Chan CU, Xiong F, Michaut A, Pourquie O, Mahadevan L. Direct Force Measurement and Loading on Developing Tissues in Intact Avian Embryos. BioRxiv. Submitted. Publisher's VersionAbstract
Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (0.1-1mm) in situ such as in early embryos require high spatial precision and minimal invasiveness. Here we report tissue force microscopy (TFM) integrating a vertical cantilever probe and live imaging to enable close-loop control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TFM quantitatively captures stress dynamics with high sensitivity. TFM also provides the capacity of applying a stable, non-invasive and physiologically relevant load to drive tissue deformation, which alters morphogenetic progression and cell movements. Together, TFM addresses a key technological gap in tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.
Kunz D, Wang A, Pritchard R, Wang W, Galo F, Bradshaw C, Terenzani E, Müller K, Huang YYS, Xiong F. Downregulation of Extraembryonic Tension Controls Body Axis Formation in Avian Embryos. BioRxiv. Submitted. Publisher's VersionAbstract
Embryonic tissues undergoing shape change draw mechanical input from extraembryonic substrates. In avian eggs, the early blastoderm disk is under the tension of the vitelline membrane (VM). Here we report that chicken VM characteristically downregulates tension and stiffness to facilitate stage-specific embryo morphogenesis. While early relaxation of the VM impairs blastoderm expansion, maintaining VM tension in later stages resists the convergence of the posterior body causing stalled elongation, open neural tube, and axis rupture. Biochemical and structural analysis shows that VM weakening follows the reduction of its outer-layer glycoprotein fibers, which is caused by an increasing albumen pH due to CO2 release from the egg. Our results identify a previously unrecognized mechanism of body axis defects through mis-regulation of extraembryonic tissue tension.
Xiong F*, Tentner AR*, Hiscock TW, Huang P, Megason SG. Heterogeneity of Sonic Hedgehog Response Dynamics and Fate Specification in Single Neural Progenitors. Submitted.Abstract
During neural tube patterning, a gradient of Sonic hedgehog (Shh) signaling specifies ventral progenitor fates. The cellular response to Shh is processed through a genetic regulatory network (GRN) to code distinct fate decisions. This process integrates Shh response level, duration and other inputs and is affected by noise in signaling and cell position. How reliably a single cell’s Shh response profile predicts its fate choice is unclear. Here we use live imaging to track neural progenitors that carry both Shh and fate reporters in zebrafish embryos. We found that there is significant heterogeneity between Shh response and fate choice in single cells. We quantitatively modeled reporter intensities to obtain single cell response levels over time and systematically determined their correlation with multiple models of cell fate specification. Our input-output analysis shows that while no single metric perfectly predicts fate choices, the maximal Shh response level correlates best overall with progenitor fate choices across the anterior-posterior axis.
Sutlive J, Xu H, Chen Y, Gou K, Xiong F, Guo M, Chen Z. Generation, Transmission, and Regulation of Mechanical Forces in Embryonic Morphogenesis. Small. 2021. Publisher's VersionAbstract
Embryonic morphogenesis is a biological process which depicts shape forming of tissues and organs during development. Unveiling the roles of mechanical forces generated, transmitted, and regulated in cells and tissues through these processes is key to understanding the biophysical mechanisms governing morphogenesis. To this end, it is imperative to measure, simulate, and predict the regulation and control of these mechanical forces during morphogenesis. This article aims to provide a comprehensive review of the recent advances on mechanical properties of cells and tissues, generation of mechanical forces in cells and tissues, the transmission processes of these generated forces during cells and tissues, the tools and methods used to measure and predict these mechanical forces in vivo, in vitro, or in silico, and to better understand the corresponding regulation and control of generated forces. Understanding the biomechanics and mechanobiology of morphogenesis will not only shed light on the fundamental physical mechanisms underlying these concerted biological processes during normal development, but also uncover new information that will benefit biomedical research in preventing and treating congenital defects or tissue engineering and regeneration.
Moon LD, Xiong F. Mechanics of neural tube morphogenesis. Seminars in Cell & Developmental Biology. 2021. Publisher's VersionAbstract
The neural tube is an important model system of morphogenesis representing the developmental module of out-of-plane epithelial deformation. As the embryonic precursor of the central nervous system, the neural tube also holds keys to many defects and diseases. Recent advances begin to reveal how genetic, cellular and environmental mechanisms work in concert to ensure correct neural tube shape. A physical model is emerging where these factors converge at the regulation of the mechanical forces and properties within and around the tissue that drive tube formation towards completion. Here we review the dynamics and mechanics of neural tube morphogenesis and discuss the underlying cellular behaviours from the viewpoint of tissue mechanics. We will also highlight some of the conceptual and technical next steps.
Oginuma* M, Harima* Y, Tarazona OA, Diaz-Cuadros M, Michaut A, Ishitani T, Xiong F, Pourquie O. Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature. 2020. Publisher's VersionAbstract
Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic β-catenin acetylation downstream of WNT signalling. As acetylated β-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.
Mechanical Coupling Coordinates the Co-elongation of Axial and Paraxial Tissues in Avian Embryos
Xiong F, Ma W, Benazeraf B, Mahadevan L, Pourquie O. Mechanical Coupling Coordinates the Co-elongation of Axial and Paraxial Tissues in Avian Embryos. Developmental Cell. 2020. Publisher's VersionAbstract
Tissues undergoing morphogenesis impose mechanical effects on one another. How developmental programs adapt to or take advantage of these effects remains poorly explored. Here, using a combination of live imaging, modeling, and microsurgical perturbations, we show that the axial and paraxial tissues in the forming avian embryonic body coordinate their rates of elongation through mechanical interactions. First, a cell motility gradient drives paraxial presomitic mesoderm (PSM) expansion, resulting in compression of the axial neural tube and notochord; second, elongation of axial tissues driven by PSM compression and polarized cell intercalation pushes the caudal progenitor domain posteriorly; finally, the axial push drives the lateral movement of midline PSM cells to maintain PSM growth and cell motility. These interactions form an engine-like positive feedback loop, which sustains a shared elongation rate for coupled tissues. Our results demonstrate a key role of inter-tissue forces in coordinating distinct body axis tissues during their co-elongation.
Mongera A, Michaut A, Guillot C, Xiong F, Pourquié O. Mechanics of Anteroposterior Axis Formation in Vertebrates. Annual Review of Cell and Developmental Biology. 2019;35 (1) :null. Publisher's VersionAbstract
The vertebrate anteroposterior axis forms through elongation of multiple tissues during embryogenesis. This process is based on tissue-autonomous mechanisms of force generation and intertissue mechanical coupling whose failure leads to severe developmental anomalies such as body truncation and spina bifida. Similar to other morphogenetic modules, anteroposterior body extension requires both the rearrangement of existing materials—such as cells and extracellular matrix—and the local addition of new materials, i.e., anisotropic growth, through cell proliferation, cell growth, and matrix deposition. Numerous signaling pathways coordinate body axis formation via regulation of cell behavior during tissue rearrangements and/or volumetric growth. From a physical perspective, morphogenesis depends on both cell-generated forces and tissue material properties. As the spatiotemporal variation of these mechanical parameters has recently been explored in the context of vertebrate body elongation, the study of this process is likely to shed light on the cross talk between signaling and mechanics during morphogenesis. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 35 is October 7, 2019. Please see for revised estimates.
Oginuma M, Moncuquet P, Xiong F, Karoly E, Chal J, Guevorkian K, Pourquié O. A Gradient of Glycolytic Activity Coordinates FGF and Wnt Signaling during Elongation of the Body Axis in Amniote Embryos. Developmental Cell. 2017;40 (4) :342-353. Publisher's VersionAbstract

Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatiotemporal regulation of glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of fibroblast growth factor (FGF) signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that glycolysis in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis.

Multibow: Digital Spectral Barcodes for Cell Tracing
Xiong F, Obholzer ND, Noche RR, Megason SG. Multibow: Digital Spectral Barcodes for Cell Tracing. PLOS ONE. 2015;10 (5) :e0127822. Publisher's VersionAbstract

We introduce a multicolor labeling strategy (Multibow) for cell tracing experiments in developmental and regenerative processes. Building on Brainbow-based approaches that produce colors by differential expression levels of different fluorescent proteins, Multibow adds a layer of label diversity by introducing a binary code in which reporters are initially OFF and then probabilistically ON or OFF following Cre recombination. We have developed a library of constructs that contains seven different colors and three different subcellular localizations. Combining constructs from this library in the presence of Cre generates cells labeled with multiple independently expressed colors based on if each construct is ON or OFF following recombination. These labels form a unique "barcode" that allows the tracking of the cell and its clonal progenies in addition to expression level differences of each color. We tested Multibow in zebrafish which validates its design concept and suggests its utility for cell tracing applications in development and regeneration.

Abstracting the principles of development using imaging and modeling
Xiong F, Megason SG. Abstracting the principles of development using imaging and modeling. Integrative Biology. 2015;7 :633-642. Publisher's VersionAbstract

Here we look at modern developmental biology with a focus on the relationship between different approaches of investigation. We argue that direct imaging is a powerful approach not only for obtaining descriptive information but also for model generation and testing that lead to mechanistic insights. Modeling, on the other hand, conceptualizes imaging data and provides guidance to perturbations. The inquiry progresses most efficiently when a trinity of approaches—quantitative imaging (measurement), modeling (theory) and perturbation (test)—are pursued in concert, but not when one approach is dominant. Using recent studies of the zebrafish system, we show how this combination has effectively advanced classic topics in developmental biology compared to a perturbation-centric approach. Finally, we show that interdisciplinary expertise and perhaps specialization are necessary for carrying out a systematic approach, and discuss the technical hurdles.

Interplay of Cell Shape and Division Orientation Promotes Robust Morphogenesis of Developing Epithelia
Xiong F, Ma W, Hiscock TW, Mosaliganti KR, Tentner AR, Brakke KA, Rannou N, Gelas A, Souhait L, Swinburne IA, et al. Interplay of Cell Shape and Division Orientation Promotes Robust Morphogenesis of Developing Epithelia. Cell. 2014;159 (2) :415-427. Publisher's VersionAbstract

Epithelial cells acquire functionally important shapes (e.g., squamous, cuboidal, columnar) during development. Here, we combine theory, quantitative imaging, and perturbations to analyze how tissue geometry, cell divisions, and mechanics interact to shape the presumptive enveloping layer (pre-EVL) on the zebrafish embryonic surface.We find that, under geometrical constraints, pre-eVL flattening is regulated by surface cell number changes following differentially oriented cell divisions. The division pattern is, in turn, determined by the cell shape distribution, which forms under geometrical constraints by cell-cell mechanical coupling. An integrated mathematical model of this shape-division feedback loop recapitulates empirical observations. Surprisingly, the model predicts that cell shape is robust to changes of tissue surface area, cell volume, and cell number, which we confirm in vivo. Further simulations and perturbations suggest the parameter linking cell shape and division orientation contributes to epithelial diversity. Together, our work identifies an evolvable design logic that enables robust cell-level regulation of tissue-level development.

Xiong F. Integrated Analysis of Patterning, Morphogenesis, and Cell Divisions in Embryonic Development by in toto Imaging and Quantitative Cell Tracking. Harvard University. 2013. Publisher's VersionAbstract

Patterning, morphogenesis, and cell divisions are distinct processes during development yet are concurrent and likely highly integrated. However, it has been challenging to investigate them as a whole. Recent advances in imaging and labeling tools make it possible to observe live tissues with high coverage and resolution. In this dissertation work, we developed a novel imaging platform that allowed us to fully capture the early neural tube formation process in live zebrafish embryos at cellular resolution. Importantly, these datasets allow us to reliably track single neural progenitors. These tracks carry information on the history of cell movement, shape change, division, and gene expression all together. By comparing tracks of different progenitor fates, we found they show a spatially noisy response to Sonic hedgehog (Shh) and become specified in a positionally mixed manner, in surprising contrast to the "French Flag" morphogen patterning model. Both cell movement and division contribute to cell mixing. In addition, we decoupled the temporal and genetic regulatory network (GRN) noises in Shh interpretation using tracks that carry both Shh signaling and cell fate reporters. Our tracks suggest that, after specification, progenitors undergo sorting to self-assemble a sharp pattern. Consistent with this hypothesis, we found ectopically induced progenitors move to correct locations. Furthermore, we show that proper adhesion is required for cell sorting to happen (Chapters 2 and 3). In the cleavage stage embryos, the cells on the surface undergo shape changes followed by lineage separation and differentiation. We quantitatively measured this morphogenesis process and tracked cell divisions. By applying a mathematical model we uncover a predictive, and perhaps general link between cell division orientation, mechanical interaction, and the morphogenetic behavior of the whole surface layer (Chapter 4). Finally, we discuss the concepts and tools of cell tracking including a multi-color cell labeling method we developed by modifying the "Brainbow" system (Chapter 5). Together this dissertation showcases the importance and promise of live observation based, quantitative and integrated analysis in our understanding of complex multi-cellular developmental processes.

Specified neural progenitors sort to form sharp domains after noisy Shh signaling.
Xiong F, Tentner AR, Huang P, Gelas A, Mosaliganti KR, Souhait L, Rannou N, Swinburne IA, Obholzer ND, Cowgill PD, et al. Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell. 2013;153 (3) :550-61. Publisher's versionAbstract

Sharply delineated domains of cell types arise in developing tissues under instruction of inductive signal (morphogen) gradients, which specify distinct cell fates at different signal levels. The translation of a morphogen gradient into discrete spatial domains relies on precise signal responses at stable cell positions. However, cells in developing tissues undergoing morphogenesis and proliferation often experience complex movements, which may affect their morphogen exposure, specification, and positioning. How is a clear pattern achieved with cells moving around? Using in toto imaging of the zebrafish neural tube, we analyzed specification patterns and movement trajectories of neural progenitors. We found that specified progenitors of different fates are spatially mixed following heterogeneous Sonic Hedgehog signaling responses. Cell sorting then rearranges them into sharply bordered domains. Ectopically induced motor neuron progenitors also robustly sort to correct locations. Our results reveal that cell sorting acts to correct imprecision of spatial patterning by noisy inductive signals.

Xu C, Fan ZP, Müller P, Fogley R, DiBiase A, Trompouki E, Unternaehrer J, Xiong F, Torregroza I, Evans T, et al. Nanog-like regulates endoderm formation through the Mxtx2-Nodal pathway. Dev Cell. 2012;22 (3) :625-38. Publisher's VersionAbstract

In mammalian embryonic stem cells, the acquisition of pluripotency is dependent on Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extraembryonic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We examined the genes that were regulated by Nanog-like and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which, in turn, specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extraembryonic tissue required for endoderm induction.

Mosaliganti KR, Noche RR, Xiong F, Swinburne IA, Megason SG. ACME: automated cell morphology extractor for comprehensive reconstruction of cell membranes. PLoS Comput Biol. 2012;8 (12) :e1002780. Publisher's VersionAbstract

The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (

Huang P, Xiong F, Megason SG, Schier AF. Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord. PLoS Genet. 2012;8 (6) :e1002762. Publisher's VersionAbstract

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.