Warren symposium follows legacy of geneticist giant

If we want to understand how the brain creates memories, and how genetic disorders distort the brain’s machinery, then the fragile X gene is an ideal place to start. That’s why the Stephen T. Warren Memorial Symposium, taking place November 28-29 at Emory, will be a significant event for those interested in neuroscience and genetics. Stephen T. Warren, 1953-2021 Warren, the founding chair of Emory’s Department of Human Genetics, led an international team that discovered Read more

Mutations in V-ATPase proton pump implicated in epilepsy syndrome

Why and how disrupting V-ATPase function leads to epilepsy, researchers are just starting to figure Read more

Tracing the start of COVID-19 in GA

At a time when COVID-19 appears to be receding in much of Georgia, it’s worth revisiting the start of the pandemic in early 2020. Emory virologist Anne Piantadosi and colleagues have a paper in Viral Evolution on the earliest SARS-CoV-2 genetic sequences detected in Georgia. Analyzing relationships between those virus sequences and samples from other states and countries can give us an idea about where the first COVID-19 infections in Georgia came from. We can draw Read more

Tamara Caspary

For genetically altered mice/rats, freeze and recharge

With a pandemic threatening the health and safety of Emory researchers in March 2020, university leadership made the difficult decision to ramp down some types of research. For investigators that use laboratory mice or rats in their research, this posed a significant challenge.

How could investigators maintain valuable, often unique, lines of genetically engineered animals for future research? The Mouse Transgenic and Gene Targeting Core (TMF) had a solution: cryopreservation. Animals’ sperm — and occasionally, embryos – can be carefully preserved in cold-resistant straws and stored in liquid nitrogen.

“Cryopreservation is a reliable and efficient method for archiving and distributing genetically engineered mouse lines,” says Karolina Piotrowska-Nitsche, PhD, director of the Core.

The TMF is located on the ground floor of the Emory Health Sciences Research Building and provides a suite of services related to transgenics and gene editing, working with tools such as CRISPR/Cas9 to make subtle or complex changes in the DNA of living animals.

Read more

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Hedgehog pathway outside cilia

Emory geneticist Tamara Caspary is an expert on the Hedgehog pathway, critical for brain development. In particular, she and her colleagues have been studying a gene that is part of the Hedgehog pathway called Arl13b, which is mutated in Joubert syndrome, affecting development of the cerebellum and brain stem.

The Arl13b protein was known to be enriched in primary cilia, tiny hair-like cellular structures with a signaling/navigation function in neuronal development. However Caspary’s lab, in a collaboration with Frederic Charron’s group in Montreal, has found that Arl13b can also function outside cilia: in axons and growth cones.

The Hedgehog pathway has several roles, some in specifying what embryonic cells will become, and others in terms of guiding growing axons, the scientists conclude in their new paper in Cell Reports.

“Arl13b regulates Shh [Sonic Hedgehog] signaling through two mechanisms: a cilia-associated one to specify cell fate and a cilia localization-independent one to guide axons,” they write.  A related preprint, confirming Arl13b’s extra-ciliary role in mouse development, has been posted on bioRxiv.

 

 

 

 

 

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Mouse version of 3q29 deletion: insights into schizophrenia/ASD pathways

Scientists at Emory University School of Medicine have created a mouse model of human 3q29 deletion syndrome, which is expected to provide insights into the genetic underpinnings of both schizophrenia and autism spectrum disorder.

In 3q29 deletion syndrome, a stretch of DNA containing several genes is missing from one of a child’s chromosomes. The deletion usually occurs spontaneously rather than being inherited. Affected individuals have a higher risk of developing intellectual disability, schizophrenia, and autism spectrum disorder. 3q29 deletion is one of the strongest genetic risk factors for schizophrenia, and the Emory researchers see investigating it as a way of unraveling schizophrenia’s biological and genetic complexity.

The results were published in Molecular Psychiatry.

“We see these mice as useful tools for understanding the parts of the brain whose development is perturbed by 3q29 deletion, and how it affects males and females differently,” says Jennifer Mulle, PhD, assistant professor of human genetics. “They are also a starting point for dissecting individual genes within the 3q29 deletion.”

Working with clinicians and psychologists at Marcus Autism Center, Mulle is leading an ongoing study of 3q29 deletion’s effects in humans, and observations from the mice are expected to inform these efforts. (More about Mulle here.) Read more

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Mother’s milk is OK, even for the in-between babies

“Stop feeding him milk right away – just to be safe” was not what a new mother wanted to hear. The call came several days after Tamara Caspary gave birth to fraternal twins, a boy and a girl. She and husband David Katz were in the period of wonder and panic, both recovering and figuring out how to care for them.

“A nurse called to ask how my son was doing,” says Caspary, a developmental biologist in Emory’s Department of Human Genetics. “She started asking about vomiting and other specific symptoms.”

Her son had tested positive by newborn screening for a rare disorder called galactosemia. Galactosemia is an inherited disease that results from inability to metabolize galactose, a component of human milk and cow-milk-based formula. If a baby with “classic” galactosemia continues to drink milk, the baby may quickly develop symptoms such as jaundice, vomiting and diarrhea, progressing to liver disease and other serious complications that can lead to infant death. If a newborn has classic galactosemia, it is critical for the baby to stop drinking milk and switch to a low-galactose formula, such as soy-based formula, as soon as possible.

Caspary and Katz, a cell biologist, learned several days later that their son did not have classic galactosemia but instead had inherited Duarte galactosemia, a milder, more common form of the metabolic disorder, affecting more than 1 in 5,000 children in the United States. But there was still a looming question.

“We needed to figure out what to feed the baby!” Katz exclaimed, recalling their confusion years later.

The looming question was: what to feed the baby?

Their pediatrician didn’t know what to recommend. Galactosemia, in whatever form, is rare enough in the US that most pediatricians don’t develop experience with it. There was no uniform standard of care, and state-level guidelines for children with Duarte galactosemia varied widely, from no dietary restrictions to banning all milk products for the first year. Some of the limited research available at the time suggested that affected children might experience developmental problems as they grew up. Read more

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Navigating monstrous anticancer obstacles

A new PNAS paper from geneticist Tamara Caspary’s lab identifies a possible drug target in medulloblastoma, the most common pediatric brain tumor. Come aboard to understand the obstacles this research seeks to navigate. Emory library link here.

Standard treatment for children with medulloblastoma consists of surgery in combination with radiation and chemotherapy. Alternatives are needed, because survivors can experience side effects such as neurocognitive impairment. One possibility has emerged in the last decade: inhibitors of the Hedgehog pathway, whose aberrant activation drives growth in medulloblastoma.

Medulloblastoma patients are caught “between Scylla and Charybdis”: facing a deadly disease, the side effects of radiation and/or existing Hedgehog inhibitors. From Wikimedia.

As this 2017 Oncotarget paper from St. Jude’s describes, Hedgehog inhibitors are no fun either. In adults, these agents cause muscle spasms, hair loss, distorted sense of taste, fatigue, and weight loss. In a pediatric clinical trial, the St. Jude group observed growth plate fusions, resulting in short stature. The drug described in the paper was approved in 2012 for basal cell carcinoma, a form of cancer whose growth is also driven by the Hedgehog pathway. Basal cell carcinoma is actually the most common form of human cancer, although it is often caught at an early stage that doesn’t require harsh treatment.

Caspary’s lab studies the Hedgehog pathway in early embryonic development. In the PNAS paper, former graduate student Sarah Bay and postdoc Alyssa Long show that targeting a downstream part of the Hedgehog pathway may be a way to avoid problems presented by both radiation/chemo and existing Hedgehog inhibitors. Read more

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Manipulating mouse genes to order, CRISPR or old-school

Just a follow-up to last week’s announcement from the Emory Transgenic Mouse and Gene Targeting core that they are offering CRISPR/Cas9 gene editing for mice. Using CRISPR/Cas9 to produce genetically altered mice is a

Knockout_mice

Gene targeting – the 20th century way

substantial advance over the old way of doing knockouts and other manipulations (which itself won a Nobel Prize in 2007), mainly because it’s faster and easier.

To appreciate the difference, consider that the old way involves introducing DNA into mouse embryonic stem cells, and then selecting for the rare cells that take up and incorporate the DNA in the right way. Then the ES cells have to be injected into a blastocyst, followed by mouse breeding to “go germline.”

With CRISPR/Cas9, it’s possible to inject pieces of RNA that target the desired genetic changes, straight into a one-cell stage mouse embryo. Not every embryo has all the right changes, but the frequency is high enough to inject and screen. As this review explains, it’s possible to introduce mutations into three genes at once and get mice quickly, rather than make each one separately and then breed the mice together, which can take many months.

Also, because of the need for drug selection, the targeting construct in old-school gene targeting has to be a blunt instrument. That can make it hard to make subtle changes to a gene — like introduce point mutations corresponding to natural variations linked with human disease — without taking a sledgehammer to the entire gene locus. CRISPR/Cas9 takes care of that problem.

Despite the advantages of this technology, three things to keep in mind:

*Many genetically altered mice are already available “off the shelf” as part of the International Knockout Mouse/Mouse Phenotyping Consortium.

*Emory’s Mouse Core has been working with the company Ingenious Gene Targeting, and has been out-sourcing some of the tedious aspects of old-school gene targeting in mice to Ingenious, starting last year. Technicians there can generate a dazzling array of conditional knockouts. If you want your favorite gene to flip around and produce a fluorescent protein when you give the mice an antibiotic, but only in some cells — Ingenious can do that. Old school is actually still the way to go for fancy stuff like this.

*Jackson Labs in Maine also works with Emory, offering similar services, and offers a guarantee. Read more

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A CRISPR way to edit DNA

The CRISPR/Cas gene editing system has a lot of buzz behind it: an amusingly crunchy name, an intriguing origin, and potential uses both in research labs and even in the clinic. We heard that Emory scientists are testing it, so an explainer was in order.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was originally discovered by dairy industry researchers seeking to prevent phages, the viruses that infect bacteria, from ruining the cultures used to make cheese and yogurt. Bacteria incorporate small bits of DNA from phages into their CRISPR region and use that information to fight off the phages by chewing up their DNA.

At Emory, infectious disease specialist David Weiss has published research on CRISPR in some types of pathogenic bacteria, showing that they need parts of the CRISPR system to evade their hosts and stay infectious. Biologist Bruce Levin has modeled CRISPR-mediated immunity’s role in bacterial evolution.

What has attracted considerable attention recently is CRISPR/Cas-derived technology, which offers the ability to dive into the genome and make a very precise change. Scientists have figured out how to retool the CRISPR/Cas machinery – the enzymes that do the chewing of the phage DNA — into enzymes that can be targeted by an external guide.

For biologists in the laboratory, this is a way to probe a gene’s function by making an animal with its genes altered in a certain way. The method is gaining popularity here at Emory. Geneticist Peng Jin reports:

“CRISPR is much more efficient and quicker than traditional homologous recombination. One can directly inject the plasmid and guide RNA into mouse embryo to make knockout mice. You can also target multiple genes at the same time.”

The traditional method Jin refers to involves taking cultured embryonic stem cells, zapping DNA carrying a modified or disabled gene into them, and hoping that the cells’ repair machinery sews the DNA into the genome in the right way. Usually they have to use antibiotics and drugs to screen out all the cells where the DNA gets jammed into the genome haphazardly. Also, Jin adds that CRISPR/Cas technology can be used for whole-genome screens.

Tamara Caspary, a developmental biologist and scientific director of Emory’s transgenic mouse and gene targeting core, says she and her core team are in the process of developing and validating CRISPR, so that the technique could be accessible to many Emory investigators.

Potential clinical uses: Japanese scientists have proposed that CRISPR/Cas be employed against HIV infection. One can envision similar gene therapy applications.

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Cilia = not silly

Please check out the news story on “Cilia guide neuronal migration in  developing brain,” illustrating the dynamic role played by cilia. Cilia are tiny hair-like structures on the surfaces of cells, but in the brain they are acting more like radio antennae.

In developing mouse embryos, Emory and UNC researchers were able to see cilia extending and retracting as neurons migrate. The cilia appear to be receiving signals needed for neurons to find their places.

The Developmental Cell paper is here. As a bonus, we have a video featuring two of the paper’s authors, geneticist Tamara Caspary and “Neurotypical?” blogger Laura Mariani, a graduate student in Caspary’s lab.

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Flexibility and forgiveness during embryonic development

Geneticist Tamara Caspary’s laboratory has a recent paper in the journal Development showing how a developing mammalian embryo can correct a mispatterned neural tube over time. Former Genetics + Molecular Biology graduate student Chen-Ying Su, now a postdoctoral fellow at the Fred Hutchinson Cancer Research Center in Seattle, is the first author of the paper.

A molecule called “Sonic Hedgehog” is needed for proper patterning of the brain, spinal cord and eyes – it provides signals to the cells in the embryo, telling them what to become. Mutations that enhance Sonic Hedgehog signaling can lead to neural tube defects, some of the most common birth defects in humans, while those that diminish it can lead to holoprosencephaly, malformations of the brain and face. However, the majority of neural tube defects such as spina bifida do not come solely as a result of genetics – doctors think that getting enough (and possibly, not too much) of the B vitamin folic acid can prevent most of them.

Red = motor neuron precursor, green = later motor neuron marker
Mutation of Arl13b causes expansion of motor neurons (B and J)
Later deletion causes temporary expansion (C), corrected two days later (K)

Su and her colleagues examined mouse development in a situation where patterning of the neural tube is disrupted for a short time, because of a deletion in a gene (Arl13b), which helps to carry out Sonic Hedgehog’s instructions.

If Arl13b is not working starting from the beginning of development, embryos have an expansion of motor neurons, at the expense of other types of cells. The mutation leads to an open neural tube as well as abnormal eye, heart and limb development. However, if the deletion of Arl13b occurs on the ninth day, the embryo can recover proper patterning over the next few days. Mouse pregnancies last roughly three weeks.

Caspary says that while the relationship between Hedgehog signaling and neural tube defects is complicated, her lab’s recent work “does help define the time window during which we could non-surgically correct neural tube defects in utero.”

“In addition, it points to the importance of what we call “plasticity”- that cells can make incorrect decisions and correct them if still in a competency window, much like we think of adolescence,” she says. “It hints at the promise of stem cell research, that cells might be coaxed into other fates even though they start expressing tissue-specific markers. And it shows that the embryo is still much better at it than we are in a tissue culture dish.”

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