I. Studies on alkaline phosphatase activity in developing sea urchin embryos. II. Studies on the activation of protein biosynthesis in sea urchin eggs at fertilization

I. Alkaline phosphatase activity in the developing sea urchin Lytechinus pictus has been investigated with respect to intensity at various stages, ionic requirements and intracellular localization. The activity per embryo remains the same in the unfertilized egg, fertilized egg and cleavage stages. At a time just prior to gastrulation (about 10 hours after fertilization) the activity per embryo begins to rise and increases after 300 times over the activity in the cleavage stages during the next 60 hours.

The optimum ionic strength for enzymatic activity shows a wide peak at 0.6 to 1.0. Calcium and magnesium show an additional optimum at a concentration in the range of 0.02 to 0.07 molar. EDTA at concentrations of 0.0001 molar and higher shows a definite inhibition of activity.

The intracellular localization of alkaline phosphatase in homogenates of 72-hour embryos has been studied employing the differential centrifugation method. The major portion of the total activity in these homogenates was found in mitochondrial and microsomal fractions with less than 5% in the nuclear fraction and less than 2% in the final supernatant. The activity could be released from all fractions by treatment with sodium deoxycholate.

II. The activation of protein biosynthesis at fertilization in eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus has been studied in both intact eggs and cell-free homogenates. It is shown that homogenates from both unfertilized and fertilized eggs are dependent on potassium and magnesium ions for optimum amino acid incorporation activity and in the case of the latter the concentration range is quite narrow. Though the optimum magnesium concentrations appear to differ slightly in homogenates of unfertilized and fertilized eggs, in no case was it observed that unfertilized egg homogenates were stimulated to incorporate at a level comparable to that of the fertilized eggs.

An activation of amino acid incorporation into protein has also been shown to occur in parthenogenetically activated non-nucleate sea urchin egg fragments or homogenates thereof. This activation resembles that in the fertilized whole egg or fragment both in amount and pattern of activation. Furthermore, it is shown that polyribosomes form in these non-nucleate fragments upon artificial activation. These findings are discussed along with possible mechanisms for activation of the system at fertilization.

Complex behavior and perception in Drosophila emerges from iterative feedback-regulated reflexes

For a hungry fruit fly, locating and landing on a fermenting fruit where it can feed, find mates, and lay eggs, is an essential and difficult task requiring the integration of both olfactory and visual cues. Understanding how flies accomplish this will help provide a comprehensive ethological context for the expanding knowledge of their neural circuits involved in processing olfaction and vision, as well as inspire novel engineering solutions for control and estimation in computationally limited robotic applications. In this thesis, I use novel high throughput methods to develop a detailed overview of how flies track odor plumes, land, and regulate flight speed. Finally, I provide an example of how these insights can be applied to robotic applications to simplify complicated estimation problems. To localize an odor source, flies exhibit three iterative, reflex-driven behaviors. Upon encountering an attractive plume, flies increase their flight speed and turn upwind using visual cues. After losing the plume, flies begin zigzagging crosswind, again using visual cues to control their heading. After sensing an attractive odor, flies become more attracted to small visual features, which increases their chances of finding the plume source. Their changes in heading are largely controlled by open-loop maneuvers called saccades, which they direct towards and away from visual features. If a fly decides to land on an object, it begins to decelerate so as to maintain a stereotypical ratio of expansion to retinal size. Once they reach a stereotypical distance from the target, flies extend their legs in preparation for touchdown. Although it is unclear what cues they use to trigger this behavior, previous studies have indicated that it is likely under visual control. In Chapter 3, I use a nonlinear control theoretic analysis and robotic testbed to propose a novel and putative mechanism for how a fly might visually estimate distance by actively decelerating according to a visual control law. Throughout these behaviors, a common theme is the visual control of flight speed. Using genetic tools I show that the neuromodulator octopamine plays an important role in regulating flight speed, and propose a neural circuit for how this controller might be implemented in the flies brain. Two general biological and engineering principles are evident across my experiments: (1) complex behaviors, such as foraging, can emerge from the interactions of simple independent sensory-motor modules; (2) flies control their behavior in such a way that simplifies complex estimation problems.

Mitochondria and the development of sea urchin embryos

After artificial activation or fertilization of non-nucleate fragments or eggs of the sea urchin, the mitochondria actively synthesize RNA. The RNA made in non-nucleate fragments is shown to be mostly single stranded and to be associated primarily with the low speed pellet of centrifuged cellular homogenates.

Protein synthesis is observed in non-nucleate fragments in the presence or absence of the mitochondrial RNA synthesis: it is found to be qualitatively similar but quantitatively less in the absence of the RNA synthesis. The continued syntheses of proteins in the non-nucleate fragments in the absence of mitochondrial RNA synthesis provides additional evidence for the presence of a stable messenger RNA component in the unfertilized sea urchin egg.

Since the uptake or actinomycin D was found to be inhibited by the presence of a fertilization membrane, ethidium bromide, at 10 μgs/ml, is used as an effective inhibitor of RNA synthesis in non-nucleate fragments and in early cleavage stage embryos. However, this same concentration of ethidium bromide is found to be only partially effective in blocking RNA synthesis at the mesenchyme blastula stage of development.

Low concentrations of ethidium bromide (2 and 5 μgs/ml) are found not to be lethal but to be capable of producing moderate developmental defects. In the presence of concentrations of ethidium bromide adequate to inhibit all the mitochondrial RNA synthesis (10 μgs/ml of ethidium bromide), from fertilization on, the embryos do not cleave beyond the 4-8 cell stages. When similar concentrations of ethidium bromide are added at an early mesenchyme blastula stage, the embryos do not gastrulate but continue to swim for more than 24 additional hours (adequate for control embryos to develop to a late prism stage). These results lead to the conclusion that mitochondrial RNA synthesis may be very essential for normal development to occur.

DNA is synthesized in the non-nucleate fragments of sea urchin eggs. None of the newly synthesized DNA is found in the closed circular form. When phenol extracted directly from the fragments, the DNA is found to sediment at approximately 38 and 27s in sucrose gradients but neither of these size classes could be found associated with the isolated mitochondria. The template for the synthesis of DNA in non-nucleate fragments remains unknown.

Identification and characterization of the egg-laying hormone from the neurosecretary bag cells of Aplysia

This investigation has resulted in the chemical identification and isolation of the egg-laying hormone from Aplysia californica, Aplysia vaccaria, and Aplysia dactylomela. The hormone, which was originally identified as the Bag Cell-Specific protein (BCS protein) on polyacrylamide gels, is a polypeptide of molecular weight ≈ 6000, which is localized in the neurosecretory bag cells of the parietovisceral ganglion and the surrounding connective tissue sheath which contains the bag cell axons. All three species produce a hormone of similar molecular weight, but varying electrophoretic mobility as determined on polyacrylamide gels. As tested, the hormone is completely cross-reactive among the three species.

Although the bag cells of sexually immature animals contain the active hormone, sexual maturation of the animal results in a 10-fold increase in the BCS protein content of these neurons.

A seasonal variation in the BCS protein content was also observed, with 150 times more hormone contained in the bag cells of Aplysia californica in August than in January. This correlates well with the variation in the animals' ability to lay eggs throughout the year (Strumwasser et al., 1969). There are some indications that the receptivity of the animal to the available hormone also fluctuates during the year, being lower in winter than in swmner. The seasonal rhythm of the other species, Aplysia vaccaria and Aplysia dactylomela, has not been investigated.

A polyacrylamide gel electrophoresis analysis of water-soluble proteins in Aplysia californica revealed several other nerve-specific proteins. One of these is also located in the bag cell somas and stains turquoise with Amido Schwarz. The function of this protein has not been investigated.