Sources for Comparative Studies of Placentation. II. Genomic Resources

Abstract

The genomes of dozens of placental mammal species are now publicly available. These genome sequences have the potential to provide insight into the development and evolution of the placenta. In particular, the variable anatomy of the placenta has likely been affected by natural selection on the genomes of living and extinct mammals. In this note the current availability of mammal genome sequences is reviewed, and strengths and limitations of these data are discussed. Additionally, museums, zoos, and commercial entities are available to provide genomic resources to the placental research community. Recommendations for tissue storage conditions of placentas in genomic research are given.

1. Introduction

The placenta is arguably the most morphologically variable mammalian organ. All eutherian mammals share the derived feature of a chorioallantoic placenta. Despite this uniting feature, the eutherian placenta varies in its shape, interface, interdigitation and other aspects. Recent work has focused on reconstructing the evolutionary history of these features [1–3]. The placenta is essential for successful reproduction, and since evolution requires successful reproduction, it is likely that differences in placental morphology have been shaped by natural selection. Natural selection leaves detectable signatures in genomes, and the burgeoning fields of comparative genomics and molecular phylogenetics provide a powerful set of tools for studying placental variation.

The purpose of this paper is to provide information on genomic resources available for those interested in studying placentation from a genetic perspective. As genomes of more species become available it will be necessary to annotate them by isolating placental mRNA and producing cDNA sequences. A reasonable goal of comparative placental genomics would be to determine which parts of the genome are expressed in the placenta in many mammal species, and to catalogue the similarities and differences in the different mammalian lineages. In addition to cDNA studies, epigenetic profiling seems to be a logical pursuit in the genomics of the placenta because of the evidence for imprinting in this organ.

2. Available genome sequences

Placental mammals

As of August 11, 2007 there were draft genome assemblies for 19 eutherian species available at the Ensembl databases (http://www.ensembl.org/index.html). Whole genome multiple sequence alignments for 18 placental mammal species are available at the University of California at Santa Cruz genome browser (http://genome.ucsc.edu). The NCBI Entrez genome project page lists whole genome sequencing projects for 41 placental species, and the National Human Genome Research Institute (NHGRI) describes genome sequence projects either finished, assembled in draft format, in progress, or approved for funding for 41 placental mammals. Table 1 lists the currently approved genome projects as determined by searching through these databases.

Table I.

Current genome projects of particular interest

Genome	Class	Clade	Order	Genus	Species	Placental Interface	Fold Coverage	Scheduled deep coverage
Human	Mammalia	Euarchontoglires	Primates	Homo	sapiens	Hemochorial	Finished
Chimpanzee	Mammalia	Euarchontoglires	Primates	Pan	troglodytes	Hemochorial	6
Gorilla	Mammalia	Euarchontoglires	Primates	Gorilla	gorilla	Hemochorial	?
Orangutan	Mammalia	Euarchontoglires	Primates	Pongo	abelli	Hemochorial	7
Gibbon	Mammalia	Euarchontoglires	Primates	Nomascus	leucogenys	Hemochorial	BAC Ends
Rhesus macaque	Mammalia	Euarchontoglires	Primates	Macaca	mulatta	Hemochorial	5.2
Cynomolgus macaque	Mammalia	Euarchontoglires	Primates	Macaca	fascicularis	Hemochorial
Baboon	Mammalia	Euarchontoglires	Primates	Papio	hamadryas	Hemochorial
Marmoset	Mammalia	Euarchontoglires	Primates	Callithrix	jacchus	Hemochorial	7
Squirrel monkey	Mammalia	Euarchontoglires	Primates	Saimiri	sp.	Hemochorial
Tarsier	Mammalia	Euarchontoglires	Primates	Tarsius	syrichta	Hemochorial	2
Bush baby	Mammalia	Euarchontoglires	Primates	Otolemur	garnetti	Epitheliochorial	1.5
Mouse lemur	Mammalia	Euarchontoglires	Primates	Microcebus	murinus	Epitheliochorial	2
Treeshrew	Mammalia	Euarchontoglires	Scandentia	Tupaia	belangeri	Endotheliochorial	2	Yes
Colugo	Mammalia	Euarchontoglires	Dermoptera	Cynocephalus	volans	Hemochorial	2
Rabbit	Mammalia	Euarchontoglires	Lagomorpha	Oryctolagus	cuniculus	Hemochorial	2	Yes
Pika	Mammalia	Euarchontoglires	Lagomorpha	Ochonta	princeps	Hemochorial	2
Rat	Mammalia	Euarchontoglires	Rodentia	Rattus	norvegicus	Hemochorial	11.9
Mouse	Mammalia	Euarchontoglires	Rodentia	Mus	musculus	Hemochorial	Finished
Kangaroo rat	Mammalia	Euarchontoglires	Rodentia	Dipodymys	panamintinus	Endotheliochorial	2
Mole rat	Mammalia	Euarchontoglires	Rodentia	Cryptomys	sp.	unknown	2
Guinea Pig	Mammalia	Euarchontoglires	Rodentia	Cavia	porcellus	Hemochorial	1.92	Yes
Squirrel	Mammalia	Euarchontoglires	Rodentia	Spermophilis	tridecemlineatus	Hemochorial	2
Dog	Mammalia	Laurasiatheria	Carnivora	Canis	familiaris	Endotheliochorial	7.6
Cat	Mammalia	Laurasiatheria	Carnivora	Felis	catus	Endotheliochorial	2	Yes
Pangolin	Mammalia	Laurasiatheria	Pholidota	Manis	pentadactyla	Epitheliochorial	2
Cow	Mammalia	Laurasiatheria	Cetartiodactyla	Bos	taurus	Epitheliochorial	7
Pig	Mammalia	Laurasiatheria	Cetartiodactyla	Sus	scrofa	Epitheliochorial	?
Alpaca	Mammalia	Laurasiatheria	Cetartiodactyla	Vicugna	pacas	Epitheliochorial	2
Dolphin	Mammalia	Laurasiatheria	Cetartiodactyla	Tursiops	truncatus	Epitheliochorial	2
Horse	Mammalia	Laurasiatheria	Perissodactyla	Equus	caballus	Epitheliochorial	7	Yes
Little brown bat	Mammalia	Laurasiatheria	Chiroptera	Myotis	lucifugus	Endotheliochorial/Hemochorial	1.7	Yes
Megabat	Mammalia	Laurasiatheria	Chiroptera	Pteropus	vampyrus	Endotheliochorial/Hemochorial	2
Hedgehog	Mammalia	Laurasiatheria	Erinaceomorpha	Erinaceus	europaeus	Hemochorial	1.86
Shrew	Mammalia	Laurasiatheria	Soricomorpha	Sorex	araneus	Endotheliochorial/Hemochorial	1.9
Tenrec	Mammalia	Afrotheria	Afrosoricida	Echinops	telfairi	Hemochorial	2
Elephant shrew	Mammalia	Afrotheria	Macroscelidea	Elephantulus	sp.	Hemochorial	2
Hyrax	Mammalia	Afrotheria	Hyracoidea	Procavia	capensis	Hemochorial	2
African elephant	Mammalia	Afrotheria	Proboscidea	Loxodonta	africana	Endotheliochorial	2	Yes
Armadillo	Mammalia	Xenarthra	Cingulata	Dasypus	novemcinctus	Hemochorial	2	Yes
Two-toed sloth	Mammalia	Xenarthra	Pilosa	Choloepus	hoffmanni	Endotheliochorial	2
Opossum	Mammalia	Marsupiala	Didelpomorphia	Monodelphis	domestica	Choriovitelline	6.8
Wallaby	Mammalia	Marsupiala	Diprotodontia	Macropus	eugenii	Choriovitelline	2
Platypus	Mammalia	Monotremata	Monotremata	Ornithorynchus	anatinus	NA	6
Chicken	Sauria	Bird	Galliformes	Gallus	gallus	NA	6.6
Lizard	Sauria	Lizard	Squamata	Anolis	carolinensis	NA	6.8

Shaded rows: approved either in progress or not yet started. Placental inference [4]

The genome sequences of seven of these species have already been published [5–11].

Outgroups

The genomes for non-placental mammals and other vertebrates provide the necessary comparative basis for understanding the evolution of the mammalian placenta from a genomic perspective. Appropriate outgroups include two marsupial genomes, the tammar wallaby (Macropus eugenii) and the gray, short-tailed opossum (Monodelphis domestica; [12]), the platypus (Ornithorhynchus anatinus), an egg-laying monotreme mammal; and two saurian non-mammals, the chicken (Gallus gallus; [13]) and the anole lizard (Anolis carolinensis).

While these genomes are a powerful resource, some limitations currently exist regarding their use. Of the 46 genomes listed in Table 1, only nine have been published. There are conditions on the fair use of publicly available but as yet unpublished genome sequences. Moreover, many of the genomes were sequenced at low fold coverage (<2x); therefore, these genomes contain a higher number of sequence errors, misassembled fragments, and gaps, than those genomes with better coverage (e.g. the rat genome has 11.9X fold coverage). Finally, expressed sequence tags (ESTs) and other expression data are unavailable for the majority of species with sequenced genomes.

Fortunately, the placentas of most of the taxa sequenced have been described in the literature. Our best estimates of mammalian phylogeny [14–17] provide the framework to reconstruct the evolutionary history of the placental interface in these species. Given this phylogenetic framework, it is possible to make testable predictions regarding the genomic changes associated with placental morphological change. For example, all recent studies agree that the epitheliochorial placenta of the strepsirrhine primates was derived on that lineage. Thus, using a phylogenomic approach [18], it is predicted that many of the genetic changes responsible for the emergence of the epitheliochorial placenta would have occurred on the stem lineage leading to extant strepsirrhines. Today we are uniquely poised to unravel placental history. Within primates, the completion of the human genome, followed by draft assemblies of the common chimpanzee, the rhesus macaque, the common marmoset, the northern greater galago, and the mouse lemur offer unprecedented opportunities to develop genomic methods to reconstruct morphological change.

3. Other genomic resources

In addition to these whole genomes, ESTs and other large collections of transcribed sequences from placenta are available for a variety of species including cow (~160,000 records), pig (~130,000), human (~240,000), rhesus macaque (~2000), mouse (~110,000), and rat (~100,000). These sequences are a valuable resource for the placental evolution community. Additional comparative gene expression information is available for placental expression in microarray databases including the SymAtlas of the Genomics Institute of the Novartis Foundation. SymAtlas (http://symatlas.gnf.org/SymAtlas/) is a web-application with a searchable database of gene expression data in multiple species and tissues including placenta. The Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) is a gene expression/molecular abundance repository, and a curated, online resource for gene expression data browsing, query and retrieval.

4. Obtaining material for further study

Museums are the repositories for many specimens, and they play an invaluable role for comparative genomic and genetic studies. Below, I describe the procedures for obtaining museum samples from three U.S. institutions. However, many others exist, and researchers are encouraged to contact officials at those institutions as well.

National Museum of Natural History, Smithsonian Institution, US

The world’s largest collection of mammal specimens is housed in the Division of Mammals. The website, http://www.nmnh.si.edu/vert/mammals/ allows users to search the database of more than 500,000 specimens. Well-established loan policies and destructive sampling policies are in place. The appropriate curators should be contacted for sample requests.

Rodents, Old World Mammals: Michael D. Carleton, carleton@si.edu

Marine Mammals: James G. Mead, meadj@si.edu

Primates, Squirrels: Richard W. Thorington, Jr., thoringtonr@si.edu

Bats, Biodiversity: Don E. Wilson, wilsond@si.edu

Division of Mammals, Smithsonian Institution, PO Box 37012, National Museum of Natural History, MRC 108, Washington, DC 20013-7012.

phone (202)633-1260, fax (202)786-2979

American Museum of Natural History

There are over 275,000 specimens in the mammal collections http://research.amnh.org/mammalogy/index.php. All mammalian tissue samples have been transferred to the Ambrose Monell Cryo Collection. There is a searchable online tissue database, and requests for tissues require approval from the Department of Mammalogy using the application form available online.

Museum of Vertebrate Zoology, UC Berkeley

The MVZ collection is the third largest in the U.S. with over 200,000 skin, skull, and fluid preserved specimens. Collections of rodents and bats are particularly strong. Specimen data are available online at http://mvzarctos.berkeley.edu/SpecimenSearch.cfm. The evolutionary genetics laboratory at MVZ houses 19,000 tissue specimens. Queries regarding the availability of samples can be directed to the following curators:

Mammal Collections, Eileen A. Lacey ealacey@berkeley.EDU

Mammal Collections, James L. Patton patton@berkeley.EDU

Tel: (510) 642-3567

fax: (510) 643-8238

Museum of Vertebrate Zoology

University of California

3101 Valley Life Sciences Building

Berkeley, California 94720-3160 USA

In addition to museums, Zoological Gardens are an important resource for tissues and DNA samples. The International Species Information System (ISIS) provides current and comprehensive information on animals and their environments for zoos, aquariums and related organizations to serve institutional, regional and global animal management and conservation goals. The ISIS web site (https://www.isis.org/CMSHOME/) provides a species holdings tool. This tool allows the user to discover how many animals of each species are currently living in ISIS member institutions worldwide, and serves as an excellent starting point for contacting individual curators.

Additional sources for tissues, cell lines, and nucleotides include the Coriell Cell Repositories, the Biochain Institute, and a host of other commercial entities that store tissues. It is important to ensure that all international, national, and local regulations are adhered to when obtaining tissues, especially from endangered and threatened species.

5. Considerations for preservation

Studies require different handling, processing, and storage of tissue and other materials depending on the goal. For example, formaldehyde- or paraformaldehyde-fixed and paraffin embedded (FFPE) sections are very useful for histological and immunohistochemical studies, but this method of preservation makes recovery of nucleic acids like DNA and RNA challenging. For nucleic acid studies samples should be snap frozen in liquid nitrogen or placed in a suitable preservative such as RNAlater® (Ambion, Austin, TX). RNAlater is a tissue storage reagent that stabilizes cellular RNA in intact, unfrozen tissue samples. It has also been shown to preserve DNA (unpublished observation). Indeed, if the goal of placental research is to separate the maternal and fetal genomes, RNAlater preservation is superior to freezing because the dissection of membranes and villous tissue is much easier from tissues that have not been frozen. Applications of RNAlater preservation include cDNA synthesis, quantitative RT-PCR, and microarray studies. In order to measure relative RNA quantities in placental tissues via RT-PCR it is important to use appropriate housekeeping genes. Comparative RT-PCR studies are challenging because of sequence mismatch between species. Commonly used control genes include beta-actin, GAPDH, 18S rRNA and RLPO.A more promising approach is sequence based quantitative studies such as those available using the 454 (454 Life Sciences; Branford CT) or Solexa (Illumina Inc.; San Diego, CA) platforms.

6. Conclusions

The comparative genomics of mammals provide a valuable resource for studying the evolution of placentation. While the genomic sequences are valuable, it will be important to annotate the genomes by sequencing the transcribed genes from placental tissue. Obtaining such material can be challenging, but with a concerted and coordinated effort from the placentology community great progress can be made.