Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Evolution of developmental gene regulatory networks in echinoids

Developmental gene regulatory networks (dGRNs) are assemblages of regulatory genes that direct embryonic development of animal body plans and their morpho-logical structures. dGRNs exhibit recursively-wired circuitry that is encoded in the genome and executed during development. Alteration to the regulatory architecture of dGRNs causes variation in developmental programs both during the development of an individual organism and during the evolution of an individual lineage. The ex-planatory power of these networks is best exemplified by the global dGRN directing early development of the euechinoid sea urchin Strongylocentrotus purpuratus. This network consists of numerous regulatory genes engaging in hundreds of genomic regulatory transactions that collectively direct the delineation of early embryonic domains and the specification of cell lineages. Research on closely-related euechi-noid sea urchins, e.g. Lytechinus variegatus and Paracentrotus lividus, has revealed marked conservation of dGRN architecture in echinoid development, suggesting little appreciable alteration has occurred since their divergence in evolution at least 90 million years ago (mya).

We sought to test whether this observation extends to all sea urchins (echinoids) and undertook a systematic analysis of over 50 regulatory genes in the cidaroid sea urchin Eucidaris tribuloides, surveing their regulatory activity and function in a sea urchin that diverged from euechinoid sea urchins at least 268 mya. Our results revealed extensive alterations have occurred to all levels of echinoid dGRN archi-tecture since the cidaroid-euechinoid divergence. Alterations to mesodermal sub-circuits were particularly striking, including functional di˙erences in specification of non-skeletogenic mesenchyme (NSM), skeletogenic mesenchyme (SM), and en-domesodermal segregation. Specification of endomesodermal embryonic domains revealed that, while their underlying network circuitry had clearly diverged, regu-latory states established in pregastrular embryos of these two groups are strikingly similar. Analyses of E. tribuloides specification leading to the estab-lishment of dorsal-ventral (aboral-oral) larval polarity indicated that regulation of regulatory genes expressed in mesodermal embryonic domains had incurred significantly more alterations than those expressed in endodermal and ectodermal domains. Taken together, this study highlights the ability of dGRN architecture to buffer extensive alterations in the evolution and early development of echinoids and adds further support to the notion that alterations can occur at all levels of dGRN architecture and all stages of embryonic development.

Analysis of a transcriptional network involving PU.1, Notch, and Gata3 in the lymphomyeloid lineage decision during early T-cell development

Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway.

Bis(thiophenolate)pyridine pincer ligands and trivalent zirconocene complexes relevant to early transition metal polymerization catalysts

Two major topics are covered: the first chapter is focused on the development of post-metallocene complexes for propylene polymerization. The second and third chapters investigate the consequences of diisobutylaluminum hydride (HAlⁱBu₂) additives in zirconocene based polymerization systems.

The synthesis, structure, and solution behavior of early metal complexes with a new tridentate LX₂ type ligand, bis(thiophenolate)pyridine ((SNS) = (2-C₆H₄S)₂-2,6-C₅H₃N) are investigated. SNS complexes of Ti, Zr, and Ta having dialkylamido coligands were synthesized and structurally characterized. The zirconium complex, (SNS)Zr(NMe₂)₂, displays C₂ symmetry in the solid state. Solid-state structures of tantalum complexes (SNS)Ta(NMe₂)₃ and (SNS)TaCl(NEt₂)₂ also display pronounced C₂ twisting of the SNS ligand. 1D and 2D NMR experiments show that (SNS)Ta(NMe₂)₃ is fluxional with rotation about the Ta N(amide) bonds occurring on the NMR timescale. The fluxional behavior of (SNS)TaCl(NEt₂)₂ in solution was also studied by variable temperature ¹H NMR. Observation of separate signals for the diastereotopic protons of the methylene unit of the diethylamide indicates that the complex remains locked on the NMR timescale in one diastereomeric conformation at temperatures below -50 °C.

Reduction of Zr(IV) metallocenium cations with sodium amalgam (NaHg) produces EPR signals assignable to Zr(III) metallocene complexes. Thus, chloro-bridged heterobinuclear ansa-zirconocenium cation [((SBI))Zr(μ-Cl)₂AlMe₂]⁺B(C₆F₅)_4¯ (SBI = rac-dimethylsilylbis(1-indenyl)), gives rise to an EPR signal assignable to the complex (SBI)Zr^III(μ-Cl)₂AlMe₂, while (SBI)Zr^III-Me and (SBI)Zr^III(-H)2AlⁱBu₂ are formed by reduction of [(SBI)Zr(μ-Me)₂AlMe₂]⁺B(C₆F₅)_4¯ and [(SBI)Zr(μ-H)₃(AlⁱBu₂)₂]⁺B(C₆F₅)4¯, respectively. These products are also formed, along with (SBI)Zr^III-ⁱBu and [(SBI)Zr^III]⁺ AlR4¯ when (SBI)ZrMe₂ reacts with HAlⁱBu₂, eliminating isobutane en route to the Zr(III) complex. Studies concerning the interconversion reactions between these and other (SBI)Zr(III) complexes and reaction mechanisms involved in their formation are also reported.

The addition of HAlⁱBu₂ to precatalyst [(SBI)Zr(µ-H)₃(AlⁱBu₂)₂]⁺ significantly slows the polymerization of propylene and changes the kinetics of polymerization from 1st to 2nd order with respect to propylene. This is likely due to competitive inhibition by HAlⁱBu₂. When the same reaction is investigated using [(ⁿBuCp)₂Zr(μ-H)₃(AlⁱBu₂)₂]⁺, hydroalumination between propylene and HAlⁱBu₂ is observed instead of propylene polymerization.

Temporal and spacial control of RNA stability in the early embryo of Drosophila melanogaster

Early embryogenesis in metazoa is controlled by maternally synthesized products. Among these products, the mature egg is loaded with transcripts representing approximately two thirds of the genome. A subset of this maternal RNA pool is degraded prior to the transition to zygotic control of development. This transfer of control of development from maternal to zygotic products is referred to as the midblastula transition (or MBT). It is believed that the degradation of maternal transcripts is required to terminate maternal control of development and to allow zygotic control of development to begin. Until now this process of maternal transcript degradation and the subsequent timing of the MBT has been poorly understood. I have demonstrated that in the early embryo there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is triggered by egg activation, and is targeted to specific classes of mRNAs through cis-acting elements in the 3' untranslated region (UTR}. The second pathway is activated 2 hr after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT. In addition, some transcripts fail to degrade at select subcellular locations adding an element of spatial control to RNA degradation. The spatial control of RNA degradation is achieved by protecting, or masking, transcripts from the degradation machinery. The RNA degradation and protection events are regulated by distinct cis-elements in the 3' untranslated region (UTR). These results provide the first systematic dissection of this highly conserved process in development and demonstrate that RNA degradation is a novel mechanism used for both temporal and spatial control of development.

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.