Sean G. Megason

Digitizing life at the level of the cell: high-performance laser-scanning microscopy and image analysis for in toto imaging of development

The field of biological imaging is progressing at an amazing rate. Advances in both laser-scanning microscopy and green fluorescent protein (GFP) technology are combining to make possible imaging-based approaches for studying developmental mechanisms that were previously impossible. Modern confocal and multi-photon microscopes are pushing the envelope of speed, sensitivity, spectral resolution, and depth resolution to allow in vivo imaging of whole, live embryos at cellular resolution over extended periods of time. In toto imaging, in which nearly every cell in an embryo or tissue can be tracked through space and time during development, may become a standard technique for small transparent embryos such as zebrafish and early stage chick and mouse embryos. GFP and its spectral variants can be used to mark a wide range of in vivo biological information for in toto imaging including gene expression patterns, mutant phenotypes, and protein subcellular localization patterns. Combining in toto imaging and GFP transgenic approaches on a large scale may usher in an explosion of in vivo, developmental data as has happened in the past several years with genomic data. There are significant challenges that must be met to reach these goals. This paper will discuss the current state-of-the-art, the challenges, and the prospects of in toto imaging in the areas of imaging, image analysis, and informatics.

Specified Neural Progenitors Sort to Form Sharp Domains after Noisy Shh Signaling

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.

RNA-seq-based mapping and candidate identification of mutations from forward genetic screens.

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 candidate lesions that may be causal for the phenotype of interest. We show that our method successfully identifies zebrafish mutations that cause nonsense or missense changes to codons, alter transcript splicing, or alter gene expression levels. Furthermore, we develop an easily accessible bioinformatics pipeline allowing for implementation of all steps of the method. Overall, we show that RNA-seq is a fast, reliable, and cost-effective method to map and identify mutations that will greatly facilitate the power of forward genetics in vertebrate models.

ACME: automated cell morphology extractor for comprehensive reconstruction of cell membranes.

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 (https://github.com/krm15/ACME).

Rapid positional cloning of zebrafish mutations by linkage and homozygosity mapping using whole-genome sequencing

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 improvements in DNA sequencing technology could dramatically improve the efficiency of positional cloning in zebrafish and other model organisms, but the best strategy for cloning by sequencing has yet to be established. Using four zebrafish inner ear mutants, we developed and compared two approaches for ‘cloning by sequencing’: one based on bulk segregant linkage (BSFseq) and one based on homozygosity mapping (HMFseq). Using BSFseq we discovered that mutations in lmx1b and jagged1b cause abnormal ear morphogenesis. With HMFseq we validated that the disruption of cdh23 abolishes the ears sensory functions and identified a candidate lesion in lhfpl5a predicted to cause nonsyndromic deafness. The success of HMFseq shows that the high intrastrain polymorphism rate in zebrafish eliminates the need for time-consuming map crosses. Additionally, we analyzed diversity in zebrafish laboratory strains to find areas of elevated diversity and areas of fixed homozygosity, reinforcing recent findings that genome diversity is clustered. We present a database of >15 million sequence variants that provides much of this approachs power. In our four test cases, only a single candidate single nucleotide polymorphism (SNP) remained after subtracting all database SNPs from a mutants critical region. The saturation of the common SNP database and our open source analysis pipeline MegaMapper will improve the pace at which the zebrafish community makes unique discoveries relevant to human health.

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

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 impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.

Towards a Synthetic Chloroplast

Background 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 biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner. Results We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages. Conclusion Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices.

Interplay of Cell Shape and Division Orientation Promotes Robust Morphogenesis of Developing Epithelia

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.

Attenuation of Notch and Hedgehog Signaling Is Required for Fate Specification in the Spinal Cord

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.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

Lattice light-sheet microscopy is used to examine two problems in membrane dynamics—molecular events in clathrin-coated pit formation and changes in cell shape during cell division. This methodology sets a new standard for imaging membrane dynamics in single cells and multicellular assemblies.