The Spemann organizer in amphibians gives rise to the anterior mesendoderm (AME) and is capable of inducing neural tissues. This inductive activity is thought to occur largely via the antagonism of Bone Morphogenetic Protein (BMP) signaling in the organizer. In the mouse, BMP antagonists Chordin and Noggin function redundantly in the AME and are required during forebrain maintenance. However, the timing of forebrain initiation and the function of BMP antagonism in forebrain initiation remained unclear prior to this study. In addition, the Transforming Growth Factor β (TGFβ) ligand Nodal patterns the forebrain via its function in the anterior primitive streak (APS), the precursor tissue of the AME. Whether BMP and Nodal signaling pathways interact has not been previously investigated. The goal of this dissertation was to investigate the cellular and molecular mechanisms involved in early mammalian forebrain establishment by embryonic and genetic manipulations. This study determined that forebrain initiation occurs during early gastrulation and requires signals from the AVE and AME. The AVE was identified as a source of active BMP antagonism in vivo, and the BMP antagonism supplied by exogenous tissues was capable to promote forebrain initiation and maintenance in the murine ectoderm. It is likely that BMP antagonism enhances forebrain gene expression via inhibiting posteriorization. This study further identified a possible crosstalk between BMP and Nodal signaling. Loss of Chordin or Noggin in combination with heterozygosity for Nodal or Smad3 results in holoprosencephaly. Molecular analyses suggest that the BMP-Nodal interaction occurs in the APS and/or the AME. Failure of this interaction results in an imbalance of BMP and Nodal signal levels that devastate APS and AME patterning during early forebrain establishment, ultimately leading to holoprosencephaly in mid-gestation. This interaction is likely to occur extracellularly, possibly by formation of a BMP-Nodal heteromeric complex. Furthermore, the spatiotemporal expression of phospho-Smad1/5/8, an effector of BMP signaling pathway, was characterized during early mouse embryogenesis. Distribution of phospho-Smad1/5/8 serves as a faithful readout of BMP signaling activity and helps to better understand how BMPs are involved in patterning early embryos. The implication of phospho-Smad1/5/8 expression in both wildtype and mutant embryos is also discussed. / Dissertation

World-wide amphibian declines sparked concern and encouraged investigation into potential causes beginning in the 1980’s. Infectious disease has been identified as one of the major potential contributors to amphibian declines. For example, Ranavirus has caused amphibian die-offs throughout the United States. Investigators isolated Ranavirus from dead or moribund amphibians during large-scale die-offs of amphibians in the Cades Cove area of Great Smoky Mountains National Park in 1999-2001. In 2009, after nearly a decade without follow-up monitoring, I undertook an investigation to determine if the virus persisted in the area, and if so, to assess spatial, temporal, and taxonomic patterns in prevalence. Three amphibian breeding ponds, including Gourley Pond, the site of these earlier mortality events, were monitored for Ranavirus during the 2009 amphibian breeding season. A peak in prevalence occurred at Gourley Pond corresponding to a massive amphibian die-off. Prevalence varied among three different taxonomic groups during this mortality event with the highest prevalence, 84%, detected in larval Ambystomatids, 44.4% prevalence in adult Newts, and no virus detected in adult Plethodontids. I did not detect virus at either of the other two breeding ponds despite equivalent sampling effort, similar community composition, and close proximity to Gourley Pond. These results suggest that the severity and spatial extent of Ranavirus in Cades Cove remains unchanged since its initial detection a decade ago. Also, despite the observed massive die-offs there is no evidence of local amphibian extinction at Gourley Pond.

Thesis (M.S.)--Mississippi State University. Department of Wildlife and Fisheries. / Title from title screen. Includes bibliographical references.

Agricultural practices have altered natural wetland habitats for thousands of years in lowland areas of Southeast Asia, and currently these highly modified wetlands constitute some of the most important remaining habitats for amphibians. However, decreasing area of arable land and increased use of chemicals may affect the persistence of lowland amphibian populations that are now dependent on these habitats. I investigated how amphibians responded to different kinds of farming treatments in a large agricultural wetland in Hong Kong. In the first part of the study, I assessed the occurrence of breeding amphibians in 53 farming plots in 2010 and 2011. Anuran call surveys were conducted at night at the plots once a week from March to August for two years. Environmental variables were measured and used to develop models to examine species presence, occupancy, and detection probabilities. Wet agricultural plots supported 10 species of amphibians and all had detection probabilities of < 1 that varied seasonally and yearly. Organically managed plots and shallow water plots yielded high species richness and particularly attracted the ornate pygmy frog (Microhyla fissipes) and the paddy frog (Fejervarya limnocharis). Air temperature and humidity were the relatively consistent predictors that influenced calling activity of the four most commonly detected species (M. fissipes; F. limnocharis; brown tree frog Polypedates megacephalus; and G?nther’s frog Hylarana guentheri). For the second part of the study, I assessed the impacts of fertilizers on amphibians. Using mesocosm experiments in the field, I compared the effects of a chemical fertilizer (granular urea) and an organic fertilizer (peanut cake) on the survival and growth of hatchlings of Polypedates megacephalus, the marbled pygmy frog (Microhyla pulchra), Asian common toad (Duttaphrynus melanostictus) and Chinese bullfrog (Hoplobatrachus rugulosus). Fertilizers were applied at low, manufacturer-recommended, and high levels, and survival and snout-vent-length were measured after 21 days. No individuals survived in the chemical fertilizer treatment at the recommended application level. Conversely, survival was high for P. megacephalus (96%), M. pulchra (54%) and D. melanostictus (90%), but relatively low for H. rugulosus (18%), at the recommended level of the organic fertilizer. P. megacephalus and M. pulchra tadpoles showed increased growth in elevated concentrations of organic fertilizer. Polypedates megacephalus tadpoles were 1.6 times longer in the low concentration and almost double in length in the high concentration treatments. Similarly, increased growth in M. pulchra in all organic treatments resulted in abbreviated time to metamorphosis. Chemical fertilizers are clearly detrimental to early life stages of these amphibians, but organic fertilizers may confer benefits including a shorter time to, and larger size at, metamorphosis. These results suggest that where amphibian conservation is a priority, shifts in the management of wet agricultural crops and limiting the use of chemical fertilizers may increase the suitability of breeding habitats and survival at early life stages. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

No description available.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1966.

Common carp (Cyprinus carpio) have been hypothesized to contribute to declines in aquatic macrophytes, waterfowl, and water clarity in Delta Marsh, an 18,500 ha freshwater coastal wetland on Lake Manitoba, Canada. Ten ponds (1-13 ha) were chosen for a two-year experimental manipulation study. Following a year of baseline monitoring, manipulations were conducted in 2002. To facilitate access by carp into isolated ponds, channels were blasted from the main marsh into two ponds. Meanwhile, to restrict or exclude carp access into ponds, channels were either screened or diked to four ponds. Two connected and two isolated ponds functioned as controls. Although common carp were the original subject of the study, it became apparent that hydrological connection to the surrounding marsh had a paramount importance on the abundance and diversity of the fish, amphibian and water-column invertebrate communities. Connectivity, or lack of connectivity, played an important role in the distribution of the fish community, and subsequently the composition and abundance of water-column invertebrates and amphibians. Ponds with direct connection had diverse, mixed-species fish assemblages, with fewer invertebrates and amphibians. Ponds with restricted connections had fish communities composed of tolerant small-sized species and increased abundance of invertebrates and amphibians. Ponds that lacked connection could freeze and lose all fish, and had higher numbers of invertebrates and amphibians. An absence of adult common carp may have been responsible for increased amphibian numbers in the screened ponds, however more study is needed. Confounding impacts of fluctuating water levels made it impossible to implicate common carp for most changes observed within ponds in Delta Marsh.

Common carp (Cyprinus carpio) have been hypothesized to contribute to declines in aquatic macrophytes, waterfowl, and water clarity in Delta Marsh, an 18,500 ha freshwater coastal wetland on Lake Manitoba, Canada. Ten ponds (1-13 ha) were chosen for a two-year experimental manipulation study. Following a year of baseline monitoring, manipulations were conducted in 2002. To facilitate access by carp into isolated ponds, channels were blasted from the main marsh into two ponds. Meanwhile, to restrict or exclude carp access into ponds, channels were either screened or diked to four ponds. Two connected and two isolated ponds functioned as controls. Although common carp were the original subject of the study, it became apparent that hydrological connection to the surrounding marsh had a paramount importance on the abundance and diversity of the fish, amphibian and water-column invertebrate communities. Connectivity, or lack of connectivity, played an important role in the distribution of the fish community, and subsequently the composition and abundance of water-column invertebrates and amphibians. Ponds with direct connection had diverse, mixed-species fish assemblages, with fewer invertebrates and amphibians. Ponds with restricted connections had fish communities composed of tolerant small-sized species and increased abundance of invertebrates and amphibians. Ponds that lacked connection could freeze and lose all fish, and had higher numbers of invertebrates and amphibians. An absence of adult common carp may have been responsible for increased amphibian numbers in the screened ponds, however more study is needed. Confounding impacts of fluctuating water levels made it impossible to implicate common carp for most changes observed within ponds in Delta Marsh.

The ING (INhibitor of Growth) tumor suppressor genes are conserved from yeast to humans and are implicated in several processes important to cell proliferation and apoptosis. ING proteins contain a plant homeodomain (PHD) finger that suggests these proteins may modulate transcription factor-mediated pathways. Little is known about the mechanism of action of INGs, especially in the context of normal development. The ING family of proteins includes at least five different genes, ING1-ING5, with evidence for alternate promoter usage and splicing that generate multiple isoforms. To elucidate the role of ING in different tissues to modulate function, I used amphibian metamorphosis as a model system in which a single stimulus, thyroid hormone (TH), initiates apoptosis, proliferation, and remodeling in the tail, hindlimb, and brain, respectively. I discovered seven ING1 and three ING2 transcript variants in Xenopus laevis and investigated their expression patterns. High expression levels of most variants were found in adult brain, testis, and eye. During natural metamorphosis or precocious metamorphosis induced by treating tadpoles with exogenous TH, ING1 and ING2 transcript variant levels were differentially regulated in a tissue-specific manner. Some variant levels increased with the induction of apoptosis of the tail, while levels of the same variants decreased upon induction of proliferation and differentiation in the hindlimb. Although levels of all INC variants were relatively high in whole brain, they did not change during metamorphosis or TH treatment. Given that ING has previously been shown to modulate apoptosis, it is likely that upregulation of specific isoforms may contribute to the tissue-specific TH-mediated response in the tail, and that downregulation facilitates proliferation of the hindlimb. To further investigate the hypothesis that ING is regulated by TH, an analysis of 1NG1 and ING2 genomic sequences was carried out. Promoter sequences for each variant were determined and putative thyroid hormone response elements (TREs) located. To test whether thyroid hormone receptors associate with these elements, chromatin immunoprecipitations (ChIP) assays were done on tail homogenates from premetamorphic tadpoles treated with TH or vehicle control. Both thyroid hormone receptor α (TRα) and thyroid hormone receptor β (TRβ) differentially associate with ING1 and ING2 promoter regions. TR association increased significantly on promoters for ING variant transcripts that increase upon TH treatment, and decreased significantly on promoters for ING variant transcripts that decrease upon TH treatment. ChIPs also showed that ING associates with TH-regulated promoters including TRβ, TH-Responsive Basic Leucine Zipper Transcription Factor (TH/bZIP), ING1 and ING2. Furthermore, TR and ING were shown to co-immunoprecipitate with both purified proteins and using total tail homogenates from metamorphic tadpoles. The antibodies used for these experiments were made against Xenopus TRβ and ING2 and were characterized as part of this thesis. Bioinformatics revealed that TREs are present in promoters of ING genes for other species including human, mouse, and a related frog species, Xenopus tropicalis; therefore, it is likely that modulation by TH is a conserved mechanism of ING regulation. These data suggest that there may be antagonistic regulation of ING transcript variants by TH that correlates with tissue fate. TRs associate with ING promoters, and ING is associated with TR-regulated promoters. Moreover, TR and ING proteins co-immunoprecipitate. It is therefore likely that TR and ING are co-regulators of gene expression during TH-dependent tadpole metamorphosis. This thesis contributes to the understanding of ING which is relevant to elucidating many disease states, as well as being critical in understanding the role of this tumor suppressor in the context of TH regulation and normal development.

Riparian management in the Pacific Northwest has become an Increasingly common way to conserve biodiversity on landscapes managed for timber production. The efficacy of two different riparian buffer widths in providing habitat for terrestrial amphibians was assessed using a Before-After-Control-Impact approach. My findings suggest that there is no global response by terrestrial amphibians to logging or to the retention of riparian management zones in the US Pacific Northwest. Rather, species showed individual responses that varied over time and between treatments and transects. Overall, a minimum riparian buffer width of 30 m was sufficient to maintain the relative abundance and richness of terrestrial amphibians at levels commensurate with pre-harvest conditions. A co-inertia analysis revealed that habitat associations changed little over time and that there were no significant differences between buffered sites suggesting that the treatments applied were biologically insignificant. The benefits of retaining riparian forest are identified and discussed in the context of maintaining biodiversity and conserving terrestrial amphibians in western Washington.