Patrick Chang

Fertilization kinetics of sea urchin eggs

Abstract Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagstrom clarifies some discrepancies between and within these papers.

Organisation of Xenopus oocyte and egg cortices

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‐defined “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. Microsc. Res. Tech. 44:415–429, 1999.

Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

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.

Polymorphic phase behavior of fluorocarbon double-chain phosphocholines derived from diaminopropanol, serine and ethanolamine and long-term shelf stability of their liposomes

Abstract This paper describes the morphological characterization, by freeze-fracture electron microscopy, and the thermotropic phase behavior, by differential scanning calorimetry, of aqueous dispersions of various fluorocarbon/fluorocarbon or mixed fluorocarbon/hydrocarbon double-chain 2,3- or 1,3-diamidopropano-, diamidoserino- or amidoethyl-phosphocholines (PC). These fluorinated PCs form lamellar phases and liposomes, as their ester or ether glycerol-based analogs. The effects on the thermodynamic parameters associated with these lamellar phases of several structural elements — chemical junction (secondary CONHR or tertiary CONRR), connecting unit (diaminopropanol, serine or ethanolamine), number and length of the perfluoroalkylated chains, length of the fluorinated tail and hydrocarbon — are discussed. These data are compared with those reported for fluorocarbon or hydrocarbon glycerophosphocholines and hydrocarbon diamidophosphocholines, and for sphingomyelins. Most of the liposomes formed from the fluorinated amido-connected PCs display a remarkable long-term shelf stability: they can be thermally sterilized and stored at room temperature for several months without any significant modification of their size and size distribution.

Les bolaphiles bi(Falkylés) de type cationique à espaceur court: aptitude à la vésiculation en solution aqueuse après sonication

Abstract The production of vesicular membranes in aqueous solution by a new series of bis (polyfluoroalkylated) bis (ammonium) compounds with a short spacer length, i.e. [R F C 2 H 4 XCOCH 2 N(CH 3 ) 2 (CH 2 ) n N(CH 3 ) 2 CH 2 OCXC 2 H 4 R F ] 2+ , 2Br − , has been studied. Of these compounds, it is shown by freeze-fracture electron microscopy that after sonication two amphiphiles produce small unilamellar vesicles in water. Dynamic light scattering (DLS) experiments have been used to characterise their mean hydrodynamic radii.

Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis.

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.

2 Cytoskeleton and Ctenophore Development

Publisher Summary Ctenophores are gelatinous marine organisms characterized by rows of beating “combs” of cilia. They are related only very distantly to other animals. Although their precise evolutionary position is controversial, it is clear that they diverged very early from other metazoans. The ctenophore egg is a beautiful eukaryotic cell containing dynamic microtubule and actin filament networks with organelles shuffling around them. During the cleavage stages of development, a stereotypic asymmetric cleavage pattern produces cells with distinct shapes, sizes, and composition. Associated with these cleavages are dramatic cytoplasmic reorganizations, which segregate components between different cells. Distinct types of blastomere from the early embryo develop autonomously to give particular adult structures, implying that different “developmental potentials” become segregated at each cleavage. The challenge is to understand the relationship between the cytoplasmic events and the attribution of developmental potential to different cells. This chapter shows that Beroe ovatu eggs and embryos are ideal for detailed microscopy, and investigates the cell biology of early developmental events. The relationship between the behavior of the microtubule network and cytoplasmic relocation is examined and the findings and outlines the possibilities for future study in the context of embryological knowledge are summarized.