In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.

Time-lapse microscopy has advanced our understanding of yolk sac and early embryonic vascularization. However, it has been difficult to assess endothelial interactions during epithelial morphogenesis of internal organs. To address this issue we have developed the first time-lapse system to study vascularization of a mammalian organ in four dimensions. We show that vascularization of XX and XY gonads is a highly dynamic, sexually dimorphic process. The XX gonad recruits vasculature by a typical angiogenic process. In contrast, the XY gonad recruits and patterns vasculature by a novel remodeling mechanism beginning with breakdown of an existing mesonephric vessel. Subsequently, in XY organs individual endothelial cells migrate and reaggregate in the coelomic domain to form the major testicular artery. Migrating endothelial cells respect domain boundaries well before they are morphologically evident, subdividing the gonad into 10 avascular regions where testis cords form. This model of vascular development in an internal organ has a direct impact on the current dogma of vascular integration during organ development and presents important parallels with mechanisms of tumor vascularization.organogenesis ͉ ovary ͉ testis I nsights into vascular development and patterning in internal organs have historically relied on xenograft models and static analysis (1-7). However, this approach does not elucidate dynamic interactions between endothelial cells and other cells of developing organs. Advances in time-lapse imaging have improved understanding of vasculogenesis, flow-induced vascular remodeling, the genetic programming of angiogenesis, and many other facets of vascular development in the mouse and chick yolk sac and during establishment of the body plan in zebrafish (8-17). However, culturing internal organs throughout critical and prolonged periods of development has been difficult. The technical challenges of organ culture, compounded by simultaneous live imaging, is a major hurdle in understanding how endothelial cells remodel and integrate in an internal organ undergoing morphogenesis.For more than a decade the urogenital ridge (UGR) explant model has provided a unique system to analyze morphogenesis of an internal organ (18,19). The fact that the UGR can be successfully explanted to culture at the critical sex-determination stage [11.5 days postcoitum (dpc)], using conditions that maintain the normal morphological structure of the gonad, has been the driving force behind its broad adoption in the sexdetermination field. However, with respect to internal organ vascularization, the UGR has received little attention as a model system. In the UGR explant model the gonad retains contact with the mesonephros, the source of the endothelium, and vascularization ex vivo occurs similarly to vascularization in vivo (20). During the period of vascularization, development of the testis and ovary diverge morphologically. Whereas the XX gonad shows few morphological changes during this period, the XY gonad undergoes dramatic epi...

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.

The genes and hormones involved in gonadal differentiation are highly conserved between eutherians and marsupials, although the timing of the developmental events differs. In marsupials, the testis develops seminiferous cords two days after birth, and the ovaries are not distinguishable until around eight days after birth. Differentiation of the internal genitalia is controlled in marsupials, as in eutherians, by testicular testosterone and Müllerian inhibiting substance, but differentiation of the scrotum in males and mammary primordia in females is hormone-independent. Since the young are easily accessible in the pouch, it is possible to administer gonadal hormones during the period of sexual differentiation. In both Australian and South American marsupials, estradiol treatment of neonatal males can induce male-to-female gonadal sex reversal. The testicular transformations range from partial suppression of seminiferous tubule development to the development of a morphologically normal ovary depending on the stage that treatment starts. The sex-reversed testes have a clearly defined cortex and medulla, and there are significantly fewer germ cells. The germ cells are surrounded by follicle-like cells and are in the early stages of meiosis, as is normal for XX germ cells in ovaries. In normal males, germ cells only enter meiosis at the onset of puberty. As in eutherians, estrogen treatment of neonatal male marsupials prevents regression of the Müllerian ducts, which are hypertrophic. Neonatal estradiol exposure also causes hypertrophy of the prostate and urogenital sinus. Estradiol treatment also inhibits transabdominal testicular descent and many animals develop inguinal hernias. The ability of estradiol to cause testis-to-ovary sex reversal in marsupials provides a new way of studying the interactions between genes and hormones in testicular differentiation.

One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development are inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Interestingly, this process occurs in the absence of other obvious features of testis differentiation, suggesting that Wnt4 specifically inhibits XY vascular development. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify sets of genes similarly upregulated in wildtype XY gonads and XX mutant gonads or upregulated in XX gonads as compared to XY gonads and XX mutant gonads. We show that several genes identified in the first set are expressed in vascular domains, and have predicted functions related to cell migration or vascular development. However, the expression patterns and known functions of other genes are not consistent with roles in these processes. This screen has identified candidates for regulation of sex specific vascular development, and has implicated a role for WNT4 signaling in the development of Sertoli and germ cell lineages not immediately obvious from previous phenotypic analyses.

This study reports the developmental anatomy of testicular descent and inguinal closure of the tammar wallaby ( Macropus eugenii ) from birth to maturity. In females the ovary migrated caudally between days 10 and 20 after birth. The gubernaculum differentiates into the round ligament in the abdomen and extra-abdominally as the ilio-marsupialis muscle of the mammary glands. In males the testes migrated to the internal inguinal ring by day 20 post partum (pp), coinciding with the enlargement of the gubernaculum, and from the internal inguinal ring to the scrotum between days 20 and 65 pp. During descent there was an increase in the hyaluronic acid concentration in cells of the gubernaculum and scrotum. Development of the cremaster muscle began by day 10 pp on the periphery of the gubernaculum and its basic structure was completed by day 60 pp. After descent the inguinal canal closed between days 50 and 60 pp, but a small irregular lumen persisted, somewhat similar to that seen in the congenital scrotal hydrocoele of humans. Tammars have a hopping mode of locomotion and, like humans, are essentially bipedal. We suggest that inguinal closure evolved in these two species because their upright posture may otherwise lead to a high incidence of inguinal hernias.

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