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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. [ABSTRACT FROM AUTHOR]

Genome-wide functional analyses require high-resolution genome assembly and annotation. We applied ChIA-PET to analyze gene regulatory networks, including 3D chromosome interactions, underlying thyroid hormone (TH) signaling in the frog Xenopus tropicalis. As the available versions of Xenopus tropicalis assembly and annotation lacked the resolution required for ChIA-PET we improve the genome assembly version 4.1 and annotations using data derived from the paired end tag (PET) sequencing technologies and approaches (e.g., DNA-PET [gPET], RNA-PET etc.). The large insert (~10Kb, ~17Kb) paired end DNA-PET with high throughput NGS sequencing not only significantly improved genome assembly quality, but also strongly reduced genome “fragmentation”, reducing total scaffold numbers by ~60%. Next, RNA-PET technology, designed and developed for the detection of full-length transcripts and fusion mRNA in whole transcriptome studies (ENCODE consortia), was applied to capture the 5' and 3' ends of transcripts. These amendments in assembly and annotation were essential prerequisites for the ChIA-PET analysis of TH transcription regulation. Their application revealed complex regulatory configurations of target genes and the structures of the regulatory networks underlying physiological responses. Our work allowed us to improve the quality of Xenopus tropicalis genomic resources, reaching the standard required for ChIA-PET analysis of transcriptional networks. We consider that the workflow proposed offers useful conceptual and methodological guidance and can readily be applied to other non-conventional models that have low-resolution genome data. [ABSTRACT FROM AUTHOR]

Gnathostome jaws derive from the first pharyngeal arch (PA1), a complex structure constituted by Neural Crest Cells (NCCs), mesodermal, ectodermal and endodermal cells. Here, to determine the regionalized morphogenetic impact of Dlx5/6 expression, we specifically target their inactivation or overexpression to NCCs. NCC-specific Dlx5/6 inactivation (NCC∆Dlx5/6) generates severely hypomorphic lower jaws that present typical maxillary traits. Therefore, differently from Dlx5/6 null-embryos, the upper and the lower jaws of NCC∆Dlx5/6 mice present a different size. Reciprocally, forced Dlx5 expression in maxillary NCCs provokes the appearance of distinct mandibular characters in the upper jaw. We conclude that: (1) Dlx5/6 activation in NCCs invariably determines lower jaw identity; (2) the morphogenetic processes that generate functional matching jaws depend on the harmonization of Dlx5/6 expression in NCCs and in distinct ectodermal territories. The co-evolution of synergistic opposing jaws requires the coordination of distinct regulatory pathways involving the same transcription factors in distant embryonic territories. [ABSTRACT FROM AUTHOR]

The discovery of adult neural stem cells (NSC) in the mammalian brain a few years ago raised the possibility of new therapeutic perspectives for the treatment of genetic or inherited neurological disorders. However, little is known about the fundamental biology of this NSC population, and functional analysis of target genes has to be done to better understand the mechanisms underlying their proliferation or differentiation pathways. The tightly localised position of these neural stem cells in the subependymal area lining the lateral ventricles in the brain make them accessible to gene transfer by stereotaxic injections in the lateral ventricles. Knocking down the expression of a target gene by RNA interference (RNAi) in the NSC population of adult mice brains could lead to the determination of the function of a given protein in its in vivo cellular context.After having demonstrated that RNAi technology can be efficient in vivo by stereotaxic injection of small interfering RNAs (siRNAs) in the lateral ventricles of newborn mice (Hassani et al., 2005), we now address the question of whether small hairpin RNAs (shRNAs) could lead to the specific inhibition of a target gene in the same conditions.The advantage in the use of shRNAs is that we actually know that linear polyethylenimine (L-PEI) is able to specifically deliver plasmids to NSC and neural progenitors located in the subventricular zone of adult mice brains (Lemkine et al., 2002) after stereotaxic injections in the lateral ventricles. However, PEI is not an efficient vector for the delivery of siRNAs in the brain in vivo (Hassani et al., 2005).On this basis we injected plasmids carrying small hairpin RNAs complexed by L-PEI to the lateral ventricles of newborn mice brains in order to specifically inhibit the expression of a target gene. Our first observation was that shRNAs under the control of a H1- or a CMV-promoter are not efficient for the inhibition of the luciferase target gene in vivo. Surprisingly, we found that an hybrid CMV-H1 promoter directing the expression of the shRNA gave significantly better inhibition of the target gene than a H1- or CMV- promoter alone.As thyroid hormone tri-iodothyronin (T3) is necessary for proliferation of NSC in vivo (Lemkine et al., 2005), we sought to identify the genes involved in T3-mediated regulation of NSC proliferation. Two genes potentially implicated in NSC proliferation were tested and found to respond to T3 in the newborn mouse brain: CyclinD1 and Sox2. We found that cyclinD1 expression was significantly decreased after T3 treatment of hypothyroid animals, and that this repression involved Thyroid hormone Receptor alpha1 (TRa1). Sox2 is a transcription factor that is a major player in embryonic neurogenesis. Its expression is limited to NSC in the adult. Sox2 was found to be activated by T3 in newborn mice brains. We now aim to determine whether Sox2 is also regul ated by T3 via TRa1 or via another TR, and we will assess this question by inhibiting TRa1 with specific shRNAs.Molecular Therapy (2006) 13, S134–S134; doi: 10.1016/j.ymthe.2006.08.409 [ABSTRACT FROM AUTHOR]

Abstract: Thyroid hormones (THs) are fundamental in regulation of growth and development, particularly of the brain. THs are required for full proliferative activity of neural stem cells in the subventricular zone (SVZ) of adult mouse brains, and also affect the normal fate of progenitor cells: apoptosis. Transthyretin (TTR) is a TH distributor protein in the blood and cerebrospinal fluid. TTR secretion by the choroid plexus is involved in transport of THs from blood into cerebrospinal fluid. We investigated the regulation of neural stem cell cycle in the SVZ of adult TTR null mice. Markers for neural stem cell mitosis that are reduced during hypothyroidism, did not differ between genotypes. However, in TTR null mice the level of apoptosis, the fate of most progenitor cells, was as low as that in brains of hypothyroid wildtype mice. Thus, lack of TTR results in reduced availability of TH to progenitor cells in the SVZ. We show that proliferation and apoptosis in the SVZ neural stem cell niche are differentially affected by the lack of TTR synthesis. [Copyright &y& Elsevier]