A fundamental question of evolutionary ecology is, what determines body size? In general, the body size of vertebrates is thought to be relatively inflexible, a product of their genes, food, environment, and stress. However, vertebrate growth can be plastic in response to population interactions such as predator-prey and competition. While these relationships can elicit plasticity of vertebrate growth, mutualistic relationships have yet to be investigated. An iconic example of mutualism involving a vertebrate is the relationship between anemone and anemonefish. In this interaction anemonefish size is often positively correlated with anemone size. Here, I test the hypothesis that anemonefish growth is a plastic response to variation in anemone size. Juvenile clownfish (Amphiprion percula) of relatively uniform size were paired with sea anemones (Entacmaea quadricolor) of variable size and monitored over three months. The average anemone size over the course of the month was then used to predict the fish growth each month. Mixed model analyses verified that anemone area is significantly associated with both change in fish standard length and change in fish body depth. Fish in larger anemones grew more than did fish in small anemones. Remarkably, individuals in large anemones achieved this despite receiving the same amount of food as individuals in small anemones. This clownfish growth plasticity in response to anemone size might be adaptive if anemone area is a good indicator of resource availability in the wild, because it would enable the fish to maximize their reproduction without compromising survival. This study extends the understanding of how plasticity of vertebrate species can be influenced by a wide variety of population interactions.

Roads cause substantial wildlife mortality, but there is currently limited understanding of the relative magnitude of this mortality source. There are also substantial gaps in knowledge concerning the ecological ramifications of carrion introduced to the environment from vehicle collisions and in particular how vertebrate scavengers may consume carrion resulting from vehicle collisions. Although a variety of factors influence scavenger use of carcasses, the mechanisms influencing competition for this resource between obligate and facultative scavengers have not been thoroughly explored. I conducted a global synthesis of mortality of terrestrial vertebrates documenting 42,755 mortalities of known cause from 120,657 individuals representing 305 vertebrate species. Overall, 28% of mortalities were directly caused by humans and 72% were from natural sources. Vehicle collisions accounted for 4% of mortality overall. Larger birds were more likely than smaller birds to die from vehicle collisions and vehicle mortality of mammals increased over time. There was no difference in proportion of rabbit carcasses scavenged or scavenger arrival time between those placed along roads, power line clearings, and forests. No species arrived at roads quicker than other treatments. Turkey vultures (Cathartes aura) and coyotes (Canis latrans) scavenged equally across treatments, whereas gray foxes (Urocyon cinereoargenteus) scavenged along roads and power lines, but not in forests. Scavenger use of carrion near roads likely relates to factors besides carrion availability, such as traffic avoidance and predation risk. Because some scavengers make substantial use of carrion on roads, this resource could be an important mechanism by which human activities impact wildlife. Scavenging by facultative scavengers did not increase in the absence of competition with vultures. I found no difference in scavenger presence between control carcasses and those from which vultures were excluded. Facultative scavengers did not functionally replace vultures during summer in this study. These results suggest that under the conditions of this study, facultative scavengers would not compensate for loss of vultures. Carcasses would persist longer in the environment and consumption of carrion would likely shift from vertebrates to decomposers. Such changes could have substantial implications for disease transmission, nutrient cycling, and ecosystem functioning.

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Previous studies on microtaphonomy have identified multiple different organic microstructures in fossil vertebrates from a variety of time periods and environmental settings. This study seeks to investigate the potential taphonomic, paleoclimatic, and geochemical controls on soft tissue and cellular microstructure preservation. To this end, fifteen vertebrate fossils were studied: eight fossils collected from the Oligocene Sharps Formation of the Arikaree Group in Badlands National Park, South Dakota, and seven fossils from formations in the underlying White River Group, including the the (Oligocene) Brule Formation of Badlands National Park, and the (Eocene) Chadron Formation of Flagstaff Rim, Wyoming; Toadstool Geologic Park, Nebraska; and Badlands National Park, South Dakota. A portion of each fossil was demineralized to identify any organic microstructures preserved within the fossils. I investigated several potential soft tissue preservation factors, including taxonomy, paleoclimate, depositional environment, and diagenetic history as shown through bone apatite crystallinity and trace element alteration. Soft tissue microstructures were preserved in all fossil samples, and cellular material was recovered from most fossil specimens. Soft tissue and cellular preservation was found to occur independent of taxonomy, paleoclimate regime, depositional environment, and apatite crystallinity. The period of fossilization and diagenetic trace element addition, as shown through rare earth element (Lanthanum) diffusion profiles, may be connected to organic microstructure preservation, as longer estimated diffusion periods were correlated with poorer preservation of bone histology and greater cellular degradation in some of the fossil samples. / Geology / Accompanied by one Excel file: Thesis XRD Driffractograms Full.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

In this work, we identified and characterized developmentally regulated aspects to cell division in the Xenopus laevis. We found that cells in the early neural plate divide in an oriented manner. This orientation is established by Cdc42 controlled maintenance of stable interactions between the spindle and the cell cortex. This role of Cdc42 is developmentally regulated and cells dividing later in a related tissue, the tail epidermis, are not under this control. Moreover, we find that the cell divisions in the early neural plate are further specialized in their mechanisms of cell division. Cells in the early neural plate exhibit exaggerated anaphase-B movements, a delayed onset of cytokinesis, low density of midzone microtubules and a rapid cytokinetic furrow ingression as compared to the late tail epidermis, another ectodermally derived tissue. These modifications to the mechanism of cell division appear to be because of a reduced level of PRC1, a microtubule bundling protein, and thus modifications to the central spindle structure. Finally, we find that cytokinetic mechanisms may be functionally related to the process of ciliogenesis. We find proteins known to localize to the central spindle localized to the rootlet of the basal body of cilia in multiciliated cells of the mucociliary epidermis. This localization may be related to vesicle transport during both these processes. This work reveals unexpected plasticity to fundamental mechanisms of cell division. / text

The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions. The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization. A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals. Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours. In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.

Vertebrate wnt8a occupies a position at a crossroads linking anteroposterior and dorsoventral axis patterning. While functional aspects of wnt8a are beginning to be understood, the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways are unclear. Three inputs that control wnt8a expression in the zebrafish embryonic margin have been identified: the Brachyury-related T-box transcription factors No tail a (Ntla) and No tail b (Ntlb, previously called Bra) and the maternal zinc-finger transcription factor Zbtb4 (previously called Kzp) are known direct regulators, and Nodal signaling is genetically upstream of wnt8a expression. The transcriptional mechanisms by which the wnt8a locus integrates these diverse temporal inputs are not yet known. We have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Using this transgenic line, we identified two phases of wnt8a transcriptional regulation in zebrafish: phase I comprises Nodal-dependent activation during early gastrulation and phase II comprises No tail (Ntl)-dependent regulation from mid to late gastrula stages onwards. These phases mirror the transition from Nodal-dependent mesoderm induction to Ntl-dependent mesoderm maintenance. To further understand how the wnt8a locus integrates these signals to achieve its transcriptional output, we analyzed upstream cis-regulatory regions through transgenic reporter assays. We identified three promoters in the bicistronic wnt8a locus, two of which drive expression of the upstream coding region (wnt8a.1). We identified two regulatory regions, proximal and distal: the proximal regulatory region contains a mesodermal enhancer with potential binding sites for FoxH1 and Ntl that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal signaling through the mesoderm enhancer in combination with the distal regulatory region, which bears a Zbtb4 consensus binding site. Phase II expression requires Ntl regulation of the mesoderm enhancer in the context of the proximal regulatory region. The distal regulatory region negatively impacts phase II expression driven by the proximal regulatory region, indicating a complex relationship of regulatory elements.

Oct4, a transcription factor belonging to the fifth class of POU proteins (POUV), plays essential roles in the maintenance of pluripotency, differentiation and the generation of induced pluripotent stem cells (iPSCs). Oct4 regulates two levels of pluripotency, which are distinguished by their gene expression profiles and epigenetic status, namely the naïve and primed state of pluripotency. Embryonic stem cells (ESCs) and embryonic germ cells (EGCs), which are isolated from inner cell mass and primordial germ cells in the embryo, respectively, are in vitro models in which the naïve state is propagated through self-renewal. Epiblast stem cells (EpiSCs) and traditional human ESCs have gene expression profiles that are closest to the post-implantation epiblast, which is closer to embryonic differentiation, and exhibit a primed state of pluripotency. As Oct4 is important for pluripotency in all these cell types, where it regulates different targets, it appears to have two distinct sets of functions, namely germ cell/naïve ESC-like activity and epiblast/primed pluripotency-like activity. Based on protein sequences and syntenic gene analysis, Oct4/POUV homologs of jawed vertebrates can be classified into two subfamilies: POU5F1 and POU5F3, which are thought to originate from a genome duplication event that occurred in a common ancestor. Most extant vertebrates have lost one of these paralogs, while a small fraction, including coelacanths, axolotls, turtles, and marsupials, retains both POUV forms. In my thesis, I investigated the gene duplication event that underlies divergence of POU5F1 and POU5F3 in both expression pattern and specialised function. In particular, I focused on species that have retained both genes and asked whether POUV functional divergence correlates with ancestral origin. To test the function of POU5F1 and POU5F3, I substituted endogenous mouse Oct4/Pou5f1 with different POUV proteins using a cell line in which endogenous Oct4 expression can be silenced with tetracycline (ZHBTc4). Results showed that POU5F1 proteins had a greater capacity to support naïve ESC pluripotency and self-renewal than POU5F3 proteins. Global transcriptome analysis of the POUV-rescued ESC lines revealed that coelacanth POU5F1 protein regulates gene expression in a similar manner to mouse Oct4, in that genes involved in stem cell maintenance, reproduction and development are upregulated in ESCs rescued by POU5F1, but not POU5F3. Coelacanth POU5F3 rescued lines, however, expressed genes involved in various cell differentiation programs, including cell adhesion (e.g. E-cadherin and N-cadherin). This suggests that POU5F3 plays a role in primed pluripotency, while POU5F1 regulates naïve pluripotency. However, there is one POU5F3 factor that rescues ESCs like Oct4, the Xenopus gene Xlpou91 (Pou5f3.1). In Xenopus, a further duplication of POU5F3 gene enabled specialization, and Xlpou91 is expressed specifically in the primordial germ cells. Xlpou25 (Pou5f3.2) exhibits epiblast-specific activities and lacks the capacity to maintain naïve ESC pluripotency, similar to other POU5F3 proteins. This functional distinction between the different Xenopus POUV paralogs enabled us to address how specific Oct4 functions (germ cell-like versus epiblast-like activity) are related to the induction of pluripotency. To address this question, mouse Oct4 was replaced by either Xlpou91 or Xlpou25 in murine cellular reprogramming using a Nanog-GFP reporter line to monitor iPSC generation. Results showed that Xlpou91 and mouse Oct4 were required at similar levels to reprogram somatic cells toward iPSCs and reprogrammed cells emerged with similar kinetics. Conversely, Xlpou25 was required at higher expression levels and the resulting iPSCs appeared at a later timepoint, while the pluripotent population in these cultures appeared to be less stable and more prone to differentiate. I found that this phenotype of enhanced differentiation in Xlpou25 reprogrammed cultures may be a product of a different set of immediate early genes induced at the first stages of differentiation. Global transcriptome analysis of the naïve ESC-like pluripotent subpopulation of these iPSC lines confirmed the capacity of all Xenopus POUVs to drive reprogramming towards the pluripotent state. However, the gene sets induced by both Xlpou91 and mouse Oct4, but not Xlpou25, were somewhat enriched for genes involved in reproduction, emphasizing the segregated role of Xlpou91 as a germ cell specific POUV protein. Lastly, I explored the evolutionary origin of these two POUV paralogs and attempted to identify a POUV-related gene in jawless vertebrate (cyclostomes). Based on in silico analysis of genomic and transcriptome databases, my collaborators and I were able to identify a single POUV gene in the Japanese/arctic lamprey, thus providing the first insight into the origin of gnathosome POUV genes.

The objectives were (1) to relate terrestrial vertebrate responses to the management practices used for lodgepole pine forests within the Barometer Watershed, Mountain View Ranger District, Wasatch National Forest, Utah . (2) To correlate terrestrial vertebrate densities or frequencies with community types, edge, major forest stand structures , and (3) to propose management plans to manipulate densities of major vertebrate species in a predetermined manner. The study was conducted between 1973-1975 and provides a detailed description of forest vegetation for 53 lodgepole pine (Pinus contorta) stands. For each of these stands, a complete summary is provided of tree populations, coverage, and frequency of major vascular undergrowth species . Eight lodgepole pine forest community types are defined. A key is provided for identification of each community type and its anticipated habitat type. The relationships between densities of major vertebrate species, forest communities, and major forest stand structures are described. Big game utilization of ecotones created by mountain meadows and the lodgepole pine forest are discussed. In addition, the response of selected small mammal and big game species to clearcut size is provided. Important research findings were: (1) In the lodgepole pine forest most vertebrates exhibit preferences for specific community types. (2) Due to the broad definition of present habitat classifications, limited predictions can be made about the general response of wildlife populations on most sites. (3) The use of park-like openings and associated peripheral timber hy big game animals is closely associated with community type, edge configuration, and historical travel lanes. (4) An abundance of downed woody material enhances big game calving and resting areas. (S) The number of bird species is closely associated with understory biomass and diversity. (6) Management schemes that speed up the rotation of lodgepole pine overstories eliminate certain vertebrate communities associated with the final successional stages. The following specific recommendations for overstory removal are suggested. (1) Timber sales should be developed by drainage, with longterm objectives that insure the distribution of a variety of communities within a drainage. (2) The addition of major stand structure information should be included in habitat classification systems. (3) Timber sales should be designed with irregular edges and buffers of standing timber which provide cover and concealment. (4) Moist sites and relic areas, representing the final stages of succession, should be planned into the overall drainage sale philosophy. {5) In relatively undisturbed areas, vehicular travel should be prohibited following overstory removal.