Danielle Hickford

Evolution of vertebrate interferon inducible transmembrane proteins

BackgroundInterferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates.ResultsFive IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein.ConclusionsLike eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

DDX4 (VASA) Is Conserved in Germ Cell Development in Marsupials and Monotremes

DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups—eutherians, marsupials, and monotremes.

The Tammar Wallaby, Macropus eugenii: A Model Kangaroo for the Study of Developmental and Reproductive Biology

Cold Spring Harb Protoc Danielle Hickford, Stephen Frankenberg and Marilyn B. Renfree Study of Developmental and Reproductive Biology : A Model Kangaroo for the Macropus eugenii The Tammar Wallaby, Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (873 articles) Laboratory Organisms, general (316 articles) Genetics, general (96 articles) Evolutionary Development (Evo-Devo) (90 articles) Evolution (283 articles) Emerging Model Organisms (548 articles) Developmental Biology (130 articles) Bioinformatics/Genomics, general

A Dual Role for SHH during Phallus Development in a Marsupial

The mammalian phallus arises from identical primordia in both sexes and is patterned in part by the key morphogen Sonic hedgehog (SHH). We have investigated SHH and other morphogens during phallus development in the tammar wallaby. In this marsupial, testis differentiation and androgen production occurs just after birth, but it takes a further 50-60 days before the phallus becomes sexually dimorphic. One day before birth, SHH was expressed in both sexes in the urethral epithelium. In males, there was a marked upregulation of SHH, GLI2, and AR at day 50 postpartum, a time when testicular androgen production falls. SHH, GLI2, and AR were downregulated in female pouch young treated with androstanediol from days 24-50, but not when treatments were begun at day 29, suggesting an early window of androgen sensitivity. SHH, GLI2, and AR expression in the phallus of males castrated at day 23 did not differ from controls, but there was an increase in SHH and GLI2 and a decrease in FGF8 and BMP4 expression when the animals were castrated at day 29. These results suggest that the early patterning by SHH is androgen-independent followed by an androgen-dependent window of sensitivity and a sharp rise in SHH expression after androgen withdrawal at day 50.

In vitro culture of peri‐gastrulation embryos of a macropodid marsupial

Peri‐gastrulation stage tammar wallaby embryos were cultured for up to 78 h in either Dulbeccos Modified Eagles Medium or Medium 199, in air/6% CO2 or 95% O2/5% CO2, and with added fetal calf or wallaby serum. There was little difference between the two media or sera sources, but development was markedly superior for embryos cultured in 95% O2/5% CO2. Many embryos survived even prolonged culture periods up to and over 70 h, and although development continued throughout the culture period, the embryos as a whole became increasingly abnormal. Embryos explanted at the primitive streak/ regressing node stages performed better in vitro than embryos explanted at earlier or later stages. The embryo that developed the furthest had a newly formed node at the initiation of culture and after 64 h in vitro it had developed forelimb ridges, fused, beating heart tubes and mesonephric ducts. Thus high oxygen appears to be the critical component of the culture system for optimal development of primitive streak stage tammar embryos. These results provide a basis for developing culture conditions for longer term development of marsupial embryos in vitro.

Collection, Handling, Fixation, and Processing of Tammar Wallaby (Macropus eugenii) Embryos

This protocol describes methods for collecting and handling tammar wallaby (Macropus eugenii) conceptuses. Zygotes, cleavage stages, diapausing blastocysts, and unexpanded blastocysts are ~250 μm in diameter and are collected by flushing either the oviduct or uterus. Later stages are collected by opening the uterus. Late preimplantation stages must be handled carefully because their large size (>2 cm in diameter) results in high surface tension within the yolk sac, especially on the delicate avascular area. When handled too roughly, the embryonic vesicle tends to split and then rapidly collapse; such embryos do not reinflate. If embryos are to be cultured, all steps during collection must be carried out aseptically. In all cases, solutions should be prewarmed to 37°C and embryos should be kept warm by use of a heating stage. Standard fixatives, fixation times, and washing protocols used for mouse embryos are also used for tammar embryos. Methods for embedding tammar conceptuses in paraffin wax depend on the size and stage of the conceptus.

Expression of STRA8 is conserved in therian mammals but expression of CYP26B1 differs between marsupials and mice.

Abstract The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown. Summary Sentence This comparative study using a marsupial species suggests that the roles of retinoic acid and STRA8 but not CYP26B1 in controlling the initiation of meiosis are conserved between different mammals.

Whole-mount immunohistochemical staining of tammar wallaby (Macropus eugenii) cleavage stages and blastocysts.

An introduction to the tammar wallaby and its use as a model organism in studies of reproductive and developmental biology is presented in The Tammar Wallaby, Macropus eugenii: A Model Kangaroo for the Study of Developmental and Reproductive Biology: A Model Kangaroo for the Study of Developmental and Reproductive Biology (Hickford et al. 2009a). Additional information on the collection of tammar embryos can be found in Collection, Handling, Fixation, and Processing of Tammar Wallaby (Macropus eugenii) Embryos (Hickford et al. 2009b). As a complement to wholemount staining, Immunohistochemical Staining of Sectioned Tammar Wallaby (Macropus eugenii) Tissue (Hickford et al. 2009c) provides details on staining slide-mounted tammar wallaby sections and offers suggestions on choosing antibodies for use on tammar tissues.