New Book on Wnt Signaling

A wide range of biological phenomena – from embryonic development to diseases such as cancer – involve Wnt proteins and their signaling pathways. Our recent book Wnt Signaling contains 24 chapters covering all aspects of Wnt biology, from the molecular mechanisms involved in Wnt signal transduction to the effects of these pathways on normal development and physiology, as well as human disease.

 One chapter describes the history of Wnt research. It was written by Roel Nusse and Harold Varmus, who discovered the first Wnt gene 30 years ago. “Since the identification of the first Wnt gene, research in the Wnt field has taken flight,” write Roel Nusse, Xi He, and Renée van Amerongen, the book’s editors. “Wnt-related investigations continue to reveal fascinating principles of embryonic patterning, cell growth and differentiation, the wiring of the nervous system, the pathogenic mechanisms underlying cancer as well as degenerative disease, stem cell and regenerative biology, and potential therapeutic applications.”

 “It is our hope that this volume serves as a stepping-stone for the reader to guide and encourage further exploration and, perhaps, to open up novel avenues of investigation, particularly applications in the fields of bioengineering, regenerative medicine, and cancer treatment,” they continue. Wnt Signaling will be a fascinating read for cell and developmental biologists, as well as those who are interested in targeting the Wnt pathway for therapeutic purposes. For more information about the book, click here.

 

Archived Guthrie cards find a new purpose

A newborn’s blood spotted onto a Guthrie card. Photo: The New York State Department of Health Newborn Screening Program.

Over the last 50 years, the spotting of newborn’s blood onto filter paper for disease screening, called Guthrie cards, has become so routine that since 2000, more than 90% of newborns in the United States have had Guthrie cards created.  In a study published online in Genome Research (www.genome.org), researchers have shown that epigenetic information stored on archived Guthrie cards provides a retrospective view of the epigenome at birth, a powerful new application for the card that could help understand disease and predict future health.

DNA methylation, an epigenetic chemical modification of DNA, is known to affect gene activity and play a role in normal development, aging, and also in diseases such as heart disease, diabetes, and cancer.  “But are these epigenetic marks involved in causing the disease, or a result of the disease itself?” asked Dr. Vardhman Rakyan of Queen Mary, University of London and co-senior author of the study.  Rakyan explained that this is impossible to know when samples are obtained after onset of the disease.  Guthrie cards, commonly used to collect blood spots from the pricked heel of newborns to screen for diseases such as phenylketonuria, cystic fibrosis, and sickle cells disorders, might offer a snapshot of the epigenome before disease develops.  Many Guthrie cards are stored indefinitely by health authorities around the world, posing a potential wealth of information about the epigenome at birth.

Rakyan and an international group of colleagues purified genomic DNA and analyzed DNA methylomes from Guthrie cards and verified that this archived DNA yields high-quality methylation data when compared to fresh samples.  The researchers then compared the DNA methylation profiles of newborns to the same healthy individuals at the age of three, looking for epigenetic variations detected in the Guthrie card sample that are stable into the early years of life.

“We found similar epigenetic differences between different people both at birth and when they were three years old,” said Rakyan, who added that these differences, already present at birth, are unlikely due solely to inherent genetic differences between the individuals, but also due to environment or random events in utero.  Furthermore, Guthrie card samples could be analyzed for both genetic and epigenetic differences together to view a more complete picture of the genome at birth.

Guthrie card methylomics is a potentially powerful new application for archived blood spots, which could provide a wealth of information about epigenetics and disease, and could give clues about health later in life.  Dr. David Leslie, co-senior author of the study, added that because national health authorities routinely make Guthrie cards available, and with the proper consent obtained from parents and children, “we are talking about an invaluable, and non-renewable, resource for millions of individuals.”

 

Differences between human twins at birth highlight importance of intrauterine environment

Your genes determine much about you, but environment can have a strong influence on your genes even before birth, with consequences that can last a lifetime.  In a study published online in Genome Research (www.genome.org), researchers have for the first time shown that the environment experienced in the womb defines the newborn epigenetic profile, the chemical modifications to DNA we are born with, that could have implications for disease risk later in life.

Epigenetic tagging of genes by a chemical modification called DNA methylation is known to affect gene activity, playing a role in normal development, aging, and also in diseases such as diabetes, heart disease, and cancer.  Studies conducted in animals have shown that the environment shapes the epigenetic profile across the genome, called the epigenome, particularly in the womb.  An understanding of how the intrauterine environment molds the human epigenome could provide critical information about disease risk to help manage health throughout life.

Twin pairs, both monozygotic (identical) and dizygotic (fraternal), are ideal for epigenetic study because they share the same mother but have their own umbilical cord and amniotic sac, and in the case of identical twins, also share the same genetic make-up.  Previous studies have shown that methylation can vary significantly at a single gene across multiple tissues of identical twins, but it is important to know what the DNA methylation landscape looks like across the genome.

In this report, an international team of researchers has for the first time analyzed genome-scale DNA methylation profiles of umbilical cord tissue, cord blood, and placenta of newborn identical and fraternal twin pairs to estimate how genes, the shared environment that their mother provides and the potentially different intrauterine environments experienced by each twin contribute to the epigenome.  The group found that even in identical twins, there are widespread differences in the epigenetic profile of twins at birth.

“This must be due to events that happened to one twin and not the other,” said Dr. Jeffrey Craig of the Murdoch Childrens Research Institute (MCRI) in Australia and a senior author of the report.  Craig added that although twins share a womb, the influence of specific tissues like the placenta and umbilical cord can be different for each fetus, and likely affects the epigenetic profile.

Interestingly, the team found that methylated genes closely associated with birth weight in their cohort are genes known to play roles in growth, metabolism, and cardiovascular disease, lending further support to a known link between low birth weight and risk for diseases such as diabetes and heart disease.  The authors explained that their findings suggest the unique environmental experiences in the womb may have a more profound effect on epigenetic factors that influence health throughout life than previously thought.

Furthermore, an understanding of the epigenetic profile at birth could be a particularly powerful tool for managing future health.  “This has potential to identify and track disease risk early in life, said Dr. Richard Saffery of the MCRI and a co-senior author of the study, “or even to modify risk through specific environmental or dietary interventions.”

 

3D Imaging of Embryos is Featured in CSH Protocols

Until recently, a common technique for creating a 3D image of an embryo was to slice it into hundreds of thin sections, photograph each section, and then computationally recombine the images to produce a 3D representation of the embryo.  But during this process, the specimen may become deformed, and information about the alignment of the sections – the third dimension – is lost.

Optical projection tomography (OPT) overcomes these problems, as Laura Quintana and James Sharpe (Centre for Genomic Regulation, Barcelona) explain in a featured article in the latest issue of Cold Spring Harbor Protocols.  OPT is ideal for analyzing the morphology of fixed embryos – especially for analyzing mutant phenotypes, for developing anatomical atlases, and for analyzing gene expression patterns.

 

CSH Protocols Features Methods for Imaging Kidney Development

The adult mouse kidney begins to develop at embryonic day 10.5, when the epithelial ureteric bud evaginates from the Wolffian duct and grows into adjacent metanephric mesenchyme.  Over the course of several days, the ureteric bud repeatedly branches, giving rise to the ureter, pelvis, calyces, and renal collecting ducts of the adult kidney.

The kidney can develop in culture, from the first stage of ureteric bud evagination through the first 8-10 rounds of branching.  These processes can therefore be visualized through time-lapse imaging, providing a greater understanding of normal kidney morphogenesis and how genetic perturbations affect kidney development.

This month’s issue of Cold Spring Harbor Protocols, out today, features an article that presents the general concepts of imaging kidney development and describes genetically modified mice that express fluorescent proteins useful for visualizing different cell lineages and developmental processes in these organ cultures.  A detailed step-by-step protocol for dissecting, culturing, and imaging embryonic mouse kidneys is also published in the issue.  Both articles were written by Frank Costantini (Columbia University Medical Center), Shankar Srinivas (University of Oxford), and colleagues.

 

Mouse Embryogenesis – Live!

Cell proliferation, migration, differentiation, and death are remarkably synchronized during embryonic development.  But mouse embryos are confined to the uterus, thus limiting our ability to observe these astonishing events in vivo.

In this month’s issue of Cold Spring Harbor Protocols, Mary Dickinson and colleagues (Baylor College of Medicine) describe methods for culturing live mouse embryos directly on a microscope stage, and for performing time-lapse imaging of embryos expressing genetically encoded fluorescent proteins.  These methods permit the visualization of mouse embryos from gastrulation until early organogenesis.  An introductory article, available here, is freely accessible to subscribers and non-subscribers alike. 

 

Using μMRI to Construct Atlases of Animal Development

The cover of the March 2011 issue of Cold Spring Harbor Protocols, out today, features several striking images of mouse and quail embryos.  The method used to produce the images, microscopic magnetic resonance imaging (μMRI), is a noninvasive imaging technique that permits the visualization of regions deep within embryos that are inaccessible using optical methods.  During μMRI, the specimens remain in near-physiological conditions, remaining anatomically unperturbed.  The method is ideal, therefore, for generating developmental atlases of these organisms.

Quail embryos in a "relaxed" posture used to construct a μMRI-based developmental atlas. (©2011, CSHL Press)

In an accompanying article, the authors, Seth Ruffins and Russell Jacobs (Caltech Biological Imaging Center), describe the preparation of specimens for μMRI and appropriate applications of μMRI for developmental biology, including the construction of atlases.  Using these methods, they have successfully generated digital anatomical atlases of both quail and mouse development (see the Caltech MRI Atlases).  These atlases, and others constructed using μMRI, will be useful references for developmental biologists, providing identifiable anatomical landmarks and standards for comparison.

 

Kidney Organ Culture

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.

 

Brainbow Fish

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.

 

New Imaging Manuals Available

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.