Sean Megason

Harvard University | Harvard · FAS Center for Systems Biology

Background Spatial mapping on the single‐cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of i...

A regular heartbeat is essential to vertebrate life. In the mature heart, this function is driven by an anatomically localized pacemaker. By contrast, pacemaking capability is broadly distributed in the early embryonic heart1–3, raising the question of how tissue-scale activity is first established and then maintained during embryonic development....

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell res...

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 a...

Much progress has been made toward generating analogs of early embryos, such as gastruloids and embryoids, in vitro. However, methods for how to fully mimic the cell movements of gastrulation and coordinate germ-layer patterning to induce head formation are still lacking. Here, we show that a regional Nodal gradient applied to zebrafish animal pole...

Brain endothelial cells (BECs) play an important role in maintaining central nervous system (CNS) homeostasis through blood-brain barrier (BBB) functions. In addition to actively regulating material exchange between the circulation and CNS, BECs express low baseline levels of adhesion receptors, which limits entry of leukocytes. Supporting cells in...

The heart is among the first organs to function in vertebrate development, but its transition from silent to beating has not been directly characterized. Using all-optical electrophysiology, we captured the very first zebrafish heartbeat and analyzed the development of cardiac excitability around this singular event. The first beats appeared sudden...

Since the proposal of the differential adhesion hypothesis, scientists have been fascinated by how cell adhesion mediates cellular self-organization to form spatial patterns during development. The search for molecular tool kits with homophilic binding specificity resulted in a diverse repertoire of adhesion molecules. Recent understanding of the d...

Morphogenesis, the process by which tissues develop into functional shapes, requires coordinated mechanical forces. Most current literature ascribes contractile forces derived from actomyosin networks as the major driver of tissue morphogenesis. Recent works from diverse species have shown that pressure derived from fluids can generate deformations...

Cell segmentation plays a crucial role in understanding, diagnosing, and treating diseases. Despite the recent success of deep learning-based cell segmentation methods, it remains challenging to accurately segment densely packed cells in 3D cell membrane images. Existing approaches also require fine-tuning multiple manually selected hyperparameters...

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, ma...

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restricts its permeability to proteins and small molecules. Classic chick-quail chimera studies showed that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here we identify Spock1 as a...

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator o...

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator o...

Nodal, as a morphogen, plays important roles in cell fate decision, pattern formation and organizer function. But because of the complex context in vivo and technology limitations, systematic studying of genes, cell types and patterns induced by Nodal alone is still missing. Here, by using a relatively simplified model, the zebrafish blastula anima...

Cellular analysis of developmental processes and toxicities has traditionally entailed bulk methods (e.g., transcriptomics) that lack single cell resolution or tissue localization methods (e.g., immunostaining) that allow only a few genes to be monitored in each experiment. Recent technological advances have enabled interrogation of genomic functio...

Convergence of paradigms yields patterns In embryo development, spatial patterns of distinct cell types arise reproducibly. In the zebrafish spinal cord, neural progenitors form stereotypic stripe patterns despite the noisy instructive signals and large-scale cellular rearrangement required during morphogenesis. Tsai et al. show that a cell type–sp...

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, ma...

An outstanding question in embryo development is how spatial patterns are formed robustly. In the zebrafish spinal cord, neural progenitors form stereotypic stripe-like patterns despite noisy morphogen signaling and large-scale cellular rearrangement required for tissue growth and morphogenesis. We set out to understand the mechanisms underlying th...

Animals make organs of precise size, shape, and symmetry but how developing embryos do this is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the developing inner ear. We find that fluid accumulation creates hydr...

As an optically transparent model organism with an endothelial blood-brain barrier (BBB), zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms governing the zebrafish BBB remain poorly characterized. Here we captur...

Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized der...

As an optically transparent model organism with an endothelial blood-brain barrier (BBB), zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms governing the zebrafish BBB remain poorly characterized. Here we find a...

Combining expansion and the lattice light sheet Optical and electron microscopy have made tremendous inroads into understanding the complexity of the brain. Gao et al. introduce an approach for high-resolution tracing of neurons, their subassemblies, and their molecular constituents over large volumes. They applied their method, which combines expa...

To enable robust patterning, morphogen systems should be resistant to variations in gene expression and tissue size. Here we explore how a Shh morphogen gradient in the ventral neural tube enables proportional patterning in embryos of varying sizes. Using a surgical technique to reduce the size of zebrafish embryos and quantitative confocal microsc...

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 pos...

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion micro...

Animals make organs of precise size, shape, and symmetry despite noise in underlying molecular and cellular processes. How developing organs manage this noise is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the...

The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and bal...

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here we examine somite scaling and f...

Mapping the vertebrate developmental landscape As embryos develop, numerous cell types with distinct functions and morphologies arise from pluripotent cells. Three research groups have used single-cell RNA sequencing to analyze the transcriptional changes accompanying development of vertebrate embryos (see the Perspective by Harland). Wagner et al....

Mapping the vertebrate developmental landscape As embryos develop, numerous cell types with distinct functions and morphologies arise from pluripotent cells. Three research groups have used single-cell RNA sequencing to analyze the transcriptional changes accompanying development of vertebrate embryos (see the Perspective by Harland). Wagner et al....

Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Whilst many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find...

Continuing the resolution revolution The living cell contains dynamic, spatially complex subassemblies that are sensitive to external perturbations. To minimize such perturbations, cells should be imaged in their native multicellular environments, under as gentle illumination as possible. However, achieving the spatiotemporal resolution needed to f...

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here we examine somite scaling and f...

A small number of signaling pathways are used ubiquitously throughout development. How can enough information be carried through such a small number of channels? Reporting in Cell, Nandagopal et al. (2018) reveal how different Notch ligands can regulate different targets through the same receptor using differences in signaling dynamics.

Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Whilst many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find...

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution...

The inner ear is a fluid-filled closed-epithelial structure whose normal function requires maintenance of an internal hydrostatic pressure and fluid composition by unknown mechanisms. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear. ES defects can cause distended ear tissue, a pathology often seen in hearing and...

Background: Paired organs in animals are largely bilaterally symmetric despite inherent noise in most biological processes. How is precise organ shape and size achieved during development despite this noise? Examining paired organ development is a challenge because it requires repeated quantification of two structures in parallel within living emb...

Membrane remodeling is an essential part for transfer of components to and from the cell surface and membrane-bound organelles, and for changes in cell shape, particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging, mostly restricted by technical necessity to the attached bottom surface, showed per...

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver a...

Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involve...

c-Myc and Fgf pathway inhibitors block HC regeneration. 5-dpf zebrafish larvae were treated with different inhibitors or DMSO after neomycin-induced HC death. 72 hrs later, the HCs were labeled with Yo-Pro-1. The pictures of whole fish (left) and enlarged neuromast L1 (right) showed the reduction of HC number in the inhibitor-treated neuromasts. Sc...

Pathways regulated by c-Myc (A) and Fgf (B) by IPA analysis. Genes up-regulated in chick BP during HC regeneration were marked in red and those down-regulated were marked in green. (A) c-Myc is a central node for the network formed by the up-regulated genes in proliferation. (B) A large number of Fgf members showed differential expression patterns...

Blockade of Fgf signaling in heat shocked transgenic fish. In situ hybridization showed Fgf targets etv4 (A,B) and spry4 (C,D) were relatively down-regulated in hsp70l:dn-fgfr1:GFP (Hsp) zebrafish neuromasts at 37°C compared to control. Scale bars: 10 μm (TIF)

Blockade of Tgf-β1 pathway with an inhibitor (TGFBR1I) has no effect on zebrafish HC regeneration. Quantification of Yo-Pro-1-labeled HCs of the 5-dpf neomycin-treated zebrafish neuromasts with different concentrations of TGFBR1I for 72 hrs showed no effect on hair cell regeneration compared to the no-treatment control (Ctr). (TIF)

GO categories enriched in up- or down-regulated genes in chick BP regeneration identified from microarray studies using DAVID analysis. (XLS)

Genes differentially expressed during chick BP HC regeneration by microarray analysis. (A) Venn Diagram of genes up- and down-regulated in chick BP 48 or 72 hrs after gentamicin injection compared with control. (B) GO categories enriched in up- (top, dark blue bars) or down-regulated genes (bottom, light blue bars) using the IPA package. Mouse orth...

The inhibition by Myc peptide inhibitor and SU5402 is reversible. 5-dpf zebrafish larvae with neomycin treatment were treated for 72 hrs with 100 nM c-MYC inhibitor Int-H1-S6A, F8A (Myc-pep) or 20 μM SU5402, followed by replacement with fresh media for additional 72 hrs. HCs were labeled with HCS1 antibody. There was no significant difference in th...

The Myc inhibitor does not induce apoptosis. 5-dpf zebrafish larvae with neomycin treatment were then treated with or without 100 nM c-MYC inhibitor Int-H1-S6A, F8A for 72 hrs (G-I, J-L). Larvae without neomycin and inhibitor treatment (No Trt) and larvae collected 1 hr after neomycin treatment were used as controls. The fish were stained with HCS1...

The inhibitors do not affect hair cell survival. 5-dpf zebrafish larvae without neomycin treatment were treated for 72 hrs with different c-Myc and Fgf inhibitors at the highest concentrations used for our experiments. There was no significant difference in the number of HCs after the inhibitor treatment in comparison to DMSO-treated (DMSO) or no-t...

Laser ablation of HCs and SCs in zebrafish neuromasts. Hybrid larvae of pou4f3:GFP and ET20 fish were used to ablate HCs alone (A,B), and HC/fgfr1a(+) SCs (C-D), or HC/fgfr1a(-) SCs (E-F). Neuromasts before (A,C,E) and after ablation (B,D,F) were shown. The red circles are the target areas for ablation. A-P, anterior-posterior; D-V, dorsal-ventral....

Genes differentially expressed in chick BP 48 or 72 hrs after gentamicin treatment. The listed included redundant probesets for some genes, i.e. genes that were represented multiple times by different probesets. (XLS)

Primers used for RT-PCR and in situ hybridization. (XLS)

Patterning of periodic stripes during development requires mechanisms to control both stripe spacing and orientation. A number of models can explain how stripe spacing is controlled, including molecular mechanisms, such as Turing’s reaction-diffusion model, as well as cell-based and mechanical mechanisms. However, how stripe orientation is controll...

Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunate...

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 i...

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 imagi...

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the o...

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the o...

How periodic patterns are generated is an open question. A number of mechanisms have been proposed - most famously, Turing's reaction-diffusion model. However, many theoretical and experimental studies focus on the Turing mechanism while ignoring other possible mechanisms. Here, we use a general model of periodic patterning to show that different t...

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,...

In the last decade, level-set methods have been actively developed for applications in image registration, segmentation, tracking, and reconstruction. However, the development of a wide variety of level-set PDEs and their numerical discretization schemes, coupled with hybrid combinations of PDE terms, stopping criteria, and reinitialization strateg...

of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

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 developin...

Forward genetic screens have elucidated molecular pathways required for innumerable aspects of life, however identifying the causal mutations from such screens has long been the bottleneck in the process, particularly in vertebrates. We have developed an RNA-seq based approach that identifies both the region of the genome linked to a mutation and c...

A single time-point of somite image data along with intermediate processing results from using ACME code. (ZIP)

Ten synthetic image sets generated with progressively higher noise parameters ( , ) and increasing cell number. (ZIP)

Tensor voting field determination. (A) 2D voting field parameters. (B) Heat map showing the stick voting field saliencies in 2D. The stick tensor is represented using line glyphs and overlaid on the figure. (C) A simple example showing two sampled intersecting circles and their reconstruction (D). (TIF)

A flowchart of processing filters and parameters with intermediate outputs. There are four filters that take the input image to produce an output segmented image. For each step on the left, the corresponding input and output image is shown on the right. (TIF)

Step-by-step instructions for downloading, compiling, and executing ACME code on provided test datasets. (PDF)

Synthetic membrane images along XY, XZ, and YZ sections. The (, ) values were sampled as (A–C) (0.01, 1.00), (D–F) (0.05, 0.6), and (G–I) (0.1, 0.1). Corresponding ground truth segmentation images (XY) are shown in (J–L). (TIF)

A compressed zipped file containing ACME source code in C++, a README file, and python scripts for generating synthetic image data. (ZIP)

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 establish...

Forward genetic screens in zebrafish have identified >9000 mutants, many of which are potential disease models. Most mutants remain molecularly uncharacterized because of the high cost, time and labor investment required for positional cloning. These costs limit the benefit of previous genetic screens and discourage future screens. Drastic improvem...

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaire...

Activation of Notch signaling by NICD does not induce the expression of gli genes. hsp-Gal4; UAS-NICD embryos and non-double-transgenic sibling controls were treated with DMSO or cyclopamine at 4.3 hpf, heat-shocked at 11 hpf, and stained at 24 hpf for the expression of gli1, gli2a, gli2b, and gli3. Note that in cyclopamine-treated embryos (right p...

Time-lapse imaging reveals different division patterns in the LFP domain. The top panel corresponds to the time-lapse sequence of the region containing cell 1, cell 2 and their daughter cells in Figure 2C and Video S1. Each frame is a projection of confocal slices containing cells of interest. The green channel was switched on after 6.5 hours. The...

Time-lapse imaging of the LFP domain. The time-lapse movie corresponds to the embryo shown in Figure 2C (left) and Figure S1 (top panels). Cell 1 to 5 and their daughter cells are indicated by arrows. Medial floor plate cells are denoted by white dots. Scale bar: 10 µm. (AVI)

LFP cells can be reliably identified based on their locations. (A) Gata2-GFP embryos were stained with the shh probe (green), the GFP antibody (red) and the Draq5 dye (blue) to label cell nuclei. Gata2-GFP-positive KA″ cells (short arrows) and Gata2-GFP-negative LFP cells (long arrows) flank the shh-expressing medial floor plate cells (arrowheads)....

Ptc1-Kaede reporter reveals Hh signaling dynamics in vivo. This movie depicts a series of confocal images through the spinal cord of Ptc1-Kaede fish that was photoconverted at 25 hpf and imaged at 36 hpf. Motor neurons, KA′ interneurons, and KA″ interneurons (indicated by arrows) express Ptc1-Kaedered, suggesting that they all terminate Hh response...

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 differenti...

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-wid...

The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we...

We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins...

In order to test our software GoFigure2 for the visualization and the processing of extremely large 3D+t microscopy images, we utilized the visual programming technology Sikuli to automate our GUI testing. We then integrated these Sikuli tests into CTest and used CDash to report the results on a dashboard with the view to test our software automati...

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic bi...

We are developing an approach called in toto imaging whose goal is to track all the cell movements and divisions that give rise to an embryo. We can also capture protein expression and localization throughout development using GFP transgenics. Our long term goal is to integrate these data into a “Digital Fish” that shows how the genetic circuits en...

This document describes a project that can compare many images simultaneously in a synchronized manner. Such an application can be used in any ITK/VTK image processing pipelines; the programmer can use it to quickly visualize and compare intermediate results of used filters within the pipeline.The goals of this project are two folds: (i) to be inte...

This paper describes our contribution of two new classes to the Insight Toolkit (ITK) community. This includes a base and derived ImageIO class for the JPEG2000 file formats based on the openjpeg-v2 libraries. In the current release of ITK, support for JPEG2000 is absent. Our current implementation of the JPEG2000 supports 2D images (8-bit, 16-bit...