Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students’ learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our “formative frustration” hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of “formative frustration” is an important aspect for a successful CURE

EYA and NITO

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.During development of an adult organism from a fertilized embryo, signaling pathways are deployed reiteratively to regulate a variety of cellular processes, including cell proliferation, specification, differentiation, migration, survival and death. These signaling pathways do not act independently, but rather are integrated to yield desired developmental outcomes. While there are a variety of mechanisms by which signaling pathways are integrated, our work has focused on signal integration at the level of transcriptional control. Signaling pathways have been demonstrated to converge at the level of transcription, either by regulating activity of a common transcription factor and affecting its transcriptional output, or by regulating activity of distinct transcription factors, which target a common set of genes. In order to understand how signaling pathways are integrated to affect developmental outcomes, it is not only necessary to investigate the regulation of these transcription factors during development, but also it is necessary to identify their downstream transcriptional targets. In this thesis we have focused on the roles of two downstream transcriptional effectors, Eyes absent (EYA) and Spenito (NITO), in Drosophila eye development.(cont.) EYA is a member of the retinal determination gene network (RDGN), a network of transcription factors and cofactors that when mutated result in defects in eye development, and when overexpressed can induce ectopic eye development. EYA functions as a part of a bipartite transcriptional complex, with EYA providing the transactivation function for the complex, and its cofactor Sine oculis (SO), providing the DNA-binding function. In addition to acting as a transcription cofactor, EYA also functions as a protein phosphatase, presenting a unique juxtaposition of functions and raising the question, how are these functions related? In order to examine how transcriptional activity is affected by mutating EYA phosphatase activity, we needed to identify additional transcriptional targets, given that only one target had been identified at the time these experiments began. Using microarray analysis, we compared expression profiles of wildtype tissue to those of tissue overexpressing a wildtype eya transgene. In parallel, we overexpressed a phosphatase mutant eya transgene, which allowed us to examine the overall effects of reducing phosphatase activity on transcriptional output.(cont.) We identified 577 genes with altered expression upon overexpression of wildtype or mutant eya and confirmed 6 genes as direct targets of EYA transcriptional regulation, including string, a cell cycle regulatory gene. Stg is a particularly intriguing target, given previously characterized proliferation defects observed in tissue misexpressing eya and so. In addition, our array results suggest that mutating EYA phosphatase activity does not globally impair EYA's transcriptional output, suggesting that these functions are somewhat independent. Further analysis of transcriptional target expression and identification of targets of EYA phosphatase activity will be necessary to understand how these two functions are integrated during development. In addition, we have examined the role of SPOC family proteins, which function as effectors of Notch, Wingless and Epidermal Growth Factor Receptor (EGFR) signaling, in Drosophila eye development. Studies of these proteins in cultured cells have demonstrated their ability to function as transcriptional coregulators. In the work presented here, we have investigated the role of the small SPOC protein, Spenito (NITO), during Drosophila eye development and analyzed its relationship to a large SPOC protein, Split ends (SPEN), which is predicted to have redundant function.(cont.) First, we characterized the eye phenotypes arising from overexpression of nito. Overexpression of nito perturbs eye development and in contrast to spen, which has been shown to act as a positive effector of EGFR signaling, appears to be a negative effector of EGFR signaling during eye development. Further genetic analysis of the relationship between spen and nito in the eye suggests they function antagonistically, possibly by targeting a common set of genes for transcriptional regulation. Further analysis of SPOC family mutants and study of the functions of these proteins at a molecular level will be necessary to understand the relationship of small and large SPOC proteins and the roles of the proteins in development and disease.by Jennifer Colleen Jemc.Ph.D

Identification of raw as a regulator of glial development.

Glial cells perform numerous functions to support neuron development and function, including axon wrapping, formation of the blood brain barrier, and enhancement of synaptic transmission. We have identified a novel gene, raw, which functions in glia of the central and peripheral nervous systems in Drosophila. Reducing Raw levels in glia results in morphological defects in the brain and ventral nerve cord, as well as defects in neuron function, as revealed by decreased locomotion in crawling assays. Examination of the number of glia along peripheral nerves reveals a reduction in glial number upon raw knockdown. The reduced number of glia along peripheral nerves occurs as a result of decreased glial proliferation. As Raw has been shown to negatively regulate Jun N-terminal kinase (JNK) signaling in other developmental contexts, we examined the expression of a JNK reporter and the downstream JNK target, matrix metalloproteinase 1 (mmp1), and found that raw knockdown results in increased reporter activity and Mmp1 levels. These results are consistent with previous studies showing increased Mmp levels lead to nerve cord defects similar to those observed upon raw knockdown. In addition, knockdown of puckered, a negative feedback regulator of JNK signaling, also causes a decrease in glial number. Thus, our studies have resulted in the identification of a new regulator of gliogenesis, and demonstrate that increased JNK signaling negatively impacts glial development

JNK reporter levels are increased upon <i>raw</i> knockdown.

<p>(A-B'') Single slices of peripheral nerves of third instar larvae co-stained for JNK Green Fluorescent Protein (GFP) reporter (green) and glial cytoplasm (Red Fluorescent Protein (RFP), red). All images acquired with 60x oil objective with same confocal settings. Scale bars are 25μm. (A-B) GFP alone. (A'-B') RFP alone. (A''-B'') Merge of GFP (green) and RFP (red) (C-D") Single slices of the ventral nerve cord of third instar larvae stained for GFP and RFP. All images acquired with 60x oil objective with same confocal settings. Scale bars are 50μm. (A-A", C-C') Control animals overexpressing <i>dcr2</i> (<i>repo-Gal4</i>> <i>dcr2</i>; n = 12). (B-B", D-D') Panglial <i>raw</i> knockdown in the presence of overexpressed <i>dcr2</i> results in an increase of GFP levels in nerves and the VNC (<i>repo-Gal4</i>><i>raw-RNAi</i>^<i>2</i>, <i>dcr2</i>; n = 13). Posterior to the right. (E) Quantitation of average GFP intensity in VNC relative to adjacent muscle tissue. **p < .0001 based on Mann-Whitney test.</p

Third instar larval nervous system structure.

<p>(A) Cross section of a peripheral nerve. Axons are in yellow, wrapping glia (WG) in purple, subperineurial glia (SPG) in light blue, perineurial glia (PG) in dark blue, septate junctions in pink, and neural lamella (NL) in gray. (B) Schematic of the third instar larval nervous system. The brain, ventral nerve cord (VNC) and peripheral nerves A1-A8/9 are illustrated with the muscle field area (MFA) boxed and the nerve extension region (NER) encircled.</p

<i>raw</i> knockdown in glia results in reduced larval crawling.

<p>(A) Crawling speed assayed in control animals (<i>repo-Gal4</i>; n = 11) and <i>raw</i> knockdown animals (<i>repo-Gal4</i>, UAS-<i>raw-RNAi</i>^<i>2</i>; n = 19) over a 30 second interval. p<0.0001 based on unpaired, two-tailed t-test. (B) Distance traveled by control animals (<i>repo-Gal4</i>; n = 11) and <i>raw</i> knockdown animals (<i>repo-Gal4</i>><i>raw-RNAi</i>^<i>2</i>; n = 19) over a 30 second interval. ***p<0.0001 based on Mann-Whitney test.</p