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- Creators: Kusumi, Kenro
- Creators: Harrington Bioengineering Program
Description
Well-established model systems exist in four out of the seven major classes of vertebrates. These include the mouse, chicken, frog and zebrafish. Noticeably missing from this list is a reptilian model organism for comparative studies between the vertebrates and for studies of biological processes unique to reptiles. To help fill in this gap the green anole lizard, Anolis carolinensis, is being adapted as a model organism. Despite the recent release of the complete genomic sequence of the A. carolinensis, the lizard lacks some resources to aid researchers in their studies. Particularly, the lack of transcriptomic resources for lizard has made it difficult to identify genes complete with alternative splice forms and untranslated regions (UTRs). As part of this work the genome annotation for A. carolinensis was improved through next generation sequencing and assembly of the transcriptomes from 14 different adult and embryonic tissues. This revised annotation of the lizard will improve comparative studies between vertebrates, as well as studies within A. carolinensis itself, by providing more accurate gene models, which provide the bases for molecular studies. To demonstrate the utility of the improved annotations and reptilian model organism, the developmental process of somitogenesis in the lizard was analyzed and compared with other vertebrates. This study identified several key features both divergent and convergent between the vertebrates, which was not previously known before analysis of a reptilian model organism. The improved genome annotations have also allowed for molecular studies of tail regeneration in the lizard. With the annotation of 3' UTR sequences and next generation sequencing, it is now possible to do expressional studies of miRNA and predict their mRNA target transcripts at genomic scale. Through next generation small RNA sequencing and subsequent analysis, several differentially expressed miRNAs were identified in the regenerating tail, suggesting miRNA may play a key role in regulating this process in lizards. Through miRNA target prediction several key biological pathways were identified as potentially under the regulation of miRNAs during tail regeneration. In total, this work has both helped advance A. carolinensis as model system and displayed the utility of a reptilian model system.
ContributorsEckalbar, Walter L (Author) / Kusumi, Kenro (Thesis advisor) / Huentelman, Matthew (Committee member) / Rawls, Jeffery (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2012
Description
While a number of vertebrates, including fishes, salamanders, frogs, and lizards, display regenerative capacity, the process is not necessarily the same. It has been proposed that regeneration, while evolutionarily conserved, has diverged during evolution. However, the extent to which the mechanisms of regeneration have changed between taxa still remains elusive. In the salamander limb, cells dedifferentiate to a more plastic state and aggregate in the distal portion of the appendage to form a blastema, which is responsible for outgrowth and tissue development. In contrast, no such mechanism has been identified in lizards, and it is unclear to what extent evolutionary divergence between amniotes and anamniotes has altered this mechanism. Anolis carolinensis lizards are capable of regenerating their tails after stress-induced autotomy or self-amputation. In this investigation, the distribution of proliferating cells in early A. carolinensis tail regeneration was visualized by immunohistochemistry to examine the location and quantity of proliferating cells. An aggregate of proliferating cells at the distal region of the regenerate is considered indicative of blastema formation. Proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2) were utilized as proliferation markers. Positive cells were counted for each tail (n=9, n=8 respectively). The percent of proliferating cells at the tip and base of the regenerating tail were compared with a one-way ANOVA statistical test. Both markers showed no significant difference (P=0.585, P=0.603 respectively) indicating absence of a blastema-like structure. These results suggest an alternative mechanism of regeneration in lizards and potentially other amniotes.
ContributorsTokuyama, Minami Adrianne (Author) / Kusumi, Kenro (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / Menke, Douglas (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
The development of skeletal muscle during embryogenesis and repair in adults is dependent on the intricate balance between the proliferation of myogenic progenitor cells and the differentiation of those cells into functional muscle fibers. Recent studies demonstrate that the Drosophila melanogaster transcription factor CG9650 is expressed in muscle progenitor cells, where it maintains myoblast numbers. We are interested in the Mus musculus orthologs Bcl11a and Bcl11b (C2H2 zinc finger transcription factors), and understanding their role as molecular switches that control proliferation/differentiation decisions in muscle progenitor cells. Expression analysis revealed that Bcl11b, but not Bcl11a, is expressed in the region of the mouse embryo populated with myogenic progenitor cells; gene expression studies in muscle cell culture confirmed Bcl11b is also selectively transcribed in muscle. Furthermore, Bcl11b is down-regulated with differentiation, which is consistent with the belief that the gene plays a role in cell proliferation.
ContributorsDuong, Brittany Bach (Author) / Rawls, Alan (Thesis director) / Wilson-Rawls, Jeanne (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
A major goal of the Center for Biosignatures Discovery Automation (CBDA) is to design a diagnostic tool that detects novel cancer biosignatures at the single-cell level. We designed the Single-cell QUantitative In situ Reverse Transcription-Polymerase Chain Reaction (SQUIRT-PCR) system by combining a two-photon laser lysis (2PLL) system with a microfluidic reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) platform. It is important to identify early molecular changes from intact tissues as prognosis for premalignant conditions and develop new molecular targets for prevention of cancer progression and improved therapies. This project analyzes RNA expression at the single-cell level and presents itself with two major challenges: (1) detecting low levels of RNA and (2) minimizing RNA absorption in the polydimethylsiloxane (PDMS) microfluidic channel. The first challenge was overcome by successfully detecting picogram (pg) levels of RNA using the Fluidigm (FD) BioMark™ HD System (Fluidigm Corporation, South San Francisco, CA) for RT-qPCR analysis. This technology incorporates a highly precise integrated fluidic circuit (IFC) that allows for high-throughput genetic screening using microarrays. The second challenge entailed collecting data from RNA flow-through samples that were passed through microfluidic channels. One channel was treated with a coating of polyethylene glycol (PEG) and the other remained untreated. Various flow-through samples were subjected to RT-qPCR and analyzed using the FD FLEXsix™ Gene Expression IFC. As predicted, the results showed that the treated PDMS channel absorbed less RNA than the untreated PDMS channel. Once the optimization of the PDMS microfluidic platform is complete, it will be implemented into the 2PLL system. This novel technology will be able to identify cell populations in situ and could have a large impact on cancer diagnostics.
ContributorsBlatt, Amy Elissa (Author) / Meldrum, Deirdre R. (Thesis director) / Tran, Thai (Committee member) / Chao, Joseph (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05
Description
Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related organism to humans that can regenerate de novo skeletal muscle, hyaline cartilage, spinal cord, vasculature, and skin. Progress in studying the cellular and molecular mechanisms of lizard regeneration has previously been limited by a lack of genomic resources. Building on the release of the genome of the green anole, Anolis carolinensis, we developed a second generation, robust RNA-Seq-based genome annotation, and performed the first transcriptomic analysis of tail regeneration in this species. In order to investigate gene expression in regenerating tissue, we performed whole transcriptome and microRNA transcriptome analysis of regenerating tail tip and base and associated tissues, identifying key genetic targets in the regenerative process. These studies have identified components of a genetic program for regeneration in the lizard that includes both developmental and adult repair mechanisms shared with mammals, indicating value in the translation of these findings to future regenerative therapies.
ContributorsHutchins, Elizabeth (Author) / Kusumi, Kenro (Thesis advisor) / Rawls, Jeffrey A. (Committee member) / Denardo, Dale F. (Committee member) / Huentelman, Matthew J. (Committee member) / Arizona State University (Publisher)
Created2015
Description
Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.
Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees, .vcf and GO enrichment files are available on Harvard Dataverse (doi:10.7910/DVN/EH2S9K).
ContributorsTollis, Marc (Author) / DeNardo, Dale F (Author) / Cornelius, John A (Author) / Dolby, Greer A (Author) / Edwards, Taylor (Author) / Henen, Brian T. (Author) / Karl, Alice E. (Author) / Murphy, Robert W. (Author) / Kusumi, Kenro (Author)
Created2017-05-31
Description
Rapid advancements in genomic technologies have increased our understanding of rare human disease. Generation of multiple types of biological data including genetic variation from genome or exome, expression from transcriptome, methylation patterns from epigenome, protein complexity from proteome and metabolite information from metabolome is feasible. "Omics" tools provide comprehensive view into biological mechanisms that impact disease trait and risk. In spite of available data types and ability to collect them simultaneously from patients, researchers still rely on their independent analysis. Combining information from multiple biological data can reduce missing information, increase confidence in single data findings, and provide a more complete view of genotype-phenotype correlations. Although rare disease genetics has been greatly improved by exome sequencing, a substantial portion of clinical patients remain undiagnosed. Multiple frameworks for integrative analysis of genomic and transcriptomic data are presented with focus on identifying functional genetic variations in patients with undiagnosed, rare childhood conditions. Direct quantitation of X inactivation ratio was developed from genomic and transcriptomic data using allele specific expression and segregation analysis to determine magnitude and inheritance mode of X inactivation. This approach was applied in two families revealing non-random X inactivation in female patients. Expression based analysis of X inactivation showed high correlation with standard clinical assay. These findings improved understanding of molecular mechanisms underlying X-linked disorders. In addition multivariate outlier analysis of gene and exon level data from RNA-seq using Mahalanobis distance, and its integration of distance scores with genomic data found genotype-phenotype correlations in variant prioritization process in 25 families. Mahalanobis distance scores revealed variants with large transcriptional impact in patients. In this dataset, frameshift variants were more likely result in outlier expression signatures than other types of functional variants. Integration of outlier estimates with genetic variants corroborated previously identified, presumed causal variants and highlighted new candidate in previously un-diagnosed case. Integrative genomic approaches in easily attainable tissue will facilitate the search for biomarkers that impact disease trait, uncover pharmacogenomics targets, provide novel insight into molecular underpinnings of un-characterized conditions, and help improve analytical approaches that use large datasets.
ContributorsSzelinger, Szabolcs (Author) / Craig, David W. (Thesis advisor) / Kusumi, Kenro (Thesis advisor) / Narayan, Vinodh (Committee member) / Rosenberg, Michael S. (Committee member) / Huentelman, Matthew J (Committee member) / Arizona State University (Publisher)
Created2015
Description
In the U.S., breast cancer (BC) incidences among African American (AA) and CA (CA) women are similar, yet AA women have a significantly higher mortality rate. In addition, AA women often present with tumors at a younger age, with a higher tumor grade/stage and are more likely to be diagnosed with the highly aggressive triple-negative breast cancer (TNBC) subtype. Even within the TNBC subtype, AA women have a worse clinical outcome compared to CA. Although multiple socio-economic and lifestyle factors may contribute to these observed health disparities, it is essential that the underlying biological differences between CA and AA TNBC are identified. In this study, gene expression profiling was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage TNBC. Initial analysis revealed a pattern of differential expression in the AA cohort compared to CA. Further molecular characterization results showed that the AA cohort segregated into 3-TNBC molecular subtypes; Basal-like (BL2), Immunomodulatory (IM) and Mesenchymal (M). Gene expression analyses resulted in 190 differentially expressed genes between the AA and CA cohorts. Pathway enrichment analysis demonstrated that differentially expressed genes were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC -cohort. Furthermore, genes in the Wnt/β-catenin pathway were over-expressed. These results were validated using RT-qPCR on an independent cohort of FFPE samples from AA and CA women with early stage TNBC, and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort. Furthermore, CAV1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, silencing of CAV1 expression by siRNA resulted in a significant decrease in proliferation in each of the TNBC cell lines. These observations suggest that CAV1 expression may contribute to the more aggressive phenotype observed in AA women diagnosed with TNBC.
ContributorsGetz, Julie (Author) / Baumbach-Reardon, Lisa L (Thesis advisor) / Lake, Douglas F (Thesis advisor) / Bussey, Kimberly (Committee member) / Kusumi, Kenro (Committee member) / Arizona State University (Publisher)
Created2015
Description
Traumatic injury to the central nervous or musculoskeletal system in traditional amniote models, such as mouse and chicken, is permanent with long-term physiological and functional effects. However, among amniotes, the ability to regrow complex, multi-tissue structures is unique to non-avian reptiles. Structural regeneration is extensively studied in lizards, with most species able to regrow a functional tail. The lizard regenerated tail includes the spinal cord, cartilage, de novo muscle, vasculature, and skin, and unlike mammals, these tissues can be replaced in lizards as adults. These studies focus on the events that occur before and after the tail regrowth phase, identifying conserved mechanisms that enable functional tail regeneration in the green anole lizard, Anolis carolinensis. An examination of coordinated interactions between peripheral nerves, Schwann cells, and skeletal muscle reveal that reformation of the lizard neuromuscular system is dependent upon developmental programs as well as those unique to the adult during late stages of regeneration. On the other hand, transcriptomic analysis of the early injury response identified many immunoregulatory genes that may be essential for inhibiting fibrosis and initiating regenerative programs. Lastly, an anatomical and histological study of regrown alligator tails reveal that regenerative capacity varies between different reptile groups, providing comparative opportunities within amniotes and across vertebrates. In order to identify mechanisms that limit regeneration, these cross-species analyses will be critical. Taken together, these studies serve as a foundation for future experimental work that will reveal the interplay between reparative and regenerative mechanisms in adult amniotes with translational implications for medical therapies.
ContributorsXu, Cindy (Author) / Kusumi, Kenro (Thesis advisor) / Newbern, Jason M (Thesis advisor) / Wilson-Rawls, Jeanne (Committee member) / Fisher, Rebecca E (Committee member) / Arizona State University (Publisher)
Created2020