Developmental Biology

Visualizing mammalian development presents an obvious problem: embryos must develop in utero. That makes them a lot more difficult to see under a microscope than a zebrafish or a frog that develops as a free-standing egg. Extensive work has been done to develop embryonic culture techniques for external development of mouse embryos, allowing imaging approaches to be applied. Early efforts by members of Scott Fraser’s lab (including myself) provided a protocol for growing d 6.5-9.5 mouse embryos on the microscope stage. The December issue of Cold Spring Harbor Protocols features Imaging Cell Movements in Egg-Cylinder Stage Mouse Embryos from Oxford University’s Shankar Srinivas. The article describes a method for isolating and culturing much earlier mouse embryos, as well as an approach for time-lapse imaging as those embryos develop. While cell movements can be followed using light microscopy alone, the increasing variety of transgenic fluorescent reporter mice makes studies of cell movement easier and more informative. As one of December’s featured articles, the protocol is freely available to subscribers and non-subscribers alike.

How do you see something smaller than the wavelength of light itself? Fluorescence microscopy is the most common optical technique used for visualizing cellular functions. The latest techniques allow labeling of specific organelles and proteins with molecular precision. But conventional microscopy cannot resolve objects closer than 200 nanometers at the focal plane. Many subcellular structures and groups of proteins occur on the 10 nanometer scale. A true understanding of cellular physiology requires new superresolution methods.

Of these methods, PALM (Photoactivated Localization Microscopy) provides the highest shown resolution in biological samples (approximately 10 nanometers) and allows for the assessment of individual molecules. In the December issue of Cold Spring Harbor Protocols, Oregon Health Science University’s Haining Zhong presents Photoactivated Localization Microscopy (PALM): An Optical Technique for Achieving ~10-nm Resolution. The article provides an overview of the basic principles of PALM, its implementation and the potential applications in neuroscience.

New imaging technologies have revolutionized the study of developmental biology. Where researchers once struggled to connect events at static timepoints, imaging tools now offer the ability to visualize the dynamic form and function of molecules, cells, tissues, and whole embryos throughout the entire developmental process. In order to observe development over time, it is necessary to grow the embryos of laboratory model organisms on the microscope stage, and keep them as healthy and in as natural a state as possible. Methods for culturing and imaging the embryos of model organisms are featured in the December issue of Cold Spring Harbor Protocols.

Caenorhabditis elegans has been a key organism for understanding cellular differentiation and development. The fate of every one of the worm’s somatic cells has been mapped out, and its short developmental time, transparent shell, and nonpigmented cells makes C. elegans an ideal subject for imaging studies. Timothy Walston from Truman State University and Jeff Hardin from the University of Wisconsin-Madison provide An Agar Mount for Observation of Caenorhabditis elegans Embryos, an easy way to prepare live C. elegans embryos for microscopic visualization. The method involves embedding the embryo in agar to hold it in place,providing a fixed orientation for consistent imaging. Embryos prepared this way are amenable to both light microscopy and confocal microscopy. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

Imaging has rapidly become a defining tool of the current era in biological research. But finding the right method and optimizing it for data collection can be a daunting process, even for an established imaging laboratory. Cold Spring Harbor Protocols is one of the world’s leading sources for detailed technical instruction for implementation of imaging methods, and the November issue features articles detailing standard and cutting-edge laboratory techniques.

The confocal microscope is a workhorse of the modern life science laboratory. Its popularity stems from its ability to permit volume objects to be imaged and rendered in three dimensions. But the confocal microscope itself does not produce three-dimensional images; in fact, it only images very thin sections of a specimen that lie within its focal region. To produce a three-dimensional image, a series of thin optical sections are collected, and computer processing is used to combine them into a volumetric rendering. In the first of November’s featured articles, Spinning-Disk Microscopy Systems, Oxford University’s Tony Wilson reviews the many methods for producing optical sections, of which the confocal optical system is just one. He also describes a number of convenient methods of implementation that can lead to, among other things, real-time image formation. The paper, like all our featured articles, is freely available to subscribers and non-subscribers alike.

Blood feeding mosquitoes transmit many of the world’s deadliest diseases, which are resurgent in developing countries and pose threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for disease control through the genetic manipulation of vector insects. To accomplish this, vector insects must be established as laboratory model organisms, allowing for a better understanding of their biology, and in particular, the genes that regulate their development. Aedes aegypti is a vector mosquito of great medical importance because it is responsible for the transmission of dengue fever and yellow fever. In the October issue of Cold Spring Harbor Protocols, Molly Duman-Scheel and colleagues present an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Protocols are provided for culturing and egg collection, fixation and tissue preparation, whole mount in situ hybridization, immunohistochemical analysis and RNA interference in Ae. aegypti. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

This article series marks the latest entrant in Cold Spring Harbor Protocols’ long-running series on Emerging Model Organisms.

Means to an End: Apoptosis and Other Cell Death Mechanisms is the new book from Douglas Green, a clear and comprehensive view of apoptosis and other death mechanisms. He examines the enzymes that perform the execution (caspases) and the molecular machinery that links their activation to signals that cause cell death, emphasizing the importance of BCL-2 proteins and cytochrome c released from mitochondria. Green also outlines the roles of cell death in embryogenesis, neuronal selection, and the development of self-tolerance in the immune system, explains how cell death defends the body against cancer, and traces the evolutionary origins of the apoptosis machinery back over a billion years.

There’s also an online companion resource that’s rapidly growing. Be sure not to miss Cell Death: The Movie.

The Drosophila neuromuscular junction (NMJ) provides a superb model system for investigating the cellular and molecular mechanisms of synaptic transmission. The NMJ is large, easily accessed and its genetics are well-characterized. It shares many structural and functional similarities to synapses in other animals, including humans. In the September issue of Cold Spring Harbor Protocols, Bing Zhang and Bryan Stewart present an essential set of primers for electrophysiological recording from the Drosophila NMJ. The issue contains a detailed explanation of the Equipment Setup necessary, as well as instructions for Fabrication of Microelectrodes, Suction Electrodes, and Focal Electrodes. Protocols for Electrophysiological Recording from a ‘Model’ Cell, Electrophysiological Recording from Drosophila Larval Body-Wall Muscles, Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction, and Focal Recording of Synaptic Currents from Single Boutons at the Drosophila Neuromuscular Junction are also included. These protocols are adapted from Drosophila Neurobiology: A Laboratory Manual. Based on Cold Spring Harbor Laboratory’s long-running course, this manual has rapidly become an important resource for any neuroscience lab.

Improvements in automation and acquisition time have made the microscope a viable platform for performing hundreds of concurrent parallel experiments. Using these sorts of tools, it is now possible to run high-throughput screens for protein function and interaction in living cells, examining dynamic cellular processes to distinguish between primary and secondary phenotypes, and to study the phenotype kinetics. In the August issue of Cold Spring Harbor Protocols, Jan Ellenberg and colleagues from the EMBL present High-Throughput Microscopy Using Live Mammalian Cells, an overview of how to screen live cells using imaging technologies. The article examines each aspect of the general screening process and considers specific examples in the processing of time-lapse experiments. The techniques discussed are based on the use of cultured mammalian cells, but the concepts are easily transferred to cultured cells from other species like Drosophila and small organisms such as C. elegans.

Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing an engineered zinc finger DNA-binding domain to the DNA cleavage domain of a restriction enzyme. ZFNs can be used to generate targeted genomic deletions of large segments of DNA in a wide variety of cell types and organisms. In the August issue of Cold Spring Harbor Protocols, Jin-Soo Kim and colleagues present Analysis of Targeted Chromosomal Deletions Induced by Zinc Finger Nucleases, a detailed protocol for the detection and analysis of large genomic deletions in cultured cells introduced by the expression of ZFNs. The method described allows researchers to detect and estimate the frequency of ZFN-induced genomic deletions by simple PCR-based methods. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

The zebrafish (Danio rerio) has rapidly become a favored model organism for studying developmental biology. One of the most commonly used methods for genetic manipulation in the zebrafish is the delivery of plasmids or oligonucleotides to cells within the living embryo via electroporation. When cells are exposed to brief electrical fields, transient membrane destabilization occurs and nucleic acids can cross the plasma membrane. When the electrical field is removed, the membrane seals and the nucleic acids are trapped inside the cell. In vivo electroporation has proven particularly effective for delivering fluorescent protein expression vectors for imaging and loss-of-function reagents such as morpholinos or RNA interference (RNAi) constructs for the knockdown of gene function. In the July issue of Cold Spring Harbor Protocols, Jack Horne and colleagues present Targeting the Zebrafish Optic Tectum Using In Vivo Electroporation, a modification of the technique that can be used to specifically target the developing optic tectum, the midbrain’s visual processing center. Instructions are given for the construction of electroporation electrodes, preparation and injection of DNA, and electroporation of the DNA into the embryonic brain.

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