Molecular neurogenetics of eye development in Drosophila

The compound eye of Drosophila melanogaster begins to differentiate during the late third larval instar in the eye-antennal imaginal disc. A wave of morphogenesis crosses the disc from posterior to anterior, leaving behind precisely patterned clusters of photoreceptor cells and accessory cells that will constitute the adult ommatidia of the retina. By the analysis of genetically mosaic eyes, it appears that any cell in the eye disc can adopt the characteristics of any one of the different cell types found in the mature eye, including photoreceptor cells and non-neuronal accessory cells such as cone cells. Therefore, cells within the prospective retinal epithelium assume different fates presumably via information present in the environment. The sevenless^+ (sev^+) gene appears to play a role in the expression of one of the possible fates, since the mutant phenotype is the lack of one of the pattern elements, namely, photoreceptor cell R7. The sev^+ gene product had been shown to be required during development of the eye, and had also been shown in genetic mosaics to be autonomous to presumptive R7. As a means of better understanding the pathway instructing the differentiation R7, the gene and its protein product were characterized.

The sev+ gene was cloned by P-element transposon tagging, and was found to encode an 8.2 kb transcript expressed in developing eye discs and adult heads. By raising monoclonal antibodies (MAbs) against a sev^+- β-galactosidase fusion protein, the expression of the protein in the eye disc was localized by immuno-electronmicroscopy. The protein localizes to the apical cell membranes and microvilli of cells in the eye disc epithelium. It appears during development at a time coincident with the initial formation of clusters, and in all the developing photoreceptors and accessory cone cells at a time prior to the overt differentiation of R7. This result is consistent with the pluripotency of cells in the eye disc. Its localization in the membranes suggests that it may receive information directing the development of R7. Its localization in the apical membranes and microvilli is away from the bulk of the cell contacts, which have been cited as a likely regions for information presentation and processing. Biochemical characterization of the sev^+ protein will be necessary to describe further its role in development.

Other mutations in Drosophila have eye phenotypes. These were analyzed to find which ones affected the initial patterning of cells in the eye disc, in order to identify other genes, like sev, whose gene products may be involved in generating the pattern. The adult eye phenotypes ranged from severe reduction of the eye, to variable numbers of photoreceptor cells per ommatidium, to sub de defects in the organization of the supporting cells. Developing eye discs from the different strains were screened using a panel of MAbs, which highlight various developmental stages. Two identified matrix elements in and anterior to the furrow, while others identified the developing ommatidia themselves, like the anti-sev MAb. Mutation phenotypes were shown to appear at many stages of development. Some mutations seem to affect the precursor cells, others, the setting up of the pattern, and still others, the maintenance of the pattern. Thus, additional genes have now been identified that may function to support the development of a complex pattern.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Mitigating Scarring and Inflammation during Corneal Wound Healing using Nanofiber-Hydrogel Scaffolds

Due to the universal lack of donor tissue, there has been emerging interest in engineering materials to stimulate living cells to restore the features and functions of injured organs. We are particularly interested in developing materials for corneal use, where the necessity to maintain the tissue’s transparency presents an additional challenge. Every year, there are 1.5 – 2 million new cases of monocular blindness due to irregular healing of corneal injuries, dwarfing the approximately 150,000 corneal transplants performed. The large gap between the need and availability of cornea transplantation motivates us to develop a wound-healing scaffold that can prevent corneal blindness.

To develop such a scaffold, it is necessary to regulate the cells responsible for repairing the damaged cornea, namely myofibroblasts, which are responsible for the disordered and non-refractive index matched scar that leads to corneal blindness. Using in vitro assays, we identified that protein nanofibers of certain orientation can promote cell migration and modulate the myofibroblast phenotype. The nanofibers are also transparent, easy to handle and non-cytotoxic. To adhere the nanofibers to a wound bed, we examined the use of two different in situ forming hydrogels: an artificial extracellular matrix protein (aECM)-based gel and a photo-crosslinkable heparin-based gel. Both hydrogels can be formed within minutes, are transparent upon gelation and are easily tunable.

Using an in vivo mouse model for epithelial defects, we show that our corneal scaffolds (nanofibers together with hydrogel) are well-tolerated (no inflammatory response or turbidity) and support epithelium regrowth. We developed an ex vivo corneal tissue culture model where corneas that are wounded and treated with our scaffold can be cultured while retaining their ability to repair wounds for up to 21 days. Using this technique, we found that the aECM-based treatment induced a more favorable wound response than the heparin-based treatment, prompting us to further examine the efficacy of the aECM-based treatment in vivo using a rabbit model for stromal wounds. Results show that treated corneas have fewer myofibroblasts and immune cells than untreated ones, indicating that our corneal scaffold shows promise in promoting a calmer wound response and preventing corneal haze formation.

The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish

Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively.