Biological Sciences Student Research Showcase 2010

Cellular and Developmental Biology Abstracts


Epidermal Growth Factor Receptor Overexpression Contributes To the Acquisition of Stem Cell like Properties In Head and Neck Squamous Cell Carcinoma And Thyroid Cancer Cell Lines

Eric Abhold
Dr. Martin Haas

Epidermal Growth Factor Receptor Overexpression Contributes To the Acquisition of Stem Cell like Properties In Head and Neck Squamous Cell Carcinoma And Thyroid Cancer Cell Lines    Eric Abhold1, Alan Kiang1, Andrew Yu1, Jessica Wang-Rodrigues2, Weg M. Ongkeko1, Martin Haas3  1Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of California, San Diego   2Department of Pathology, University of California, San Diego   3Moores Cancer Center, University of California, San Diego  The cancer stem cell hypothesis proposes that a small subpopulation of cells exists in cancers capable of the initiation and maintenance of malignant tumors. These cancer stem cells are thought to arise from progenitor cells that have acquired stem cell properties through dysregulation of key regulatory genes, proto-oncogenes and tumor suppressors.  Proto-oncogenes, including those that play a key role in head and neck squamous cell carcinoma (HNSCC) and thyroid carcinoma tumorigenesis such as the EGFR and Akt, are thought to promote regenerative capacity by promoting stem cell function.  Conversely, tumor suppressors inhibit regenerative capacity by promoting cell death or senescence in stem cells, but also protect against cancer.    Members of the EGFR/ErbB family of receptor tyrosine kinases are proto-oncogenes found to be highly expressed in many cancers including HNSCC and thyroid carcinoma.  Studies have indicated EGFR as a key player in tumorigenesis and that elevated EGFR kinase activity induces cell proliferation and promotes cell migration and invasion, thus contributing to cancer metastasis. Consequently, the ErbB family has been highly targeted in cancer therapies. A recent clinical study (Schmidt, C., JNCI 100: 694-695, 2008) demonstrated a link between EGFR and ErbB2 inhibition and reduced CD44+/CD24low expression, the cancer stem cell marker in breast cancer. This observation has subsequently been partially confirmed at the basic science level as ErbB2 has been demonstrated to regulate the mammary/stem progenitor cell population.    In this study, we demonstrate that in HNSCC, activation of EGFR results in induction of cancer stem cell genes BMI-1, CD44, Nanog, Oct4, CXCR-4, and SDF-1 by qPCR and western blot analysis.  Conversely, inhibition of EGFR using small molecule inhibitors resulted in downregulation of these markers.    Our results further indicate that EGFR activation results in increased self-renewal capacity.  In addition, we demonstrate that EGFR activation or overexpression results in increased ability to form spheres in suspension, both in terms of size and number, while the inhibition of EGFR prevents sphere formation in HNSCC cancer stem cells derived from fresh tissue.  Taken together, these results suggest that EGFR activation is critical in the acquisition of stem cell like properties in HNSCC.


A Temporal Code in the Dentate Gyrus: A Unique Role for Adult-Born Granule Cells in the Formation of Memories

Stephanie Alfonso
Dr. Fred Gage

Adult-born neurons in the dentate gyrus (DG) of the hippocampus show increased plasticity and are discretely regulated by network activity making them likely contributors to learning and memory. The formation of memories in the dentate gyrus has been modeled as a sparse code which dictates that the output firing patterns of a network are less similar to one another than the input firing patterns; this reduces interference and facilitates the consolidation of separate memories. However, adult-born dentate granule cells (DGCs) are preferentially active as compared to their mature counterparts. This hyperexcitability, in turn, makes them less selective. A new computational model endows these immature cells with the ability to provide an association between events that are temporally proximal owing to their indiscriminate responses to inputs- a phenomenon termed pattern integration. To test this model, we have exposed neurogenesis knockdown (TMZ) and control rats to a task which requires the temporal association of object locations. We hypothesize that neurogenesis knockdown animals will show a deficit in temporal integration and will not associate events close in time.


Type-I IFN signaling is required for the maintenance of Foxp3 expression and Treg cell function

Chang Kyung Kim
Dr. Eyal Raz

The immune cells of the gastrointestinal tract have to execute unique regulatory and physiological tasks to prevent intestinal inflammation in this microbial rich environment. In this study, we uncovered that type-I IFN signaling is essential for Treg phenotype and function in vivo. IFN-abR-/- (KO) Treg cells failed to suppress experimental colitis in an adoptive transfer model. Foxp3 expression in the transferred KO Treg cells was significantly reduced. Furthermore, the administration of rec. IFNα ameliorated experimental colitis by increasing the number of Foxp3+ cells and Treg function. Our subsequent studies revealed that type-I IFN was independent of IL-10 signaling in the inhibition of experimental colitis. Our data demonstrate a novel role for type-I IFN in immune regulation of adaptive immunity through the induction of Foxp3 expression and Treg cell function in vivo.


DSCAM and it's role in the developing nervous system

Eunice Kym
Dr. Eduardo Macagno

As the nervous system develops neurons must be able to extend their axons into the correct locations, form synapses with the correct targets, avoid self-dendritic interactions, and defasciculate appropriately from nascent axonal branches. One molecule that has been implicated in these processes is Down Syndrome Cell Adhesion Molecule (DSCAM in vertebrates and Dscam in invertebrates).  Here we have sequenced part of the Dscam homolog in the medicinal leech Hirudo medicinalis (HmDscam). Using a portion of the 3’ end of the HmDscam gene taken from an EST library we have sequenced more of the gene towards the 5’ end. To do this, we designed primers generated from a conserved amino acid search on translated genomic contigs. We have shown that HmDscam is missing one of the 6 fibronectin type III domains that are conserved across all species. Furthermore, using techniques such as in situ hybridization we have shown that HmDscam is expressed in sensory neurons and the eye regions of the head. Future studies will focus on describing phenotypes of HmDscam knock down, characterizing HmDscam expression patterns at different stages of development, and elucidating downstream mechanisms of HmDscam signaling.


Canonical Wnt Signaling and Quiescence in Embryonic Murine Mammary Gland Branching Morphogenesis and Stem Cell Function

Justin La
Dr. Geoffrey Wahl

In mammalian organisms, mammary gland development begins during embryogenesis. Mammary gland development in mice initiates with epithelial cell migration to form mammary placodes on the surface of the skin. The placodes then invaginate into the mesenchymal layer underneath the skin to form mammary buds. Mammary buds are seen in both males and females, but on embryonic day 14, androgen signaling retards male bud development leading to their eventual destruction. On embryonic day 16, female buds further sprout and branch to form rudimentary ductal trees.   Many molecular pathways are known to direct embryonic mammary gland development. It has been shown that mouse mammary placode formation requires canonical Wnt signaling and it has been proposed that stem cell populations may maintain a dormant subset of cells in a quiescent cell cycle state.  These data suggests that Wnt signaling may be important in order to acquire mammary identity. In addition, Wnt signaling has been shown to play a role in mouse models of breast cancer. We hypothesize that canonical Wnt signaling may play a critical role in the maintenance of normal embryonic ductal outgrowths and elementary branching. We are analyzing the effects of Wnt signaling in embryonic mouse mammary buds with an in vitro organ explant method. Using small molecule inhibitors and genetic tools to up and down-regulate Wnt signaling, we are investigating the role Wnt signaling plays in branching morphogenesis.   Label retention is a way of detecting infrequently cycling, dormant cells. This method utilizes fluorescent histone proteins to distinguish quiescent cells from dividing cells. Because some populations of tissue stem cells are thought to maintain a dormant population, we predict that mammary stem cell populations may contain some label retaining cells. To test this hypothesis, we will utilize previous work in our lab that demonstrated that the CD24+ cells in embryonic day 18.5 mammary rudiments contain all the embryonic mammary stems cells. Our main goal is to determine whether mammary stem cells maintain a dormant, quiescent subset or are all proliferative by identifying whether label retaining cells exist and if so, whether they are within the CD24+ population.   It has long been suggested that Wnt signaling and quiescence are important for maintaining a stem cell identity, but this has yet to be definitively shown. Our in vitro data will give us insight as to whether Wnt signaling and label retention correlate with mammary stem cell function during development. The results of this work will establish whether using these properties may improve the isolation of mammary stem cells such that better comparisons can be made in the future between stem cells and cancer.


Functional Characterization of Zebrafish Granulocyte-Colony Stimulating Factor

Ryan Lau
Dr. David Traver

Granulocyte Colony-Stimulating Factor (G-CSF) is a polypeptide growth factor essential for the differentiation of neutrophilic granulocytes from hematopoietic progenitor cells. First identified and studied in mice and humans, two gene candidates for G-CSF were recently identified in zebrafish. These evolutionarily conserved genes were cloned and recombinantly generated to test their function in vitro. When whole kidney marrow (WKM), the primary site of adult teleost hematopoietic progenitors, was isolated and cultured in methylcellulose with either variant, an increase in colony forming units was observed. When isolated, these colonies were comprised of myeloid cells. This is the first time that G-CSF has been shown to be functionally active and effective for the differentiation of myeloid progenitors in vitro in zebrafish cultures. To support our in vitro findings, mRNA was transcribed and injected into single-cell embryos of transgenic zebrafish with cell lineage-specific fluorescent markers. Over-expression of both variants caused an increase in definitive myeloid cells in these live embryos. Based on these functional studies, these two putative G-CSF cytokines appear to functionally recapitulate the activity of G-CSF in mammals, bolstering the further importance of the zebrafish as a model of hematopoietic system evolution, development, and dysregulation. Further development of these in vitro functional assays should prove to be helpful in discovering developmental defects in mutant animals, as well as allow further dissection of genetic programs subverted in myeloid diseases.


Detecting Novel Protein Interactions with Desmoplakin using Yeast 2 Hybrid Protocols

Patrick Lee
Dr. Farah Sheikh

I am currently enrolled in BISP 196 and will be presenting my data on novel protein interactions with Desmoplakin. I have used a Yeast 2 Hybrid system from Clontech to identify these protein interactions. Desmoplakin is an integral part of the desmosome that is found in the cell to cell junction. It has been found that in Desmoplakin KO mice the animals exhibit ARVC (Arrhythmogenic Right Ventricular Cardiomyopathy) which is characterized by a thinning of the right ventricular wall as well as fatty deposits. ARVC accounts for 17% of all sudden cardiac deaths in the young and the incidence of ARVC ranges from 1/10,000 to 1/1,000.   In my experiments the desmoplakin gene has been divided up into 6 fragments and transformed into pGBKT7 vector. The 6 fragments will then be mated with a cDNA library from a Yeast 2 Hybrid system from clontech to determine interacting proteins with each specific fragment. So far we have been able to screen the first two fragments of desmoplakin and have identified several new interesting interacting proteins such as Cop Signalosome Subunit 6, Cyclin D, Cereblon, andNAD(P)H dehydrogenase quinone 2. Further research will be carried out by the post-docs in the lab on the importance of these interactions. As of now fragment 6 will be mated with the cDNA library soon to discover interactors on the C-terminus of the desmoplakin protein.


Identification of Mechanosensory Genes in Drosophila Melanogaster

Anh Nguyen
Dr. Boaz Cook

Sensing mechanical forces is important for hearing, touch, balance, and pain. Receptor neurons sense mechanical stimuli and encode them into electrical signals that are relayed to the central nervous system. There is little known about the molecular mechanism that transduces mechanical stimuli to electrical signals. In order to understand the process, I chose to identify genes that are essential for mechnical transduction and study their functions. I used Drosophila Melanogaster as a model animal since they enable genetic screening and functional analysis. A chemical mutagenesis screen was used for identifying temperature sensitive (ts) uncoordinated flies. One of the mutants, line 2883, was isolated from a collection of F3 mutagenized lines that caused temperature sensitive uncoordination. Using recombination mapping, the region of the suspected mutation was found to be in the range from 92 to 94 on the third chromosome. A secondary screen for suppressor mutants identified 8 lines. Six of them showed complete rescue and two are hypermorphs. I used a combination of genetic tools to demonstrate that an elimination of the gain of function gene in 2883 does not result in a discernable phenotype. Therefore, the gene affected 2883 is not essential for mechnosensation and only its dominant form affects the process. Further experiments are being conducted to identify the 2883 gene and other genes that genetically interacting with it.


The role of Dynamin in flies as a model for human centronuclear myopathy

Jen Nguyen
Dr. Amy Kiger

Muscles are large, multinucleated cells with highly compartmentalized membrane domains that serve in contraction. Small myofibers with central nuclei characterize a group of muscle diseases called centronuclear myopathies (CNMs). Mutant genes associated with CNM point to possible defects in membrane trafficking. An autosomal dominant form of CNM is associated with mutations in dynamin 2 (DNM2), a large GTPase shown to function during membrane scission in many cell types at different membrane compartments. To investigate the roles of DNM2 in muscle, the Drosophila system offers several advantages.  For example, there are three forms of human dynamin, while flies only have one—shibire (shi). Here, we show that Drosophila muscles expressing shi RNAi phenocopy muscles expressing myotubularin RNAi.  A phosphoinositide phosphate phosphatase, myotubularin-1 (MTM1) also functions in membrane trafficking and MTM1 mutants have been found in X-linked forms of CNM. We thus propose that DNM2 and MTM1 may function in a common pathway to regulate muscle maintenance. In Drosophila muscles, we show that a loss of shibire results in detached or misaligned persistent larval muscles, which suggests that shi is required for maintaining muscle attachments. At a molecular level, we clone GFP-and FLAG-tagged wild-type shi and find GFP-shi co-localizing with Kc cell golgi. In the presence of mtm RNAi, a change in GFP-shi localization would serve as evidence for a shared pathway between mtm and shi.


Functional Significance of Mtm's Substrate Selectivity

Vignesh Raman
Dr. Amy Kiger

Phosphoinositides, the phosphorylated forms of phosphatidylinositol (PI), are highly regulated to mediate localized cellular processes such as signaling and membrane trafficking. Myotubularins (MTMs) encode phosphoinositide 3-phosphate phosphatases that dephosphorylate the D-3 position phosphate of PI(3)P and PI(3,5)P2 to yield PI and PI(5)P, respectively. The Drosophila MTM1/MTMR2 ortholog, mtm, has been shown to downregulate PI(3)P in cells and play an important role in cell morphology and membrane trafficking. However, little is known about the functional significance of Mtm activity for PI(3,5)P2. To address this, we attempted to generate Mtm mutants inhibited for PI(3,5)P2 but that remain selectively active for PI(3)P to assay their relative contributions to cell functions, as compared with catalytically inactive mutants. While biochemical assays of immunoprecipitated proteins verified the inactivity of the phosphatase ‘dead’ mutants, the hypothetical PI(3)P-selective mutants showed a 50% decrease in both PI(3)P and PI(3,5)P2 activity, indicating that the residues we mutated are important for the dephosphorylation of both PI(3)P and PI(3,5)P2 . Surprisingly, the phosphatase ‘dead’ mutants do not demonstrate a dominant negative phenotype in PI(3)P localization in Drosophila cell cultures. Moreover, a previously published PI(3)P-selective mutation (H337N) which did not demonstrate PIP selectivity in our biochemical assays displays a wild type Mtm phenotype in PI(3)P localization in cell cultures. This indicates that other determinants may affect Mtm activity in cells. We are currently performing functional studies on these same mutants in Drosophila immune cells to evaluate effects on Mtm-related roles in cell shape and endolysosome membrane flux.

POSTER # 11:

tbx20 as a downstream target of bone-morphogenetic protein (BMP) signaling in zebrafish heart development

Richard Shehane
Dr. Deborah Yelon

BMP signaling has been shown to influence heart development in zebrafish.  lost-a-fin (laf) zebrafish mutants lack the BMP receptor Alk8 and exhibit decreased atrial size.  The requirement of BMP signaling for regulating atrial size has also been shown to be time-sensitive.  Blockade of BMP signaling during early gastrulation using the drug dorsomorphin leads to a decrease in atrial size, while blockade of BMP signaling in later stages has no effect on atrial size.  However, the downstream targets of the BMP pathway that control atrial size remain largely unknown.  Previous studies in other model organisms such as mouse and Xenopus have shown strong evidence for the roles of the T-box genes in heart development.   In particular tbx20 may be a potential downstream target since tbx20 expression is reduced in laf mutants.  To test the hypothesis that tbx20 is a downstream target of BMP signaling in this context, I will block BMP activity using dorsomorphin during different points in zebrafish embryonic development.  I will then examine tbx20 gene expression via in-situ hybridization and immunohistochemistry and compare these findings to control embryos.

POSTER # 12:

The Role of TRP Channel Activaton in Gut Mucosal Inflammation and Tumorigenesis

Amy Triano
Dr. Eyal Raz

The primary focus of our research is to determine the role that Transient Receptor Potential (TRP) channels play in the coordination and control of intestinal inflammation and colitis-associated cancer. TRP channels are pain receptors (nociceptors) that can detect noxious stimuli of thermal, mechanical, and chemical origin. Present in the connective tissue of the gut are neurons containing TRP ion channels that detect inflammatory damage. These cation channels can be activated by food components, cytokines, lipid mediators, and H+ ions. The role of TRP activation on sensory nerves in the gastro-intestinal tissues is currently unknown. Our initial studies indicate that TRP signaling plays a significant role in maintaining gut homeostasis. The release of neuropeptides mediated by TRP signaling could thus potentially have an impact on inflammatory diseases of the gut. Inflammation of the gut mucosa is associated with an increased risk of tumor development and growth in the colon (colon carcinoma). Our research is especially concerned with the subfamily V1 (TRPV1) and the signaling pathway it mediates during conditions of inflammation and tumorigenesis. Our specific aim is the activation of TRP channels on regulation of the growth and malignancy of colorectal carcinomas induced by azoxymethane (AOM) and repeated administration of DSS (colitis-associated cancer.

POSTER # 13:

SON DNA-binding Protein Homologues is crucial in Zebrafish Embryo Development


Xu Yao
Dr. Dong-Er Zhang


In recent studies, SON, a large Ser/Arg (SR)-related protein, has been reported to be specifically required for cell cycle progression. Down-regulation of SON in human cells induced mitotic arrest due to severe defects in centrosome organization, microtubule dynamics, and chromosome segregation.  These results reveal SON as a key specificity mediator for coordinated regulation of the cell cycle machinery. SON is also conserved in human, chimpanzee, dog, mouse, rat, fruit fly, mosquito, and C.elegans. Therefore, we hypothesize that the knockdown of SON in vertebrate embryos will lead to large-scale developmental defects. Zebrafish was chosen to be our model organism in consideration of its rapid development. The LOC565999 gene in zebrafish is predicted to be similar to the SON DNA-binding protein isoform F in having the G-Patch and the DSRM. In this experiment, we confirmed the presence of the LOC565999 gene in zebrafish via PCR. Knock-down of the LOC565999 gene by morpholino resulted in significant growth retardation in 24hpf embryos. Observable phenotypic abnormalities include significantly smaller brains, slit-like and closely spaced eyes, shortened trunks, lack of yolk extension and bent tails. RT-PCR revealed the reduction in the quantity of LOC565999 mRNA in mutant embryos. These results confirmed the importance of SON DNA-binding protein in embryogenesis and opened up the possibilities of SON as a crucial protein for brain, neural, eye and muscle development. Our future study includes the identification of specific SON target genes vital to embryo development.


POSTER # 14:

Investigation of the intracellular trafficking of ENaC subunits in colonic cell lines and dysfunctions of intracellular trafficking in cells exposed to the Forskolin.


Angela Yu
Dr. Kim Barrett

Diarrheal diseases are the major cause of childhood and pregnant women hospitalization, mostly for dehydration. According to the Institute of One World Health, the pediatric death toll due to diarrheal diseases exceeds that of AIDS, tuberculosis and malaria combined. Diarrhea, which can occur from excessive fluid secretion, deficient fluid absorption or even a combination of both, is the major symptom of many chronic intestinal diseases, and it is in large part due to defective electrogenic sodium absorption in the colon. A dysfunction in the absorptive mechanism of water from the intestinal lumen can result in the symptoms of diarrhea by altering the Na+ gradient and eliminating the driving force of water absorption. The epithelial sodium channel (ENaC) is one of the channels responsible for the absorption of Na+ out of the lumen. In case of inflammatory bowel disease (IBD), enterocytes exposed to cytokines are thought to exhibit an altered intracellular trafficking of alpha-ENaC. This altered intracellular trafficking prevents movement to and insertion into the apical membrane. My aim is to investigate the intracellular trafficking of ENaC subunits (alpha-ENac, beta-ENac and gamma ENac) in colonic cell lines and dysfunctions of intracellular trafficking in cells exposed to the Forskolin.

POSTER # 15:

Bnip3 induces mitochondrial fragmentation via down regulation of mitochondrial fusion proteins

Rita Hanna
Dr. Asa Gustafsson

Bcl-2 family proteins are important regulators of mitochondrial integrity and apoptosis. Bnip3 is a pro-apoptotic BH3-only protein which mediates mitochondrial dysfunction and cell death in response to stress. In addition, Bnip3 is known to induce mitochondrial fragmentation (fission) and autophagy but the functional roles of these two processes are unknown. In this study, we have investigated the mechanisms by which Bnip3 mediates mitochondrial fragmentation. Western blot analysis of lysates prepared from isolated adult cardiac myocytes revealed that overexpression of Bnip3 caused down regulation of mitochondrial fusion proteins Mitofusion1 and Opa1. In contrast, Bnip3 induced upregulation of the mitochondrial fission protein Drp-1. Under normal conditions, there is a balance between fission and fusion of mitochondria but overexpression of Bnip3 shifts this balance toward fission which would promote mitochondrial fragmentation. Moreover, Parkin is a ubiquitin ligase that is selectively recruited to dysfunctional mitochondrial fragments which are then removed by autophagosomes. We found that Bnip3 induced translocation of Parkin to mitochondria in myocytes and Parkin positive mitochondria co-localized with autophagosomes.  The translocation of Parkin correlated with an increase in ubiquitinated mitochondrial proteins in cells overexpressing Bnip3.  Interestingly, Mitofusion was recently shown to be ubiquitinated by Parkin which led to its degradation. We plan to investigate whether Bnip3 induces degradation of Mitofusin1 via activation and recruitment of Parkin to mitochondria. We will also investigate whether Bnip3-mediated mitochondrial fragmentation is a protective or detrimental response in the cell.