TRIP6 promotes neural stem cell maintenance through YAP-mediated Sonic Hedgehog activation.

In the adult mammalian brain, new neurons are continuously generated from neural stem cells (NSCs) in the subventricular zone (SVZ)-olfactory bulb (OB) pathway. YAP, a transcriptional co-activator of the Hippo pathway, promotes cell proliferation and inhibits differentiation in embryonic neural progenitors. However, the role of YAP in postnatal NSCs remains unclear. Here, we showed that YAP was present in NSCs of the postnatal mouse SVZ. Forced expression of Yap promoted NSC maintenance and inhibited differentiation, whereas depletion of Yap by RNA interference or conditional knockout led to the decline of NSC maintenance, premature neuronal differentiation, and collapse of neurogenesis. For the molecular mechanism, thyroid hormone receptor-interacting protein 6 (TRIP6) recruited protein phosphatase PP1A to dephosphorylate LATS1/2, therefore inducing YAP nuclear localization and activation. Moreover, TRIP6 promoted NSC maintenance, cell proliferation, and inhibited differentiation through YAP. In addition, YAP regulated the expression of the Sonic Hedgehog (SHH) pathway effector Gli2 and Gli1/2 mediated the effect of YAP on NSC maintenance. Together, our findings demonstrate a novel TRIP6-YAP-SHH axis, which is critical for regulating postnatal neurogenesis in the SVZ-OB pathway.

Ophthalmic characteristics and retinal vasculature changes in Williams syndrome, and its association with systemic diseases.

BACKGROUND: We aim to characterise the ophthalmic findings and retinal vasculature changes in patients with WS, and to analyse the correlation between ophthalmic manifestations and the associated systemic diseases. METHODS: This retrospective case-control study included 27 WS patients and 28 age-matched healthy participants. Stellate pattern of iris, central macular thickness (CMT), foveal width, retinal vessel diameter, superficial vascular density (SVD) of macula and foveal avascular zone (FAZ) were compared between WS patients and healthy participants. RESULTS: Twenty-five patients (93%) had the classic stellate iris presentation. Compared with healthy controls, WS patients had decreased CMT, increased foveal width and a lower SVD of macula (all P < 0.001). Significantly decreased mean retinal arterial (117.9 ± 9.9 µm vs. 133.0 ± 6.7 µm in WS and controls, respectively; p < 0.001) and venous (158.9 ± 11.2 µm vs. 174.0 ± 8.0 µm in WS and controls, respectively; p < 0.001) outer diameters, as well as mean arterial wall thickness (11.2 ± 1.3 µm vs. 12.2 ± 0.8 µm in WS and controls, respectively; p < 0.01) were found in WS. Stellate iris grading was significantly associated with CMT, foveal width, retinal vessel diameter (all p < 0.05), and a significant increase in the odds of having hypertension (Odds ratio (OR), 5.63; P < 0.05). The severity of stellate iris in WS seemed to have the trend of increasing risk of having pulmonary stenosis, tricuspid regurgitation and mitral regurgitation. CONCLUSIONS: This study provides the first in vivo evidence reflecting current knowledge on vessel morphology in WS patients that deficient circumferential growth is the predominant pathophysiologic changes resulting from elastin deficiency. The ophthalmic characteristics may serve as a complementary tool to diagnose and follow-up patients suffering from WS.

Candidate Modifier Genes for the Penetrance of Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease caused by mitochondria DNA (mtDNA) mutation. It is characterized by acute and subacute visual loss predominantly affecting young men. The mtDNA mutation is transmitted to all maternal lineages. However, only approximately 50% of men and 10% of women harboring a pathogenic mtDNA mutation develop optic neuropathy, reflecting both the incomplete penetrance and its unexplained male prevalence, where over 80% of patients are male. Nuclear modifier genes have been presumed to affect the penetrance of LHON. With conventional genetic methods, prior studies have failed to solve the underlying pathogenesis. Whole exome sequencing (WES) is a new molecular technique for sequencing the protein-coding region of all genes in a whole genome. We performed WES from five families with 17 members. These samples were divided into the proband group (probands with acute onset of LHON, n = 7) and control group (carriers including mother and relative carriers with mtDNSA 11778 mutation, without clinical manifestation of LHON, n = 10). Through whole exome analysis, we found that many mitochondria related (MT-related) nuclear genes have high percentage of variants in either the proband group or control group. The MT genes with a difference over 0.3 of mutation percentage between the proband and control groups include AK4, NSUN4, RDH13, COQ3, and FAHD1. In addition, the pathway analysis revealed that these genes were associated with cofactor metabolism pathways. Family-based analysis showed that several candidate MT genes including METAP1D (c.41G > T), ACACB (c.1029del), ME3 (c.972G > C), NIPSNAP3B (c.280G > C, c.476C > G), and NSUN4 (c.4A > G) were involved in the penetrance of LHON. A GWAS (genome wide association study) was performed, which found that ADGRG5 (Chr16:575620A:G), POLE4 (Chr2:7495872T:G), ERMAP (Chr1:4283044A:G), PIGR (Chr1:2069357C:T;2069358G:A), CDC42BPB (Chr14:102949A:G), PROK1 (Chr1:1104562A:G), BCAN (Chr 1:1566582C:T), and NES (Chr1:1566698A:G,1566705T:C, 1566707T:C) may be involved. The incomplete penetrance and male prevalence are still the major unexplained issues in LHON. Through whole exome analysis, we found several MT genes with a high percentage of variants were involved in a family-based analysis. Pathway analysis suggested a difference in the mutation burden of MT genes underlining the biosynthesis and metabolism pathways. In addition, the GWAS analysis also revealed several candidate nuclear modifier genes. The new technology of WES contributes to provide a highly efficient candidate gene screening function in molecular genetics.

YAP maintains the production of intermediate progenitor cells and upper-layer projection neurons in the mouse cerebral cortex.

BACKGROUND: The Hippo pathway is conserved through evolution and plays critical roles in development, tissue homeostasis and tumorigenesis. Yes-associated protein (YAP) is a transcriptional coactivator downstream of the Hippo pathway. Previous studies have demonstrated that activation of YAP promotes proliferation in the developing brain. Whether YAP is required for the production of neural progenitor cells or neurons in vivo remains unclear. RESULTS: We demonstrated that SATB homeobox 2 (SATB2)-positive projection neurons (PNs) in upper layers, but not T-box brain transcription factor 1-positive and Coup-TF interacting protein 2-positive PNs in deep layers, were decreased in the neonatal cerebral cortex of Yap conditional knockout (cKO) mice driven by Nestin-Cre. Cell proliferation was reduced in the developing cerebral cortex of Yap-cKO. SATB2-positive PNs are largely generated from intermediate progenitor cells (IPCs), which are derived from radial glial cells (RGCs) during cortical development. Among these progenitor cells, IPCs but not RGCs were decreased in Yap-cKO. We further demonstrated that cell cycle re-entry was reduced in progenitor cells of Yap-cKO, suggesting that fewer IPCs were generated in Yap-cKO. CONCLUSION: YAP is required for the production of IPCs and upper-layer SATB2-positive PNs during development of the cerebral cortex in mice.

ßPS-Integrin acts downstream of Innexin 2 in modulating stretched cell morphogenesis in the Drosophila ovary.

During oogenesis, a group of specialized follicle cells, known as stretched cells (StCs), flatten drastically from cuboidal to squamous shape. While morphogenesis of epithelia is critical for organogenesis, genes and signaling pathways involved in this process remain to be revealed. In addition to formation of gap junctions for intercellular exchange of small molecules, gap junction proteins form channels or act as adaptor proteins to regulate various cellular behaviors. In invertebrates, gap junction proteins are Innexins. Knockdown of Innexin 2 but not other Innexins expressed in follicle cells attenuates StC morphogenesis. Interestingly, blocking of gap junctions with an inhibitor carbenoxolone does not affect StC morphogenesis, suggesting that Innexin 2 might control StCs flattening in a gap-junction-independent manner. An excessive level of ßPS-Integrin encoded by myospheroid is detected in Innexin 2 mutant cells specifically during StC morphogenesis. Simultaneous knockdown of Innexin 2 and myospheroid partially rescues the morphogenetic defect resulted from Innexin 2 knockdown. Furthermore, reduction of ßPS-Integrin is sufficient to induce early StCs flattening. Taken together, our data suggest that ßPS-Integrin acts downstream of Innexin 2 in modulating StCs morphogenesis.

Ginkgolide B promotes neuronal differentiation through the Wnt/ß-catenin pathway in neural stem cells of the postnatal mammalian subventricular zone.

Chinese herbal medicines (CHMs) have been used to treat human diseases for thousands of years. Among them, Ginkgo biloba is reported to be beneficial to the nervous system and a potential treatment of neurological disorders. Since the presence of adult neural stem cells (NSCs) brings hope that the brain may heal itself, whether the effect of Ginkgo biloba is on NSCs remains elusive. In this study, we found that Ginkgo biloba extract (GBE) and one of its main ingredients, ginkgolide B (GB) promoted cell cycle exit and neuronal differentiation in NSCs derived from the postnatal subventricular zone (SVZ) of the mouse lateral ventricle. Furthermore, the administration of GB increased the nuclear level of ß-catenin and activated the canonical Wnt pathway. Knockdown of ß-catenin blocked the neurogenic effect of GB, suggesting that GB promotes neuronal differentiation through the Wnt/ß-catenin pathway. Thus, our data provide a potential mechanism underlying the therapeutic effect of GBE or GB on brain injuries and neurodegenerative disorders.

The Hippo pathway acts downstream of the Hedgehog signaling to regulate follicle stem cell maintenance in the Drosophila ovary.

The Hippo pathway is conserved and plays important roles in organ size control. The core components of the Hippo pathway are two kinases Hippo (Hpo), Warts (Wts), and a transcription-co-activator Yorkie (Yki). Yki activity is regulated by phosphorylation, which affects its nuclear localization and stability. To determine the role of the Hippo pathway in stem cells, we examine follicle stem cells (FSCs) in the Drosophila ovary. Yki is detected in the nucleus of FSCs. Knockdown of yki in the follicle cell lineage leads to a disruption of the follicular epithelium. Mitotic clones of FSCs mutant for hpo or wts are maintained in the niche and tend to replace the other FSCs, and FSCs mutant for yki are rapidly lost, demonstrating that the Hippo pathway is both required and sufficient for FSC maintenance. Using genetic interaction analyses, we demonstrate that the Hedgehog pathway acts upstream of the Hippo pathway in regulating FSC maintenance. The nuclear localization of Yki is enhanced when the Hedgehog signaling is activated. Furthermore, a constitutively active but not a wild-type Yki promotes FSC maintenance as activation of the Hedgehog signaling does, suggesting that the Hedgehog pathway regulates Yki through a post-translational mechanism in maintaining FSCs.

Ascl1 promotes tangential migration and confines migratory routes by induction of Ephb2 in the telencephalon.

During development, cortical interneurons generated from the ventral telencephalon migrate tangentially into the dorsal telencephalon. Although Achaete-scute family bHLH transcription factor 1 (Ascl1) plays important roles in the developing telencephalon, whether Ascl1 regulates tangential migration remains unclear. Here, we found that Ascl1 promoted tangential migration along the ventricular zone/subventricular zone (VZ/SVZ) and intermediate zone (IZ) of the dorsal telencephalon. Distal-less homeobox 2 (Dlx2) acted downstream of Ascl1 in promoting tangential migration along the VZ/SVZ but not IZ. We further identified Eph receptor B2 (Ephb2) as a direct target of Ascl1. Knockdown of EphB2 disrupted the separation of the VZ/SVZ and IZ migratory routes. Ephrin-A5, a ligand of EphB2, was sufficient to repel both Ascl1-expressing cells in vitro and tangentially migrating cortical interneurons in vivo. Together, our results demonstrate that Ascl1 induces expression of Dlx2 and Ephb2 to maintain distinct tangential migratory routes in the dorsal telencephalon.

Modulation of cell morphogenesis by tousled-like kinase in the Drosophila follicle cell.

BACKGROUND: Tousled-like kinase (Tlk) is a conserved serine/threonine kinase regulating DNA replication, chromatin assembly, and DNA repair. Previous studies have suggested that Tlk is involved in cell morphogenesis in vitro. In addition, tlk genetically interact with Rho1, which encodes a key regulator of the cytoskeleton. However, whether Tlk plays a physiological role in cell morphogenesis and cytoskeleton rearrangement remains unknown. RESULTS: In tlk mutant follicle cells, area of the apical domain was reduced. The density of microtubules was increased in tlk mutant cells. The density of actin filaments was increased in the apical region and decreased in the basal region. Because area of the apical domain was reduced, we examined the levels of proteins located in the apical region by using immunofluorescence. The fluorescence intensities of two adherens junction proteins Armadillo (Arm) and DE-cadherin (DE-cad), atypical protein kinase C (aPKC), and Notch, were all increased in tlk mutant cells. The basolateral localized Discs large (Dlg) shifted apically in tlk mutant cells. CONCLUSIONS: Increase of protein densities in the apical region might be resulted from disruption of the cytoskeleton and shrinkage of the apical domain. Together, these data suggest a novel role of Tlk in maintaining cell morphology, possibly through modulating the cytoskeleton.

The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary.

The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator, Yorkie (Yki). Yes-associated protein (YAP, a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. Here, we use border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. We find that hpo and wts are required for migration of the border cell cluster. In outer border cells, overexpression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo and wts or overexpression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, our data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: (1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; (2) in polar cells, it regulates upd expression to control border cell induction and migration.

Foxp2 regulates neuronal differentiation and neuronal subtype specification.

Mutations of the transcription factor FOXP2 in humans cause a severe speech and language disorder. Disruption of Foxp2 in songbirds or mice also leads to deficits in song learning or ultrasonic vocalization, respectively. These data suggest that Foxp2 plays important roles in the developing nervous system. However, the mechanism of Foxp2 in regulating neural development remains elusive. In the current study, we found that Foxp2 increased neuronal differentiation without affecting cell proliferation and cell survival in primary neural progenitors from embryonic forebrains. Foxp2 induced the expression of platelet-derived growth factor receptor α, which mediated the neurognic effect of Foxp2. In addition, Foxp2 positively regulated the differentiation of medium spiny neurons derived from the lateral ganglionic eminence and negatively regulated the formation of interneurons derived from dorsal medial ganglionic eminence by interacting with the Sonic hedgehog pathway. Taken together, our results suggest that Foxp2 regulates multiple aspects of neuronal development in the embryonic forebrain.

Effects of maternal immune activation on adult neurogenesis in the subventricular zone-olfactory bulb pathway and olfactory discrimination.

Maternal infection and maternal immune activation (MIA) during pregnancy increase risks for psychiatric disorders such as schizophrenia and autism. Many deficits related to psychiatric disorders are observed in adult offspring of MIA animal models, including behavioral abnormalities, morphological defects in various brain regions, and dysregulation of neurotransmitter systems. It has previously been shown that MIA impairs adult neurogenesis in the dentate gyrus of the hippocampus. In this study, we examined whether MIA affects adult neurogenesis in the subventricular zone (SVZ)-olfactory bulb (OB) pathway. Polyinosinic-polycytidylic acid (PolyI:C), a synthetic analog of double-stranded RNA mimicking viral infection, was injected into pregnant mice on gestation day 9.5 to activate immune systems. In the SVZ-OB pathway of adult offspring, different cell types of the neural stem cell lineage responded differently to MIA. Neural stem cells and neuroblasts were decreased. Cell proliferation of transit-amplifying cells was impaired. Consequently, newborn neurons were reduced in the OB. Olfactory deficiency has been suggested as a biomarker for schizophrenia. Here we found that olfactory discrimination was compromised in adult MIA offspring. Taken together, these findings show that MIA leads to defective adult neurogenesis in the SVZ-OB pathway, and the impairment of adult neurogenesis may lead to deficits in olfactory functions.

Pheromones from males of different familiarity exert divergent effects on adult neurogenesis in the female accessory olfactory bulb.

Pheromones from urine of unfamiliar conspecific male animals can reinitiate a female's estrus cycle to cause pregnancy block through the vomeronasal organ (VNO)-accessory olfactory bulb (AOB)-hypothalamic pathway. This phenomenon is called the Bruce effect. Pheromones from the mate of the female, however, do not trigger re-entrance of the estrus cycle because an olfactory memory toward its mate is formed. The activity of the VNO-AOB-hypothalamic pathway is negatively modulated by GABAergic granule cells in the AOB. Since these cells are constantly replenished by neural stem cells in the subventricular zone (SVZ) of the lateral ventricle throughout adulthood and adult neurogenesis is required for mate recognition and fertility, we tested the hypothesis that pheromones from familiar and unfamiliar males may have different effects on adult AOB neurogenesis in female mice. When female mice were exposed to bedding used by a male or lived with one, cell proliferation and neuroblast production in the SVZ were increased. Furthermore, survival of newly generated cells in the AOB was enhanced. This survival effect was transient and mediated by norepinephrine. Interestingly, male bedding-induced newborn cell survival in the AOB but not cell proliferation in the SVZ was attenuated when females were subjected to bedding from an unfamiliar male. Our results indicate that male pheromones from familiar and unfamiliar males exert different effects on neurogenesis in the adult female AOB. Given that adult neurogenesis is required for reproductive behaviors, these divergent pheromonal effects may provide a mechanism for the Bruce effect. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 632-645, 2013.

YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway.

Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation.

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis.

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior-posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.

Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation.

Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.

microRNAs regulate human embryonic stem cell division.

microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells.

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila melanogaster germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. We report that intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, we show that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. We further demonstrate that the miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling.

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation.

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated alpha-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5' base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.

Stage-specific differences in the requirements for germline stem cell maintenance in the Drosophila ovary.

In this study, we uncover a role for microRNAs in Drosophila germline stem cell (GSC) maintenance. Disruption of Dicer-1 function in GSCs during adult life results in GSC loss. Surprisingly, however, loss of Dicer-1 during development does not result in a GSC maintenance defect, although a defect is seen if both Dicer-1 and Dicer-2 function are disrupted. Loss of the bantam microRNA mimics the Dicer-1 maintenance defect when induced in adult GSCs, suggesting that bantam plays a key role in GSC self-renewal. Mad, a component of the TGF-beta pathway, behaves similarly to Dicer-1: adult GSC maintenance requires Mad if it is lost during adult life, but not if it is lost during pupal development. Overall, these results show stage-specific differential sensitivity of GSC maintenance to certain perturbations, and suggest that there may be Dcr-2 dependent redundancy of GSC maintenance mechanisms during development that is lost in later life.