Timely Schwann cell division drives peripheral myelination in vivo via the laminin/cAMP pathway.

Schwann cells (SCs) migrate along peripheral axons and divide intensively to generate the right number of cells prior to axonal ensheathment; however, little is known regarding the temporal and molecular control of their division and its impact on myelination. We report that Sil, a spindle pole protein associated with autosomal recessive primary microcephaly, is required for temporal mitotic exit of SCs. In sil-deficient cassiopeia (csp-/-) mutants, SCs fail to radially sort and myelinate peripheral axons. Elevation of cAMP, but not Rac1 activity, in csp-/- restores myelin ensheathment. Most importantly, we show a significant decrease in laminin expression within csp-/- posterior lateral line nerve and that forcing Laminin 2 expression in csp-/- fully restores the ability of SCs to myelinate. Thus, we demonstrate an essential role for timely SC division in mediating laminin expression to orchestrate radial sorting and peripheral myelination in vivo.

Pals1a and aPKCλ are not essential for Schwann cell migration, division or myelination in zebrafish.

BACKGROUND: Schwann cells (SCs) are specialized glial cells of the peripheral nervous system that produce myelin and promote fast action potential propagation. In order to myelinate, SCs engage in a series of events that include migration and division along axons, followed by extensive cytoskeletal rearrangements that ensure axonal ensheathment and myelination. SCs are polarized and extend their processes along an abaxonal-adaxonal axis. Here, we investigate the role of the apical polarity proteins, Pals1a, and aPKCλ, in SC behavior during zebrafish development. RESULTS: We analyzed zebrafish nok and has mutants deficient for pals1a and aPKCλ function respectively. Using live imaging, transmission electron microscopy and whole mount immunostaining, we show that SCs can migrate and divide appropriately, exhibit normal radial sorting, express myelin markers and ensheath axons on time in has and nok mutants. CONCLUSIONS: Pals1a and aPKCλ are not essential for SC migration, division or myelination in zebrafish.

Elmo1 function, linked to Rac1 activity, regulates peripheral neuronal numbers and myelination in zebrafish.

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.

Long-lasting masculinizing effects of postnatal androgens on myelin governed by the brain androgen receptor.

The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.

Neuronal Ndrg4 Is Essential for Nodes of Ranvier Organization in Zebrafish.

Axon ensheathment by specialized glial cells is an important process for fast propagation of action potentials. The rapid electrical conduction along myelinated axons is mainly due to its saltatory nature characterized by the accumulation of ion channels at the nodes of Ranvier. However, how these ion channels are transported and anchored along axons is not fully understood. We have identified N-myc downstream-regulated gene 4, ndrg4, as a novel factor that regulates sodium channel clustering in zebrafish. Analysis of chimeric larvae indicates that ndrg4 functions autonomously within neurons for sodium channel clustering at the nodes. Molecular analysis of ndrg4 mutants shows that expression of snap25 and nsf are sharply decreased, revealing a role of ndrg4 in controlling vesicle exocytosis. This uncovers a previously unknown function of ndrg4 in regulating vesicle docking and nodes of Ranvier organization, at least through its ability to finely tune the expression of the t-SNARE/NSF machinery.

The Chromatin Regulator BRPF3 Preferentially Activates the HBO1 Acetyltransferase but Is Dispensable for Mouse Development and Survival.

To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of ß-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2.

Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation.

CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.

miR-98 delays skeletal muscle differentiation by down-regulating E2F5.

A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase in terminal myogenic differentiation. In the present paper, we report that a transcriptomic analysis of human myoblasts, where miR-98 was knocked down, revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5 and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation of skeletal muscle differentiation. We subsequently show that E2F5 can bind to the promoters of two inhibitors of terminal muscle differentiation, ID1 (inhibitor of DNA binding 1) and HMOX1 (heme oxygenase 1), which decreases their expression in skeletal myoblasts. We conclude that miR-98 regulates muscle differentiation by altering the expression of the transcription factor E2F5 and, in turn, of multiple E2F5 targets.

Interplay between androgen and CXCR4 chemokine signaling in myelin repair.

In men, reduced levels of testosterone are associated with the prevalence and progression of multiple sclerosis (MS), a chronic and disabling demyelinating disorder. Testosterone has been shown to promote myelin repair. Here, we demonstrate that the cooperation between testosterone and CXCR4 signaling involving astrocytes is required for myelin regeneration after focal demyelination produced in the ventral mouse spinal cord by the infusion of lysolecithin. The testosterone-dependent remyelination of axons by oligodendrocytes was accompanied by an increase in astrocytes expressing CXCR4, its ligand CXCL12 and the androgen receptor (AR) within the demyelinated area. Depriving males of their testosterone or pharmacological inhibition of CXCR4, with the selective antagonist AMD3100, prevented the appearance of astrocytes expressing CXCR4, CXCL12 and AR within the demyelinated area and the concomitant recruitment of myelin forming oligodendrocytes. Conditional genetic ablation of either CXCR4 or AR in astrocytes also completely blocked the formation of new myelin by oligodendrocytes. Interestingly, the gain of function mutation in CXCR4 causing WHIM syndrome allows remyelination to take place, even in the absence of testosterone, but its potentiating effects remained observable. After testosterone deprivation or CXCR4 inhibition, the absence of astrocytes within the demyelinated area led to the incursion of Schwann cells, most likely derived from spinal nerves, and the formation of peripheral nerve type myelin. In patients with progressive MS, astrocytes expressing CXCR4 and AR surrounded myelin lesions, and their presence opposed the incursion of Schwann cells. These results highlight a mechanism of promyelinating testosterone signaling and the importance of normalizing its levels in combined myelin repair therapies.

Zebrafish as an emerging model to study estrogen receptors in neural development.

Estrogens induce several regulatory signals in the nervous system that are mainly mediated through estrogen receptors (ERs). ERs are largely expressed in the nervous system, yet the importance of ERs to neural development has only been elucidated over the last decades. Accumulating evidence shows a fundamental role for estrogens in the development of the central and peripheral nervous systems, hence, the contribution of ERs to neural function is now a growing area of research. The conservation of the structure of the ERs and their response to estrogens make the zebrafish an interesting model to dissect the role of estrogens in the nervous system. In this review, we highlight major findings of ER signaling in embryonic zebrafish neural development and compare the similarities and differences to research in rodents. We also discuss how the recent generation of zebrafish ER mutants, coupled with the availability of several transgenic reporter lines, its amenability to pharmacological studies and in vivo live imaging, could help us explore ER function in embryonic neural development.

Rgs4 is a regulator of mTOR activity required for motoneuron axon outgrowth and neuronal development in zebrafish.

The Regulator of G protein signaling 4 (Rgs4) is a member of the RGS proteins superfamily that modulates the activity of G-protein coupled receptors. It is mainly expressed in the nervous system and is linked to several neuronal signaling pathways; however, its role in neural development in vivo remains inconclusive. Here, we generated and characterized a rgs4 loss of function model (MZrgs4) in zebrafish. MZrgs4 embryos showed motility defects and presented reduced head and eye sizes, reflecting defective motoneurons axon outgrowth and a significant decrease in the number of neurons in the central and peripheral nervous system. Forcing the expression of Rgs4 specifically within motoneurons rescued their early defective outgrowth in MZrgs4 embryos, indicating an autonomous role for Rgs4 in motoneurons. We also analyzed the role of Akt, Erk and mechanistic target of rapamycin (mTOR) signaling cascades and showed a requirement for these pathways in motoneurons axon outgrowth and neuronal development. Drawing on pharmacological and rescue experiments in MZrgs4, we provide evidence that Rgs4 facilitates signaling mediated by Akt, Erk and mTOR in order to drive axon outgrowth in motoneurons and regulate neuronal numbers.

[Small non-coding RNAs: new targets and new therapeutic possibilities]. / Les petits ARN non codant, nouvelles cibles et nouvelles approches thérapeutiques.

The discovery of regulatory small non-coding RNAs represents a revolution in our understanding of gene regulation. These small non-coding RNAs are powerful tools for exploring cellular pathways and for artificially controlling gene expression. Natural small RNAs also represent potential therapeutic targets.

Rho GTPases Signaling in Zebrafish Development and Disease.

Cells encounter countless external cues and the specificity of their responses is translated through a myriad of tightly regulated intracellular signals. For this, Rho GTPases play a central role and transduce signals that contribute to fundamental cell dynamic and survival events. Here, we review our knowledge on how zebrafish helped us understand the role of some of these proteins in a multitude of in vivo cellular behaviors. Zebrafish studies offer a unique opportunity to explore the role and more specifically the spatial and temporal dynamic of Rho GTPases activities within a complex environment at a level of details unachievable in any other vertebrate organism.

ERM transcription factor contains an inhibitory domain which functions in sumoylation-dependent manner.

ERM, PEA3 and ETV1 belong to the PEA3 group of ETS transcription factors. They are involved in many developmental processes and are transcriptional regulators in metastasis. The PEA3 group members share an N-terminal transactivation domain (TAD) whose activity is inhibited by a flanking domain named the negative regulatory domain (NRD). The mechanism of this inhibition is still unknown. Here we show that the NRD maps to residues 73 to 298 in ERM and contains three of the five SUMO sites previously identified in the protein. We demonstrate that these three SUMO sites are responsible for NRD's inhibitory function in the Gal4 system. Although the presence of the three sites is required to obtain maximal inhibition, only one SUMO site is sufficient to repress transcription whatever its localization within the NRD. We also show that NRD is a SUMO-dependent repression domain that can act in cis and in trans to downregulate the powerful TAD of the VP16 viral protein. In addition, we find that the SUMO sites outside the NRD also play a role in the negative regulation of full-length ERM activity. We thus postulate that each SUMO site in ERM may function as an inhibitory motif.

The Ets transcription factors of the PEA3 group: transcriptional regulators in metastasis.

The PEA3 group is composed of three highly conserved Ets transcription factors: Erm, Er81, and Pea3. These proteins regulate transcription of multiple genes, and their transactivating potential is affected by post-translational modifications. Among their target genes are several matrix metalloproteases (MMPs), which are enzymes degrading the extracellular matrix during normal remodelling events and cancer metastasis. In fact, PEA3-group genes are often over-expressed in different types of cancers that also over-express these MMPs and display a disseminating phenotype. Experimental regulation of the synthesis of PEA3 group members influences the metastatic process. This suggests that these factors play a key role in metastasis.

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M.

MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3' untranslated region (3'UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3'UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.

TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.

Genome-wide exploration of miRNA function in mammalian muscle cell differentiation.

MiRNAs impact on the control of cell fate by regulating gene expression at the post-transcriptional level. Here, using mammalian muscle differentiation as a model and a phenotypic loss-of-function screen, we explored the function of miRNAs at the genome-wide level. We found that the depletion of a high number of miRNAs (63) impacted on differentiation of human muscle precursors, underscoring the importance of this post-transcriptional mechanism of gene regulation. Interestingly, a comparison with miRNA expression profiles revealed that most of the hit miRNAs did not show any significant variations of expression during differentiation. These constitutively expressed miRNAs might be required for basic and/or essential cell function, or else might be regulated at the post-transcriptional level. MiRNA inhibition yielded a variety of phenotypes, reflecting the widespread miRNA involvement in differentiation. Using a functional screen (the STarS--Suppressor Target Screen--approach, i. e. concomitant knockdown of miRNAs and of candidate target proteins), we discovered miRNA protein targets that are previously uncharacterized controllers of muscle-cell terminal differentiation. Our results provide a strategy for functional annotation of the human miRnome.

Tandem PHD fingers of MORF/MOZ acetyltransferases display selectivity for acetylated histone H3 and are required for the association with chromatin.

MORF [MOZ (monocytic leukemia zinc-finger protein)-related factor] and MOZ are catalytic subunits of histone acetyltransferase (HAT) complexes essential in hematopoiesis, neurogenesis, skeletogenesis and other developmental programs and implicated in human leukemias. The canonical HAT domain of MORF/MOZ is preceded by a tandem of plant homeodomain (PHD) fingers whose biological roles and requirements for MORF/MOZ activity are unknown. Here, we demonstrate that the tandem PHD1/2 fingers of MORF recognize the N-terminal tail of histone H3. Acetylation of Lys9 (H3K9ac) or Lys14 (H3K14ac) enhances binding of MORF PHD1/2 to unmodified H3 peptides twofold to threefold. The selectivity for acetylated H3 tail is conserved in the double PHD1/2 fingers of MOZ. This interaction requires the intact N-terminus of histone H3 and is inhibited by trimethylation of Lys4. Biochemical analysis using NMR, fluorescence spectroscopy and mutagenesis identified key amino acids of MORF PHD1/2 necessary for the interaction with histones. Fluorescence microscopy and immunoprecipitation experiments reveal that both PHD fingers are required for binding to H3K14ac in vivo and localization to chromatin. The HAT assays indicate that the interaction with H3K14ac may promote enzymatic activity in trans. Together, our data suggest that the PHD1/2 fingers play a role in MOZ/MORF HATs association with the chromatic regions enriched in acetylated marks.