Graduate Summer Colloquium 2007:

Hua Wang

“The Effect of Genetic Background on Tumor Development in Apc^Min/+ Mice ”

Apc^Min, a dominant allele of Apc, results in a predisposition to intestinal tumors and mammary tumors in mice that is affected by genetic background. FVBxB6 F1 Apc^Min/+ mice develop significantly fewer mammary tumors and intestinal tumors than B6-Apc^Min/+ mice. We transferred Apc^Min onto the FVB background to assess the effect of a homozygous FVB background on tumor development. Upon treatment with the alkylating agent, ENU, FVB-Apc^Min/+ female mice develop mammary adenocarcinomas, in contrast to squamous cell carcinomas (SCC) in B6-Apc^Min/+ mice. Immunohistochemical analysis was used to characterize the molecular difference between the adenocarcinomas and SCC. While FVB-Apc^Min/+ mice are resistant to intestinal tumor development in most regions of the intestine compared to B6-Apc^Min/+ mice, FVB-Apc^Min/+ mice are specifically susceptible to the development of proximal colon tumors, a phenotype seen in human AFAP. Backcross analysis identified a new modifier affecting the development of proximal colon tumors. The identification of the gene underlying this modifier locus may provide insight into the mechanisms of tumor development in various regions of the intestinal tract.

Lisa Johns

“C. elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons”

Messenger RNAs containing premature translation termination codons (PTCs) are selectively degraded in all eukaryotes tested; a phenomenon termed "nonsense-mediated mRNA decay" (NMD). We examined protein:protein and protein:RNA interactions among C. elegans SMG proteins and investigated potential roles of SMG-2 phosphorylation in these interactions. Co-immunoprecipitation data indicates that SMG-2, SMG-3 and SMG-4 form a complex in vivo and these interactions neither require SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the interaction of SMG-2 with endogenous mRNAs of five genes that either contain or do not contain PTCs due to alternative splicing of their pre-mRNAs. SMG-2 co-immunoprecipitates with both PTC-free and PTC-containing mRNAs, but it preferentially precipitates those containing PTCs. Preferential association of SMG-2 with PTC-containing mRNA requires SMG-3 and SMG-4, but does not require SMG-1 or SMG-5. Thus neither phosphorylation nor dephosphorylation of SMG-2 is required for its persistent association with PTC-containing mRNA. We also monitored the interaction of SMG-3 and SMG-4 with the same five mRNAs, but neither SMG-3 nor SMG-4 is detectably associated with those mRNAs. We interpret these results to indicate that (i) SMG-3 and SMG-4 function to selectively remodel PTC-containing mRNPs, such that they stably contain SMG-2; (ii) phosphorylation of SMG-2 is neither required for nor inhibits its preferential marking of PTC-containing mRNPs, and (iii) phosphorylation of SMG-2 by SMG-1 occurs only after premature terminations have been discriminated from normal terminations.

Laura Vaughn

“Quantitative analysis of Arabidopsis root behavior on a tilted hard agar surface”

Carolyn Neal

“Defining the role of Sec62L in Root Growth Behavior”

Andrei Avanesov

“Extracellular regulation of Hedgehog signaling in the wing of a fruit fly”

Drosophila wing arises from a much simpler structure – the wing imaginal disc. There, similarly to higher organisms, a handful of secreted proteins pattern the adult organ. Using the landmarks in the fly wing, such as wing veins, one can study the mechanisms by which secreted signaling molecules inform surrounding cells of their future fates. Of interest is the extracellular regulation of Hedgehog signaling by another secreted protein, Shifted (Shf). A vertebrate ortholog of Shf, Wnt Inhibitory Factor-1 (WIF-1) has no known role in promoting Hedgehog signaling. Instead, WIF-1 binds an unrelated class of secreted molecules, Wnts and inhibits their activities, hence the name. This seminar will show the current progress for understanding Shf function in more detail. The functional comparison of Drosophila Shf and zebrafish WIF-1 will also be presented.

Katie Clark

“A Comprehensive Genetic Approach to Analyzing the Functions of the MAPK Gene Family in Arabidopsis thaliana”

Anongpat Suttangkakul

“Molecular Analysis of the ATG1 Regulatory Complex in Arabidopsis thaliana”

Autophagy is a eukaryotic mechanism for bulk protein degradation that involves the engulfment of cytoplasm and cellular constituents into double membrane-bound vesicles followed by the deposition of these vesicles into the lytic vacuole for breakdown. This degradative process allows for nutrient remobilization during periods of starvation and facilitates developmental remodeling via programmed cell death. In budding yeast, the formation of a kinase complex containing ATG1 and ATG13 plays an important role in the induction and regulation of autophagy. We have identified orthologs of ATG1 and ATG13 in Arabidopsis, suggesting that a similar complex exists in plants. Three isoforms of ATG1 and two isoforms of ATG13 are expressed (ATG1a-c and ATG13a, b). In addition, a truncated form of ATG1 is synthesized (ATG1t); it contains the kinase domain but is missing the regulatory domain. Similar truncations are also present in rice and maize, but not in animals, suggesting that ATG1t serves a novel plant-specific function. Insertional mutations in Arabidopsis ATG1 and ATG13 have been acquired and characterized molecularly.

Andy Klocko

“Dissecting the interaction between 6S RNA and σ⁷⁰-RNA polymerase”

Xinjie Xu

“From coat color dilution to neurodegeneration”

Our goal is to understand the molecular mechanism for regulation of vesicles and their associated proteins in neuronal cells. Coat color dilution can be a good indicator for defects in biogenesis and trafficking of vesicles (melanosomes). It is known that coat color dilution is accompanied by abnormal phenotypes in other organs in many mutants, suggesting common pathways underlying the regulation of vesicles. Screening for mice showing the coat color dilution with a neuronal defect such as ataxia will give us a unique opportunity to identify new molecules associated with the regulation of vesicles in the nervous system.

nur17 mice generated by ENU mutagenesis show ataxia and coat color dilution. We observed progressive Purkinje cell degeneration in nur17, which accounts for ataxia movement. Histological analysis showed abundant filamentous tau lesioins, ER stress and abnormal ER morphology and dendritic spine size, which suggest that nur17 mice may have a defect in regulation of vesicles. Positional cloning effort is underway to identify the nur17 gene. We have mapped the mutation on chromosome 9 and have found several candidate genes. We are in the process of narrowing down the genetic region and testing the candidate genes.

Chris Mayne

“Systemic Autoimmunity in BAFF-R Mutant A/WySnJ Mice ”

Lupus, an autoimmune disease of unknown etiology, affects five million individuals worldwide. This systemic disease is thought to reflect autoantibody-mediated damage due to a failure of B lymphocyte tolerance. Since excessive BAFF expression is linked to human and murine lupus, and BAFF signals B cell survival through the BAFF receptor (BAFF-R), excessive BAFF-R signaling is currently thought to subvert B cell tolerance and facilitate lupus development. Therefore, it was surprising that the BAFF-R signaling defective, B cell-deficient A/WySnJ mice develop a lupus-like syndrome. This strain has a high incidence of IgM (80%) and IgG (73%) Ab to dsDNA as well as a dramatic increase in the frequency of antibody secreting cells to dsDNA. Additionally, 50% of these mice display proteinuria (?100 mg/dL urinary protein) suggesting end-organ damage. Furthermore, most A/WySnJ kidneys show renal pathology characteristic of lupus, including PAS-positive deposits, capillary bed destruction, and immune complex deposition in the glomeruli. Importantly, we genetically linked this autoimmune phenotype to the Baffr locus, since BAFF-R mutant A/WySnJ mice developed IgM, IgG, and antibody secreting cells to dsDNA in addition to kidney pathology but congenic AW.Baffr-wt mice did not. Our data provide the first evidence relating disrupted BAFF-R signaling to a lupus-like condition.

Min Ni

“A novel regulator couples sporogenesis and trehalose biogenesis in Aspergillus nidulans ”

Trehalose is a compatible osmolyte produced by bacteria, fungi, insects and plants to protect the integrity of cells against various environmental stresses. Spores, the reproductive, survival and infection bodies of fungi require high amounts of trehalose for long-term survival. Here, via a gain-of-function genetic screen, we identify the novel regulator VosA that couples the formation of spores and focal trehalose biogenesis in the model fungus Aspergillus nidulans. The vosA gene is expressed specifically during the formation of both sexual and asexual spores (conidia). Levels of vosA mRNA and protein are high in both types of spore. The deletion of vosA results in the lack of trehalose in spores, a rapid loss of the cytoplasm, organelles and viability of spores, and a dramatic reduction in tolerance of conidia to heat and oxidative stress. Moreover, the absence of vosA causes uncontrolled activation of asexual development, whereas the enhanced expression of vosA blocks sporulation, suggesting that VosA also functions in negative-feedback regulation of sporogenesis. VosA localizes in the nucleus of mature conidia and its C-terminal region contains a potential transcription activation domain, indicating that it may function as a transcription factor primarily controlling the late process of sporulation including trehalose biogenesis. VosA is conserved in most fungi and may define a new fungus-specific transcription factor family.

Zhen Zhang

“Investigating the roles of Etv5 and Pea3 in mouse limb development”

Etv5 and Pea3 are members of the PEA3 group of ETS transcriptional factors. Their expressions in vertebrate embryos are regulated by Fibroblast Growth Factors (FGFs); therefore they are supposed to be general transcriptional mediators of Fgf pathway. To test if Etv5 and Pea3 play essential roles in Fgf pathways, we knockout both genes in mouse limbs that serve as an excellent model to dissect Fgf pathway since the function of FGF for limb development is well characterized. Surprisingly, the mutant limbs exhibit no defects in proximal-distal (PD) outgrowth that are usually observed in Fgf mutants. The only phenotype observed is preaxial polydactyly (PPD), probably due to ectopic expression of Sonic hedgehog (Shh), a critical molecule for limb anterior-posterior patterning. The result suggests that Etv5 and Pea3 play minor roles downstream of Fgf pathway in limb development, but function as Shh negative regulators.

Kelley Harris

“An Investigation of the role of Dicer in Lung Branching Morphogenesis”

Dicer is a cytoplasmic, ribonuclease III enzyme that processes microRNAs (miRNAs) and small inhibitory RNAs (siRNAs) to their mature forms. In general, mature miRNAs bind with imperfect complementarity within the 3’UTR of their target genes to illicit translational repression. Conversely, mature siRNAs form perfect duplexes with their targets and direct cleavage of the target mRNAs. In mouse, a null allele of Dicer exhibits an embryonic lethal phenotype prior to gastrulation. Here we bypass this early lethality and investigate the role of DICER in lung formation using a conditional knockout approach.

Lung branching morphogenesis is a major event during lung development that produces the characteristic arborization of the respiratory tree. We found that inactivating Dicer specifically in lung epithelium leads to an arrest of the branching process. We will present data toward understanding the cellular and molecular mechanism of Dicer function in lung formation.

This research is significant for a number of reasons. Namely, a basic understanding of mammalian lung development is at the foundation for future therapeutic measures aimed at treating asthmas and lung cancer. In addition, this study is novel because our data indicates that DICER may regulate a key branching morphogenesis gene through miRNA regulation.

Sarah Duellman

“Investigating Epstein-Barr Virus EBNA1 Phosphorylation by Electron Transfer Dissociation Mass Spectrometry”

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor in a variety of malignancies, including Burkitt’s lymphoma, some Hodgkins disease cases, and nasopharyngeal carcinoma. EBV is capable of immortalizing the host cell and in doing so predisposes the cell to malignant transformation. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It is thought to play a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, latent viral DNA replication, and viral genome maintenance, but the regulation of EBNA1 is poorly understood. We hypothesize that post-translational modifications influence EBNA1 function. We were the first to overexpress and purify EBNA1 from E. coli and made a panel of monoclonal antibodies (mAbs) against this protein. We are now using these mAbs and a new mass spectrometry technique, electron transfer dissociation, to investigate EBNA1 phosphorylation. Utilizing this approach, we have been able to determine a new phosphorylation site which has provided insight into the functional regulation of EBNA1.

Justin Schleede

“A mosaic screen in the posterior compartment of the Drosophila wing to identify genes necessary for crossvein development”

David Berry

“Acquired Stress Resistance in Saccharomyces cerevisiae”

Acquired stress resistance is a phenomenon in which cells exposed to a mild dose of one stress can survive an otherwise lethal dose of the same or a different stress. This response has been observed across kingdoms, however its underlying mechanism is not understood in eukaryotes. We characterized acquired stress resistance in yeast by investigating the dose of mild stress required, the specificity of stress protection across 5 different stresses, and the kinetics of the phenomenon. We show that acquired resistance is widespread but not universal for all pairs of stress. Stress-specific differences in the timing and levels of acquired stress resistance suggest that 'same-stress' protection likely occurs via a distinct mechanism compared to 'cross-stress' protection. Cross-stress protection was previously proposed to depend on the yeast environmental stress response (ESR), a gene expression program triggered by diverse types of stress. We provide evidence for a role of the ESR in acquired stress resistance but show that the initiation of the ESR cannot explain the phenomenon. Cross-stress protection was dependent on the ESR regulators Msn2p or Msn4p, which played different roles depending on the conditions used. Cells with acquired stress resistance showed altered genomic expression in response to stress, particularly for genes in the ESR. Our results suggest that although the ESR contributes to acquired stress resistance, the phenomenon cannot be determined by a universal 'general stress' response in yeast.

Britt Johnson

“Identification of a major QTL, which modifies the schisis and layer disorganization phenotypes in Rs1h^tmgc1 mice”

Our goal is to understand what genes, molecules, and mechanisms are necessary for the proper development and maintenance of synapses. X-linked retinoschisis (XLRS) is an inherited form of macular degeneration that affects juvenile males. Mutations in the RS1 gene cause splitting of the retinal layers (schisis) and a loss in synaptic transmission as indicated by a loss in the electroretinogram b-wave. Interestingly, family members with the same RS1 mutation show phenotypic variability, suggesting the existence of genetic modifiers. We observed a wide range in phenotypic severity in mice harboring a mutation in the murine homologue of RS1, Rs1h^tmgc1, on a mixed genetic background. We have identified a major QTL on Chr 7 (LOD score=24.6, p<1x10-6). The AKR/J allele rescues the Rs1h^tmgc1 retinal phenotypes in a recessive fashion. We have named this QTL Modifier of retinoschisis 1 (Mor1). N5 congenic mice, B6.Cg-Rs1h^tmgc1Mor1^AKR/J, show complete rescue of Rs1h^tmgc1 phenotypes. Identification of Mor1 may not only provide insight into the molecular mechanism of RS1H mediated cell adhesion, but may also provide valuable information for developing therapeutic interventions for XLRS.

Erkang Ai

“A novel function for RACK-1 in endosomal recycling during cytokinesis”

Membrane trafficking is required for cytokinesis to add new membranes during cleavage furrow progression and abscission. In animal cells, recycling endosomes acts as a major source of the additional membranes. However, the mechanisms and factors that regulate endosomal recycling during the cell cycle remain poorly understood. We show here that RACK-1 is required during cytokinesis and mediates RAB-11 endosome recruitment to centrosomes. RACK-1 localizes to centrosomes, kinetochores, the cleavage furrow and midbody during the cell cycle. Reducing the levels of RACK-1 leads to failures in both furrow initiation and completion events. We conclude that the WD40 repeat protein, RACK-1, mediates RAB-11 endosomal recycling during cytokinesis.

Jun Chen

“crossveinless d likely encodes a vitellogenin domain lipoprotein required for the formation of the posterior crossvein in Drosophila”

The BMP-like signaling mediated by the ligands Dpp and Gbb is required to reinforce the development of most veins in the Drosophila wing. The formation of the crossveins is especially sensitive to reductions in BMP-like signaling. Of the four classical crossveinless loci in Drosophila, only crossveinless d (cv-d) has not been cloned. cv-d[1] homozygotes are viable and lack all or part of the posterior crossvein. I mapped cv-d[1] to the CG31150 locus using deficiency mapping, female recombination and P-element mediated male-specific recombination. CG31150 encodes a lipoprotein containing a vitellogenin domain and a von Willebrand Factor type D domain. cv-d[1] has a deletion within the vitellogenin domain and also generates an abnormally spliced CG31150 mRNA. I also isolated two more cv-d alleles, and each contains mutations in CG31150. Thus it highly likely that CG31150 is cv-d, and I am investigating its role in the formation of the posterior crossvein.

Lisa Farmer

“Regulation of Plant Development and Abscisic Acid Signaling by the Ubiquitin/26S Proteasome System in Arabidopsis thaliana”

Plants have developed adaptive traits to optimize growth under unfavorable conditions and ensure their survival. The stress-induced phytohormone abscisic acid (ABA) is an endogenous mechanism by which plants regulate important aspects of development, including tolerance to seed dormancy, water, salt and cold stresses, drought or osmotically-induced stomatal closure, wounding and pathogens. Responses to ABA are facilitated by important regulators such as the b-zip transcription factor ABSCISIC ACID INSENSITIVE 5 (ABI5), which is necessary to initiate post-germinative growth arrest under adverse growth conditions. We and others have found that while ABI5 expression is stimulated by ABA biosynthesis, ABI5 half-life is regulated by the Ubiquitin/26S Proteasome System (UPS), in which transient regulatory proteins are selectively ubiquitinated and degraded. ABI5 stability appears to be regulated by phosphorylation, and we hypothesize that its subsequent ubiquitination is de-phosphorylation dependent. Our research centers on characterizing how the post-translational modification and degradation of ABI5 by the UPS influences plant development.

Adeline Veillet

“The mammary carcinogenesis susceptibility locus Mcs5c has been mapped to a region devoid of known genes”

Breast cancer is a polygenic disease. It is thought to be due in part to both high penetrance alleles that are rare (such as BRCA1 and BRCA2 functional mutations) and low penetrance alleles that are more common in the population. To identify low penetrance breast cancer modifier genes, we performed a linkage analysis using the Wistar-Furth (WF) and Wistar-Kyoto (WKy) rat strains. Four significant quantitative trait loci (QTL) were identified: mammary carcinoma susceptibility (Mcs) 5, 6, 7 and 8. Mcs5, 6 and 8 are associated with resistance to 7,12-dimethylbenz-[a] anthracene (DMBA)-induced mammary carcinogenesis, while Mcs7 is associated with increased susceptibility. Congenic analysis demonstrated that having the WKy allele in the Mcs5 locus decreased tumor numbers by 80% compared to the WF genotype after DMBA exposure. Fine-mapping of Mcs5 revealed three loci: Mcs5a, Mcs5b and Mcs5c. Mcs5a and Mcs5c decrease susceptibility while Mcs5b increases susceptibility to mammary cancer compared to the WF controls.

The current study focuses on the fine-mapping and characterization of the Mcs5c locus. Rats homozygous for the WKy allele of the Mcs5c locus average 4.9 + 0.5 tumors compared to 8.0 + 0.6 tumors for the WF homozygous animals (p-value <0.001, n=32 and 31 respectively). Using a number of congenic lines, the Mcs5c locus has been mapped to a 410 Kb region of rat chromosome 5 devoid of known genes. The resistant phenotype attributed to the Mcs5c locus could be due to uncharacterized transcripts in the region or to genetic elements that regulate genes outside this interval. Further characterization of the locus is needed to determine which of the two above hypotheses can best be rejected.

Erin Patterson

“Determining the consequences of increased dosage of the Forkhead Homolog One (FKH1) gene Saccharomyces cerevisiae”

University of Chicago

Mike White

“Genetics of speciation in house mice”

Bob Schmitz

“Epigenetic Reprogramming in Arabidopsis: How is the Vernalization-Induced Epigenetic Silencing of FLC Reset each Generation?”

Leah Frater-Rubsam

“The smg connection to RNAi in C. elegans”

Angela Verdoni

“The Identification of Factors Involved in the Corneal Neovascularization in Dstn^corn1 Mice”

Destrin (DSTN; also known as actin depolymerizing factor or ADF) is a member of the ADF/cofilin family and an essential actin regulatory protein. DSTN influences actin filament turnover by binding to actin subunits in filamentous actin (F-actin), resulting in destabilization of the filament and enhancement of subunit dissociation. Mice homozygous for the spontaneous null mutation of Dstn (Dstn^corn1) develop a thickened corneal epithelium due to hyperproliferation of the epithelial cells, followed by stromal neovascularization. Consistent with a lack of destrin function, F-actin accumulates in the corneal epithelium of Dstn^corn1 mice. Our goal is to determine the factors that are involved in the development of neovascularization in the Dstn^corn1 cornea. To identify genes that are differentially expressed in the cornea of Dstn^corn1 mice compared to wild-type mice, we performed a microarray analysis. Through this study, we were able to identify up-regulated genes involved in inflammation and transcription. Inflammation has been shown to induce blood vessel growth in several mouse models of neovascularization, and therefore is a potential cause of neovascularization in the Dstn^corn1 cornea. We also observed up-regulation of a group of genes in the Dstn^corn1 cornea that are targets of serum response factor (SRF). SRF is a transcription factor whose activation is dependent upon the status of the cytoskeleton, which is affected in the corneal epithelium of Dstn^corn1 mice. Several SRF target genes have functions that are consistent with the development of the Dstn^corn1 phenotypes. We are attempting to determine if these factors are contributing to neovascularization in the Dstn^corn1 cornea.

Nick Sanek

“Zic2a, Hedgehog Signaling, and the Genetic Control of Forebrain Development”

Raul Correa

“Evolutionary Transgenomics: A New Screen to Identify Genes that Explain Phenotypic Differences Between Species”

An evolutionary transgenomic screen entails the introduction of genomic inserts from a donor species into a recipient species followed by phenotypic screening of the transgenic lines. Phenotypic effects caused by trangenes can theoretically be due to either gene dosage or evolutionary divergence of the donor and recipient species, all of which are of potential developmental and/or evolutionary interest. Dosage effects can be identified by seeing if the homologous region of the recipient species’ genome can cause the same effect when introduced as a supernumerary copy. Evolutionary divergence can be imperfectly divided into two main kinds: phenotypic divergence and developmental system drift. Phenotypic divergence refers to cases in which a major gene has contributed to the phenotypic divergence of the donor and recipient species, in which case an insert containing this gene could convert the recipient species to resemble the donor species for this phenotype. In cases where the transformants manifest a phenotype seen in neither species, developmental system drift is implied: divergence in genetic interactions such that genes from one species behave as gain-of-function mutant alleles in the other genetic background. We will present one such case that illustrates the potential of transgenomics to discover formerly cryptic developmental roles for even well-characterized genes. Among the many interesting phenotypes that can be discovered by such an approach, cases of dominant sterility or lethality have important implications for our understanding of the evolution of reproductive isolation in allopatry. We present results from a proof-of-concept transgenomic screen of Leavenworthia alabamica in an Arabidopsis thaliana background.