Growth of Blood Vessels Precedes
That of Neurons in Developing Brain

The brain is surrounded by layers of protective membranes, the innermost being infiltrated with a nourishing network of blood vessels, known as pial vessels. It has been assumed that during development, blood vessels in this pial network simply extend deeper into the brain to areas of increased metabolic need. Yet new findings reported by HMS researchers in the March 16 Nature Neuroscience have challenged this assumption, showing that vessels in the developing brain do not arise on demand from pial vessels but, instead, emerge in an autonomous manner based on an internal program.

Courtesy Anju Vasudevan

Vessel navigation. Labeling for blood vessels (green), proliferating cells (pink) and undifferentiated stem cells (blue) in an embryonic mouse brain section revealed two independent sets of vasculature. The pial vessels are present early on and encircle the entire brain while a separate set of deeper blood vessels develops later and systematically migrates (clockwise in image) from the front (F) to the back (B) of the brain.

The research team, led by Pradeep Bhide, HMS associate professor of neurology and director of neurology research at Massachusetts General Hospital, was initially interested in observing the anatomical and temporal relationship between blood vessel and brain cell growth. They started by looking at brain sections from mouse embryos at different stages of development and staining for blood vessels. Intriguingly, the team discovered that rather than developing in response to increased neuronal growth, blood vessel—or endothelial—cells actually emerged ahead of neurons and systematically migrated from the front to the back of the brain. Moreover, they displayed a unique structure—“like diamonds organized in a necklace,” said HMS instructor in neurology and first author Anju Vasudevan. “They were obviously regulated by something.”

A leading clue for what was regulating these endothelial cells came from the observation that their migration showed marked similarities to neuronal migration, which also extends from front to back, albeit a little later. Neuronal migration was known to be under the control of regulatory transcription factors. “So the first question that came to mind was, maybe the same transcription factors that regulate neuron migration regulate endothelial cell migration,” said Bhide.

Using a variety of tissue culture techniques and transgenic mice, the team subsequently demonstrated that not only did this necklacelike array of endothelial cells emerge and migrate independently but that it was, indeed, under the control of the same transcription factors that regulate neuron migration.

Bhide and his colleagues speculate that these endothelial cells are pioneers, traversing the brain in a regulated fashion, prior to other important events in brain development. The cells might even orchestrate these later events. “It opens up a lot of fundamental issues in neuroscience,” he said.

Knowing that endothelial cell growth is governed intrinsically could open up possibilities for genetic manipulation. Depending on the clinical condition, suppressing or improving vessel development could have applications for disorders of brain development, brain tumors, and conditions of interrupted blood flow such as stroke and ischemia. “This is like a key,” Vasudevan said. “There are now so many doors to open.”

—Yvonna Reekie

Insulation Ensures Transgenes Thrive

In some ways, transgenes resemble foster kids who are expected to thrive and express themselves after being plucked from one location and deposited in another. They flourish or flounder, depending on the new surroundings. A novel technique now ensures their success in Drosophila,

nsulating the introduced DNA from the disruptive influences of nearby chromatin.
Working in the lab of Howard Hughes investigator and HMS professor of genetics Norbert Perrimon, postdoctoral researcher Michele Markstein sandwiched transgenes between protective stretches of DNA before inserting them into the germ cells of fruit flies. The resulting animals expressed the transgenes optimally in every tissue tested. The method appears in the April issue of Nature Genetics.

“Classically, transgenes are randomly integrated into the DNA of the host cell, often landing in areas where the surrounding chromatin prevents them from being optimally expressed,” explained Markstein. “We’ve insulated these transgenes, so they’re expressed at high levels, regardless of their integration spot along the chromosome.”

“Using Michele’s method, we can create quality transgenic flies on the first try,” added Perrimon.

Initially, Markstein played caseworker for the transgenes. She searched for a perfect home in the DNA of the host cells, a location where the transgenes would always be nurtured rather than a random residence where they might be silenced.

“Michele tried about 20 integration sites, but none of them proved optimal in every tissue of the fly,” explained Perrimon. If the transgene worked in muscle, for example, it might be silent in the brain.

Markstein decided to take a different approach. Seeking to safeguard the transgenes from trouble in their new homes, she borrowed a trick from the gypsy retrovirus, which infects fruit flies. This pathogen uses insulator sequences to protect its own genetic code from the DNA of the fly. Markstein took these insulator sequences and added them to her transgenes before unleashing them on the germ cells of flies.

“In a sense, the transgene travels with the perfect environment, enabling it to function optimally wherever it lands,” said Markstein.

According to Perrimon, the new method could be applied to other species, though labs must first identify an appropriate insulator sequence, since the gypsy retrovirus is specific to flies. In the short term, the method will advance Perrimon’s goal of creating a massive RNAi library of transgenic fruit flies for use in experiments. Each line of flies will contain a single transgene, coding for a short interfering RNA that disrupts the expression of an endogenous gene.

“The library should be complete in three to four years,” said Perrimon. “These fruit flies will allow us to probe developmental and physiological processes at a systems level in organisms.”

—Alyssa Kneller

Binding Proteins Fix Transcription Factor Role in Immunity

The attack of the immune system on normal healthy cells and tissues in the body causes a range of autoimmune diseases. The recent discovery of the role of a ligand-activated transcription factor, aryl hydrocarbon receptor (AHR), has shed light on how two key immune cell types can control experimental autoimmune encephalomyelitis (EAE), an animal model of one of these diseases, multiple sclerosis.

The yin and yang regulation of autoimmunity is managed by regulatory T cells (Tregs), responsible for inhibiting the immune response against healthy cells, and pro-inflammatory IL-17–producing T cells (TH17s), which mediate the destruction of cells infected with pathogens. A tip in the balance in favor of Treg activation can inhibit autoimmune disease. Since the discovery that Foxp3 promotes Treg differentiation, there has been a quest to determine what regulates this transcription factor.

The laboratory of Howard Weiner, the Robert L. Kroc professor of neurology at Brigham and Women’s Hospital, identified AHR as a central T cell regulator that not only mediates the production of Tregs but also promotes the activation of TH17 cells. The surprising finding was that AHR can mediate opposite roles in the immune response and the induction of Treg or TH17 cells was dependent on the ligand used to activate AHR.

In the study, published online on March 23 in Nature, Weiner’s group demonstrated that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed EAE while another AHR ligand, 6-formylindolo[3,2-b]carbazole (FICZ), aggravated it. These opposing effects were attributed to the distinct T cell and cytokine profile induced by each ligand.

Treg cells mediated EAE protection in mice injected with TCDD. A significant increase in the number of Treg cells isolated from the lymph nodes was observed in TCDD-treated mice. Furthermore, by transferring these cells to animals that were not exposed to TCDD, the protection was passed on. In contrast, exacerbation of EAE in FICZ-injected mice was the result of an upregulation in TH17 cells and related cytokines and a decrease in Treg production.

“The ability of AHR to regulate Treg and TH17 differentiation in a ligand-specific manner makes it a unique target for the therapeutic manipulation of the immune response,” said Francisco Quintana, HMS instructor in neurology at BWH and the first author of the study.

—Kafi Meadows

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