Sexual Reproduction and Early Development in the Estuarine Sea Anemone, Nematostella vectensis Stephenson, 1935

This dissertation: 1) determines the factor(s) responsible for spawning induction in NematosteJla vectensis; 2) isolates, describes, and documents the source of jelly from egg masses of N. vectensis; and 3) describes N. vectensis' early development. Namatostella vectensis were maintained on a 7-day mussel feeding/water change regime over 159 days. Within 36 hours of mussel feeding/water change. 69.1% of females and 78.5% of males spawned reliably. Through manipulation of feeding, water change, oxygen and nitrogenous waste concentrations, spawning induction was found to be triggered by the oxygen concentration associated with water change, and not by feeding. Ammonia, anemones' major waste product, inhibited this induction in a concentration-dependent manner. Female N. vectensis release eggs in a persistent jellied egg mass which is unique among the Actiniaria. The major component of this egg mass jelly was a positive periodic acid-Schiffs staining, 39.5-40.5 kD glycoprotein. Antibodies developed in rabbits against this glycoprotein bound to jelly of intact egg masses and to granules (~ 2.8 IJm in diameter) present in female anemone mesenteries and their associated filaments. Antibodies did not label male tissues. Nematostella vecfensis embryos underwent first karyokinesis -60 minutes following the addition of sperm to eggs. Second nuclear division took place, followed by first cleavage, 90-120 minutes later. Each of the 4 blastomeres that resulted from first cleavage contained a single nucleus. Arrangement of these blastomeres ranged from radial to pseudospiral. Embryonic development was both asynchronous and holoblastic. Following formation of the 4-cell stage, 71% of embryos proceeded to cleave again to form an 8-cell stage. In each of the remaining 29% of embryos, a fusion of from 2-4 blastomeres resulted in 4 possible patterns which had no affect on either cleavage interval timing or subsequent development. The fusion event was not due to ooplasmic segregation. Blastomeres isolated from 4-celled embryos were regulative and developed into normal planula larvae and juvenile anemones that were 1/4 the size of those that developed from intact 4-celled embryos. Embryos exhibiting the fusion phenomenon were examined at the fine structural level. The fusion phenomenon resulted in formation of a secondary syncytium and was not a mere compaction of blastomeres.

Mantenimiento en el laboratorio del pez cebra

El pez cebra (Danio rerio) se utiliza como organismo modelo en distintos campos como la biomedicina o la toxicogenómica. Las ventajas que ofrece frente a otros modelos animales, hace que se haya convertido en los últimos años en uno de los organismos modelo más utilizado en experimentación. La similitud de su desarrollo embrionario con el de otros vertebrados superiores o la semejanza de su genoma con la del ser humano lo han colocado en el punto de mira de las principales investigaciones. Pero disponer de ejemplares óptimos tanto de embriones como de adultos para su utilización en investigación, requiere de unos cuidados y de una atención adecuada. Para su mantenimiento se deberán tener en cuenta el tipo de instalaciones para su cría, la calidad del agua donde se encuentran, la alimentación que reciben o los procesos de reproducción utilizados. El objetivo de este estudio ha sido elaborar un protocolo de mantenimiento del pez cebra, que optimice los cuidados necesarios para que el número de embriones viables sea máximo y también poder mantener un número de ejemplares adultos en buenas condiciones.

Marine Ranching: Proceedings of the Eighteenth U.S.-Japan Meeting on Aquaculture Port Ludlow, Washington, 18-19 September 1989

Female reproduction in penaeid shrimp is carefully regulated by several different endocrine factors. Their precise modes of action have not yet been fully elucidated. Three endocrine factors, each representing a different chemical class of hormones, have been investigated in the penaeid shrimp Sicyonia ingentis in our laboratory: ecdysteroids, vitellogenesis-inhibiting hormone (VIH) , and methyl farnesoate (MF). Ecdysteroids (the steroid molting hormones of arthropods; predominantly 20-hydroxyecdysone), are initially present in low levels (<10 ng/mg) in shrimp embryos. As development of the embryos nears time of hatch, the ecdysteroid levels increase to approximately 150 ng/ mg, indicating that they may be of embryonic origin and involved in embryonic development. An assay was developed for shrimp VIH, which presumably is a protein. Delay of onset of the next reproductive cycle was observed following injection of sinus gland extracts into shrimp that had previously had their eyestalks removed. A photoaffinity analog was synthesized for the putative shrimp reproductive hormone MF-a terpenoid. This analog, farnesyl diazomethyl ketone (FDK) , was used to demonstrate the presence of specific binding proteins for MF in shrimp hemolymph. (PDF file contains 136 pages.)

Additions to a revision of the shark genus Carcharhinus: Synonymy of Aprionodon and Hypoprion, and description of a new species of Carcharhinus (Carcharhinidae)

Features of the valid nominal species of Aprionodon Gill (isodon Valenciennes) and Hypoprion Muller and Henle (hemiodon Valenciennes, macloti Muller and Henle, and signatus Poey), plus those of a previously unrecognized species here described as Carcharhinus leiodon n.sp., are examined and compared with those of Carcharhinus Blainville. Features studied include morphometrics, vertebral numbers and other vertebral characteristics, tooth numbers, color pattern, and some other aspects of external morphology. It is concluded that on these features C. leiodon n.sp. is entirely encompassed within the parameters of Carcharhinus, and that, although A. isodon, H. hemiodon, H. macloti, and H. signatus each extend the range of diversity of Carcharhinus in one or more features, A. isodon is not uniquely different from Carcharhinus, and there is no common pattern of difference between the three species of Hypoprion and Carcharhinus. Accordingly, and because the nature of the teeth of Aprionodon and Hypoprion has been found insufficient to warrant generic distinction from Carcharhinus, the genera Aprionodon and Hypoprion are synonymised with Carcharhinus. A diagnosis and description are given for each of the above species. The descriptions include measurements, counts, and line illustrations that show the whole shark in lateral view, underside of head, nostril, and teeth. The geographic distribution is summarized, as are also the meager biological data available on number of embryos, size at birth, size at sexual maturity, and maximum size. (PDF file contains 32 pages.)

Auswirkungen der Wassertemperatur auf die Missbildungshäufigkeit pelagischer Fischembryonen

Malformation rates in embryos of dab, whiting, cod, flounder and plaice have been monitored for several years (1984-2006) in the Southern North Sea. For embryos of all species investigated trends for the fluctuation of malformation rates over the time were registered in the areas showing intermediate prevalences at the beginning of the studies in 1984 and maxima in 1987. Thereafter for all species a decrease of malformation rates was found until 2006 excepting an increase in 1996. A significant negative correlation existed between surface water temperature and prevalences of malformed embryos of dab and other species.

Der Fischei-Test: Ein Toxizitätstest für ökotoxikologische Untersuchungen

Zebrafish (Danio rerio) embryos have been used to quantify the teratogenic potential of environmental samples and harmful substances respectively. The short spawning interval renders this species a good test organism in toxicological research. Due to the transparency of the eggs several lethal and non-lethal endpoints can be detected in parallel after 48 h of embryonic development. Zebrafishembryos have been shown to be sensitive to a number of environmental relevant contaminants, as well as to ex-tracts from polluted sediments

Zusammenhang zwischen Schadstoffen aus der Elbe und missgebildeten Fischembryonen in der Deutschen Bucht.

The trends of malformation prevalence in embryos of dab, Limanda limanda, in the southern North Sea after the year 1990 mirrored the drop in major pollutants in the rivers draining into the German Bight. Despite this general decline we detected a pollution event in the southern North Sea in winter 1995/1996 employing the prevalence of malformations in dab embryos as an indicator. An abrupt rise in malformation prevalence in the embryos of dab, corresponded to a dramatic increase in DDT levels in parent fish from the same area, indicating a hitherto unnoticed introduction of considerable quantities of DDT into the system. This input could be traced back to discharges of unknown origen into the River Elbe.

Embryonic development in Clarias gariepinus under laboratory conditions

The embryonic development in Clarias gariepinus was studied under laboratory conditions. The developmental stages of eggs starting from first cleavage were examined microscopically. Photomicroscope was used to take important stages of segmentation, blastulation, differentiation of embryo and hatching. The films of the photograph were developed and printed for each stage produced. The accurate timing and detailed description of each stage was done. The results show that the blastodisc (Polar cap) appeared about 35 minutes after fertilization and the first cleavage dividing the blastodisc into two blastomeres occurs 15 minutes after polar cap formation. Details of the developmental stages of embryos and the timing from one stage to the other were described. The larva shook off the shell and emerged completely from the egg case about 22 hours after fertilization at a water temperature of 25.1 degree C. The accurate determination of the time of initiation of first mitosis is of great importance in fish culture and breeding especially in the production of tetraploids

Some potential occupational and environmental hazards associated with fish pond production in Nigeria

The paper highlights the concept of information and the significance of environmental and occupational hazards associated with pond fish production in Nigeria and discuss the possible options for the ways forward. The major raw material used in fish production system is the organic manure (cow dung, poultry droppings, porcine manure etc) that serves as substrate for heterotrophic production of bacteria and protozoa, which act as food for zooplankton and the fish. The pathogenic organisms (viruses, bacteria, protozoa's, and parasites), are noted for the potential hazard to the fish handlers and consumers. Nine species from seven genera of bacteria associated with fish diseases are found to have association with diseases of human such as typhoid fever, bacillary dysentery and other gastrointestinal tract related problems. Also the environmental contaminants in pond fish production become important because of its significance to consumers' acceptance of the fish products

Characterization and developmental regulation of a gene expressed specifically in the skeletogenic lineage of the sea urchin embryo

The sea urchin embryonic skeleton, or spicule, is deposited by mesenchymal progeny of four precursor cells, the micromeres, which are determined to the skeletogenic pathway by a process known as cytoplasmic localization. A gene encoding one of the major products of the skeletogenic mesenchyme, a prominent 50 kD protein of the spicule matrix, has been characterized in detail. cDNA clones were first isolated by antibody screening of a phage expression library, followed by isolation of homologous genomic clones. The gene, known as SM50, is single copy in the sea urchin genome, is divided into two exons of 213 and 1682 bp, and is expressed only in skeletogenic cells. Transcripts are first detectable at the 120 cell stage, shortly after the segregation of the skeletogenic precursors from the rest of the embryo. The SM50 open reading frame begins within the first exon, is 450 amino acids in length, and contains a loosely repeated 13 amino acid motif rich in acidic residues which accounts for 45% of the protein and which is possibly involved in interaction with the mineral phase of the spicule.

The important cis-acting regions of the SM50 gene necessary for proper regulation of expression were identified by gene transfer experiments. A 562 bp promoter fragment, containing 438 bp of 5' promoter sequence and 124 bp of the SM50 first exon (including the SM50 initiation codon), was both necessary and sufficient to direct high levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene specifically in the skeletogenic cells. Removal of promoter sequences between positions -2200 and -438, and of transcribed regions downstream of +124 (including the SM50 intron), had no effect on the spatial or transcriptional activity of the transgenes.

Regulatory proteins that interact with the SM50 promoter were identified by the gel retardation assay, using bulk embryo mesenchyme blastula stage nuclear proteins. Five protein binding sites were identified and mapped to various degrees of resolution. Two sites are homologous, may be enhancer elements, and at least one is required for expression. Two additional sites are also present in the promoter of the aboral ectoderm specific cytoskeletal actin gene CyIIIa; one of these is a CCAA T element, the other a putative repressor element. The fifth site overlaps the binding site of the putative repressor and may function as a positive regulator by interfering with binding of the repressor. All of the proteins are detectable in nuclear extracts prepared from 64 cell stage embryos, a stage just before expression of SM50 is initiated, as well as from blastula and gastrula stage; the putative enhancer binding protein may be maternal as well.

Wassertemperaturen und Häufigkeiten von Mißbildungen pelagischer Fischembryonen

Malformation rates in fish embryos have been monitored for several years in the Southern North Sea. Their occurrence was interpreted to be related to pollution because malformation rates were highest in near coastal waters known to receive high pollution loads. For embryos of all species investigated synchronous trends for the fluctuation of malformation rates over the time were registered in the areas covered with intermediate prevalences at the beginning of the studies in 1984 and maxima in 1987. Thereafter malformation rates of all species decreased significantly followed by an increase in 1996. It was found that a significant negative correlation between surface water temperature and prevalences of malformed embryos of dab (Limanda limanda) and other species existed over time and space. These correlations became increasingly visible with decreasing concentrations of organochlorines in livers of dab. From these findings it is concluded that temperatures possibly predispose developing fish embryos to the impact of pollutants.

Experimente über die Wirkung von UV-B auf pelagische Fischembryonen in einem Sonnenscheinsimulator

One of the supposed effects of the observed ozone depletion is the increase of solar UV-B irradiation at the seasurface. This will cause an impact on certain compartments of marine ecosystems. Especially, sensitive developmental stages of pelagic fish embryos might be affected. Embryos of dab (Limanda limanda) and plaice (Pleuronectes plalessa) were experimentally exposed 10 different amounts of UVB irradiation in a sunshine simulator. This programmable device allows the dosage of realistic solar irradiation in quality and guantity. Experiments were carried out in March 1995 and February 1996. Either artificially inserninated and reared emhryos of dab and plaice or embryos caught in the German Bight were exposed to simulated solar irradiation. The 1995 experiments served to identify the effective irradiation dosages. For the 1996 experiments irradiation applied was much lower, being dose to realistic valucs expected over the North Sea as a consequence of ozone depletion. The following end points were studied: 1. Mortality, 2. sublethal morphological effects (malformations), 3. DNA damage, 4. changes in buoyancy of embryos measured as changes in osmolarity of the perivitelline fluid. Conditions for the simulation of daylight were a c1oudless sky with a solar zenith distance of 34 % (air mass 1.2). The adopted ozone depletion was 40 % corresponding to 180 DU (Dobson Units) instead of 300 DU. In the 1995 experiments time and dosage dependent influenccs on mortality and buoyancy of embryos of dab and plaice were found. Even in those embryos which were protected from the UV-B spectral range a loss of buoyancy was registered after 12 hours in the simulator. No diffcrences in DNA integrity as determined by DNA unwinding of exposed and control embryos were found. Also with lower amounts of irradiation in the 1996 experiments dosage dependent acute mortality, malformations, and impact on the buoyancy of the emhryos was registered. Sublethal effects occurred as well in embryos protected against UV-B in the exposure chambers, but were not found in the dark controls. The impact of low dosages of UV-B on the buoyancy of pelagic fish embryos might indicate an important ecological threat and deserves further studies.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

MEMS for Diabetic Retinopathy

As the worldwide prevalence of diabetes mellitus continues to increase, diabetic retinopathy remains the leading cause of visual impairment and blindness in many developed countries. Between 32 to 40 percent of about 246 million people with diabetes develop diabetic retinopathy. Approximately 4.1 million American adults 40 years and older are affected by diabetic retinopathy. This glucose-induced microvascular disease progressively damages the tiny blood vessels that nourish the retina, the light-sensitive tissue at the back of the eye, leading to retinal ischemia (i.e., inadequate blood flow), retinal hypoxia (i.e., oxygen deprivation), and retinal nerve cell degeneration or death. It is a most serious sight-threatening complication of diabetes, resulting in significant irreversible vision loss, and even total blindness.

Unfortunately, although current treatments of diabetic retinopathy (i.e., laser therapy, vitrectomy surgery and anti-VEGF therapy) can reduce vision loss, they only slow down but cannot stop the degradation of the retina. Patients require repeated treatment to protect their sight. The current treatments also have significant drawbacks. Laser therapy is focused on preserving the macula, the area of the retina that is responsible for sharp, clear, central vision, by sacrificing the peripheral retina since there is only limited oxygen supply. Therefore, laser therapy results in a constricted peripheral visual field, reduced color vision, delayed dark adaptation, and weakened night vision. Vitrectomy surgery increases the risk of neovascular glaucoma, another devastating ocular disease, characterized by the proliferation of fibrovascular tissue in the anterior chamber angle. Anti-VEGF agents have potential adverse effects, and currently there is insufficient evidence to recommend their routine use.

In this work, for the first time, a paradigm shift in the treatment of diabetic retinopathy is proposed: providing localized, supplemental oxygen to the ischemic tissue via an implantable MEMS device. The retinal architecture (e.g., thickness, cell densities, layered structure, etc.) of the rabbit eye exposed to ischemic hypoxic injuries was well preserved after targeted oxygen delivery to the hypoxic tissue, showing that the use of an external source of oxygen could improve the retinal oxygenation and prevent the progression of the ischemic cascade.

The proposed MEMS device transports oxygen from an oxygen-rich space to the oxygen-deficient vitreous, the gel-like fluid that fills the inside of the eye, and then to the ischemic retina. This oxygen transport process is purely passive and completely driven by the gradient of oxygen partial pressure (pO₂). Two types of devices were designed. For the first type, the oxygen-rich space is underneath the conjunctiva, a membrane covering the sclera (white part of the eye), beneath the eyelids and highly permeable to oxygen in the atmosphere when the eye is open. Therefore, sub-conjunctival pO₂ is very high during the daytime. For the second type, the oxygen-rich space is inside the device since pure oxygen is needle-injected into the device on a regular basis.

To prevent too fast or too slow permeation of oxygen through the device that is made of parylene and silicone (two widely used biocompatible polymers in medical devices), the material properties of the hybrid parylene/silicone were investigated, including mechanical behaviors, permeation rates, and adhesive forces. Then the thicknesses of parylene and silicone became important design parameters that were fine-tuned to reach the optimal oxygen permeation rate.

The passive MEMS oxygen transporter devices were designed, built, and tested in both bench-top artificial eye models and in-vitro porcine cadaver eyes. The 3D unsteady saccade-induced laminar flow of water inside the eye model was modeled by computational fluid dynamics to study the convective transport of oxygen inside the eye induced by saccade (rapid eye movement). The saccade-enhanced transport effect was also demonstrated experimentally. Acute in-vivo animal experiments were performed in rabbits and dogs to verify the surgical procedure and the device functionality. Various hypotheses were confirmed both experimentally and computationally, suggesting that both the two types of devices are very promising to cure diabetic retinopathy. The chronic implantation of devices in ischemic dog eyes is still underway.

The proposed MEMS oxygen transporter devices can be also applied to treat other ocular and systemic diseases accompanied by retinal ischemia, such as central retinal artery occlusion, carotid artery disease, and some form of glaucoma.