Nathalie Oulhen

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ‐line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ‐line determination in echinoderms, an early‐branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ‐line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ‐line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser‐known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ‐line fate determination. This enables a strongly contrasted picture for germ‐line determination in this phylum, but one for which transitions between different modes of germ‐line determination might now be experimentally addressed. Mol. Reprod. Dev. 81: 679–711, 2014.

Deadenylase depletion protects inherited mRNAs in primordial germ cells

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a ‘time capsule’ model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

Dysferlin is essential for endocytosis in the sea star oocyte

Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function.

Simple perfusion apparatus for manipulation, tracking, and study of oocytes and embryos

OBJECTIVE To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow rates. Each oocyte and resultant embryo must remain spatially separated and defined. DESIGN Experimental laboratory study. SETTING University and academic center for reproductive medicine. PATIENT(S)/ANIMAL(S) Women with eggs retrieved for intracytoplasmic sperm injection (ICSI) cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. INTERVENTION(S) Real-time, longitudinal imaging of oocytes after fluorescent labeling, insemination, and viability tests. MAIN OUTCOME MEASURE(S) Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantification, precise metrics of fertilization, and embryonic development. RESULT(S) Single oocytes were longitudinally imaged after significant changes in media, markers, endocytosis quantification, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. CONCLUSION(S) We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus provides for careful, precise, and flexible handling of precious samples facilitating clinical IVF approaches.

Multidrug-resistant transport activity protects oocytes from chemotherapeutic agents and changes during oocyte maturation

OBJECTIVE To determine the multidrug-resistant transporter (MDR) activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. DESIGN Experimental laboratory study. SETTING University and academic center for reproductive medicine. SUBJECT(S) Women with eggs retrieved for intracytoplasmic sperm injection cycles and adult female FVBN and B6C3F1 mouse strains. INTERVENTION(S) Inhibition of MDR activity in oocytes. MAIN OUTCOME MEASURE(S) Efflux activity of MDRs with the use of quantitative fluorescent dye efflux, and oocyte cell death when exposed to chemotherapy. RESULT(S) Oocytes effluxed fluorescent reporters, and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. Geminal vesicle oocytes were more efficient at efflux than metaphase 2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death with the use of two different viability assays compared with control samples and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes, with the greatest accumulation in the geminal vesicle stage. CONCLUSION(S) Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis and that these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up-regulation of these transporters during gonadotoxic therapy.

Diversity in the fertilization envelopes of echinoderms.

Cell surface changes in an egg at fertilization are essential to begin development and for protecting the zygote. Most fertilized eggs construct a barrier around themselves by modifying their original extracellular matrix. This construction usually results from calcium‐induced exocytosis of cortical granules, the contents of which in sea urchins function to form the fertilization envelope (FE), an extracellular matrix of cortical granule contents built upon a vitelline layer scaffold. Here, we examined the molecular mechanism of this process in sea stars, a close relative of the sea urchins, and analyze the evolutionary changes that likely occurred in the functionality of this structure between these two organisms. We find that the FE of sea stars is more permeable than in sea urchins, allowing diffusion of molecules in excess of 2 megadaltons. Through a proteomic and transcriptomic approach, we find that most, but not all, of the proteins present in the sea urchin envelope are present in sea stars, including SFE9, proteoliaisin, and rendezvin. The mRNAs encoding these FE proteins accumulated most densely in early oocytes, and then beginning with vitellogenesis, these mRNAs decreased in abundance to levels nearly undetectable in eggs. Antibodies to the SFE9 protein of sea stars showed that the cortical granules in sea star also accumulated most significantly in early oocytes, but different from sea urchins, they translocated to the cortex of the oocytes well before meiotic initiation. These results suggest that the preparation for cell surface changes in sea urchins has been shifted to later in oogenesis, and perhaps reflects the meiotic differences among the species—sea star oocytes are stored in prophase of meiosis and fertilized during the meiotic divisions, as in most animals, whereas sea urchins are one of the few taxons in which eggs have completed meiosis prior to fertilization.

English translation of Heinrich Anton de Bary’s 1878 speech, ‘Die Erscheinung der Symbiose’ (‘De la symbiose’)

Die Erscheinung der Symbiose, meaning “the phenomenon of symbiosis” in English or “de la symbiose” in French, is a transcription of the 1878 lecture by the German botanist and mycologist Heinrich Anton de Bary in which he first used the term ‘symbiosis’ in a biological context. De Bary’s speech was published in 1879 in German, later to be translated into French; though only fragments of his speech are available in English. Translating de Bary’s lecture is timely because the field of symbiosis, especially with respect to microorganisms, is expanding and the importance of symbiosis is now recognized across the biological sciences. Researchers have now begun to sort through the early literature to uncover original thoughts pertaining to symbiotic interactions. We believe that having de Bary’s lecture accessible to researchers in English will help enhance interest in the history of symbioses, document de Bary’s pioneering contribution, and aid in establishing an understanding for whom the lecture was intended and when biological symbioses were first recognized. We present a short biography of Heinrich Anton de Bary, a full translation of his lecture, and conclude by briefly highlighting current endeavors in symbiosis research.

Retention of exogenous mRNAs selectively in the germ cells of the sea urchin requires only a 5′‐cap and a 3′‐UTR

The abundance of an mRNA in a cell depends on its overall rates of synthesis and decay. RNA stability is an important element in the regulation of gene expression, and is achieved by a variety of processes including specific recruitment of nucleases and RNAi‐associated mechanisms. These mechanisms are particularly important in stem cells, which, in many cases, have attenuated transcription. Here we report that exogenous mRNA injected into fertilized eggs of the sea urchin is selectively retained in the small micromeres, which contribute to the germ line in this organism, beginning in blastulae, when compared to adjacent somatic cells. We show that modification of this exogenous RNA using cap analogs and poly‐adenosine tail deletions do not affect its selective retention in the small micromeres, but removal of the cap or of the 3′‐untranslated region eliminates any selective mRNA retention in the presumptive germ line. Our results illuminate a likely ancient mechanism used by stem cells to prolong the lifespan of RNAs—either through RNA protection or by the absence of basic RNA degradation mechanisms, which are employed by most other cells of an organism. Mol. Reprod. Dev. 80: 561–569, 2013.

Albinism as a visual, in vivo guide for CRISPR/Cas9 functionality in the sea urchin embryo.

Understanding reproduction and development benefits enormously from a comparative approach. Sea urchins are important for many types of studies, including cell fate determination, morphogenesis, and gene regulatory networks. The CRISPR/Cas9 system of targeted DNA modification provides experimental opportunities for studying animal function in otherwise non-traditional genetic model systems, such as the sea urchin – indeed, CRISPR/Cas9 was recently used to inform how signaling networks lead to left-right asymmetry (Lin and Su, 2016). Here, we streamlined the genomic RNA (gRNA) construction step by targeting a non-essential gene that enables quick assessment of successful CRISPR/Cas9 function in the embryo, thus allowing for a simple visual readout of functional gene disruption. This article is protected by copyright. All rights reserved

Migration of sea urchin primordial germ cells.

Background: Small micromeres are produced at the fifth cleavage of sea urchin development. They express markers of primordial germ cells (PGCs), and are required for the production of gametes. In most animals, PGCs migrate from sites of formation to the somatic gonad. Here, we investigated whether they also exhibit similar migratory behaviors using live‐cell imaging of small micromere plasma membranes. Results: Early in gastrulation, small micromeres transition from non‐motile epithelial cells, to motile quasi‐mesenchymal cells. Late in gastrulation, at 43 hr post fertilization (HPF), they are embedded in the tip of the archenteron, but remain motile. From 43–49 HPF, they project numerous cortical blebs into the blastocoel, and filopodia that contact ectoderm. By 54 HPF, they begin moving in the plane of the blastoderm, often in a directed fashion, towards the coelomic pouches. Isolated small micromeres also produced blebs and filopodia. Conclusions: Previous work suggested that passive translocation governs some of the movement of small micromeres during gastrulation. Here we show that small micromeres are motile cells that can traverse the archenteron, change position along the left‐right axis, and migrate to coelomic pouches. These motility mechanisms are likely to play an important role in their left‐right segregation. Developmental Dynamics 243:917–927, 2014.