Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell-death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in testis after sex determination, we have analyzed mice lacking NR5A1 in Sertoli cells (SC) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impairs the expression of genes characteristic of the SC identity (e.g., Sox9, Amh), causes SC death from E14.5 through a Trp53-independent mechanism related to anoikis, and induces disorganization of the testis cords. Together, these effects cause germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SC change their molecular identity: some acquire a "pre-granulosa-like" identity, while other revert to a "supporting progenitor-like" cell identity, most of them being "intersex" because they express both testicular and ovarian genes. Fetal Leydig cells (LC) do not display significant changes, indicating that SC are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LC were absent from the postnatal testes. In addition, adult mutant males display persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which can be explained by the loss of AMH and testosterone synthesis due to SC failure.

Cell cycle gene regulation dynamics revealed by RNA velocity and deep-learning.

Despite the fact that the cell cycle is a fundamental process of life, a detailed quantitative understanding of gene regulation dynamics throughout the cell cycle is far from complete. Single-cell RNA-sequencing (scRNA-seq) technology gives access to these dynamics without externally perturbing the cell. Here, by generating scRNA-seq libraries in different cell systems, we observe cycling patterns in the unspliced-spliced RNA space of cell cycle-related genes. Since existing methods to analyze scRNA-seq are not efficient to measure cycling gene dynamics, we propose a deep learning approach (DeepCycle) to fit these patterns and build a high-resolution map of the entire cell cycle transcriptome. Characterizing the cell cycle in embryonic and somatic cells, we identify major waves of transcription during the G1 phase and systematically study the stages of the cell cycle. Our work will facilitate the study of the cell cycle in multiple cellular models and different biological contexts.

Meiosis occurs normally in the fetal ovary of mice lacking all retinoic acid receptors.

Gametes are generated through a specialized cell differentiation process, meiosis, which, in ovaries of most mammals, is initiated during fetal life. All-trans retinoic acid (ATRA) is considered as the molecular signal triggering meiosis initiation. In the present study, we analyzed female fetuses ubiquitously lacking all ATRA nuclear receptors (RAR), obtained through a tamoxifen-inducible cre recombinase-mediated gene targeting approach. Unexpectedly, mutant oocytes robustly expressed meiotic genes, including the meiotic gatekeeper STRA8. In addition, ovaries from mutant fetuses grafted into adult recipient females yielded offspring bearing null alleles for all Rar genes. Thus, our results show that RAR are fully dispensable for meiotic initiation, as well as for the production of functional oocytes. Assuming that the effects of ATRA all rely on RAR, our study goes against the current model according to which meiosis is triggered by endogenous ATRA in the developing ovary. It therefore revives the search for the meiosis-inducing substance.

Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons.

Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55-200 CGG repeats in the 5' UTR of FMR1. These expanded CGG repeats are transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.

Reevaluation of dystrophin localization in the mouse retina.

PURPOSE. The roles of dystrophins in retinal physiology remain elusive. The lack of proper clustering of the potassium channel Kir4.1 and of the aquaporin AQP4 was proposed to be the basis of the ERG abnormality observed in many Duchenne muscular dystrophy (DMD) patients. However, the electroretinogram of Dp71-null mice, in which this clustering is disrupted, shows only a moderate reduction of the b-wave with no change in the implicit times. Additionally, the deficit in color discrimination found in DMD patients is hard to explain through the known expression of DMD gene products. The authors thus decided to reexamine their distribution in the mouse retina. METHODS. Messenger RNA distribution was assessed by PCR coupled to laser microdissection of the outer and inner nuclear layers and by in situ hybridization for Dp427. Mouse retinas were double labeled for dystrophins versus presynaptic and postsynaptic proteins or antibodies specific for Dp427 or Dp427+Dp260. RESULTS. Messengers for Dp427, Dp260, and Dp140 were present in the inner nuclear layer. Dp427 mRNA was further detected in bipolar cells and in some amacrine cells by in situ hybridization. Comparative labeling in wild-type and mdx(5Cv) retinas (lacking Dp427) indicated a differential distribution of Dp427 and Dp260 between rod and cone terminals. CONCLUSIONS. In addition to their localization in photoreceptor terminals, Dp427, Dp260, and Dp140 are expressed in inner nuclear layer neurons, notably in bipolar cells for Dp427. Dp427 was proportionally more expressed in cone- than in rod-associated synapses compared with Dp260.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.