Development in the annual killifish Austrofundulus limnaeus can follow two distinct developmental trajectories. Typical development includes the entrance of embryos into a state of metabolic and developmental arrest termed diapause. Alternately, embryos can escape diapause and develop directly without pause. These two trajectories are characterized by differences in the rate and timing of developmental, morphological, and physiological traits. Insulin and Insulin-like growth factor (IGF) signaling (IIS) is known to regulate entrance into diapause in a variety of invertebrates. In this thesis I explore the possible role of IGFs in the regulation of development and diapause in embryos of A. limnaeus. Here I report stage-specific expression of IGF-I and II proteins and their associated mRNA transcripts. Patterns of IGF-I protein expression are consistent with IGF signaling playing a major role in supporting the escape trajectory. In addition, treatment of embryos with a potent inhibitor of the IGF-I receptor (IGF1R) mimics the diapause developmental pattern even under conditions that should favor direct development. Evaluation of mRNA gene expression patterns in the two developmental trajectories suggests a role for IGF-I signaling through the RAS-MAPK-ERK pathway, which may be promoting the escape phenotype. Additionally, IGF-I activity may be enhanced in escape trajectory embryos though upregulation of IGF binding protein 2 (IGFBP-2) mRNA. These data suggest a major role for IGF signaling in the promotion of the escape trajectory, and thus we predict that specific mechanisms are in place in diapause-bound embryos that block IGF signaling and thus promote entrance into diapause. The data presented here suggest that blocking IGF signaling is critical for induction of diapause, but also suggests that other signaling pathways are likely also at play. Other pathways such at the TGF-beta signaling molecules and SMAD pathway, may also be involved in the direct regulation of the diapause phenotype, as has been shown for other animal models of developmental arrest.

The dispersion and reaggreation [sic] stages have been described as a unique feature of embryonic development in annual killifish such as Austrofundulus limnaeus, a killifish that inhabits ephemeral ponds in the Maracaibo basin of Venezuela. These stages have previously been described as an atypical developmental progression in which blastomeres completely disperse on the surface of the yolk and then reaggregate into a mass of cells to form the embryo. Temperature affects the onset as well as the duration of this stage in related annual fishes. We have undertaken this study to show in detail the behavior of blastomere cells and their distributions as a function of developmental temperature. Embryos incubated at either 25 or 30°C were fixed and stained with Hoescht dye to allow visualization and quantification of cell number during the dispersion and reaggregation phases of development. The location of every cell nucleus on the embryo was assessed through photomicroscopy using inverted epifluorescent microsopy [sic]. This analysis revealed that the rate of cell division during the process of dispersion/reaggergation [sic] has a typical sensitivity to temperature with Q10 values of about 2-3. There is no indication that the pattern of blastomere movement and distribution is different in embryos incubated at 25°C versus 30°C. The primary developmental difference was observed as a temporary plateau in blastomere counts at 25°C followed by great variation of blastomere numbers in subsequent timepoints compared to the degree of variation observed in embryos incubated at 30°C. This trend fits the model that embryos developing at 25°C enter into a brief diapause-like event at the dispersion stage from which they emerge at a variable rate. Of great interest, at both temperatures examined, the majority of the dispersed blastomeres do not reaggregate and contribute to the formation of the primary embryonic axis. Prior studies have overemphasized blastomere reaggregation in A. limnaeus due to the limitations of the sampling methods used as well as overdependence upon a statistical test, the coefficient of dispersion. Thus, the present study sheds light on these early mischaracterizations of A. limnaeus development.