PROJECTS
Photo © Will Wrobel
Research or education?
It’s all too often an either-or decision for academic institutions and their faculty. Professing philosophical support for the teacher-scholar model or advertising hands-on learning on a website is easy. But from promotion and tenure criteria to curriculum design, those stated values don’t always hold up in practice.
In the biomedical sciences, laboratory research is usually done by experienced professionals. It’s not uncommon for undergraduates to spend the large majority of their time in lecture-intensive classes. When they do get a chance to venture into the lab, it’s as likely to shadow the pros or conduct canned experiments with predetermined outcomes as to participate in true discovery-based research.
We’re taking a different approach.
By integrating original research projects into classes and putting undergraduates in charge of the federally funded lines of investigation in our lab, we are deliberately blurring the distinction between student learning and professional scientific inquiry. We reject the pervasive notion that taking such a student-centered approach must come at the expense of doing the kind of high-impact science more commonly associated with labs at doctoral institutions.
Our current work focuses on three primary projects:
Project 1 - molecular mechanisms of regeneration
We study aquatic flatworms called planarians renowned for their ability to regenerate lost body parts. Not only can a decapitated animal regenerate its head, a tiny fragment removed from almost anywhere in the body can form a complete new individual in just a little over a week!
This process depends on a large population of adult stem cells known as ‘neoblasts’. In response to amputation, neoblasts migrate to the wound site, increase their rate of division, and give rise to a mass of new tissue called the regeneration blastema. Cells in the blastema then differentiate to restore a complete and functional anatomy. At the same time, uninjured tissues are remodeled through a process involving increased cell death to restore anatomical scale and proportion.
What are the molecular mechanisms of the regenerative response?
We are addressing this question through a combination of in-class
and independent study projects. In the upper-level elective course Developmental Biology, students use RNA interference (RNAi) to screen for novel regeneration genes. This semester-long project begins with a mini bioinformatics unit involving BLAST searches to identify planarian homologs of genes of interest. Students then clone identified sequences by RT-PCR, use RNAi to knock down expression of their chosen genes, and finally, amputate their RNAi animals and screen for regeneration defects.
Head regeneration in a decapitated planarian. Following amputation, a purse-string mechanism reduces the surface area of the wound site. Adult stem cells then give rise to a mass of new tissue called the blastema (white). Cells in the blastema eventually adopt specialized fates to form new organs like the light-sensing photoreceptors (black spots). The pictures in this series were taken immediately after amputation, and after three and seven days of regeneration.
To date, we have screened dozens of genes through this approach,
and identified three required for blastema formation. One of these is
a homolog of Drosophila mago nashi, named for its discovery in a maternal-effect sterile screen (“mago nashi” is Japanese for “without grandchildren”). mago nashi encodes one of four core subunits of the Exon Junction Complex (EJC), an evolutionarily conserved regulator of RNA biochemistry that assembles just upstream of exon-exon junctions during pre-mRNA splicing.
The EJC controls splicing, export of spliced mRNAs from the nucleus, nonsense-mediated mRNA decay (degradation of transcripts harboring premature termination codons), and translation. Despite these broad ‘housekeeping’ roles, disruption of EJC function has been linked to surprisingly specific stem cell defects in flies and mice. We found that RNAi knockdown of core EJC subunits leads to rapid stem cell loss in planarians, explaining their inability to regenerate after amputation.
Our findings are detailed in a recent publication in Developmental Biology. We are currently exploiting the experimentally accessible planarian system to explore possible roles for nonsense-mediated decay in regulation of the planarian stem cell lineage.
Project 2 - an animal model of acute porphyrias
Named after the ancient Greek word for purple ("porphura"), the porphyrias are a group of inherited metabolic disorders involving an accumulation of toxic intermediates in heme biosynthesis; these compounds can impart a characteristic port-wine color to the urine and cause symptoms including sunlight-induced skin damage and neuropsychiatric issues ranging from mild confusion to seizures or paralysis.
Porphyrias affect an estimated 1:10,000 people. Some have speculated the disorder afflicted historical figures like the painter Vincent van Gogh.
What do these rare human diseases have to do with planarians? Interesting story . . .
In addition to incorporating planarian research projects into upper-level science classes like Developmental Biology, we have exploited the dramatic and easily observable regenerative response to engage non-science majors in original, discovery-based research. Students in the general education course Stem Cells and Regeneration conduct science fair-style experiments examining the effects of environmental variables on regeneration to learn about the scientific method.
Several years ago, a group of students investigating the impacts of light exposure (planarians are normally in a dark environment in the wild and in the lab) made the surprising discovery that intense light can induce complete bodily depigmentation.
To investigate the basis for this light-induced depigmentation response, we conducted an RNA-Seq analysis in collaboration with the Pearson Lab at The Hospital for Sick Children in Toronto. This revealed that the first three enzymes in the eight-step heme biosynthesis pathway exhibit enriched expression in the subepithelial pigment cells; the remaining five pathway enzymes are expressed predominantly (or exclusively) in other cell types. The resulting bottleneck effect causes a buildup of porphyrins, sensitizing the pigment cells to light. Remarkably, this is the same biochemistry underlying the light sensitivity observed in many porphyria patients.
We published results from this work in the open-access journal eLife, establishing a new animal model for research on the basic science of porphyrias. The first six authors are former undergraduates in our lab.
Porphyrias are rare diseases that can involve life-threatening neurological issues and sunlinght-induced skin damage. The latter symptom is caused by a buildup of ring-shaped molecules called porphyrins in the skin. When exposed to light, porphyris react with oxygen to generate harmful reactive oxygen species (ROS), causing tissue damage.
Photo credit: International Porphyria Patient Network
A particularly interesting feature of our planarian porphyria model emerged from the observation that starvation enhances the rate of light-induced depigmentation.
In the ‘acute’ porphyrias, patients are usually asymptomatic, but can experience a sudden onset of disease symptoms in response to
various triggers that include dieting or fasting. This has been traced to upregulation of the first, and rate-limiting pathway enzyme, ALAS. In the presence of an inherited mutation in a downstream enzyme, ALAS upregulation exacerbates the accumulation of porphyrins and other toxic heme precursors.
We found that induction of ALAS by starvation is conserved in planarians, and are now investigating the mechanisms responsible for metabolic control of porphyrin/heme biosynthesis. This work has the potential to reveal new targets for therapeutic intervention in the acute porphyrias.
Ultimately, we hope our work will not only provide new insight into the basic science of porphyrias, but might also accelerate the development of new disease treatments.
Project 3 - coevolution of flatworms and ciliates
Our newest line of research, a collaboration with Ryan Woodcock at Trocaire College, is focused on eukaryotic microbes called ciliates that parasitize a wide variety of flatworms. Ciliates exhibit a remarkable and underappreciated degree of biodiversity. The genus Tetrahymena, for example, includes more than 80 known species found from the Arctic to the Antarctic. Some of these are free-living pond dwellers that consume bacteria; others are ‘histophages’ that feed on the tissues of diverse animal hosts. Histophagous Tetrahymena and related ciliates are a major problem in the ornamental fish trade and can reduce the viability of ecologically important host species like freshwater mussels.
In addition to isolating and culturing ciliates from multiple planarians, we developed a novel bioinformatic pipeline to search flatworm transcriptomes for sequences that are in fact of ciliate origin. From more than 2.5 million screened ESTs, we identified nearly 6,000 high-confidence ciliate transcripts, supporting parasitism of seven additional flatworm species. Our follow-up analysis of these sequences provides strong evidence for coevolution of histophagous ciliates and their flatworm hosts. Importantly, some of the planarians we found to be infected are invasive species, raising the prospect they contribute to the geographic dispersal of histophagous ciliates.
Photo © Will Wrobel
Brad Stubenhaus '14 did much of the initial work in characterizing the biochemistry and molecular biology of our planarian porphyria model. After generating two first-author publications as an undergraduate, he went on to earn a master's degree from the Johns Hopkins University School of Medicine.
