Robert W. Zeller

The nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyury-regulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3′UTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development.

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ∼15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ∼20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating.

Here we present the ascidian Ciona intestinalis as an alternative invertebrate system to study Alzheimer’s disease (AD) pathogenesis. Through the use of AD animal models, researchers often attempt to reproduce various aspects of the disease, particularly the coordinated processing of the amyloid precursor protein (APP) by α-, β- and γ-secretases to generate amyloid beta (Aβ)-containing plaques. Recently, Drosophila and C. elegans AD models have been developed, exploiting the relative simplicity of these invertebrate systems, but they lack a functional Aβ sequence and a β-secretase ortholog, thus complicating efforts to examine APP processing in vivo. We propose that the ascidian is a more appropriate invertebrate AD model owing to their phylogenetic relationship with humans. This is supported by bioinformatic analyses, which indicate that the ascidian genome contains orthologs of all AD-relevant genes. We report that transgenic ascidian larvae can properly process human APP₆₉₅ to generate Aβ peptides. Furthermore, Aβ can rapidly aggregate to form amyloid-like plaques, and plaque deposition is significantly increased in larvae expressing a human APP₆₉₅ variant associated with familial Alzheimer’s disease. We also demonstrate that nervous system-specific Aβ expression alters normal larval behavior during attachment. Importantly, plaque formation and alterations in behavior are not only observed within 24 hours post-fertilization, but anti-amyloid drug treatment improves these AD-like pathologies. This ascidian model for AD provides a powerful and rapid system to study APP processing, Aβ plaque formation and behavioral alterations, and could aid in identifying factors that modulate amyloid deposition and the associated disruption of normal cellular function and behaviors.

Background: The draft genome sequence of the ascidian Ciona intestinalis, along with associated gene models, has been a valuable research resource. However, recently accumulated expressed sequence tag (EST)/cDNA data have revealed numerous inconsistencies with the gene models due in part to intrinsic limitations in gene prediction programs and in part to the fragmented nature of the assembly. Results: We have prepared a less-fragmented assembly on the basis of scaffold-joining guided by paired-end EST and bacterial artificial chromosome (BAC) sequences, and BAC chromosomal in situ hybridization data. The new assembly (115.2 Mb) is similar in length to the initial assembly (116.7 Mb) but contains 1,272 (approximately 50%) fewer scaffolds. The largest scaffold in the new assembly incorporates 95 initial-assembly scaffolds. In conjunction with the new assembly, we have prepared a greatly improved global gene model set strictly correlated with the extensive currently available EST data. The total gene number (15,254) is similar to that of the initial set (15,582), but the new set includes 3,330 models at genomic sites where none were present in the initial set, and 1,779 models that represent fusions of multiple previously incomplete models. In approximately half, 5'-ends were precisely mapped using 5'-full-length ESTs, an important refinement even in otherwise unchanged models. Conclusion: Using these new resources, we identify a population of non-canonical (non-GT-AG) introns and also find that approximately 20% of Ciona genes reside in operons and that operons contain a high proportion of single-exon genes. Thus, the present dataset provides an opportunity to analyze the Ciona genome much more precisely than ever.

Two customized electroporators were specifically designed for creating transgenic ascidian embryos. These electroporators were simple to build, inexpensive, and produced transgenic embryos with efficiencies that equaled or rivaled commercially available machines. A key design feature of these machines resulted in the generation of consistent electroporation pulses providing repeatability between experiments. These devices were used to optimize experimental parameters allowing for the creation of transient transgenic embryos with predictable patterns of mosaic transgene expression. We used these new electroporators to examine the expression of two different fluorescent protein reporter genes with regard to embryonic cell lineage. In general, transgene expression followed the embryonic cell lineage and coelectroporated transgenes were always expressed in the same embryonic cells. Our analysis also indicated that, during development, transgenes could be lost from embryonic cells, suggesting that transgenes may be present in extrachromosomal arrays, as has been observed in other organisms. Our new electroporator designs will allow ascidian researchers to inexpensively produce transgenic ascidians and should prove useful for adapting the electroporation technique to other marine embryo systems. Developmental Dynamics 235:1921–1932, 2006.

The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been “codon optimized” for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456–467, 2006.