Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary.

R-spondin1 is a secreted regulator of WNT signaling, involved in both embryonic development and homeostasis of adult organs. It can have a dual role, acting either as a mitogen or as a tumor suppressor. During ovarian development, Rspo1 is a key factor required for sex determination and differentiation of the follicular cell progenitors, but is downregulated after birth. In human, increased RSPO1 expression is associated with ovarian carcinomas, but it is not clear whether it is a cause or a consequence of the tumorigenic process. To address the role of Rspo1 expression in adult ovaries, we generated an Rspo1 gain-of-function mouse model. Females were hypofertile and exhibited various ovarian defects, ranging from cysts to ovarian tumors. Detailed phenotypical characterization showed anomalies in the ovulation process. Although follicles responded to initial follicle-stimulating hormone stimulation and developed normally until the pre-ovulatory stage, they did not progress any further. Although non-ovulated oocytes degenerated, the surrounding follicular cells did not begin atresia. RSPO1-induced expression not only promotes canonical WNT signaling but also alters granulosa cell fate decisions by maintaining epithelial-like traits in these cells. This prevents follicle cells from undergoing apoptosis, leading to the accumulation of granulosa cell tumors that reactivates the epithelial program from their progenitors. Taken together, our data demonstrate that activation of RSPO1 is sufficient in promoting ovarian tumors and thus supports a direct involvement of this gene in the commencement of ovarian cancers.

Loss of R-spondin1 and Foxl2 amplifies female-to-male sex reversal in XX mice.

In vertebrates, 2 main genetic pathways have been shown to regulate ovarian development. Indeed, a loss of function mutations in Rspo1 and Foxl2 promote partial female-to-male sex reversal. In mice, it has been shown that the secreted protein RSPO1 is involved in ovarian differentiation and the transcription factor FOXL2 is required for follicular formation. Here, we analysed the potential interactions between these 2 genetic pathways and have shown that while Rspo1 expression seems to be independent of Foxl2 up-regulation, Foxl2 expression partly depends of Rspo1 signalisation. This suggests that different Foxl2-positive somatic cell lineages exist within the ovaries. In addition, a combination of both mutated genes in XX Foxl2(-/-)/Rspo1(-/-) gonads promotes sex reversal, detectable at earlier stages than in XX Rspo1(-/-) mutants. Ectopic development of the steroidogenic lineage is more pronounced in XX Foxl2(-/-)/Rspo1(-/-) gonads than in XX Rspo1(-/-) embryos, suggesting that Foxl2 is involved in preventing ectopic steroidogenesis in foetal ovaries.

R-spondin1 deficiency in mice improves glycaemic control in association with increased beta cell mass.

AIMS/HYPOTHESIS: Roof plate-specific spondin (R-spondin1; RSPO1) is a modulator of canonical Wg (wingless) plus Int1 (chromosomal integration site of mouse mammary tumour virus on mouse chromosome 15) (cWNT) signalling that induces cWNT target genes. We have demonstrated that Rspo1 is expressed in murine beta cells, and that it stimulates proliferation and insulin secretion, and inhibits cytokine-induced apoptosis, in mouse insulinoma (MIN6) and beta cells. We thus investigated the role of RSPO1 in beta cells in vivo using Rspo1 ( -/- ) mice. METHODS: The effects of Rspo1 deficiency were assessed by determination of cWNT signalling, glucose tolerance and beta cell mass. RESULTS: Rspo1 ( -/- ) mice demonstrated an 82% reduction in RSPO1 transcripts and a 61% reduction in the signal detected by an RSPO1 antibody, as well as a 47% decrease in islet cWNT signalling. Despite no differences in body and pancreatic weights or in fasting glycaemia and insulinaemia compared with Rspo1 (+/+) mice, Rspo1 ( -/- ) animals had improved glycaemic control after oral glucose challenge (p < 0.05), with no difference in insulin sensitivity, but an enhanced insulin response over 30 min (p < 0.05); glucagon responses were normal. Rspo1 deficiency also resulted in a twofold increase in beta cell mass (p < 0.05) in association with 2- and 12-fold increases in the number of beta cells positive for antigen identified by monoclonal antibody Ki67 (Ki67) (p < 0.01) and insulin-positive ductal cells (p < 0.05), respectively. No change in the number of TUNEL-positive beta cells was detected. Islets isolated from Rspo1 ( -/- ) animals displayed no differences in glucose-induced insulin secretion or in glucose suppression of glucagon. CONCLUSIONS/INTERPRETATION: The present study reveals an unexpected role for RSPO1 as a regulator of both beta cell proliferation and neogenesis in vivo, and reinforces the importance of cWNT signalling for the maintenance of normal pancreatic beta cell behaviour.

Genetics of ovarian differentiation: Rspo1, a major player.

In mammals, the sex of the embryo is determined during development by its commitment either to the male or female genetic program regulating testicular or ovarian organogenesis. Major steps towards unraveling sex determination in mammals are achieved by the identification of key genes involved in human pathologies and the application of mouse genetics to analyze their function. While the expression of Sry and Sox9 is sufficient to induce the male developmental program, the molecular pathways that specify ovarian differentiation were unclear before the recent demonstration that mutations in the RSPO1 gene induce female-to-male sex reversal in XX patients. By generating the corresponding mouse model, we have shown that Rspo1 is so far the earliest known gene controlling the female genetic developmental program. Rspo1 activates the canonical beta-catenin signaling pathway required for female somatic cell differentiation and germ cell commitment into meiosis. The aim of this review is to describe the roles of R-spondins (Rspo)in developmental processes and disorders and the current knowledge obtained from murine models. A particular focus will be on Rspo1 and its crucial function in sex determination.

Molecular cloning, expression and regulation of the avian tubby-like protein 1 (tulp1) gene.

The tubby-like protein 1 (tulp1) gene is a member of the tubby multigene family which includes tub, tulp1, tulp2 and tulp3. Human and mouse tulp1 genes were cloned and mutations in tulp1 have been implicated in retinitis pigmentosa in man. Here we report on the cDNA cloning of the chicken tulp1 homologue and its protein product deduced from the nucleotide sequence. The chicken Tulp1 protein comprises 358 amino acids with a calculated molecular mass of 40 kDa. The overall structure of Tub and Tulp proteins, exemplified by the highly conserved C-terminal domain of 255 amino acids and the signature motif KLACE, is also preserved in chicken Tulp1. Phylogenetic analysis demonstrates that chicken tulp1 cDNA and protein are closely related to human and mouse tulp1. In addition, chicken tulp1 mRNA is abundantly expressed in retina similar to tulp1 expression in human and mouse. Two tulp1-specific transcripts of 2 and 3 kb in size were identified that showed differential regulation during embryonic and postnatal development. Finally, tulp1 mRNA was found to be expressed in chicken erythroid cells and upregulated by ligand-activated thyroid hormone receptor (TR alpha/c-erbA).

Sox9 induces testis development in XX transgenic mice.

Mutations in SOX9 are associated with male-to-female sex reversal in humans. To analyze Sox9 function during sex determination, we ectopically expressed this gene in XX gonads. Here, we show that Sox9 is sufficient to induce testis formation in mice, indicating that it can substitute for the sex-determining gene Sry.

Retrotransposition of the I factor, a non-long terminal repeat retrotransposon of Drosophila, generates tandem repeats at the 3' end.

Non-long terminal repeat (LTR) retrotransposons or LINEs transpose by reverse transcription of an RNA intermediate and are thought to use the 3' hydroxyl of a chromosomal cleavage to initiate synthesis of the first strand of the cDNA. Many of them terminate in a poly(dA) sequence at the 3' end of the coding strand although some, like the I factor of Drosophila melanogaster, have 3' ends formed by repeats of the trinucleotide TAA. We report results showing that I factor transcripts end a few nucleotides downstream of the TAA repeats and that these extra nucleotides are not integrated into chromosomal DNA during retrotransposition. We also show that the TAA repeats are not required for transposition and that I elements containing mutations affecting the TAA sequences generate transposed copies ending with tandem repeats of various types. Our results suggest that during integration the 3' end of the I factor RNA template can pair with nucleotides at the target site and that tandem duplications are generated by the reverse transcriptase of the I factor in a manner that is reminiscent of the activity of the reverse transcriptases of telomerases. Reverse transcriptases of other non-LTR retrotransposons may function in a similar way.

Copy number control of a transposable element, the I factor, a LINE-like element in Drosophila.

The I factor is a LINE-like transposable element in Drosophila. Most strains of Drosophila melanogaster, inducer strains, contain 10-15 copies of the I factor per haploid genome located in the euchromatic regions of the chromosome arms. These are not present in a few strains known as reactive strains. I factors transpose at low frequency in inducer strains but at high frequency in the female progeny of crosses between reactive and inducer flies. We have found that the activity of the I factor promoter is sensitive to the number of copies of the first 186 nucleotides of the I factor sequence, which constitutes the 5'-untranslated region. The activity of the I factor decreases as the copy number of this sequence increases.

A genetically marked I element in Drosophila melanogaster can be mobilized when ORF2 is provided in trans.

I factors in Drosophila melanogaster are non-LTR retrotransposons similar to mammalian LINEs. They transpose at very high frequencies in the germ line of SF females resulting from crosses between reactive females, devoid of active I factors, and inducer males, containing active I factors. The vermilion marked IviP2 element was designed to allow easy phenotypical screening for retrotransposition events. It is deleted in ORF2 and therefore cannot produce reverse transcriptase. IviP2 can be mobilized at very low frequencies by actively transposing I factors in the germ line of SF females. This paper shows that IviP2 can be mobilized more efficiently in the germ line of strongly reactive females in the absence of active I factors, when it is trans-complemented by the product of ORF2 synthesized from the hsp70 heat-shock promoter. This represents a promising step toward the use of marked I elements to study retrotransposition and as tools for mutagenesis.

A genetically tagged, defective I element can be complemented by actively transposing I factors in the germline of I-R dysgenic females in Drosophila melanogaster.

Non-LTR retrotransposons, also known as LINEs, transpose by reverse transcription of an RNA intermediate. Their mechanism of transposition is apparently different from that of retrotransposons and similar to that of proviruses of retroviruses. The I factor is responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. Inducer strains contain several functional I factors whereas reactive strains do not. Transposition of I factors can be experimentally induced: they are stable in inducer strains, but transpose at high frequency in the germline of females, known as SF females, produced by crossing reactive females and inducer males. We have constructed an I element, called IviP2, marked with the vermilion gene, the coding sequence of which was interrupted by an intron. Splicing of the intron can only occur in the transcript initiated from the I element promoter. Transposed copies expressing a wild-type vermilion phenotype were recovered in the germline of SF females in which I factors were actively transposing. This indicates that trans-complementation of a defective I element, deficient for the second open reading frame, by functional I factors can occur in the germline of dysgenic females.

IR hybrid dysgenesis increases the frequency of recombination in Drosophila melanogaster.

The I factor is a LINE-like transposable element responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. Inducer strains of this species contain several I factors whereas reactive strains do not. I factors are stable in inducer strains, but transpose at high frequency in the germ-line of females, known as SF females, produced by crossing reactive females and inducer males. Various abnormalities occur in SF females, most of which result from this high rate of transposition. We report here that recombination is increased in the germ-line of these females. This is a new characteristic of the I-R system of hybrid dysgenesis that might also be associated with transposition of the I factor.

I factors in Drosophila melanogaster: transposition under control.

I factors are responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. They belong to the LINE class of mobile elements, which transpose via reverse transcription of a full-length RNA intermediate. I factors are active members of the I element family, which also contains defective I elements that are immobilized within peri-centromeric heterochromatin and represent very old components of the genome. Active I factors have recently invaded natural populations of Drosophila melanogaster, giving rise to inducer strains. Reactive strains, devoid of active I factors, derive from old laboratory stocks established before the invasion. Transposition of I factors is activated at very high frequencies in the germline of hybrid females issued from crosses between females from reactive strains and males from inducer strains. It results in the production of high rates of mutations and chromosomal rearrangements as well as in a particular syndrome of sterility. The frequency of transposition of I factors is dependent on the amount of full-length RNA that is synthesized from an internal promoter. This full-length RNA serves both as an intermediate of transposition and presumably as a messenger for protein synthesis. Regulators of transposition apparently affect transcription initiation from the internal promoter. The data presented here lead to the proposal of a tentative model for transposition.

The Doc transposable element in Drosophila melanogaster and Drosophila simulans: genomic distribution and transcription.

The mobile element Doc is similar in structure and coding potential to the LINE families found in various organisms. In this paper, we analyze the insertional and structural polymorphism of this element and show that it appears to have a long evolutionary history in the genome of D. melanogaster. Like the family of I elements, the Doc family seems to display three types of elements: full length elements, defective members that have recently transposed and long since immobilized members common to each D. melanogaster strain. These three classes of Doc elements seem to be present in D. simulans, a closely related species to D. melanogaster. Furthermore, we show that Doc is transcribed as a polyadenylated RNA of about 5 kb in length, presumed to be a full length RNA. This transcript is present in different tissues and at different stages of Drosophila development. These results are compared with previous records on the chromosomal distribution of LINEs or other transposable element families. Doc transcription is analyzed in an attempt to understand the link between Doc transcription and transposition.

I elements and the Drosophila genome.

LINEs are a large class of transposable elements in eukaryotes. They transpose by reverse transcription of an RNA intermediate. I elements of Drosophila melanogaster belong to this class and are responsible for the I-R system of hybrid dysgenesis. Many results indicate that at the beginning of the century natural populations of this species were devoid of active I elements and that they were invaded by functional I elements in the last decades. Many Drosophila species contain both defective and active I elements. It seems that the latter were lost in Drosophila melanogaster before its spread throughout the world, and that the recent invasion results from the spread of functional elements originating either from another species by horizontal transfer or from an isolated population of the same species. These data are discussed, as well as their significance in evolutionary processes.

Identification of a potential RNA intermediate for transposition of the LINE-like element I factor in Drosophila melanogaster.

The I factor, a transposable element related to mammalian LINEs, controls the I-R system of hybrid dysgenesis in Drosophila melanogaster. It transposes at high frequency in the germ-line of the female progeny of crosses between females of the reactive class of strains and males of the inducer class. The structure and DNA sequence of the I factor suggest that it transposes by reverse transcription of an RNA intermediate. Northern blot and S1 mapping experiments show that a full-length RNA of the I factor is synthesized specifically in the conditions of which I factors transpose. This RNA has all characteristics of a transposition intermediate. It is only found in the ovaries of dysgenic females suggesting that I factor activity is restricted to this tissue because of regulation at the level of the initiation of transcription or RNA stability.

A long interspersed repetitive element--the I factor of Drosophila teissieri--is able to transpose in different Drosophila species.

Long interspersed repetitive elements (LINEs) are transposable elements present in many species. In mammals they are difficult to study because most of them are defective and their transposition frequency is low. The I factor of Drosophila melanogaster is a LINE element that is particularly interesting because its transposition occurs at high frequency during I-R hybrid dysgenesis. This phenomenon occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several complete 5.4-kilobase I factors at various sites on the chromosomal arms. Reactive strains are devoid of complete I factors. Many results indicate that active I factors have invaded the D. melanogaster genome recently. To study the evolutionary history of I elements, we have cloned and sequenced a potentially active I factor from Drosophila teissieri. It is flanked by a target-site duplication and terminates at the 3' end by tandem repeats of the sequence TAA. When introduced into the germ line of a reactive strain of D. melanogaster by P element-mediated transformation, it is able to transpose and induces hybrid dysgenesis. This strengthens the hypothesis of a recent reinvasion of the D. melanogaster genome by active I factors giving rise to the inducer strains. They could have originated by horizontal transfer from another species. Such events also could occur for other LINE elements and might explain the spread of new variants in mammalian genomes. Moreover, the results give a further insight into I factor functional organization.