Adrian Reich

PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells

Significance The P-element–induced wimpy testis (PIWI) proteins and their bound small RNAs (PIWI-interacting RNAs, piRNAs) are known to repress transposon expression in the germline, yet they likely have broader regulatory functions. We show that the PIWI–piRNA pathway functions in the stem cells of an early diverging animal. We demonstrate that Hydra has two PIWI proteins that are localized in the cytoplasm of all adult stem/progenitor cell types. We identified putative targets of the pathway, both transposon and nontransposon, by sequencing piRNAs and mapping them to a newly assembled Hydra transcriptome. Finally we demonstrate that Hydra PIWI is essential in the somatic lineages. This study supports the existence of a common regulatory pathway ancestral to both stem and germ cells. PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI–piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality.

The Transcriptome of a Human Polar Body Accurately Reflects Its Sibling Oocyte

Background: Clinicians need additional metrics for predicting quality of human oocytes for IVF procedures. Results: Human polar bodies reflect the oocyte transcript profile. Conclusion: Quantitation of polar body mRNAs could allow for both oocyte ranking and embryo preferences in IVF applications. Significance: The transcriptome of a polar body has never been reported in any organism. Improved methods are needed to reliably and accurately evaluate oocyte quality prior to fertilization and transfer into the woman of human embryos created through in vitro fertilization (IVF). All oocytes that are retrieved and matured in culture are exposed to sperm with little in the way of evaluating the oocyte quality. Furthermore, embryos created through IVF are currently evaluated for developmental potential by morphology, a criterion lacking in quantitation and accuracy. With the recent successes in oocyte vitrification and storage, clear metrics are needed to determine oocyte quality prior to fertilizing. The first polar body (PB) is extruded from the oocyte before fertilization and can be biopsied without damaging the oocyte. Here, we tested the hypothesis that the PB transcriptome is representative of that of the oocyte. Polar body biopsy was performed on metaphase II (MII) oocytes followed by single-cell transcriptome analysis of the oocyte and its sibling PB. Over 12,700 unique mRNAs and miRNAs from the oocyte samples were compared with the 5,431 mRNAs recovered from the sibling PBs (5,256 shared mRNAs or 97%, including miRNAs). The results show that human PBs reflect the oocyte transcript profile and suggests that mRNA detection and quantification through high-throughput quantitative PCR could result in the first molecular diagnostic for gene expression in MII oocytes. This could allow for both oocyte ranking and embryo preferences in IVF applications.

Phylogenomic Analyses of Echinodermata Support the Sister Groups of Asterozoa and Echinozoa

Echinoderms (sea urchins, sea stars, brittle stars, sea lilies and sea cucumbers) are a group of diverse organisms, second in number within deuterostome species to only the chordates. Echinoderms serve as excellent model systems for developmental biology due to their diverse developmental mechanisms, tractable laboratory use, and close phylogenetic distance to chordates. In addition, echinoderms are very well represented in the fossil record, including some larval features, making echinoderms a valuable system for studying evolutionary development. The internal relationships of Echinodermata have not been consistently supported across phylogenetic analyses, however, and this has hindered the study of other aspects of their biology. In order to test echinoderm phylogenetic relationships, we sequenced 23 de novo transcriptomes from all five clades of echinoderms. Using multiple phylogenetic methods at a variety of sampling depths we have constructed a well-supported phylogenetic tree of Echinodermata, including support for the sister groups of Asterozoa (sea stars and brittle stars) and Echinozoa (sea urchins and sea cucumbers). These results will help inform developmental and evolutionary studies specifically in echinoderms and deuterostomes in general.

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.

Use of Sea Stars to Study Basic Reproductive Processes

Echinoderms are closely related to chordates and comprise a major group of invertebrate deuterostomes. They are broadcast spawners and as such, each female accumulates millions of eggs and oocytes. These cells are readily isolated, and are often large, clear, and surrounded by accessory cells and extracellular coverings that do not prevent access to the oocyte. Sea star oocytes are stored in prophase of meiosis, and since the natural meiotic stimulus has been identified as 1-methyladenine, these cells can be induced to complete meiotic maturation as individuals, or synchronously en masse. Microinjection and culture of these cells is feasible using quantitative or repetitive methods so that hundreds of oocytes and eggs can be modified each hour. Experimentation on this organism is extensive over a rich history of reproductive and developmental biology so that new investigators can easily incorporate this organism into their repertoire of research. This review will highlight the fundamental protocols to enable a new investigator to perform an array of approaches on this organism, including oocyte isolation, microinjection, and even single cell quantitative PCR.

Selective accumulation of germ-line associated gene products in early development of the sea star and distinct differences from germ-line development in the sea urchin.

Background: Echinodermata is a diverse phylum, a sister group to chordates, and contains diverse organisms that may be useful to understand varied mechanisms of germ‐line specification. Results: We tested 23 genes in development of the sea star Patiria miniata that fall into five categories: (1) Conserved germ‐line factors; (2) Genes involved in the inductive mechanism of germ‐line specification; (3) Germ‐line associated genes; (4) Molecules involved in left–right asymmetry; and (5) Genes involved in regulation and maintenance of the genome during early embryogenesis. Overall, our results support the contention that the posterior enterocoel is a source of the germ line in the sea star P. miniata. Conclusions: The germ line in this organism appears to be specified late in embryogenesis, and in a pattern more consistent with inductive interactions amongst cells. This is distinct from the mechanism seen in sea urchins, a close relative of the sea star clad. We propose that P. miniata may serve as a valuable model to study inductive mechanisms of germ‐cell specification and when compared with germ‐line formation in the sea urchin S. purpuratus may reveal developmental transitions that occur in the evolution of inherited and inductive mechanisms of germ‐line specification. Developmental Dynamics 243:568–587, 2014.

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.

Transcriptome variance in single oocytes within, and between, genotypes.

The zygote of sexually reproducing organisms contains a combination of parental genomes, and all subsequent cells of the embryo are derived from this original genotype. Although clonal, it is not known how much genetic variation exists in progeny of this original cell, or between cells of the same lineage resulting from this zygote. Oocytes in mammals, especially humans, have prolonged developmental histories and each may be quite different in terms of gene expression. It is clear that oocyte quality can differ significantly within a cohort, and the variation in early developmental success from each oocyte can be dramatic. Oocyte quality is ultimately best measured by success of the embryo, but other features, such as normalcy of the mRNA population, may be important criteria to identify such potential. Here we test the variation in steady-state levels of mRNAs in mouse oocytes to establish a baseline of “normal” variation, and compare it mRNA levels of individual oocytes of poor quality. We sequenced to saturation the mRNA from 5 wildtype oocyte samples (three individual oocytes, and 2 pools of 5 oocytes each from 2 wildtype mice) and 16 Taf4b-deficient oocyte samples (12 individual oocytes and 4 pools of 5 or 10 oocytes each from 2 Taf4b-deficient mice). The Taf4b-deficient mice are known to have oocytes that appear morphologically normal (Figure 1A and B), but are of poor quality with regards to successful embryogenesis. This genotype was selected as a model for human premature ovarian insufficiency (POI; Lovasco et al., 2010). Taf4b-null animals are viable as adults, but the oocytes they make die prematurely in adults, leading to a POI phenotype, and any oocytes that mature and are fertilized do not develop past the 2–4-cell stage (Falender et al., 2005; Lovasco et al., 2010). Figure 1 A, B) Photos of sequenced wildtype and Taf4b-knockout oocytes The hypothesis tested here is that the transcriptome of the Taf4b-deficient oocyte differs significantly from that of the wildtype oocyte. To properly assess this, we also needed to determine the variance between individual oocytes to ascribe significance to the comparison. This data set was generated by high throughput DNA sequencing following transcriptome amplification (Reich et al., 2011) and compared within and between genotypes to determine the variance. To test the fidelity of the amplification process for this protocol, prior to and independent of high-throughput DNA sequencing, oocytes from a wildtype mouse were isolated and pooled before lysing. Following DNase treatment, one oocyte-equivalent was isolated and the cDNA library was synthesized. The resulting library was diluted 100 times, the approximate volume of a single polar body, which is important if a polar body were to be used to determine the oocyte quality without harming the oocyte (Reich et al., 2011). Three samples from this pool were independently amplified, and each technical replicate was tested by qPCR as a measure of the fidelity of the amplification procedure (Reich et al., 2011). Overall, low technical variation was detected, providing confidence in the protocol (Figure 1C). We do not know what kinds of bias the amplification procedure may have, but based on these results, the amplification appears to be consistent. The starting material for a polar body is so limiting, however, that even with this cDNA amplification, qPCR is only able to consistently amplify some transcripts – most rare transcripts have high Ct values, thus the sensitivity of sequencing is therefore preferred. In order to test the inter- and intra-genotype variation, we collected oocytes from Taf4b-null and wildtype oviducts after ovulation, mechanically and enzymatically stripped of all granulosa cells, and processed the cells for cDNA synthesis and amplification for sequencing as described (Reich et al., 2011). The libraries were sequenced on a HiSeq 2000, and the reads were mapped to the mouse genome (mm9) using TopHat (Trapnell et al, 2009), yielding an average of 219,207 (std. 138,190) mappable reads per sample. These reads were tested for differential expression using edgeR (Robinson, and Smyth, 2007). A total of 11,373 genes were detected across all 21 samples that were also above a filter threshold of greater than 20 raw counts across all 21 libraries, and a total of 3,242 genes were differentially expressed with a false discovery rate (FDR) of <0.05 (Supplemental Table 1). A large number of genes are upregulated in the Taf4b mutant samples, including 3,465 genes undetected in the wildtype samples; 1,037 of these genes achieve significance (Supplemental Table 1 and Figure 1D). The gene-by-gene average of the RPKM (Reads Per Kilobase of transcript per Million mapped reads) from one genetic background is very similar to the average RPKM from another background (Figure 1D). The log-transformed standard deviations of the RPKMs of wildtype and knockout samples (Supplemental Figure 2) closely mirrors the graph of the means of the RPKMs (Figure 1D), suggesting: a) as genes become more abundant, the variation increases, b) different genomic backgrounds have similar rates of variation, and c) assuming the qPCR results from (Figure 1C) represent the technical variability of all genes, then any bias introduced by the amplification process appears significantly less than the biological variability within a population. Although the gene-by-gene standard deviation of the RPKM scales linearly with the abundance of the gene, suggesting that samples within a background are similar, we compared how the entire gene set of a sample compared with another sample within the same genetic background and also across backgrounds. The 5 samples isolated from the two wildtype mice (WT1 and WT2) and 16 samples from the two Taf4b mutant mice (KO1 and KO2) clearly segregate by genotype into two main groups; within a group, the samples segregate by mouse to a great degree (Figure 2, and Supplemental Figure 1). Only one of the wildtype samples clustered together with the knockout samples, although the distance between this wildtype sample and all knockout samples (cophenetic distance) is larger than any of the other samples within this group; this indicates that its transcriptional profile is intermediate between the two genotypes. Figure 2 Dendogram of 5 wildtype samples and 16 Taf4b-knockout samples showing the high sample-to-sample relatedness within genotypes and even within individual mice (see Supplemental Figure 1 for full details). Each sample name also describes the type of sample ... We conclude that the biological variability of transcriptomes can be quantified between single cells within a genotype, and the comparison between genotypes can reveal genes that are differentially expressed in a robust manner. This approach may help reveal oocyte quality by use of the polar body metric without harm to the oocyte (Reich et al., 2011).

Two-pore channels function in calcium regulation in sea star oocytes and embryos

Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities.