderm undergoes a genuine rigidity PT, brought about by a small reduction in adhesion-dependent cell connectivity below a critical value. We quantitatively predict and experimentally verify hallmarks of PTs, including power-law exponents and associated discontinuities of macroscopic observables. Finally, we show that this uniform PT depends on blastoderm cells undergoing meta-synchronous divisions causing random and, consequently, uniform changes in cell connectivity. Collectively, our theoretical and experimental findings reveal the structural basis of material PTs in an organismal context