Prenatal postcranial development in two species of sympatric Japanese wood mice (Apodemus argenteus and A. speciosus): a comparison of arboreal versus terrestrial congeners.

Habitats of two closely related Japanese field mice, Apodemus argenteus and A. speciosus, broadly overlap in many Japanese forests. A. argenteus being more arboreal and A. speciosus being more terrestrial, it is thought that such ecological segregation allows their sympatric distribution. Comparing these two congeners, whether ecological difference is reflected in postcranial development was examined. Although overall ossification sequences were virtually identical, development of the caudal vertebrae was remarkably earlier in A. argenteus. One of the clearest morphological differences between the two species is the relative length of the tail, which is arguably related to the degree of arboreality. I suggest that accelerated ossification of the caudal vertebrae found in A. argenteus is related to its elongation of the tail.

Developmental mechanisms of stripe patterns in rodents.

Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.

Ontogeny of the adrenal gland in the spiny mouse, with particular reference to production of the steroids cortisol and dehydroepiandrosterone.

Synthesis of the androgen dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental estrogen production and may also be important for modulating the effects of glucocorticoids on the developing brain. The presence of cortisol in spiny mouse (Acomys cahirinus) blood led us to determine whether the adrenal gland of this precocial rodent also synthesized DHEA. Cytochrome P450 enzyme 17α-hydroxylase/17,20-lyase (P450c17), cytochrome-b5 (Cytb5), and 3ß-hydroxysteroid dehydrogenase (3ßHSD) were detected in the adrenal gland from 30 days gestation (term = 39 days), and DHEA, cortisol, and aldosterone were detected in fetal plasma from this time. Plasma DHEA concentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from exogenous pregnenolone, and thus, the DHEA in the fetal circulation is likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and coexpression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3ßHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. These results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and DHEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species.

Multiple mesodermal lineage differentiation of Apodemus sylvaticus embryonic stem cells in vitro.

BACKGROUND: Embryonic stem (ES) cells have attracted significant attention from researchers around the world because of their ability to undergo indefinite self-renewal and produce derivatives from the three cell lineages, which has enormous value in research and clinical applications. Until now, many ES cell lines of different mammals have been established and studied. In addition, recently, AS-ES1 cells derived from Apodemus sylvaticus were established and identified by our laboratory as a new mammalian ES cell line. Hence further research, in the application of AS-ES1 cells, is warranted. RESULTS: Herein we report the generation of multiple mesodermal AS-ES1 lineages via embryoid body (EB) formation by the hanging drop method and the addition of particular reagents and factors for induction at the stage of EB attachment. The AS-ES1 cells generated separately in vitro included: adipocytes, osteoblasts, chondrocytes and cardiomyocytes. Histochemical staining, immunofluorescent staining and RT-PCR were carried out to confirm the formation of multiple mesodermal lineage cells. CONCLUSIONS: The appropriate reagents and culture milieu used in mesodermal differentiation of mouse ES cells also guide the differentiation of in vitro AS-ES1 cells into distinct mesoderm-derived cells. This study provides a better understanding of the characteristics of AS-ES1 cells, a new species ES cell line and promotes the use of Apodemus ES cells as a complement to mouse ES cells in future studies.

Application of clinical indices of fetal growth and wellbeing to a novel laboratory species, the spiny mouse.

Ultrasound was used to measure growth of the spiny mouse fetus throughout gestation and to record Doppler measurements of heart rate and umbilical blood flow to monitor fetal blood supply and wellbeing. Female spiny mice were anesthetized on 6 occasions throughout pregnancy. Ultrasound was performed with a Philips HDI 5000 machine using a compact linear CL15-7 transducer. Fetal heart rate and growth parameters increased across gestation. Blood flow through the umbilical artery and vein showed increasing velocity over gestation, and reduced resistance index. Blood flow through the ductus venosus also increased in velocity over gestation; however the resistance index remained constant. We have determined changes in umbilical blood flow throughout pregnancy in the spiny mouse, which resemble those seen in human pregnancy. We also confirm that ultrasound can be used as a valuable, non-invasive technique for measuring fetal growth and wellbeing in the spiny mouse.

Extensive contribution of embryonic stem cells to the development of an evolutionarily divergent host.

The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.