Cell and Developmental Biology Commons^™

Nel Positively Regulates The Genesis Of Retinal Ganglion Cells By Promoting Their Differentiation And Survival During Development, Chizu Nakamoto, Soh-Leh Kuan, Amy Findlay, Elaine Durward, Zhufeng Ouyang, Masaru Nakamoto, Ewa Zakrewska, Takuma Endo

Biology Faculty Publications

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine …

Novel Neuroprotective Function Of Apical-Basal Polarity Genecrumbs In Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration, Andrew Steffensmeier, Meghana Tare, Oorvashi Roy Puli, Rohan Modi, Jaison Nainaparampil, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Alzheimer's disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophilaeye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, …

Recurrent Modification Of A Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity, William A. Rogers, Joseph R. Salomone, David J. Tacy, Eric M. Camino, Kristen A. Davis, Mark Rebeiz, Thomas M. Williams

Biology Faculty Publications

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. InDrosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development …

Germline Transgenic Methods For Tracking Cells And Testing Gene Function During Regeneration In The Axolotl, Shahryar Khattak, Maritta Schuez, Tobias Richter, Dunja Knapp, Saori L. Haigo, Tatiana Sandoval-Guzmán, Kristyna Hradlikova, Annett Duemmler, Ryan R. Kerney, Elly M. Tanaka

Biology Faculty Publications

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly …

Use Of Image Cytometry For Quantification Of Pathogenic Fungi In Association With Host Cells, Charlotte A. Berkes, Leo Li-Ying Chan, Alisha Wilkinson, Benjamin Paradis

Biology Faculty Publications

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast …

Species Boundaries And Biogeography Of East African Torrent Frogs Of The Genus Petropedetes (Amphibia: Anura: Petropeditidae), Simon P. Loader, F. Sara Ceccarelli, Mark Wilkinson, Michele Menegon, Jean Mariaux, Rafael O. De Sá, Et Al.

Biology Faculty Publications

Based on morphology there are currently three described East African species of torrent frogs belonging to the tropical African genus Petropedetes (P. dutoiti, P. martiennseni and P. yakusini). Taxonomic uncertainties are evident within this group: a putative new species reported from the Nguru Mountains and the species P. yakusini have a fragmented distribution across the Southern Tanzanian highlands. We conducted a molecular systematic study, sampling populations of East African petropedetids occurring in the mountains of Tanzania (P. martiennseni and P. yakusini), to investigate species boundaries and biogeography. Data provide evidence for the recognition of …

Unexpected Phylogenetic Positions Of The Genera Rupirana And Crossodactylodes Reveal Insights Into The Biogeography And Reproductive Evolution Of Leptodactylid Frogs, Antoine Foquet, Boris Leonardo Blotto, Maximiliano Manuel Maronna, Vanessa Kruth Verdade, Flora Acuña Juncá, Rafael O. De Sá, Miguel Trefaut Rodrigues

Biology Faculty Publications

Despite major progress in deciphering the amphibian tree of life by molecular phylogenetics, we identified two questions remaining to be answered regarding relationships within Hyloidea, the clade of South American origin that comprises most extant anuran diversity. A few genera like Rupirana and Crossodactylodes have enigmatic phylogenetic positions, and relationships among major lineages within some families like Leptodactylidae remain ambiguous. To resolve these specific questions we used two approaches (1) a complete matrix approach representing >6.6 kb, including most major Hyloidea lineages (61 terminals) combining different methods of phylogenetic reconstruction and measures of node support; and (2) a supermatrix approach …

Long-Term Data For Endemic Frog Genera Reveal Potential Conservation Crisis In The Bale Mountains, Ethiopia, David J. Gower, Roman K. Aberra, Silvia Schwaller, Malcolm J. Largen, Ben Collen, Stephen Spawls, Michele Menegon, Breda M. Zimkus, Rafael O. De Sá, Et Al.

Biology Faculty Publications

Populations of many frogs have declined alarmingly in recent years, placing nearly one third of the > 6,000 species under threat of extinction. Declines have been attributed largely to habitat loss, environmental degradation and/or infectious diseases such as chytridiomycosis. Many frogs undergo dramatic natural population fluctuations such that long-term data are required to determine population trends without undue influence of stochastic factors. We present long-term quantitative data (individuals encountered per person hour of searching) for four monotypic frog genera endemic to an Afromontane region of exceptional importance but growing conservation concern: one endemic to the Ethiopian highlands (Spinophrynoides osgoodi) and three …

Pushing Taxonomy To Extiction?, Alessandro Minelli, Annemarie Ohler, Erna Aescht, Aaron Bauer, Lucio Bonato, Roger Bour, Marcelo Rodrigues De Carvalho, Rafael O. De Sá, Et Al.

Biology Faculty Publications

Can we describe all species on Earth before they disappear? We argue that this is possible only by endowing taxonomy with professional manpower and appropriate material resources as required by big science.

Contrary to Costello et al.’s (CMS) (1) statements, taxonomy is not an easy discipline accessible to all through a smartphone. It requires exhaustive training and long familiarity with field, specimens and literature (2).

CMS’s argument is framed in terms of species numbers, but different, non-overlapping species concepts apply to bacteria, brambles and birds (3,4,5): “the species” as common unit of biodiversity does not exist (6).

Even ignoring this …

Scribble Acts In The Drosophila Fat-Hippo Pathway To Regulate Warts Activity, Shilpi Verghese, Indrayani Waghmare, Hailey Kwon, Katelin Hanes, Madhuri Kango-Singh

Biology Faculty Publications

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target …

Cartilage On The Move: Cartilage Lineage Tracing During Tadpole Metamorphosis, Ryan R. Kerney, Alison L. Brittain, Brian K. Hall, Daniel R. Buchholz

Biology Faculty Publications

The reorganization of cranial cartilages during tadpole metamorphosis is a set of complex processes. The fates of larval cartilage-forming cells (chondrocytes) and sources of adult chondrocytes are largely unknown. Individual larval cranial cartilages may either degenerate or remodel, while many adult cartilages appear to form de novo during metamorphosis. Determining the extent to which adult chondrocytes/cartilages are derived from larval chondrocytes during metamorphosis requires new techniques in chondrocyte lineage tracing. We have developed two transgenic systems to label cartilage cells throughout the body with fluorescent proteins. One system strongly labels early tadpole cartilages only. The other system inducibly labels forming …

Rapid Range Expansion In The Great Plains Narrow-Mouthed Toad (Gastrophryne Olivacea) And A Revised Taxonomy For North American Microhylids, Jeffrey W. Streicher, Christian L. Cox, Jonathan A. Campbell, Eric N. Smith, Rafael O. De Sá

Biology Faculty Publications

We investigated genetic variation within the Great Plains narrow-mouthed toad, Gastrophryne olivacea, across its geographic range in the United States and Mexico. An analysis of mitochondrial DNA (mtDNA) from 105 frogs revealed remarkably low levels of genetic diversity in individuals inhabiting the central United States and northern Mexico. We found that this widespread matrilineal lineage is divergent (ca. 2% in mtDNA) from haplotypes that originate from the western United States and western coast of Mexico. Using a dataset that included all five species of Gastrophryne and both species of the closely related genus Hypopachus, we investigated the phylogenetic …

Fluorescence-Based Reporter For Gauging Cyclic Di-Gmp Levels In Pseudomonas Aeruginosa, Morten T. Rybtke, Bradley R. Borlee, Keiji Murakami, Yasuhiko Irie, Morten Hentzer, Thomas E. Nielsen, Michael Givskov, Matthew R. Parsek, Tim Tolker-Nielsen

Biology Faculty Publications

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge …

Neurodegeneration - A Means To An End, Amit Singh

Biology Faculty Publications

Cell death, a global phenomenon found throughout the animal kingdom, is a mechanism to maintain tissue homeostasis and for adaptation to changes in the environment [1,2]. Millions of cells die in our body daily- they succumb to stress and commit suicide by a mechanism referred to as cell death or apoptosis [2-4]. Under normal conditions cells are continuously replaced by new cells from the stemor progenitor- cells. For example, an optimum balance in shedding of dead cells from the skin and their replenishment by new ones maintain our health and hygiene. In this context, apoptosis is a mechanism to eliminate …

Tumor Suppression By Cell Competition Through Regulation Of The Hippo Pathway, Chiao-Lin Chen, Molly C. Schroeder, Madhuri Kango-Singh, Chunyao Tao, Georg Halder

Biology Faculty Publications

Homeostatic mechanisms can eliminate abnormal cells to prevent diseases such as cancer. However, the underlying mechanisms of this surveillance are poorly understood. Here we investigated how clones of cells mutant for the neoplastic tumor suppressor gene scribble (scrib) are eliminated from Drosophila imaginal discs. When all cells in imaginal discs are mutant for scrib, they hyperactivate the Hippo pathway effector Yorkie (Yki), which drives growth of the discs into large neoplastic masses. Strikingly, when discs also contain normal cells, the scrib− cells do not overproliferate and eventually undergo apoptosis through JNK-dependent mechanisms. However, induction of apoptosis does …

Structure-Function Analysis Of Nel, A Thrombospondin-1-Like Glycoprotein Involved In Neural Development And Functions, Masaru Nakamoto

Biology Faculty Publications

Nel (neural epidermal growth factor (EGF)-like molecule) is a multimeric, multimodular extracellular glycoprotein with heparin-binding activity and structural similarities to thrombospondin-1. Nel is predominantly expressed in the nervous system and has been implicated in neuronal proliferation and differentiation, retinal axon guidance, synaptic functions, and spatial learning. The Nel protein contains an N-terminal thrombospondin-1 (TSP-N) domain, five cysteine-rich domains, and six EGF-like domains. However, little is known about the functions of specific domains of the Nel protein. In this study, we have performed structure-function analysis of Nel, by using a series of expression constructs for different regions of the Nel protein. …

Cytoplasmic Sequestration Of The Tumor Suppressor P53 By A Heat Shock Protein 70 Family Member, Mortalin, In Human Colorectal Adenocarcinoma Cell Lines, Erin E. Gestl, S. Anne Boettger

Biology Faculty Publications

No abstract provided.

Protein Trap Lines Of Drosophila To Demonstrate Spatio-Temporal Localization Of Proteins In An Undergraduate Lab, Oorvashi Roy Puli, Amit Singh

Biology Faculty Publications

The objective of this teaching note is to generate a laboratory exercise, which allows students to get a hands-on experience of a cell biology technique. The short duration of the laboratory classes is the biggest challenge with the development of a cell biology lab for an undergraduate curriculum. Therefore, it is necessary to design a laboratory exercise that enables the students to carry out cell biological assays in the desired time. This laboratory exercise focuses on tracking protein expression levels along a spatial (space) and temporal (time) axis in developing Drosophila melanogaster organ primordium. Here we use the protein trap …

Quantitative Comparison Of Cis-Regulatory Element (Cre) Activities In Transgenic Drosophila Melanogaster, William A. Rogers, Thomas M. Williams

Biology Faculty Publications

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism′s program for development¹, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution^2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs …

Molecular Systematics Of The Middle American Genus Hypopachus (Anura: Microhylidae), Eli Greenbaum, Eric N. Smith, Rafael O. De Sá

Biology Faculty Publications

We present the first phylogenetic study on the widespread Middle American microhylid frog genus Hypopachus. Partial sequences of mitochondrial (12S and 16S ribosomal RNA) and nuclear (rhodopsin) genes (1275 bp total) were analyzed from 43 samples of Hypopachus, three currently recognized species of Gastrophryne, and seven arthroleptid, brevicipitid and microhylid outgroup taxa. Maximum parsimony (PAUP), maximum likelihood (RAxML) and Bayesian inference (MrBayes) optimality criteria were used for phylogenetic analyses, and BEAST was used to estimate divergence dates of major clades. Population-level analyses were conducted with the programs NETWORK and Arlequin. Results confirm the placement of Hypopachus …

Laboratory Diagnosis Of Clostridium Difficile Infection: Can Molecular Amplification Methods Move Us Out Of Uncertainty?, Fred C. Tenover, Ellen Jo Baron, Lance R. Peterson, David Persing

Biology Faculty Publications

The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. …

Activation Of Jnk Signaling Mediates Amyloid-Ss- Dependent Cell Death, Meghana Tare, Rohan Modi, Jaison Nainaparampil, Oorvashi Roy Puli, Shimpi Bedi, Pedro Fernandez-Funez, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

Background: Alzheimer's disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimer's neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood.

Methodology/Principal Findings: We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that …

Origin Of Amphibian And Avian Chromosomes By Fission, Fusion, And Retention Of Ancestral Chromosomes, Stephen R. Voss, D. Kevin Kump, Srikrishna Putta, Nathan Pauly, Anna Reynolds, Rema J. Henry, Saritha Basa, John A. Walker, Jeramiah J. Smith

Biology Faculty Publications

Amphibian genomes differ greatly in DNA content and chromosome size, morphology, and number. Investigations of this diversity are needed to identify mechanisms that have shaped the evolution of vertebrate genomes. We used comparative mapping to investigate the organization of genes in the Mexican axolotl (Ambystoma mexicanum), a species that presents relatively few chromosomes (n = 14) and a gigantic genome (>20 pg/N). We show extensive conservation of synteny between Ambystoma, chicken, and human, and a positive correlation between the length of conserved segments and genome size. Ambystoma segments are estimated to be four to 51 times longer than homologous …

Mechanical Test Methods For Assessing Porcine Carotid And Uterine Artery Burst Pressure Following Ex Vivo Ultrasonic Ligature Seal And Transection, Carissa M. Krane, Margaret Pinnell, Courtney Gardner, Mercedes Thompson, James Coleman, Robert Wilkens

Biology Faculty Publications

A test method was developed to identify those variables important for assessing the performance of ultrasonic surgical devices in ex vivo ligature sealing of porcine carotid and uterine arteries. Ruggedness testing using a small sample size in pilot experiments was conducted using a newly developed test method in an effort to assess the usefulness of this methodology and to identify test variables that might warrant further testing. The development of this test method included the use of a custom-designed prototypic tension device for load-controlled ex vivo vessel stretching during saline perfusion and subsequent seal and transection of porcine arteries with …

Design Of A Factorial Experiment With Randomization Restrictions To Assess Medical Device Performance On Vascular Tissue, Wiebke Diestelkamp, Carissa M. Krane, Margaret Pinnell

Biology Faculty Publications

Background: Energy-based surgical scalpels are designed to efficiently transect and seal blood vessels using thermal energy to promote protein denaturation and coagulation. Assessment and design improvement of ultrasonic scalpel performance relies on both in vivo and ex vivo testing. The objective of this work was to design and implement a robust, experimental test matrix with randomization restrictions and predictive statistical power, which allowed for identification of those experimental variables that may affect the quality of the seal obtained ex vivo.

Methods: The design of the experiment included three factors: temperature (two levels); the type of solution used to perfuse the …

Endo-Porter–Mediated Delivery Of Phosphorodiamidate Morpholino Oligos (Pmos) In Erythrocyte Suspension Cultures From Cope's Gray Treefrog Hyla Chrysoscelis, Venkateshwar Mutyam, Matthew V. Puccetti, James Frisbie, David L. Goldstein, Carissa M. Krane

Biology Faculty Publications

Cope's gray treefrog, Hyla chrysoscelis, is a freeze-tolerant anuran that accumulates cryoprotective glycerol during cold acclimation. H. chrysoscelis erythrocytes express the aquaglyceroporin HC-3, which facilitates transmembrane glycerol and water movement. Aquaglyceroporins have no pharmacological inhibitors, and no genetic knockout tools currently exist for H. chrysoscelis. A phosphorodiamidate morpholino oligo (PMO)–mediated expression knockdown approach was therefore pursued to provide a model for testing the role of HC-3. We describe a novel procedure optimized for specific, efficient knockdown of HC-3 expression in amphibian erythrocyte suspensions cultured at nonmammalian physiological temperatures using Endo-Porter. Our protocol includes three critical components: pre-incubation at 37°C, …

Dynamic Glucoregulation And Mammalian-Like Responses To Metabolic And Developmental Disruption In Zebrafish, Agata Jurczyk, Nicole M. Roy, Rabia Bajwa, Philipp Gut, Kathryn Lipson, Chaoxing Yang, Laurence Covassin, Waldemar J. Racki, Aldo A. Rossini, Nancy Phillips, Didier Y. R. Stainier, Dale L. Greiner, Michael A. Brehm, Rita Bortell, Philip Diiorio

Biology Faculty Publications

Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is …

Variation, Systematics, And Relationships Of The Leptodactylus Bolivianus Complex (Amphibia: Anura: Leptodactylidae), W. Ronald Heyer, Rafael O. De Sá

Biology Faculty Publications

Heyer, W. Ronald, and Rafael O. de Sá. Variation, Systematics, and Relationships of the Leptodactylus bolivianus Complex (Amphibia: Anura: Leptodactylidae). Smithsonian Contributions to Zoology, number 635, viii + 58 pages, 21 figures, 20 tables, 2011.—The Leptodactylus bolivianuscomplex has been considered to consist of one or two species, L. bolivianus alone or L. bolivianus and L. insularum. Detailed morphological analyses were undertaken to evaluate variation in the complex, which ranges from Costa Rica through Panama, across northern South America in the river valleys draining to the Caribbean, and throughout much of the Amazon basin with southern limits in Bolivia. Members of …

Description Of The Tadpole Of Proceratophrys Renalis (Miranda-Ribeiro, 1920) (Anura: Cycloramphidae), Filipe Augusto C. Do Nascimento, Barnagleison S. Lisboa, Gabriel O. Skuk, Rafael O. De Sá

Biology Faculty Publications

The tadpole of Proceratophrys renalis is described based on specimens from Maceió, State of Alagoas, northeastern Brazil. At stage 35 the body is slightly dorso-ventrally depressed, ovoid in lateral, dorsal, and ventral views. Oral disc is ventral with lateral emarginations, surrounded by a single row of marginal papillae with a large gap on the upper labium. Labial tooth row formula is 2(2)/3(1). The analysis of internal oral anatomy revealed two possible characters that readily distinguish P. renalis from P. boiei, supporting the recent ressurection of P. renalis. Comparisons with available descriptions of the larvae for other species in the …

Methods For Karyotyping And For Localization Of Developmentally Relevant Genes On The Chromosomes Of The Purple Sea Urchin Strongylocentrotus Purpuratus, Celeste C. Eno, Stefanie A. Boettger, Charles W. Walker

Biology Faculty Publications

No abstract provided.