Optimizing microCT for phenotyping
Study offers standards for skeletal analysis of model organisms.
Many of the genes underpinning muscle and brain tumors also can cause overt or mild birth defects. Detection of birth defects and tumors, therefore, can significantly broaden our understanding of the genetic mechanisms of childhood cancers.
Dr. Charles Keller’s laboratory at the University of Texas Health Science Center in San Antonio has long studied childhood cancers using mouse models, through the lens of developmental genetics. The researchers rely especially on microCT — a miniature version of the commonly used CT technology — for their investigations and have reported use of the method for various applications, including phenotyping of soft tissue and skeletal defects (see “Virtual histology method bolsters visualization of mouse embryos,” July 2006, page 24).
The story does not end there, of course. “Once we optimized our methodologies for studying bones (a metastatic site of tumors) and skeletal birth defects, our colleagues in other fields of development challenged us to detect important skeletal features in their models,” Keller said. For this reason, in the May issue of The Anatomical Record, the researchers published a paper that establishes standards for phenotyping a wide array of genetic model organisms. The standards include sample preparation and scanner acquisition parameters, but also the use of specialized open source software tools developed by a team headed by Christopher R. Johnson at the University of Utah in Salt Lake City.
Researchers have reported guidelines for phenotyping model organisms with microCT. They also showed how the optimized technique could contribute to various applications, including comparative avian fetus studies (left) and investigations of bats’ skeletal features (right).
Phenotyping involves intentional genomic modification of organisms, producing phenotypic variations that help to shed light on the function of the gene or gene subset. Early approaches to such analysis were often laborious, but the development of microCT opened the field to rapid analysis of particular phenotypes: for example, skeletal defects and defects of the vascular system, as reported by Keller and colleagues in 2005 (see “Contrast agents improve CT imaging of the liver and vasculature,” July 2005, page 16). The incorporation of metal-containing contrast agents the following year extended the potential of the technique by allowing observation and characterization of the fine structure of soft tissues.
In the Anatomical Record paper, the researchers offered guidelines for species-specific phenotypic analysis of several genetic model organisms: mouse, frog, zebra fish, bat, opossum, chicken, duck and mouse lemur. With an eye toward detecting features of particular interest in each of these, they optimized the guidelines for resolution, signal-to-noise and artifact reduction, noting that investigators can tweak the settings further to meet their specific needs.
They used two microCT instruments to scan the organisms and thus to optimize the settings, depending on the resolution required. They used an eXplore Locus in vivo microCT scanner from GE Healthcare of London, Ontario, Canada, for scans of 27-, 46- and 93-mm resolution; for scans with resolutions of 6 to 36 mm, they used a μCT40 specimen scanner from Scanco USA Inc. of Southeastern, Pa.
They also demonstrated the use of specialized software tools to enhance the information content from the microCT scans. For data acquired from opossum, mouse, bat and lemur scans, they used open-source software with optional bone analysis plug-ins, also from GE Healthcare.
In addition to presenting the guidelines, the researchers demonstrated specific applications of microCT for several model organisms. For example, they showed the potential of the technique for avian fetus studies by comparing the overall morphology of the wild-type fetal chick and duck. Here they noted morphological differences in ovo after obtaining whole-body renderings of both animals: for instance, the scleral ring was apparent in the duck rendering but not in the chick rendering. The scleral ring is an evolutionary adaptation that is widely held to be more pronounced in flying and diving birds.
The study of bats’ skeletal features is also likely to benefit from use of the optimized technique. Typically, such study requires disarticulation of the bone and socket. The investigators compared microCT scans with results from traditional light microscopy and found that the former were as effective as, if not more effective than, the latter, given that the resolution chosen is appropriate for the region of interest in the bat — thus helping to avoid disarticulation.
Keller noted that the most challenging part of the study was, ironically, validation of the methods using traditional tools such as calipers. “It felt a little backwards,” he said, “to do repeated measures using calipers to demonstrate that the scans were significantly more precise. But it was a necessary [and important] exercise.”
The researchers hope to see, and indeed are working hard to facilitate, widespread implementation of the method. Keller talks about the possibility of what he calls Museum-Aid, a consortium of imaging facilities that uses the phenotyping methods and techniques described in the Anatomical Record paper and that offers free or discounted scanner access to natural history museums with unique and generally nonreproducible collections. “Imagine, for instance, what could be learned about comparative genetics from semi- or fully automated software shape analysis of bones from species such as whales, apes … maybe even early hominids.”
Of course, their cancer research continues as well. They are currently working to integrate into the National Cancer Institute’s Pediatric Preclinical Testing Program to test new drugs from the pharmaceutical industry using their mouse models of cancer. Determining the extent to which drugs prevent or treat metastasis to bone is an important outcome measure when selecting drugs to move into clinical trials, Keller said.
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