Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3′UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA.

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.

Light:dark (12:12 h) quantification of cellular carbohydrate fluxes was conducted with the unicellular, N2-fixing cyanobacterium Crocosphaera watsonii WH8501 grown in continuous cultures under conditions of diazotrophy. As commonly observed in photoautotrophs, C. watsonii accumulates photosynthetic products as carbohydrates, which thereafter serve as an internal energy reserve for energy-demanding processes. Our goal was to further discriminate between cellular pools with different sizes to observe their respective dynamics. Total cellular carbohydrate as well as the cellular carbohydrate pools showed a diel cycle of accumulation and decrease. Total cellular carbohydrate contents represented 45 and 32% of the cellular carbon content at the end of the light and dark period, respectively. The observed decrease in cellular carbohydrate was not only due to carbohydrate catabolism but also to their release in the environment as extracellular polymeric substances (EPS), including soluble EPS and transparent exopolymeric particles (TEP). The occurrence of such excretion during the exponential growth of C. watsonii appears as part of the metabolic regulations that participate in maintaining a daily carbon:nitrogen balanced growth. About 10% of the total carbon cell content is excreted daily as soluble EPS. TEP, which are formed from dissolved precursors (including some soluble EPS) released by C. watsonii, increased during the dark period, when they represented up to 3.6% of total carbon cell content. Such recurrent release of carbohydrate by populations of C. watsonii is expected to stimulate the microbial loop in warm, oligotrophic oceans.

Background Myosin II (or Myosin Heavy Chain II, MHCII) is a family of molecular motors involved in the contractile activity of animal muscle cells but also in various other cellular processes in non-muscle cells. Previous phylogenetic analyses of bilaterian MHCII genes identified two main clades associated respectively with smooth/non-muscle cells (MHCIIa) and striated muscle cells (MHCIIb). Muscle cells are generally thought to have originated only once in ancient animal history, and decisive insights about their early evolution are expected to come from expression studies of Myosin II genes in the two non-bilaterian phyla that possess muscles, the Cnidaria and Ctenophora. Results We have uncovered three MHCII paralogues in the ctenophore species Pleurobrachia pileus . Phylogenetic analyses indicate that the MHCIIa / MHCIIb duplication is more ancient than the divergence between extant metazoan lineages. The ctenophore MHCIIa gene ( PpiMHCIIa ) has an expression pattern akin to that of "stem cell markers" ( Piwi, Vasa …) and is expressed in proliferating cells. We identified two MHCIIb genes that originated from a ctenophore-specific duplication. PpiMHCIIb1 represents the exclusively muscular form of myosin II in ctenophore, while PpiMHCIIb2 is expressed in non-muscle cells of various types. In parallel, our phalloidin staining and TEM observations highlight the structural complexity of ctenophore musculature and emphasize the experimental interest of the ctenophore tentacle root, in which myogenesis is spatially ordered and strikingly similar to striated muscle formation in vertebrates. Conclusion MHCIIa expression in putative stem cells/proliferating cells probably represents an ancestral trait, while specific involvement of some MHCIIa genes in smooth muscle fibres is a uniquely derived feature of the vertebrates. That one ctenophore MHCIIb paralogue ( PpiMHCIIb2 ) has retained MHCIIa-like expression features furthermore suggests that muscular expression of the other paralogue, PpiMHCIIb1 , was the result of neofunctionalisation within the ctenophore lineage, making independent origin of ctenophore muscle cells a likely option.

Stem cells are essential for animal development and adult tissue homeostasis, and the quest for an ancestral gene fingerprint of stemness is a major challenge for evolutionary developmental biology. Recent studies have indicated that a series of genes, including the transposon silencer Piwi and the translational activator Vasa, specifically involved in germline determination and maintenance in classical bilaterian models (e.g., vertebrates, fly, nematode), are more generally expressed in adult multipotent stem cells in other animals like flatworms and hydras. Since the progeny of these multipotent stem cells includes both somatic and germinal derivatives, it remains unclear whether Vasa, Piwi, and associated genes like Bruno and PL10 were ancestrally linked to stemness, or to germinal potential. We have investigated the expression of Vasa, two Piwi paralogues, Bruno and PL10 in Pleurobrachia pileus, a member of the early-diverging phylum Ctenophora, the probable sister group of cnidarians. These genes were all expressed in the male and female germlines, and with the exception of one of the Piwi paralogues, they showed similar expression patterns within somatic territories (tentacle root, comb rows, aboral sensory complex). Cytological observations and EdU DNA-labelling and long-term retention experiments revealed concentrations of stem cells closely matching these gene expression areas. These stem cell pools are spatially restricted, and each specialised in the production of particular types of somatic cells. These data unveil important aspects of cell renewal within the ctenophore body and suggest that Piwi, Vasa, Bruno, and PL10 belong to a gene network ancestrally acting in two distinct contexts: (i) the germline and (ii) stem cells, whatever the nature of their progeny.

We have recently started to develop the hydrozoan jellyfish Clytia hemisphaerica as an experimental model for studying oogenesis. Clytia offers a fresh perspective on regulation of oogenesis and on its evolutionary history because of its phylogenetic position in the basally branching phulum Cnidaria, and by the simplicity, transparency and experimental accessibility of the female gonad. Here we describe the basic features of the Clytia system and our initial studies to characterise oogenesis. We also summarise recent studies concerning maternal mRNA localisation in the oocyte and the role of the Mos-MAP kinase pathway in oocyte maturation, as illustrations of the experimental possibilities offered by this promising system.

The active transport of iodide from the bloodstream into thyroid follicular cells is mediated by the Na+/I− symporter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference, we compared 125I uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2.5-fold lower for hNIS than mNIS. We also performed immunocytolocalization studies and observed that the subcellular distribution of the two orthologs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortholog was intracellular in ∼40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2.5-fold higher than that of the human protein, and therefore cannot alone account for the difference in the obtained Vmax values. We reasoned that the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein.

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.

We used gene over expression and gene knockdown to study the functional properties of CiMDF, the single myogenic regulatory factor gene (MRF) of Ciona intestinalis. Overexpression was achieved by injecting eggs with synthetic CiMDF mRNA. RNA-injected eggs did not develop typical tadpole morphology, but instead formed flattened balls of cells. Expression of the four muscle-specific genes assayed was strongly upregulated in muscle cells of injected embryos. Notably, these muscle-specific genes were also expressed in non-muscle lineage cells of injected embryos. This was especially apparent for troponin I, which was typically detected in all vegetal hemisphere cells of mRNA-injected embryos. Animal hemisphere cells of mRNA-injected embryos did not express muscle genes. We knocked down CiMDF expression by injecting eggs with antisense morpholino oligonucleotides (MO). MO-injected eggs often developed into quite normal tadpoles with clearly recognizable heads and tails; nevertheless, muscle-specific gene expression was reduced in these embryos compared to controls. Moreover, MO-treated larvae were almost always paralyzed and never showed the frenzied tail movements of normal larvae. Electron microscopy revealed that ‘‘muscle’’ cells of MO-treated larvae lacked the myofibrils seen in normal larval muscle. We conclude that CiMDF is a bona fide MRF because it elicits ectopic myogenesis, and that it is essential for tail muscle development. Awards from the NIH (1 R15 HD047357-01) and Rhode Island College to THM, and the Association Francaise contre les Myopathies (AFM) to HY supported this work.

In many animals, including Drosophila, C. elegans, zebrafish and Xenopus, the germ line is specified by maternal determinants localised in a distinct cytoplasmic structure called the germ plasm. This is consists of dense granules, mitochondria, and specific localised RNAs. We have characterised the expression and properties of the protein encoded by Xpat, an RNA localised to the germ plasm of Xenopus. Immunofluorescence and immunoblotting showed that this novel protein is itself a major constituent of germ plasm throughout oogenesis and early development, although it is also present in other regions of oocytes and embryos, including their nuclei. We found that an Xpat-GFP fusion protein can localise correctly in cultured oocytes, in early oocytes to the ‘mitochondrial cloud’, from which germ plasm originates, and in later oocytes to the vegetal cortex. The localisation process was microtubule-dependent, while cortical anchoring required microfilaments. Xpat-GFP expressed in late stage oocytes assembled into circular fields of multi-particulate structures resembling endogenous fields of germ plasm islands. Furthermore these structures could be induced to form at ectopic sites by manipulation of culture conditions. Ectopic Xpat-GFP islands were able to recruit mitochondria, a major germ plasm component. These data suggest that Xpat protein has an important role in Xenopus germ plasm formation, positioning and maintenance.

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.

WHAT IS THE CORTEX? The Egg Cortex The Scope of This Review STORING INFORMATION AND BUILDING THE CORTEX DURING OOGENESIS Localizing Maternal Determinants in the Cortex Structuring and Positioning Information in the Cortex Cortical Polarity and Storage of Information in Other Oocytes The Cortex during Vitellogenesis MATURATION PREPARES THE CORTEX FOR FERTILIZATION Restructuring Cortical Endoplasmic Reticulum and Organelles during Maturation Restructuring the Cytoskeleton during Maturation Relocalization of Cortical mRNAs and Domains during Maturation The Cortex as the Site of Attachment and Fusion of Sperm and Egg FERTILIZATION TRANSFORMS THE EGG CORTEX Sperm-Induced Calcium Waves and Membranes Waves of Exocytosis and Endocytosis at Fertilization Fertilization-Induced Microfilament Reorganizations The Fertilized Egg Cortex WHAT THE CORTEX IS The Cortex as a Repository for Spatiotemporal Information The Questions That Remain

The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accummulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-de fined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood. (C) 1999 Wiley-Liss, Inc.

Neuronal intranuclear inclusions are found in the brains of patients with Huntington’s disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin’s role in cell death.