Responses of chromatophores to physiological and pharmacological agents

Thesis (M.A.)--Boston Universit

USING THE FROG EPIDERMIS TO UNCOVER DESMOSOME FUNCTION AND REGULATION IN THE DEVELOPING EMBRYO

The desmosome is one of the major cell adhesion junctions found in the epithelia, heart, and hair follicle. Described as a “rivet” that hold cells together, it provides these tissues with the integrity to withstand the tremendous forces they face in everyday life. Defects in this junction can lead to devastating diseases where patients are susceptible to skin infections and cardiovascular defects. Limited treatments exist for diseases of the desmosome, and strategies do not target all symptoms. Therefore, delineating the function and regulation of desmosomes is of paramount importance for the development of prevention and treatment strategies. The Xenopus laevis has been utilized for the study of embryonic development and tissue movements. This study takes advantage of the frog model to study a key desmosomal protein, desmoplakin (Dsp), in the epidermal development of the embryo. First, Xenopus embryonic epidermis has junctional desmosomes as early as the blastula stages. Desmosomes numbers per junction increase as the embryo develops. Dsp is present in many epidermally-derived structures in the embryo at varying levels. Xenopus embryos deficient in desmoplakin have phenotypic defects in epidermal structures and the heart, mimicking mammalian models. Embryos with reduced Dsp exhibit an increased susceptibility to epidermal damage under applied mechanical forces. Assays also reveal a potential role for desmosomes in radial intercalation, a process through which cells move from the inner to the outer epidermal layers. Embryos with reduced Dsp exhibit a slight reduction in intercalation and defects in intercalating cell types, including multiciliated cells and small secretory cells. Finally, c-Jun N-terminal kinase (JNK) may have a potential role in the regulation of desmosome assembly and adhesion. Embryos with deficient Dsp display a partial recovery of mechanical integrity when treated with a JNK inhibitor

The Role of Metabolism in Embryonic Development: Mitochondria Determine the Fate of the Spemann-Mangold Organizer

Carbon metabolism is critical for complex multicellular life. Therefore, mitochondria, and the aerobic metabolism that they perform, have long been a focus of developmental biologists. The discovery of the Spemann-Mangold Organizer, a powerful embryonic patterning center in the dorsal region of the vertebrate embryo, first inspired the idea that embryonic patterning and aerobic metabolism may be connected almost 100 years ago. However, the advent of modern molecular genetics overshadowed this work, and metabolism in development has only recently resurfaced as a focus in the field. A permissive role for mitochondria has been well studied in development. However, it remains unclear whether a mechanism exists by which mitochondrial metabolism can instruct cell fate and embryonic patterning. Here, we demonstrate that mitochondrial oxidative metabolism regulates cell fate and patterning of the blastula via Hif-1α. Genetic disruption of the mitochondrial and patterning disease gene LRPPRC or exposure to hypoxia induces alteration in cell fates and expands the Spemann-Mangold Organizer, leading to downstream patterning defects of the heart and other tissues. Based on the hypoxia phenotype, we report that Hif-1α itself is sufficient to drive Organizer gene expression. Unexpectedly, oxygen consumption in the Organizer is 20% higher, rather than lower, than that of the ventral mesoderm, suggesting an increase in mitochondrial activity rather than regional hypoxia. We find that the increase in oxygen consumption is due to an inner mitochondrial membrane proton leak which decouples oxygen consumption from ATP production in the dorsal mesoderm. The proton leak is due to dorsal enrichment of the free c-subunit ring of F1Fo ATP synthase, which acts as an uncoupling channel to drive the increased rate of respiration. Overexpression of the free c-subunit is sufficient to induce Organizer cell fates via Hif-1α and can even induce a secondary, ectopic, body axis. Taken together, we have established that mitochondrial uncoupling in the dorsal mesoderm, driven by the ATP synthase free c-subunit, activates Hif-1α which determines the Spemann-Mangold Organizer, a tissue essential for global embryonic patterning

Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives

To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems

Early development and the honesty of aposematic signals in a poison frog

Doctoral scholarship program 2005-2010The causes and consequences of variation in aposematic signals during immature stages are not clearly understood. This thesis explores the effects of early environment on the expression of aposematic signals in the green and black poison frog (Dendrobates auratus), and the consequences of variation in such components in the wild. It also explores how aposematic expression relates to levels of chemical defences in immature froglets. Embryos and larvae of poison frogs in the genus Dendrobates are known to be darkly pigmented. This thesis reports for the first time polymorphism in egg pigmentation in D. auratus and ontogenetic colour change through development reverting to a normally pigmented phenotype; however whether this pigmentation results from constraints or has adaptive consequences remains unclear. Evidence on how immature individuals allocate resources to growth and warning signalling is scarce. Experimental results in this thesis show that food supply during early environment affected body size and signal luminance in post-metamorphic froglets. Therefore the relative importance of these traits in relation to predation risk was further tested, using artificial prey in a field experiment. The results indicated that rates of attack by birds correlated negatively with body size, and on the contrary survival of artificial prey was independent of signal luminance. I therefore tested the hypothesis that in the wild larger, relatively well-nourished juvenile frogs are chemically better defended. I found that in fact larger juveniles are at a selective advantage conferred by their greater foraging efficiency and their superior levels of chemical defences. Overall, these results shows plasticity in aposematic traits in relation to early environmental nutrition in D. auratus; and suggests that acquiring large body size and similar integument colour as to adults are key determinants for survival during the early stages of their terrestrial life.IFARHU-SENACY