BackgroundIn recent years, the phenotypes of leukodystrophies linked to mutations in the eukaryotic initiation factor 2B genes have been extended, classically called CACH/VWM (Childhood ataxia with cntral hypomyélination/vanishing white matter disorder). The large clinical spectrum observed from the more severe antenatal forms responsible for fetal death to milder adult forms with an onset after 16 years old and restricted to slow cognitive impairment have lead to the concept of eIF2B-related disorders. The typical MRI pattern with a diffuse CSF-like aspect of the cerebral white matter can lack particularly in the adult forms whereas an increasing number of patients with clinical and MRI criteria for CACH/VWM disease but without eIF2B mutations are found. Then we propose the use of biochemical markers to help in this difficult diagnosis. The biochemical diagnosis of eIF2B-related disorder is difficult as no marker, except the recently described asialotransferrin/transferrin ratio measured in cerebrospinal fluid, has been proposed and validated until now. Decreased eIF2B GEF activity has been previously reported in lymphoblastoid cell lines from 30 eIF2B-mutated patients. Our objective was to evaluate further the utility of this marker and to validate eIF2B GEF activity in a larger cohort as a specific diagnostic test for eIF2B-related disorders.Methodology/Principal FindingsWe performed eIF2B GEF activity assays in cells from 63 patients presenting with different clinical forms and eIF2B mutations in comparison to controls but also to patients with defined leukodystrophies or CACH/VWM-like diseases without eIF2B mutations. We found a significant decrease of GEF activity in cells from eIF2B-mutated patients with 100% specificity and 89% sensitivity when the activity threshold was set at ≤77.5%.ConclusionThese results validate the measurement of eIF2B GEF activity in patients' transformed-lymphocytes as an important tool for the diagnosis of eIF2B-related disorders.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/β and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/β and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/β and stabilize β-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.Animal studies. Teratoma assays were performed with 7-week-old severe combined immunodeficient (SCID) male mice (CB17/SCID, Charles River). Ntn1 βgeo reporter and Netrin-1 conditional-KO mESCs were derived from C57/bl6 mixed-background pregnant females at 8-15 weeks of age. Blastocyst injections were done using BALB/cANRj embryos. Embryos were flushed with M2 medium (Sigma) and grown overnight in KSOM (Sigma) or sequential blast (Origio) medium. Genotyping of Ntn1 fl/fl embryos was performed as previously described 44 . X-gal was detected in blastocysts using secondary antibodies coupled with biotin, the Vectastain ABC kit and DAB (Vector System). Teratoma-formation assays were performed by injecting 1 × 10 6 mESCs in the testes of 7-week-old SCID mice. After 3-4 weeks, the mice were euthanized and lesions were surgically removed and fixed in formalin or in 4% paraformaldehyde for sections. For blastocyst injections, Ntn1 fl/fl , untreated or treated with TAM for 48 h, were injected into BALB/cANRj blastocysts. The day before injection, frozen BALB/cANRj morulas from Quickblasto (Janvier) were thawed according to the manufacturer's instructions and incubated overnight in KSOM medium (Millipore) at 37 °C with 5% CO 2 . Between 5 and 15 cells were injected into expanded blastocysts in M2 medium (Sigma) using standard blastocyst injection techniques. Blastocysts were then allowed to recover for a period of 1-3 h prior to being implanted into pseudopregnant females. All animal procedures were performed in accordance with institutional guidelines (French ceccapp project 01369.01).Cell culture. The following cell lines were used in the study. Cgr8 ES cells (ECACC 07032901) were provided by the B. Pain laboratory (SBRI, Bron, France). E14Tg2a ES cells (ATCC CRL 1821) were provided by the M. E. Torres Padilla laboratory (IES, Munchen, Germany). Ntn1 βgeo reporter 27 and Netrin-1 conditional-KO (Ntn1 fl/fl ) 44 mESC...

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the β-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation.

BackgroundEukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.MethodsWe used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of ATF4, and transcriptional induction of stress-specific mRNAs (ATF4, CHOP, ASNS, GRP78) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).ResultsDespite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.ConclusionsTaken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.

Leukodystrophies (LD) are rare inherited disorders that primarily affect the white matter (WM) of the central nervous system. The large heterogeneity of LD results from the diversity of the genetically determined defects that interfere with glial cells functions. Astrocytes have been identified as the primary target of LD with cystic myelin breakdown including those related to mutations in the ubiquitous translation initiation factor eIF2B. EIF2B is involved in global protein synthesis and its regulation under normal and stress conditions. Little is known about how eIF2B mutations have a major effect on WM. We performed a transcriptomic analysis using fibroblasts of 10 eIF2B-mutated patients with a severe phenotype and 10 age matched patients with other types of LD in comparison to control fibroblasts. ANOVA was used to identify genes that were statistically significantly differentially expressed at basal state and after ER-stress. The pattern of differentially expressed genes between basal state and ER-stress did not differ significantly among each of the three conditions. However, 70 genes were specifically differentially expressed in eIF2B-mutated fibroblasts whatever the stress conditions tested compared to controls, 96% being under-expressed. Most of these genes were involved in mRNA regulation and mitochondrial metabolism. The 13 most representative genes, including genes belonging to the Heterogeneous Nuclear Ribonucleoprotein (HNRNP) family, described as regulators of splicing events and stability of mRNA, were dysregulated during the development of eIF2B-mutated brains. HNRNPH1, F and C mRNA were over-expressed in foetus but under-expressed in children and adult brains. The abnormal regulation of HNRNP expression in the brain of eIF2B-mutated patients was concomitant with splicing dysregulation of the main genes involved in glial maturation such as PLP1 for oligodendrocytes and GFAP in astrocytes. These findings demonstrate a developmental deregulation of splicing events in glial cells that is related to abnormal production of HNRNP, in eIF2B-mutated brains.

Coordinated changes of cellular plasticity and identity are critical for pluripotent reprogramming and oncogenic transformation. However, the sequences of events that orchestrate these intermingled modifications have never been comparatively dissected. Here, we deconvolute the cellular trajectories of reprogramming (via Oct4/Sox2/Klf4/c-Myc) and transformation (via Ras/c-Myc) at the single-cell resolution and reveal how the two processes intersect before they bifurcate. This approach led us to identify the transcription factor Bcl11b as a broad-range regulator of cell fate changes, as well as a pertinent marker to capture early cellular intermediates that emerge simultaneously during reprogramming and transformation. Multiomics characterization of these intermediates unveiled a c-Myc/Atoh8/Sfrp1 regulatory axis that constrains reprogramming, transformation and transdifferentiation. Mechanistically, we found that Atoh8 restrains cellular plasticity, independent of cellular identity, by binding a specific enhancer network. This study provides insights into the partitioned control of cellular plasticity and identity for both regenerative and cancer biology.

Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key regulatory pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression of over 300 genes involved in ribosome biogenesis in mouse ESCs and identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.

MicroRNAs are important regulators of gene expression and are involved in cellular processes such as proliferation or differentiation, particularly during development of numerous organs including the inner ear. However, it remains unknown if miRNAs are required during the earliest stages of otocyst and cochlear duct development. Here, we report that a conditional loss of Dicer expression in the otocyst impairs the early development of the inner ear as a result of the accumulation of DNA damage that trigger p53-mediated apoptosis. Moreover, cochlear progenitors in the prosensory domain do not exit the cell cycle. Our unbiased approach identified ItgA3 as a target of miR-183, which are both enriched in the otic vesicle. We observed that the repression of integrin alpha 3 by miR-183 controls cell proliferation in the developing cochlea. Collectively, our results reveal that Dicer and miRNAs play essential roles in the regulation of early inner ear development.