Roberta L. Hannibal

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.

A prominent requirement for single-minded and the ventral midline in patterning the dorsoventral axis of the crustacean Parhyale hawaiensis

In bilaterians, establishing the correct spatial positioning of structures along the dorsoventral (DV) axis is essential for proper embryonic development. Insects such as Drosophila rely on the Dorsal activity gradient and Bone morphogenetic protein (BMP) signaling to establish cell fates along the DV axis, leading to the distinction between tissues such as mesoderm, neurogenic ectoderm and dorsal ectoderm in the developing embryo. Subsequently, the ventral midline plays a more restricted role in DV patterning by establishing differential cell fates in adjacent regions of the neurogenic ectoderm. In this study, we examine the function of the ventral midline and the midline-associated gene single-minded (Ph-sim) in the amphipod crustacean Parhyale hawaiensis. Remarkably, we found that Ph-sim and the ventral midline play a central role in establishing proper fates along the entire DV axis in this animal; laser ablation of midline cells causes a failure to form neurogenic ectoderm and Ph-sim RNAi results in severely dorsalized embryos lacking both neurogenic ectoderm and the appendage-bearing lateral ectoderm. Furthermore, we hypothesize that this role of midline cells was present in the last common ancestor of crustaceans and insects. We predict that the transition to a Dorsal-dependent DV patterning system in the phylogenetically derived insect lineage leading to Drosophila has led to a more restricted role of the ventral midline in patterning the DV axis of these insects.

The crustacean Parhyale hawaiensis: a new model for arthropod development.

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. In addition to its phylogenetically strategic position, P. hawaiensis has proven to be highly amenable to experimental manipulation, is straightforward to rear in the laboratory, and has large numbers of embryos that are available year-round. A detailed staging system has been developed to characterize P. hawaiensis embryogenesis. Robust protocols exist for the collection and fixation of all embryonic stages, in situ hybridization to study mRNA localization, and immunohistochemistry to study protein localization. Microinjection of blastomeres enables detailed cell-lineage analyses, transient and transgenic introduction of recombinant genetic material, and targeted knockdowns of gene function using either RNA interference (RNAi) or morpholino methods. Directed genome sequencing will generate important data for comparative studies aimed at understanding cis-regulatory evolution. Bacterial artificial chromosome (BAC) clones containing genes of interest to the developmental and evolutionary biology communities are being targeted for sequencing. An expressed sequence tag (EST) database will facilitate discovery of additional developmental genes and should broaden our understanding of the genetic controls of body patterning. A reference genome from the related amphipod crustacean Jassa slatteryi will shortly be available.

Selective Amplification of the Genome Surrounding Key Placental Genes in Trophoblast Giant Cells

While most cells maintain a diploid state, polyploid cells exist in many organisms and are particularly prevalent within the mammalian placenta [1], where they can generate more than 900 copies of the genome [2]. Polyploidy is thought to be an efficient method of increasing the content of the genome by avoiding the costly and slow process of cytokinesis [1, 3, 4]. Polyploidy can also affect gene regulation by amplifying a subset of genomic regions required for specific cellular function [1, 3, 4]. This mechanism is found in the fruit fly Drosophila melanogaster, where polyploid ovarian follicle cells amplify genomic regions containing chorion genes, which facilitate secretion of eggshell proteins [5]. Here, we report that genomic amplification also occurs in mammals at selective regions of the genome in parietal trophoblast giant cells (p-TGCs) of the mouse placenta. Using whole-genome sequencing (WGS) and digital droplet PCR (ddPCR) of mouse p-TGCs, we identified five amplified regions, each containing a gene family known to be involved in mammalian placentation: the prolactins (two clusters), serpins, cathepsins, and the natural killer (NK)/C-type lectin (CLEC) complex [6-12]. We report here the first description of amplification at selective genomic regions in mammals and present evidence that this is an important mode of genome regulation in placental TGCs.

In Situ Hybridization of Labeled RNA Probes to Fixed Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes in situ hybridization of fluorescein- or digoxigenin (DIG)-labeled RNA probes to fixed P. hawaiensis embryos. Standard techniques of molecular biology should be used to produce an appropriate template for generation of antisense RNA probes. RNA-labeling mixes designed to produce fluorescein- or DIG-labeled RNA probes using T3, T7, or SP6 RNA polymerases are commercially available. Probes should be purified using QIAGEN RNeasy columns or similar means. Considerations for double-labeling experiments using both fluorescein- and DIG-labeled RNA probes are included.

Fixation and Dissection of Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes the dissection and fixation of P. hawaiensis embryos. Embryonic tissue fixed in the following manner is suitable for in situ hybridization experiments to study mRNA expression or for immunocytochemistry to study protein localization.

Antibody Staining of Parhyale hawaiensis Embryos

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol provides a simplified protocol for antibody staining of P. hawaiensis embryos. The method also works well for other arthropods and phyla. Fixed embryos are rehydrated, washed, blocked with normal goat serum, and incubated overnight with primary antibody. Embryos are then washed and incubated with a peroxidase-conjugated secondary antibody that binds to the primary antibody. A subsequent histochemical reaction produces a black stain in those cells where antibodies have localized.

Analysis of snail genes in the crustacean Parhyale hawaiensis : insight into snail gene family evolution

The transcriptional repressor snail was first discovered in Drosophila melanogaster, where it initially plays a role in gastrulation and mesoderm formation, and later plays a role in neurogenesis. Among arthropods, this role of snail appears to be conserved in the insects Tribolium and Anopheles gambiae, but not in the chelicerates Cupiennius salei and Achaearanea tepidariorum, the myriapod Glomeris marginata, or the Branchiopod crustacean Daphnia magna. These data imply that within arthropoda, snail acquired its role in gastrulation and mesoderm formation in the insect lineage. However, crustaceans are a diverse group with several major taxa, making analysis of more crustaceans necessary to potentially understand the ancestral role of snail in Pancrustacea (crustaceans + insects) and thus in the ancestor of insects as well. To address these questions, we examined the snail family in the Malacostracan crustacean Parhyale hawaiensis. We found three snail homologs, Ph-snail1, Ph-snail2 and Ph-snail3, and one scratch homolog, Ph-scratch. Parhyale snail genes are expressed after gastrulation, during germband formation and elongation. Ph-snail1, Ph-snail2, and Ph-snail3 are expressed in distinct patterns in the neuroectoderm. Ph-snail1 is the only Parhyale snail gene expressed in the mesoderm, where its expression cycles in the mesodermal stem cells, called mesoteloblasts. The mesoteloblasts go through a series of cycles, where each cycle is composed of a migration phase and a division phase. Ph-snail1 is expressed during the migration phase, but not during the division phase. We found that as each mesoteloblast division produces one segment’s worth of mesoderm, Ph-snail1 expression is linked to both the cell cycle and the segmental production of mesoderm.

Injection of Parhyale hawaiensis Blastomeres with Fluorescently Labeled Tracers

The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes the injection of P. hawaiensis blastomeres with fluorescently labeled tracers for the purpose of cell-lineage analysis. The total (holoblastic) cleavages that characterize early embryogenesis in P. hawaiensis generate an eight-cell embryo with a stereotypical arrangement of blastomeres, each of which already possesses an invariant cell fate. Fluorochrome-conjugated dextran solutions, mRNAs encoding fluorescent proteins, and biotin-dextran have all proven to be useful lineage markers. The relative merits of various tracers are considered.

Evolutionary perspectives into placental biology and disease.

In all mammals including humans, development takes place within the protective environment of the maternal womb. Throughout gestation, nutrients and waste products are continuously exchanged between mother and fetus through the placenta. Despite the clear importance of the placenta to successful pregnancy and the health of both mother and offspring, relatively little is understood about the biology of the placenta and its role in pregnancy-related diseases. Given that pre- and peri-natal diseases involving the placenta affect millions of women and their newborns worldwide, there is an urgent need to understand placenta biology and development. Here, we suggest that the placenta is an organ under unique selective pressures that have driven its rapid diversification throughout mammalian evolution. The high divergence of the placenta complicates the use of non-human animal models and necessitates an evolutionary perspective when studying its biology and role in disease. We suggest that diversifying evolution of the placenta is primarily driven by intraspecies evolutionary conflict between mother and fetus, and that many pregnancy diseases are a consequence of this evolutionary force. Understanding how maternal–fetal conflict shapes both basic placental and reproductive biology – in all species – will provide key insights into diseases of pregnancy.