Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item GENETIC AND MOLECULAR ANALYSIS OF GERMLINE SEX DETERMINATION IN CAENORHABDITIS BRIGGSAE, A MODEL FOR THE CONVERGENT EVOLUTION OF HERMAPHRODITISM(2010) Doty, Alana V.; Haag, Eric S.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Though sex determination and differentiation are critical biological processes, genetic mechanisms that specify sex have undergone profound and rapid evolutionary change across taxa. We may be able to infer processes that generate sex determination diversity by examining closely related species. Within the nematode genus Caenorhabditis, two species, C. elegans and C. briggsae, are androdioecious, producing self-fertile hermaphrodites and males; other Caenorhabditis species generate males/females. Interestingly, phylogenies reveal that C. elegans and C. briggsae independently acquired hermaphroditism, a relatively rare adaptation among animals. In this work, I describe differences in germline sex determination between C. elegans and C. briggsae that may help reveal the molecular basis of their convergent evolution of hermaphroditism. I first describe mutations in the pleiotropic, STAR family RNA-binding protein Cbr-GLD-1 that affect germline sex in C. briggsae. I find that C. briggsae gld-1 mutant hermaphrodites have a sex determination phenotype opposite to that of C. elegans: masculinized versus feminized germlines. I demonstrate that Cbr-GLD-1 coding-plus-regulatory sequences can rescue Ce-gld-1 null animals, arguing that this change in sex determination is not due to changes in GLD-1 function or expression. I further show that gld-1's role in regulating oogenesis is conserved across the Elegans group of Caenorhabditis, demonstrating that the oogenesis function of gld-1 is likely ancient, whereas its sperm-repressing role in C. briggsae has evolved recently. To identify mRNA targets of Cbr-GLD-1 that might be responsible for its sex determination function in C. briggsae, I use an in vivo genome-wide approach to isolate mRNAs associated with Cbr-GLD-1, including potential sex determination targets. I identify 800 putative mRNA targets and confirm specificity of this gene set via qRT-PCR and RNAi. Next, to reveal the roles of GLD-1 in evolutionary context, I create a phylogeny of STAR proteins across metazoans. Finally, I characterize a single feminizing allele recovered through forward screens in C. briggsae for germline sex determination mutants. This work thus begins to dissect the molecular and genetic basis of hermaphroditism in C. briggsae and contributes to a growing body of research on the evolution of germline sex determination differences between C. elegans and C. briggsae.Item Myosins IIIa and IIIb regulate stereocilia length by transporting espin 1 to the polymerizing end of actin filaments(2009) Merritt, Raymond Clyde; Kachar, Bechara; Popper, Arthur N; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mutations in the myosin IIIa gene are linked to DFNB30 late onset deafness, in which those affected individuals can hear for the first twenty years of life. Mutations in the espin gene are linked to DFNB36 congenital deafness. Both myosin IIIa and espin 1 are expressed in the inner ear hair cells and colocalize at stereocilia tips, the site of actin polymerization. Overexpression of these proteins in hair cells produce an increase in stereocilia length, and when both are co-expressed together they produce a length increase greater than when each one are overexpressed individually. These results suggest that these proteins interact and influence stereocilia length regulation. We confirmed the interaction of these two proteins in heterologous COS-7 cells. We observed that when co-expressed, these proteins promote elongation of filopodial actin protrusions in a synergistic manner. Mutational analyses show that myosin IIIa3THDI binds to the ankyrin repeats of espin 1. Live and fixed cell imaging shows that myosin IIIa transports espin 1 to the filopodia tips where espin 1 promotes actin polymerizations through its WH2 domain. Because of the late onset of deafness associated with myosin IIIa, it has been speculated that the lack of myosin IIIa function is partially compensated by the paralogous protein, myosin IIIb. Myosin IIIb, encoded by a distinct gene, lacks a C-terminal actin-binding domain shown to be essential for myosin IIIa filopodia tip localization. We observed that myosin IIIb localizes at stereocilia tips and is expressed at an earlier stage than myosin IIIa. We confirmed that myosin IIIb transports espin 1 to stereocilia tips and promotes actin polymerization, consistent with the hypothesis that it partially compensates for myosin IIIa. We found that binding to espin 1 is required for myosin IIIb movement and localization. We observed that myosin IIIb can downregulate the myosin IIIa localization in vestibular, but not in cochlear, hair cells. The interplay of myosins IIIa, IIIb, and espin 1 and their influence on stereocilia length unravels a novel molecular complex at the polymerizing end of F-actin and a framework to understand the cause of DFNB30 and DFNB36 deafnesses.Item Behavioral and Neuroendocrine Correlates of Sex Change in the Gilthead Seabream (Sparus aurata)(2009) Reyes-Tomassini, Jose J.; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sequential hermaphroditism is the most radical form of environmental sex determination observed in fish: functional adult males or females retain their ability to change sex even as adults. Among the factors that affect sex change in these species, the least understood is the social environment. Here, I studied the influences of social context on sex change in the Gilthead Seabream, Sparus aurata, by using the individual‟s dominance rank as an indicator of social status. To understand the role that the brain might play in sex change, I also studied the two main neuroendocrine factors that serve as the sexually differentiated axes of neural plasticity in most teleost species: AVT and GnRH. To do this, I first developed a set of tools designed to address the challenges associated with observing the behavior of aquacultured species. Using these tools, I provide the first in-depth study of seabream captive behavior, including the results of size-matched and sex-matched paired encounters. I found that females are more aggressive than males, but this difference is influenced by gonadal developmental status. I also showed that small but young males are more aggressive than bigger but older females. I cloned the AVT mRNA in seabream, and validated a quantitative assay to measure total brain AVT levels together with GnRH-1, GnRH-2, and GnRH-3 levels. I found that AVT and GnRH-3 levels rise during the onset of the hypothesized sex-change window, and drop to pre-quiescent levels until spawning, when all of these factors seem to increase their expression levels again. I also show for the first time, that GnRH-2 and dominance rank are strongly correlated in seabream during the spawning season but not during quiescence. GnRH-1 was strongly correlated to rank during quiescence but not during spawning. Finally, neither dominance rank nor size were a good predictor of the outcome of sex change, which seems to contradict what has been documented in sequential hermaphrodite reef fishes. I provide a model that accounts for this apparent contradiction and conclude that the Gilthead seabream remains true to the size-advantage hypothesis of sex allocation theory, if size and dominance are seen as proximate, rather than ultimate, factors.Item USE OF MOLECULAR TECHNIQUES TO ADDRESS THE EVOLUTION OF DISPLAY TRAITS IN THE PTILONORHYNCHIDAE AND OTHER PASSERIFORM SPECIES(2009) Zwiers, Paul; Borgia, Gerald; Fleischer, Robert; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tests of hypotheses addressing the evolution of complex traits have greatly benefited from advances in the field of molecular genetics. Current molecular techniques allow for the identification of genetic variability, useful in estimating genetic relatedness and potentially explaining phenotypic variation. Here I use molecular data to address the evolution of complex traits within the Ptilonorhynchidae and other Passeriformes. My estimation of a bowerbird molecular phylogeny suggests two highly unlikely scenarios of complex trait evolution (i.e. polygyny, bower construction, decoration use, etc.); either polygyny and complex display traits evolved in parallel, or monogamy evolved from non-resource based polygyny, a transition for which no unambiguous examples could be found, and complex traits evolved once and were lost. Molecular evidence also supports the existence of four Sericulus species, dating the radiation to coincide with the upheaval of the central New Guinea mountain range, and suggests plumage coloration may be a labile trait within this group and therefore a poor indicator of species relatedness. Use of ultraviolet (UV) signals in birds is hypothesized to associate with the ability to see UV wavelengths, a trait with a well-documented genetic basis (replacements at key amino acid positions in the short-wavelength-sensitive 1 (SWS1) opsin pigment influence pigment sensitivity and potentially color discrimination). UV signal use may alternatively evolve to match the local light environment. Results from bowerbirds suggest extreme differences in UV reflectance are due to light availability and not differences in UV vision because amino acid sequences in the bowerbirds were nearly identical. Expanding upon this study, I compared SWS1 opsin gene sequences from 134 passeriform species and plumage UV reflectance measurements from 91 of these species. Results from the molecular data are unprecedented; replacements at five amino acid positions are predicted to have occurred nearly simultaneously, suggesting a constraint on UV vision evolution. Additionally, species reflect most intensely in wavelengths to which they are predicted to be sensitive. These results suggest a constraint on UV vision may also constrain the evolution of UV signals in the Passeriformes. These studies highlight the usefulness of molecular data when testing hypothesis of species and trait evolution.Item Reproductive Physiology of the Female Blue Crab, Callinectes sapidus: Spawning Induction and Vitellogenesis(2009) Bembe, Sarah Elizabeth; Chung, J. Sook; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In aquaculture, spawning is the baseline for production; therefore, the optimization of spawning conditions will directly increase production. The current study aims to optimize spawning conditions for Callinectes sapidus using environmental manipulations of photoperiod and temperature for induction while monitoring the physiological vitellogenin (VtG) levels during ovarian development and maturation. The photothermal manipulations for this study resulted in increased spawning events in 21°C temperatures (compared to 11°C and 15°C) and complete darkness (0L:24D; compared to 8L:16D, 16L:8D, and 24L:0D) while 24L:0D and 11°C suppressed spawning. When assessing the VtG levels in the hemolymph prior to, during, and after all spawning events, the VtG showed a decrease prior to spawning, and significant VtG activity was seen in 21°C for all photoperiods. Overall, spawning and vitellogenesis are temperature dependent events with 67% of the females spawning in 21°C. Photoperiod also has an effect on spawning, but not on vitellogenesis.Item Archaeal Transcriptional Regulation of Catabolic Carbon Monoxide Dehydrogenase in Methanosarcina species(2009) Anderson, Kimberly Lynn; Sowers, Kevin R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In Archaea, the basal transcription machinery is eukaryotic-like, but some components, such as activator and repressor proteins, are bacteria-like. To further gain knowledge into cellular processes of Archaea, the genome of Methanosarcina thermophila was searched for helicase genes. A homolog of yeast RAD25, a gene with helicase and nucleotide excision repair (NER) abilities, was isolated. M. thermophila rad25 has the domains for helicase activity, but the C-terminal end is truncated, indicating that this protein mostly likely does not function in NER. After overexpression, helicase activity assays of Rad25 indicated that it might have helicase activity; however, there appeared to be contaminating proteins in the purification, so it was not possible to assign the activity only to Rad25. Additional work is necessary to characterize this protein. To investigate transcription, catabolic gene regulation was studied, specifically regulation of carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) from Methanosarcina species. The regions upstream of the transcriptional start site, as well as the 5' leader region of cdhA, were investigated for trans factors and cis elements that might be involved in regulation. Experiments revealed that regulation of cdhABCDE does not appear to involve trans factors upstream of the transcriptional start site. However, deletion analysis indicated that the 5' leader region does have a role in regulation. Comparing the protein levels to the mRNA levels revealed there was no significant difference between the two, indicating that translational regulation was not a factor. Other experiments ruled out differential mRNA stability as a factor in regulation. A region located between +358 and +405 was important in transcriptional regulation, indicating that regulation occurred at the level of transcription elongation. A model for regulation of catabolic CODH/ACS by differential elongation is proposed. Although 5' leader regions identified for other archaeal genes have been postulated to be involved in regulation, this was the first study to demonstrate a regulatory role by an archaeal leader sequence for differential elongation. Identifying regulatory mechanism(s) of catabolic genes such as CODH/ACS is critical for understanding the regulatory strategies employed by the methanoarchaea to efficiently direct carbon and electron flow during biomass conversion to methane.Item Differential requirements of the hindbrain and mesenchyme on inner ear patterning(2009) Liang, Jennifer Kimiko; Lee, Hey-Kyoung; Wu, Doris K; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microsurgical manipulations were performed in ovo to identify the tissues that are required for conferring inner ear patterning. Our results show that the hindbrain, namely rhombomeres 5 and 6, are required for the formation and patterning of the cochlear duct (basilar papilla). Rhombomere 5 and its underlying notochord appear to be important for the growth of the cochlear duct, whereas rhomobomere 6 and its respective notochord are required for cochlear patterning. Rotating the segment of hindbrain from rhombomere 5 to rhombomere 6 along the anteroposterior axis affects cochlear duct formation but has no effect on the development of vestibular structures. The signaling molecules intrinsic to these tissues are distinct from Sonic Hedgehog, which has been shown to be required for cochlear duct outgrowth. In contrast, otic mesenchyme adjacent to the developing inner ear provides anteroposterior axial information to pattern the anterior and posterior canals and ampullae.Item The Gonadotropin Releasing Hormone-3 System in Zebrafish: Early Development and Regulation(2008-12-15) Abraham, Eytan; Zohar, Yonathan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective of this study was to expand our understanding of the early development of forebrain Gonadotropin Releasing Hormone (GnRH) neurons in vertebrates in general and in fish in particular. The correct migration during early development of the hypophysiotropic GnRH neurons from the olfactory region to the hypothalamus is crucial for normal gonadal development and reproduction. We developed a Tg(GnRH3:EGFP) zebrafish line in which EGFP is specifically expressed in GnRH3 neurons. Using this line, we have studied in detail the early spatiotemporal development of the GnRH3 system in vivo. In addition, we have studied various factors, including GnRH3, Netrins and Hedgehog to better understand some of the mechanisms that mediate this complex axophilic neuron migration event. Lastly, we have conducted targeted GnRH3 neuron ablation experiments in view of determining the embryonic origin of POA-hypothalamic GnRH3 neurons and the effect of lack of GnRH3 neurons in the CNS. Our findings show that: 1) GnRH neurons first differentiate and express GnRH3 at 24-26 hours post fertilization (hpf) and immediately thereafter begin to extend fibers. 2) GnRH3 neurons project a complex network of fibers, prior the GnRH3 soma migration, to various CNS regions, and to the pituitary. 3) GnRH3 soma begin migrating towards the hypothalamus at 3 days post fertilization (dpf), passing through the terminal nerve (TN), lateral telencephalon, and reaching the hypothalamus by 12 dpf. 4) expression of GnRH3 itself is necessary for the normal early differentiation and fiber extensions of GnRH3 neurons. 5) Netrin1a is directly involved as a chemoattractant in GnRH3 fiber organization and subsequently, in GnRH3 soma migration to the hypothalamus. 6). Netrin2 is required for normal early ZF embryogenesis. 7). Sonic hedgehog a does not serve as a specific factor in the development of the GnRH3 system. 8). GnRH3 neuron regeneration capacity is temporally limited. 9). Successful ablation of olfactory GnRH3 neurons during development results in lack of GnRH3 neurons in the entire sexually mature brain as well as abnormal gonadal development and inability to reproduce. This study expands our understanding vis-à-vis the early events that occur during GnRH3 system development and that regulate this complex process. In a broader sense these findings augment current knowledge regarding the regulation of long range tangential neuron migration during development.Item Chitinase-An Evolutionary Duality(2008-08-29) Tang, Qiong; Place, Allen R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The purpose of this thesis was to characterize the molecular properties, gene copy number and gene organization for the chitinase enzyme in a lower vertebrate. Rainbow trout (Oncorhynchus mykiss) has one of the highest chitinase activities in all the fishes examined. I also aimed to clarify the evolutionary origin and associated regulatory control of the chitinase gene in this species. The cDNAs from two chitinases (Onmy-Chit.01 and Onmy-Chit.02) were successfully cloned and characterized from rainbow trout tissues. Onmy-Chit.01 is predominately expressed in the stomach, with high mRNA expression in the gastric gland. Its protein secretes along mucosal folds to the stomach lumen. Onmy-Chit.02 is primarily expressed in immunity related organs, such as kidney, spleen, and liver, as well as in reproductive organs. From in situ hybridization and flow cytometry analysis, I show that Onmy-Chit.02 is constitutively expressed in the myeloid cell lineage of the rainbow trout immune system. These two enzymes share many similarities with their mammalian orthologs. Their predicted proteins all have classic chitinase protein structures. In addition, they all have O-glycosylation sites but with different pH optimas. Fluorescent in situ hybridization (FISH) shows that both chitinases are located on Chromosome 17 of the rainbow trout genome. Upon full sequencing of two BACs containing Onmy-Chit.01 or .02, I found two copies of Onmy-Chit.01with almost identical coding regions, but with very different promoter regions. The two copies are apporoximately 9Kb apart and sit in a head to tail arrangement. Only one copy of Onmy-Chit.02 was found in the rainbow trout genome, and its distinct promoter regions are distinct from both copies of Onmy-Chit.01. Phylogenetic analyses revealed that the chitinase gene family fits an evolutionary birth and death model with the chitinase genes derived by duplication of an ancestral chitinase gene. Further gene duplication and loss of chitinolytic activity in mammals gave rise to chitolectins. Hence, I postulate that the function of chitinase is two fold: 1) it is a key element in the first line defense of the innate immunity repertoire; and 2) it serves as a gastric digestive enzyme for chitin containing food items.Item Characterization of the Role of the fem genes in the Sex Determination Pathway of Caenorhabditis briggsae(2008-08-11) Hill, Robin; Haag, Eric S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the genus Caenorhabditis, self-fertile hermaphrodites in C.elegans and C.briggsae evolved from females by developing the ability to generate a limited number of self sperm. The fem genes are crucial for spermatogenesis and the sperm-to-oocyte switch in C.elegans hermaphrodites. RNAi results of the fem genes in C.briggsae hermaphrodites differed from results in C.elegans, suggesting regulation of germ line sex determination pathway differs between the two species. To more definitively address this possibility, and to further investigate the role of the fem genes in the sex determination pathway of C.briggsae, deletion mutants of Cbr-fem-2 and Cbr-fem-3 were generated and characterized. Double Cbr-tra-1;Cbr-fem-2 and Cbr-tra-1;Cbr-fem-3 were also generated to further characterize the role of the fem genes and their relationship to tra-1. Our results show that while the somatic role of the fem genes have been conserved in both species, their germline role differs. Males of both species require the fem genes for somatic development and to suppress oocyte production. However, C.briggsae hermaphrodites do not require the fem genes for spermatogenesis or the sperm-to-ooycte switch. The double mutant analysis results suggest that, unlike C.elegans, Cbr-tra-1 remains epistatic to the Cbr-fem genes in the germline sex determination system in C.briggsae. While there is overall similarity in phenotypic categories between the double mutants, the percentages within each category differs. The double tra-1;fem3 mutant phenotype differs significantly from the single tra-1 mutant, suggesting a role for Cbr-fem-3 in regulating Cbr-tra-1 activity. A previously undescribed Emo phenotype was also discovered in both single and double mutants in C.briggsae and in C.elegans tra-1 mutant alleles e1099 and e1781. The overall results of this study are consistent with the convergent evolution of hermaphroditism within the genus Caenorhabditis and suggest considerable genetic flexibility in this developmental pathway.