Cell differentiation

A recessive mutant gene, termed "c", for cardiac non-function in the Mexican axolotl, Ambystoma mexicanum, is responsible for the failure of myofibrillogenesis in cardiac nonfunction mutant embryonic hearts. Animals that are homozygous for the mutation (c/c) fail to develop beating hearts and consequently die. Thus, the Mexican axolotl has been a useful animal model to study embryonic heart development. Recently, the cardiac troponin T (cTnT) gene, along with three additional shorter isoforms of the gene, were cloned from normal embryonic hearts. These isoforms are believed to be the alternately spliced forms of the full length gene. One of the isoforms cloned is missing a cardiac-specific exon. Real-time PCR reveals that homozygous recessive mutant embryos (c/c) exhibit a lower transcription level of the cTnT gene than wildtype animals (+/+ or +/c). Expression levels of each of the isoforms are compared in normal and mutant hearts using quantitative real-time PCR.

Follistatin (FS) proteins are highly conserved inhibitors of Activins, members of the Transforming Growth Factor beta (TGF-beta) family, which play prominent roles in patterning and cell proliferation, and can contribute to tumor formation. Comparison of FS from Drosophila (dFS) and humans (hFS) in flies shows that hFS is less active. The goal of this thesis is to test three possible mechanisms: dFS might be more stable and turn over at a lower rate, exhibit a stronger affinity for ligands, or diffuse less because of stronger interaction with the extracellular matrix. We generated chimeric proteins of dFS and hFS by exchanging individual protein domains. Our results suggest that the increased activity is likely due to ligand binding. Based on the recent structure of the hFS-Activin complex, we speculate that stronger interactions with heparin sulfate in the extracellular matrix may also contribute to the increased activity of dFS.

The lens is an avascular organ that focuses light onto the retina where neural signals are transmitted to the brain and translated into images. Lens transparency is vital for maintaining function. The lens is formed through a transition from organelle-rich epithelial cells to organelle-free fiber cells. Lens cell differentiation, leading to the lack of organelles, provides an environment optimal for minimizing light scatter and maximizing the ability to focus light onto the retina. The process responsible for orchestrating lens cell differentiation has yet to be elucidated. In recent years, data has emerged that led our lab to hypothesize that autophagy is likely involved in lens cell maintenance, cell differentiation, and maintenance of lens transparency. As a first step towards testing this hypothesis, we used RT-PCR, western blot analysis, immunohistochemistry, confocal microscopy, and next generation RNA-Sequencing (RNA-Seq) to examine autophagy genes expressed by the lens to begin mapping their lens function.

The assembly and maintenance of central synapses is a complex process, requiring myriad genes and their products. Highwire is a large gene containing a RING domain, characteristic of ubiquitin E3 ligases. Highwire has been shown to restrain axon growth and control synaptogenesis at a peripheral synapse. Here I examine the roles of Highwire at a central synapse in the adult Drosophila Giant Fiber System (GFS). Highwire is indeed necessary for proper axonal growth as well as synaptic transmission in the GFS. Differences arise between the central synapse and the neuromuscular junction (NMJ), where highwire was initially characterized : expresion from the postsynaptic cell can rescue highwire synaptic defects, which is not seen at the NMJ. In addition, a MAP kinase signaling pathway regulated by highwire at the NMJ has differing roles at a central synapse. Wallenda MAPK can rescue not only the highwire anatomical phenotype but also the defects seen in transmission. Another distinction is seen here : loss of function basket and Dfos enhance the highwire anatomical phenotype while expression of dominant negative basket and Dfos suppress the highwire phenotype. As a result we have compared the signaling pathway in flies and worms and found that the NMJ in the two organisms use a parallel pathway while the central synapse uses a distinct pathway.

Key to our understanding of growth regulation in Drosophila would be discovering a ligand that could regulate steroid synthesis. Activins are involved in regulating steroid hormone release in vertebrates. In invertebrates, they most likely function to keep ecdysone levels low to allow the larvae more time to achieve critical weight in order to initiate the metamorphic process. TGF-B(Transforming Growth Factor Beta) is a family of cytokine growth factors. We find that two members of the TGF-B signaling pathway Drosophila Activin (dACT) and Activin-like ligand Dawdle (DAW) signal through the type I receptor Baboon (BABO) and the type II receptor PUNT to primarily activate the transcription factor dSMAD2 and MAD to a lesser extent. One transcription factor brinker (brk) appears to be central to dACT signaling.

Tumor cells are characterized by an increase in genomic instability, brought about by both chromosomal rearrangement and chromosomal instability. Both of these broad changes can be induced by exposure to carcinogens. During mitosis, cells can exhibit early and late lagging chromosomes, multipolar spindles or anaphase bridges, all of which contribute to genomic rearrantement. We have studied the link between exposure to carcinogen and prevalence of mitotic defect in both chromosomally stable and unstable cell lines as well as ecamined the restorative effects of antioxidants in preventing mitotic defects. We have exposed MES-SA uterine cancer cells to vinyl chloride followed by exposure to an antioxidant : ascorbic acid, B-carotene, or lycopene. Treated cells were then scored for the prevalence of mitotic defects within the population and compared to controls. We have also investigated whether pre-treatment with the antioxidants will weaken the effects of carcinogen exposure in these cell lines.