ADAMTSL5 is an epigenetically activated gene underlying tumorigenesis and drug resistance in hepatocellular carcinoma.

BACKGROUND & AIMS: The tumour microenvironment shapes tumour growth through cellular communications that include both direct interactions and secreted factors. The aim of this study was to characterize the impact of the secreted glycoprotein ADAMTSL5, whose role in cancer has not been previously investigated, on hepatocellular carcinoma (HCC). METHODS: ADAMTSL5 methylation status was evaluated through bisulfite sequencing, and publicly available data analysis. ADAMTSL5 RNA and protein expression were assessed in mouse models and HCC patient samples and compared to data from published datasets. Functional studies, including association of ADAMTSL5 depletion with responsiveness to clinically relevant drugs, were performed in cellular and in vivo models. Molecular alterations associated with ADAMTSL5 targeting were determined using proteomics, biochemistry, and reverse-transcription quantitative PCR. RESULTS: Methylome analysis revealed hypermethylated gene body CpG islands at the ADAMTSL5 locus in both mouse and human HCC, correlating with higher ADAMTSL5 expression. ADAMTSL5 targeting interfered with tumorigenic properties of HCC cells in vitro and in vivo, whereas ADAMTSL5 overexpression conferred tumorigenicity to pre-tumoural hepatocytes sensitized to transformation by a modest level of MET receptor expression. Mechanistically, ADAMTSL5 abrogation led to a reduction of several oncogenic inputs relevant to HCC, including reduced expression and/or phosphorylation levels of receptor tyrosine kinases MET, EGFR, PDGFRß, IGF1Rß, or FGFR4. This phenotype was associated with significantly increased sensitivity of HCC cells to clinically relevant drugs, namely sorafenib, lenvatinib, and regorafenib. Moreover, ADAMTSL5 depletion drastically increased expression of AXL, accompanied by a sensitization to bemcentinib. CONCLUSIONS: Our results point to a role for ADAMTSL5 in maintaining the function of key oncogenic signalling pathways, suggesting that it may act as a master regulator of tumorigenicity and drug resistance in HCC. LAY SUMMARY: The environment of cancer cells has profound effects on establishment, progression, and response of a tumour to treatment. Herein, we show that ADAMTSL5, a protein secreted by liver cancer cells and overlooked in cancer so far, is increased in this tumour type, is necessary for tumour formation and supports drug resistance. Adamtsl5 removal conferred sensitivity of liver cancer cells to drugs used in current treatment. This suggests ADAMTSL5 as a potential marker in liver cancer as well as a possible drug target.

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis.

BACKGROUND & AIMS: The variety of alterations found in hepatocellular carcinoma (HCC) makes the identification of functionally relevant genes and their combinatorial actions in tumorigenesis challenging. Deregulation of receptor tyrosine kinases (RTKs) is frequent in HCC, yet little is known about the molecular events that cooperate with RTKs and whether these cooperative events play an active role at the root of liver tumorigenesis. METHODS: A forward genetic screen was performed using Sleeping Beauty transposon insertional mutagenesis to accelerate liver tumour formation in a genetic context in which subtly increased MET RTK levels predispose mice to tumorigenesis. Systematic sequencing of tumours identified common transposon insertion sites, thus uncovering putative RTK cooperators for liver cancer. Bioinformatic analyses were applied to transposon outcomes and human HCC datasets. In vitro and in vivo (through xenografts) functional screens were performed to assess the relevance of distinct cooperative modes to the tumorigenic properties conferred by RTKs. RESULTS: We identified 275 genes, most of which are altered in patients with HCC. Unexpectedly, these genes are not restricted to a small set of pathway/cellular processes, but cover a large spectrum of cellular functions, including signalling, metabolism, chromatin remodelling, mRNA degradation, proteasome, ubiquitination, cell cycle regulation, and chromatid segregation. We validated 15 tumour suppressor candidates, as shRNA-mediated targeting confers tumorigenicity to RTK-sensitized cells, but not to cells with basal RTK levels. This demonstrates that the context of enhanced RTK levels is essential for their action in tumour initiation. CONCLUSION: Our study identifies unanticipated genetic interactions underlying gene cooperativity with RTKs in HCC. Moreover, these results show how subtly increased levels of wild-type RTKs provide a tumour permissive cellular environment allowing a large spectrum of deregulated mechanisms to initiate liver cancer. LAY SUMMARY: Receptor tyrosine kinases (RTKs) are among signals frequently deregulated in patients with hepatocellular carcinoma and their deregulation confers essential biological properties to cancer cells. We have applied a genetic method to randomly mutate large numbers of genes in the context of a mouse model with increased RTK levels, predisposed to develop liver cancer. We identified mechanisms that accelerate tumour formation in cooperation with enhanced RTK levels. The wide array of cellular functions among these cooperators illustrates an extraordinary capability of RTKs to render the liver more vulnerable to additional alterations, by priming cells for tumour initiation.

A phosphokinome-based screen uncovers new drug synergies for cancer driven by liver-specific gain of nononcogenic receptor tyrosine kinases.

Genetic mutations leading to oncogenic variants of receptor tyrosine kinases (RTKs) are frequent events during tumorigenesis; however, the cellular vulnerability to nononcogenic RTK fluctuations has not been characterized. Here, we demonstrated genetically that in the liver subtle increases in wild-type Met RTK levels are sufficient for spontaneous tumors in mice (Alb-R26Met ), conceptually illustrating how the shift from physiological to pathological conditions results from slight perturbations in signaling dosage. By analyzing 96 different genes in a panel of tumor samples, we demonstrated that liver tumorigenesis modeled by Alb-R26Met mice corresponds to a subset of hepatocellular carcinoma (HCC) patients, thus establishing the clinical relevance of this HCC mouse model. We elucidated the regulatory networks underlying tumorigenesis by combining a phosphokinome screen with bioinformatics analysis. We then used the signaling diversity results obtained from Alb-R26Met HCC versus control livers to design an "educated guess" drug screen, which led to the identification of new, deleterious synthetic lethal interactions. In particular, we report synergistic effects of mitogen-activated protein kinase kinase, ribosomal S6 kinase, and cyclin-dependent kinase 1/2 in combination with Bcl-XL inhibition on a panel of liver cancer cells. Focusing on mitogen-activated protein kinase kinase and Bcl-XL targeting, we mechanistically demonstrated concomitant down-regulation of phosphorylated extracellular signal-regulated kinase and myeloid cell leukemia 1 levels. Of note, a phosphorylated extracellular signal-regulated kinase+/BCL-XL+ /myeloid cell leukemia 1+ signature, deregulated in Alb-R26Met tumors, characterizes a subgroup of HCC patients with poor prognosis. CONCLUSION: Our genetic studies highlight the heightened vulnerability of liver cells to subtle changes in nononcogenic RTK levels, allowing them to acquire a molecular profile that facilitates the full tumorigenic program; furthermore, our outcomes uncover new synthetic lethal interactions as potential therapies for a cluster of HCC patients. (Hepatology 2017;66:1644-1661).

A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: Application to the ascidian Ciona intestinalis.

Genome-wide resources, such as collections of cDNA clones encoding for complete proteins (full-ORF clones), are crucial tools for studying the evolution of gene function and genetic interactions. Non-model organisms, in particular marine organisms, provide a rich source of functional diversity. Marine organism genomes are, however, frequently highly polymorphic and encode proteins that diverge significantly from those of well-annotated model genomes. The construction of full-ORF clone collections from non-model organisms is hindered by the difficulty of predicting accurately the N-terminal ends of proteins, and distinguishing recent paralogs from highly polymorphic alleles. We report a computational strategy that overcomes these difficulties, and allows for accurate gene level clustering of transcript data followed by the automated identification of full-ORFs with correct 5'- and 3'-ends. It is robust to polymorphism, includes paralog calling and does not require evolutionary proximity to well annotated model organisms. We developed this pipeline for the ascidian Ciona intestinalis, a highly polymorphic member of the divergent sister group of the vertebrates, emerging as a powerful model organism to study chordate gene function, Gene Regulatory Networks and molecular mechanisms underlying human pathologies. Using this pipeline we have generated the first full-ORF collection for a highly polymorphic marine invertebrate. It contains 19,163 full-ORF cDNA clones covering 60% of Ciona coding genes, and full-ORF orthologs for approximately half of curated human disease-associated genes.

Rapid evolutionary responses of life history traits to different experimentally-induced pollutions in Caenorhabditis elegans.

BACKGROUND: Anthropogenic disturbances can lead to intense selection pressures on traits and very rapid evolutionary changes. Evolutionary responses to environmental changes, in turn, reflect changes in the genetic structure of the traits, accompanied by a reduction of evolutionary potential of the populations under selection. Assessing the effects of pollutants on the evolutionary responses and on the genetic structure of populations is thus important to understanding the mechanisms that entail specialization to novel environmental conditions or resistance to novel stressors. RESULTS: Using an experimental evolution approach we exposed Caenorhabditis elegans populations to uranium, salt and alternating uranium-salt environments over 22 generations. We analyzed the changes in the average values of life history traits and the consequences at the demographic level in these populations. We also estimated the phenotypic and genetic (co)variance structure of these traits at different generations. Compared to populations in salt, populations in uranium showed a reduction of the stability of their trait structure and a higher capacity to respond by acclimation. However, the evolutionary responses of traits were generally lower for uranium compared to salt treatment; and the evolutionary responses to the alternating uranium-salt environment were between those of constant environments. Consequently, at the end of the experiment, the population rate of increase was higher in uranium than in salt and intermediate in the alternating environment. CONCLUSIONS: Our multigenerational experiment confirmed that rapid adaptation to different polluted environments may involve different evolutionary responses resulting in demographic consequences. These changes are partly explained by the effects of the pollutants on the genetic (co)variance structure of traits and the capacity of acclimation to novel conditions. Finally, our results in the alternating environment may confirm the selection of a generalist type in this environment.

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

M1BP is an essential transcriptional activator of oxidative metabolism during Drosophila development.

Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, we show that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. We further demonstrate using high-resolution imaging, transcriptomic and biochemical analyses that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.

A live-imaging protocol to track cell movement in the Xenopus embryo.

Tracking individual cell movement during development is challenging, particularly in tissues subjected to major remodeling. Currently, most live imaging techniques in Xenopus are limited to tissue explants and/or to superficial cells. We describe here a protocol to track immature multiciliated cells (MCCs) moving within the inner epidermal layer of a whole embryo. In addition, we present a data processing protocol to uncouple the movements of individual cells from the coplanar drifts of the tissue in which they are embedded. For complete details on the use and execution of this protocol, please refer to Chuyen et al. (2021).

The Scf/Kit pathway implements self-organized epithelial patterning.

How global patterns emerge from individual cell behaviors is poorly understood. In the Xenopus embryonic epidermis, multiciliated cells (MCCs) are born in a random pattern within an inner mesenchymal layer and subsequently intercalate at regular intervals into an outer epithelial layer. Using video microscopy and mathematical modeling, we found that regular pattern emergence involves mutual repulsion among motile immature MCCs and affinity toward outer-layer intercellular junctions. Consistently, Arp2/3-mediated actin remodeling is required for MCC patterning. Mechanistically, we show that the Kit tyrosine kinase receptor, expressed in MCCs, and its ligand Scf, expressed in outer-layer cells, are both required for regular MCC distribution. Membrane-associated Scf behaves as a potent adhesive cue for MCCs, while its soluble form promotes their mutual repulsion. Finally, Kit expression is sufficient to confer order to a disordered heterologous cell population. This work reveals how a single signaling system can implement self-organized large-scale patterning.

Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle.

Complex animals build specialised muscles to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres and mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we use Drosophila muscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selector spalt instructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated leg muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this causes the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream of spalt synchronizing mitochondria with myofibril morphogenesis.

Endogenous neural stem cells modulate microglia and protect against demyelination.

In response to corpus callosum (CC) demyelination, subventricular zone-derived neural progenitors (SVZdNPs) are mobilized and generate new myelinating oligodendrocytes (OLG). Here, we examine the putative immunomodulatory properties of endogenous SVZdNPs during demyelination in the cuprizone model. SVZdNP density was higher in the lateral and rostral CC regions, and demyelination was inversely correlated with activated microglial density and pro-inflammatory cytokine levels. Single-cell RNA sequencing showed that CC areas with high levels of SVZdNP mobilization were enriched in a microglial cell subpopulation with an immunomodulatory signature. We propose MFGE8 (milk fat globule-epidermal growth factor-8) and ß3 integrin as a ligand/receptor pair involved in dialogue between SVZdNPs and microglia. Immature SVZdNPs mobilized to the demyelinated CC were found highly enriched in MFGE8, which promoted the phagocytosis of myelin debris in vitro. Overall, these results demonstrate that, in addition to their cell replacement capacity, endogenous progenitors have immunomodulatory properties, highlighting a new role for endogenous SVZdNPs in myelin regeneration.

Modeling Heterogeneity of Triple-Negative Breast Cancer Uncovers a Novel Combinatorial Treatment Overcoming Primary Drug Resistance.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterized by a remarkable molecular heterogeneity. Currently, there are no effective druggable targets and advanced preclinical models of the human disease. Here, a unique mouse model (MMTV-R26Met mice) of mammary tumors driven by a subtle increase in the expression of the wild-type MET receptor is generated. MMTV-R26Met mice develop spontaneous, exclusive TNBC tumors, recapitulating primary resistance to treatment of patients. Proteomic profiling of MMTV-R26Met tumors and machine learning approach show that the model faithfully recapitulates intertumoral heterogeneity of human TNBC. Further signaling network analysis highlights potential druggable targets, of which cotargeting of WEE1 and BCL-XL synergistically kills TNBC cells and efficiently induces tumor regression. Mechanistically, BCL-XL inhibition exacerbates the dependency of TNBC cells on WEE1 function, leading to Histone H3 and phosphoS33RPA32 upregulation, RRM2 downregulation, cell cycle perturbation, mitotic catastrophe, and apoptosis. This study introduces a unique, powerful mouse model for studying TNBC formation and evolution, its heterogeneity, and for identifying efficient therapeutic targets.

Drosophila Aversive Behavior toward Erwinia carotovora carotovora Is Mediated by Bitter Neurons and Leukokinin.

The phytopathogen Erwinia carotovora carotovora (Ecc) has been used successfully to decipher some of the mechanisms that regulate the interactions between Drosophila melanogaster and bacteria, mostly following forced association between the two species. How do Drosophila normally perceive and respond to the presence of Ecc is unknown. Using a fly feeding two-choice assay and video tracking, we show that Drosophila are first attracted but then repulsed by an Ecc-contaminated solution. The initial attractive phase is dependent on the olfactory Gr63a and Gαq proteins, whereas the second repulsive phase requires a functional gustatory system. Genetic manipulations and calcium imaging indicate that bitter neurons and gustatory receptors Gr66a and Gr33a are needed for the aversive phase and that the neuropeptide leukokinin is also involved. We also demonstrate that these behaviors are independent of the NF-κB cascade that controls some of the immune, metabolic, and behavioral responses to bacteria.

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

BACKGROUND: The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. RESULTS: We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. CONCLUSIONS: In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.

Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors.

It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. We previously showed that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation (Narbonne-Reveau et al., 2016). Using single-cell transcriptomics, clonal analysis and numerical modeling, we now identify a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin.

Publisher Correction: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer.

In the original version of this Article, the sixth sentence of the abstract incorrectly read 'Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change.', and should have read 'Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change in a large proportion of HCC patients.'. This has been corrected in both the PDF and HTML versions of the Article.

Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer.

Epigenetic modifications such as aberrant DNA methylation reshape the gene expression repertoire in cancer. Here, we used a clinically relevant hepatocellular carcinoma (HCC) mouse model (Alb-R26Met) to explore the impact of DNA methylation on transcriptional switches associated with tumorigenesis. We identified a striking enrichment in genes simultaneously hypermethylated in CpG islands (CGIs) and overexpressed. These hypermethylated CGIs are located either in the 5'-UTR or in the gene body region. Remarkably, such CGI hypermethylation accompanied by gene upregulation also occurs in 56% of HCC patients, which belong to the "HCC proliferative-progenitor" subclass. Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26Met HCC model undergo the same change. Among reprogrammed genes, several are well-known oncogenes. For others not previously linked to cancer, we demonstrate here their action together as an "oncogene module". Thus, hypermethylation of gene body CGIs is predictive of elevated oncogene levels in cancer, offering a novel stratification strategy and perspectives to normalise cancer gene dosages.

Combined drug action of 2-phenylimidazo[2,1-b]benzothiazole derivatives on cancer cells according to their oncogenic molecular signatures.

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by "RTK swapping" by interfering with PDGFRß phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

Time-lapse imaging of live Phallusia embryos for creating 3D digital replicas.

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article provides a method to fluorescently label and image structures of interest that will subsequently be reconstructed, such as cell membranes or nuclei. The protocol describes live imaging of Phallusia mammillata embryos, which are robust, colorless, and optically transparent with negligible autofluorescence. Their diameter ranges from 100 µm to 120 µm, which allows time-lapse microscopy of whole embryos using two-photon microscopy with a high-resolution objective. Although two-photon imaging is described in detail, any imaging technology that results in a z-stack may be used. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes.

Imaging of fixed ciona embryos for creating 3D digital replicas.

During embryonic development, cell behaviors that are tightly coordinated both spatially and temporally integrate at the tissue level and drive embryonic morphogenesis. Over the past 20 years, advances in imaging techniques, in particular, the development of confocal imaging, have opened a new world in biology, not only giving us access to a wealth of information, but also creating new challenges. It is sometimes difficult to make the best use of the recordings of the complex, inherently three-dimensional (3D) processes we now can observe. In particular, these data are often not directly suitable for even simple but conceptually fundamental quantifications. This article presents a method for imaging embryonic development with cellular resolution in fixed ascidian embryos. A large fraction of the ascidian community primarily studies the development of the cosmopolitan ascidian Ciona intestinalis. Because the embryos of this species are insufficiently transparent and show significant autofluorescence, live imaging is difficult. Thus, whole embryos are fixed and optically cleared. They are then stained and imaged on a regular or two-photon confocal microscope. The resulting image stacks can subsequently be digitalized and segmented to produce 3D embryo replicas that can be interfaced to a model organism database and used to quantify cell shapes.