Developmental Biology


New technologies and methods are spurring a renaissance in the study of organogenesis. Organogenesis, essentially the process through which a group of cells becomes a functioning organ, has important connections to biological processes at the cellular and developmental levels, and its study offers great potential for medical treatments through tissue engineering approaches. The January issue of Cold Spring Harbor Protocols features a method from Washington University’s Hila Barak and Scott Boyle for Organ Culture and Immunostaining of Mouse Embryonic Kidneys. The kidney is particularly interesting as it also serves as a model for branching morphogenesis. The protocol describes the isolation, culture and fluorescent immunostaining of mouse embryonic kidneys. As one of January’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

The “Brainbow” strategy was originally developed in mice, as a system for labeling neurons in a variety of different colors, allowing one to follow multiple cells regardless of their proximity. Brainbow uses a construct that carries sequences for red, blue and green fluorescent proteins in tandem array, with two pairs of lox sites flanking the first two fluorescent proteins. Recombination occurs in the presence of the Cre recombinase, and one gets a variety of outcomes, resulting in the production of a red, blue or green label. When more than one copy of the Brainbow cassette exists within a cell, the primary tones can be mixed, providing more possible color combinations. This provides a powerful platform for studying neuronal morphology and cell movements. In the January issue of Cold Spring Harbor Protocols, Alex Schier and colleagues offer Multicolor Brainbow Imaging in Zebrafish. This protocol translates the system for use in zebrafish, which offer the advantages of easy visualization of transparent embryos and efficient generation of labeled subjects.

For those looking to add to their arsenal of laboratory techniques, Cold Spring Harbor Laboratory Press has just released a new series of Imaging manuals.

I had a hand in putting these books together, and I’m always pleased when we manage to publish books that I know I would have found incredibly helpful in my previous incarnation as a bench scientist. These two hit home as I was a postdoc in an imaging lab. While that was ten (10!!!) years ago, it’s almost shocking to realize that there weren’t any comprehensive lab manuals out there that really covered the whole of bio-imaging, from the basics of optics to the most current, bleeding-edge techniques. Consider that problem solved, courtesy of series editor Rafa Yuste.

The new series spins off from a previous set of publications. In 2000, CSHL Press published Imaging Neurons, based on a CSHL laboratory course. The book was a few years ahead of its time, and the methods had really caught on by the time the sequel, Imaging in Neuroscience and Development was released in 2005. Five years later, and there’s been far too many new applications developed to fit into one volume, hence the release of the new series.

Imaging: A Laboratory Manual is the flagship of the series. It offers all the basics: optics, confocal, multi-photon, lasers, cameras, staining cells, etc. The manual goes on from there though, through labeling and indicators to advanced techniques like photoactivation, light sheet imaging, array tomography, fast imaging, molecular imaging, superresolution imaging and every acronym you can think of (FRET, FLIM, FRAP, FIONA, PALM, STORM, BiFC, AFM, TIRFM to name a sampling). If you have a microscope in your lab or if you spend any time in your local imaging center, this is the book you need.

Imaging in Developmental Biology: A Laboratory Manual is the second book in the series, just released. We old-school developmental biologists used to have to look at fixed sectioned specimens taken from different time points, and try to piece together the big picture of what was really happening as an embryo developed. New techniques have revolutionized our understanding of dynamic processes, as they allow for real-time imaging, often over the entire course of an organism’s development. Like the preceding volume, the book starts with the basics, methods for visualizing development in laboratory standard model organisms (C. elegans, Drosophila, zebrafish, Xenopus, avians and mouse) and then step by step brings the reader to the cutting edge of imaging technology.

The supplemental movies from both books are freely available through Cold Spring Harbor Protocols. Look for a third volume in March, on Imaging in Neuroscience, which will offer an astounding 90-plus chapters for analyzing every aspect of the nervous system in detail.

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.

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