Dlx5 and Dlx6 control uterine adenogenesis during post-natal maturation: possible consequences for endometriosis.

Dlx5 and Dlx6 are two closely associated homeobox genes which code for transcription factors involved in the control of steroidogenesis and reproduction. Inactivation of Dlx5/6 in the mouse results in a Leydig cell defect in the male and in ovarian insufficiency in the female. DLX5/6 are also strongly expressed by the human endometrium but their function in the uterus is unknown. The involvement of DLX5/6 in human uterine pathology is suggested by their strong downregulation in endometriotic lesions and upregulation in endometrioïd adenocarcinomas. We first show that Dlx5/6 expression begins in Müllerian ducts epithelia and persists then in the uterine luminal and glandular epithelia throughout post-natal maturation and in the adult. We then use a new mouse model in which Dlx5 and Dlx6 can be simultaneously inactivated in the endometrium using a Pgr(cre/+) allele. Post-natal inactivation of Dlx5/6 in the uterus results in sterility without any obvious ovarian involvement. The uteri of Pgr(cre/+); Dlx5/6(flox/flox) mice present very few uterine glands and numerous abnormally large and branched invaginations of the uterine lumen. In Dlx5/6 mutant uteri, the expression of genes involved in gland formation (Foxa2) and in epithelial remodelling during implantation (Msx1) is significantly reduced. Furthermore, we show that DLX5 is highly expressed in human endometrial glandular epithelium and that its expression is affected in endometriosis. We conclude that Dlx5 and Dlx6 expression determines uterine architecture and adenogenesis and is needed for implantation. Given their importance for female reproduction, DLX5 and DLX6 must be regarded as interesting targets for future clinical research.

Etiology of craniofacial malformations in mouse models of blepharophimosis, ptosis and epicanthus inversus syndrome.

Blepharophimosis, ptosis, epicanthus-inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by narrow palpebral fissures and eyelid levator muscle defects. BPES is often associated to premature ovarian insufficiency (BPES type I). FOXL2, a member of the forkhead transcription factor family, is the only gene known to be mutated in BPES. Foxl2 is essential for maintenance of ovarian identity, but the developmental origin of the facial malformations of BPES remains, so far, unexplained. In this study, we provide the first detailed account of the developmental processes leading to the craniofacial malformations associated to Foxl2. We show that, during development, Foxl2 is expressed both by Cranial Neural Crest Cells (CNCCs) and by Cranial Mesodermal Cells (CMCs), which give rise to skeletal (CNCCs and CMCs) and muscular (CMCs) components of the head. Using mice in which Foxl2 is selectively inactivated in either CNCCs or CMCs, we reveal that expression of Foxl2 in CNCCs is essential for the development of extraocular muscles. Indeed, inactivation of Foxl2 in CMCs has only minor effects on muscle development, whereas its inactivation in CNCCs provokes a severe hypoplasia of the levator palpabrae superioris and of the superior and inferior oblique muscles. We further show that Foxl2 deletion in either CNCCs or CMCs prevents eyelid closure and induces subtle skeletal developmental defects. Our results provide new insights in the complex developmental origin of human BPES and could help to understand the origin of other ocular anomalies associated to this syndrome.

Role of Foxl2 in uterine maturation and function.

Foxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women.

Allelic reduction of Dlx5 and Dlx6 results in early follicular depletion: a new mouse model of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by the loss of ovarian function before the age of 40 in humans. Although most cases of POI are idiopathic, many are familial, underlying a genetic origin of the disease. Mutations in genes involved in the control of steroidogenesis, such as NR5A1 (SF-1, Steroidogenic Factor 1), CYP17, CYP19A1 (aromatase), StAR (Steroidogenic Acute Regulatory), and the forkhead transcription factor FOXL2 have been associated with different forms of POI. In males, the homeobox transcription factors Dlx5 and Dlx6 are involved in the control of steroidogenesis through the activation of GATA4-induced-StAR transcription. Here, we analyze the potential involvement of Dlx5 and Dlx6 in female reproduction. To this end, we make use of an existing mouse model in which Dlx5 and Dlx6 are simultaneously disrupted. We show that: (i) allelic reduction of Dlx5 and Dlx6 in the mouse results in a POI-like phenotype, characterized by reduced fertility and early follicular exhaustion; (ii) in granulosa cell lines, a reciprocal regulation exists between Dlx5 and Foxl2; (iii) in the mouse ovary, allelic reduction of Dlx5/6 results in the upregulation of Foxl2. We propose that the mutual regulation between Dlx5/6 and Foxl2 and their opposite effects on StAR expression might contribute to determine the homeostatic control of steroidogenesis within the ovary. Dysregulation of this homeostatic control would result in abnormal follicular maturation and reduced fertility. Our results bring new elements to our conceptual model of follicle maturation and maintenance and provide new potential genetic targets for cases of familial POI.

Distal-less homeobox genes Dlx5/6 regulate Müllerian duct regression.

Dlx5 and Dlx6 encode distal-less homeodomain transcription factors that are present in the genome as a linked pair at a single locus. Dlx5 and Dlx6 have redundant roles in craniofacial, skeletal, and uterine development. Previously, we performed a transcriptome comparison for anti-Müllerian hormone (AMH)-induced genes expressed in the Müllerian duct mesenchyme of male and female mouse embryos. In that study, we found that Dlx5 transcripts were nearly seven-fold higher in males compared to females and Dlx6 transcripts were found only in males, suggesting they may be AMH-induced genes. Therefore, we investigated the role of Dlx5 and Dlx6 during AMH-induced Müllerian duct regression. We found that Dlx5 was detected in the male Müllerian duct mesenchyme from E14.5 to E16.5. In contrast, in female embryos Dlx5 was detected in the Müllerian duct epithelium. Dlx6 expression in Müllerian duct mesenchyme was restricted to males. Dlx6 expression was not detected in female Müllerian duct mesenchyme or epithelium. Genetic experiments showed that AMH signaling is necessary for Dlx5 and Dlx6 expression. Müllerian duct regression was variable in Dlx5 homozygous mutant males at E16.5, ranging from regression like controls to a block in Müllerian duct regression. In E16.5 Dlx6 homozygous mutants, Müllerian duct tissue persisted primarily in the region adjacent to the testes. In Dlx5-6 double homozygous mutant males Müllerian duct regression was also found to be incomplete but more severe than either single mutant. These studies suggest that Dlx5 and Dlx6 act redundantly to mediate AMH-induced Müllerian duct regression during male differentiation.

Decreased rhythmic GABAergic septal activity and memory-associated theta oscillations after hippocampal amyloid-beta pathology in the rat.

The memory deficits associated with Alzheimer's disease result to a great extent from hippocampal network dysfunction. The coordination of this network relies on theta (symbol) oscillations generated in the medial septum. Here, we investigated in rats the impact of hippocampal amyloid beta (Abeta) injections on the physiological and cognitive functions that depend on the septohippocampal system. Hippocampal Abeta injections progressively impaired behavioral performances, the associated hippocampal theta power, and theta frequency response in a visuospatial recognition test. These alterations were associated with a specific reduction in the firing of the identified rhythmic bursting GABAergic neurons responsible for the propagation of the theta rhythm to the hippocampus, but without loss of medial septal neurons. Such results indicate that hippocampal Abeta treatment leads to a specific functional depression of inhibitory projection neurons of the medial septum, resulting in the functional impairment of the temporal network.

Comparative analysis of molecular signatures suggests the use of gabapentin for the management of endometriosis-associated pain.

BACKGROUND: It has been repetitively shown that the transcription factors DLX5 and DLX6 are drastically downregulated in endometriotic lesions when compared with eutopic endometrium. These findings suggest that regulatory cascades involving DLX5/6 might be at the origin of endometriosis symptoms such as chronic pelvic pain (CPP). We have shown that inactivation of Dlx5 and Dlx5/6 in the mouse uterus results in an endometrial phenotype reminiscent of endometriosis. METHODS: We focused on genes that present a similar deregulation in endometriosis and in Dlx5/6-null mice in search of new endometriosis targets. RESULTS: We confirmed a strong reduction of DLX5 expression in endometriosis implants. We identified a signature of 30 genes similarly deregulated in human endometriosis implants and in Dlx5/6-null mouse uteri, reinforcing the notion that the downregulation of Dlx5/6 is an early event in the progress of endometriosis. CACNA2D3, a component of the α2δ family of voltage-dependent calcium channel complex, was strongly overexpressed both in mutant mouse uteri and in endometriosis implants, were also CACNA2D1 and CACNA2D2, other members of the α2δ family involved in nociception, are upregulated. CONCLUSION: Comparative analysis of gene expression signatures from endometriosis and mouse models showed that calcium channel subunits α2δ involved in nociception can be targets for the treatment of endometriosis-associated pain. CACNA2D3 has been associated with pain sensitization and heat nociception in animal models. In patients, CACNA2D3 variants were associated with reduced sensitivity to acute noxious stimuli. As α2δs were targets of gabapentinoid analgesics, the results suggested the use of these drugs for the treatment of endometriosis-associated pain. Indeed, recent small-scale clinical studies have shown that gabapentin could be effective in women with CPP. The findings of this study reinforce the need for a large definitive trial.

A new neuronal target for ß-amyloid peptide in the rat hippocampus.

In Alzheimer's disease, amyloid beta peptide (Aß) accumulation is associated with hippocampal network dysfunction. Intrahippocampal injections of Aß induce aberrant inhibitory septohippocampal (SH) network activity in vivo and impairment of memory processing. In the present study, we observed, after hippocampal Aß treatment, a selective loss of neurons projecting to the medial septum (MS) and containing calbindin (CB) and/or somatostatin (SOM). Other GABAergic neuronal subpopulations were not altered. Thus, the present study identifies hippocamposeptal neuron populations as specific targets for Aß deposits. We observed that in Aß-treated rats but not in controls, glutamate agonist application induced rhythmic bursting in 55% of the slow-firing neurons in the medial septum. This suggests that hippocampal Aß can trigger modifications of the septohippocampal pathway via the alteration of a specific neuronal population. Long-range hippocamposeptal GABA/calbindin neurons, targets of hippocampal amyloid deposits, are implicated in supporting network synchronization. By identifying this target, we contribute to the understanding of the mechanisms underlying deleterious effects of Aß, one of the main agents of dementia in Alzheimer's disease.