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Interstitial axon branching is an essential step during the establishment of neuronal connectivity. However, the exact mechanisms on how the number and position of branches are determined are still not fully understood. Here, we investigated the role of Arl8B, an adaptor molecule between lysosomes and kinesins. In chick retinal ganglion cells (RGCs), downregulation of Arl8B reduces axon branch density and shifts their location more proximally, while Arl8B overexpression leads to increased density and more distal positions of branches. These alterations correlate with changes in the location and density of lysosomes and autophagosomes along the axon shaft. Diminishing autophagy directly by knock-down of atg7, a key autophagy gene, reduces branch density, while induction of autophagy by rapamycin increases axon branching, indicating that autophagy plays a prominent role in axon branch formation.In vivo, local inactivation of autophagy in the retina using a mouse conditional knock-out approach disturbs retino-collicular map formation which is dependent on the formation of interstitial axon branches. These data suggest that Arl8B plays a principal role in the positioning of axon branches by spatially controlling autophagy, thus directly controlling formation of neural connectivity in the brain.SIGNIFICANCE STATEMENTThe formation of interstitial axonal branches plays a prominent role in numerous places of the developing brain during neural circuit establishment. We show here that the GTPase Arl8B controls density and location of interstitial axon branches, and at the same time controls also density and location of the autophagy machinery. Upregulation or downregulation of autophagyin vitropromotes or inhibits axon branching. Local disruption of autophagyin vivodisturbs retino-collicular mapping. Our data suggest that Arl8B controls axon branching by controlling locally autophagy. This work is one of the first reports showing a role of autophagy during early neural circuit development and suggests that autophagy in general plays a much more prominent role during brain development than previously anticipated.

The small RNA regulatory molecules called microRNAs (miRNAs) play key roles in the development of most organisms. The expression of many different miRNAs has been described in the developing and mature vertebrate retina. The ability of miRNAs to regulate a diversity of messenger RNA targets allows them to have effects on many different developmental processes, but the functions of only a few miRNAs have been documented to date. Developmental transitions between cell states appear to be particularly sensitive to miRNA loss of function, as evidenced by specific miRNA knockdowns or from global perturbations in miRNA levels (e.g., Dicer deletion). However, we are still in only the very early stages of understanding the range of cellular functions miRNAs control during development.

Background The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. Results It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. Conclusions miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

SummaryPlanar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood [1]. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways [1–3]. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration [4]. Here, we investigate the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. We find that Fat4 and Dchs1 are expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. Our results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.

Summary A striking feature of the CNS is the precise wiring of its neuronal connections. During vertebrate visual system development, different subtypes of retinal ganglion cells (RGCs) form specific connections with their corresponding synaptic partners. However, the underlying molecular mechanisms remain to be fully elucidated. Here, we report that the cell-adhesive transmembrane protein Teneurin-3 (Tenm3) is required by zebrafish RGCs for acquisition of their correct morphological and functional connectivity in vivo. Teneurin-3 is expressed by RGCs and their presynaptic amacrine and postsynaptic tectal cell targets. Knockdown of Teneurin-3 leads to RGC dendrite stratification defects within the inner plexiform layer, as well as mistargeting of dendritic processes into outer portions of the retina. Moreover, a subset of RGC axons exhibits tectal laminar arborization errors. Finally, functional analysis of RGCs targeting the tectum reveals a selective deficit in the development of orientation selectivity after Teneurin-3 knockdown. These results suggest that Teneurin-3 plays an instructive role in the functional wiring of the vertebrate visual system., Graphical Abstract, Highlights • Teneurin-3 is expressed by RGCs, amacrine cells, and tectal neurons • Teneurin-3 is required by RGC dendrites for correct IPL stratification • A subset of RGC axons show laminar arborization defects upon Teneurin-3 knockdown • Orientation-, but not direction-selective, RGCs are impaired in Teneurin-3 morphants, In the vertebrate visual system, different subtypes of retinal ganglion cells (RGCs) form specific connections with their corresponding synaptic partners. In this study, Hindges and colleagues show that knockdown of the cell-adhesive transmembrane protein Teneurin-3 leads to structural defects in subsets of zebrafish RGCs in both their pre- and postsynaptic target regions. Moreover, Teneurin-3 is required for functional development of orientation-selective RGCs. These findings suggest that Teneurin-3 plays an instructive role in wiring cell-subtype-specific visual circuits.

The larval zebrafish (Danio rerio) is an excellent vertebrate model for in vivo imaging of biological phenomena at subcellular, cellular and systems levels. However, the optical accessibility of highly pigmented tissues, like the eyes, is limited even in this animal model. Typical strategies to improve the transparency of zebrafish larvae require the use of either highly toxic chemical compounds (e.g. 1-phenyl-2-thiourea, PTU) or pigmentation mutant strains (e.g. casper mutant). To date none of these strategies produce normally behaving larvae that are transparent in both the body and the eyes. Here we present crystal, an optically clear zebrafish mutant obtained by combining different viable mutations affecting skin pigmentation. Compared to the previously described combinatorial mutant casper, the crystal mutant lacks pigmentation also in the retinal pigment epithelium, therefore enabling optical access to the eyes. Unlike PTU-treated animals, crystal larvae are able to perform visually guided behaviours, such as the optomotor response, as efficiently as wild type larvae. To validate the in vivo application of crystal larvae, we performed whole-brain light-sheet imaging and two-photon calcium imaging of neural activity in the retina. In conclusion, this novel combinatorial pigmentation mutant represents an ideal vertebrate tool for completely unobstructed structural and functional in vivo investigations of biological processes, particularly when imaging tissues inside or between the eyes.

Background Knocking down neuronal LINGO-1 using short hairpin RNAs (shRNAs) might enhance axon regeneration in the central nervous system (CNS). Integration-deficient lentiviral vectors have great potential as a therapeutic delivery system for CNS injuries. However, recent studies have revealed that shRNAs can induce an interferon response resulting in off-target effects and cytotoxicity. Methods CNS neurones were transduced with integration-deficient lentiviral vectors in vitro. The transcriptional effect of shRNA expression was analysed using quantitative real time-polymerase chain reaction and northern blots were used to assess shRNA production. Results Integration-deficient lentiviral vectors efficiently transduced CNS neurones and knocked down LINGO-1 mRNA in vitro. However, an increase in cell death was observed when lentiviral vectors encoding an shRNA were applied or when high vector concentrations were used. We demonstrate that high doses of vector or the use of vectors encoding shRNAs can induce an up-regulation of interferon-stimulated genes (2′,5′-oligoadenylate synthase 1 and protein kinase R although not myxovirus resistance 1) and a down-regulation of off-target genes (including p75NTR and Nogo receptor 1). Furthermore, the northern blot demonstrated that these negative consequences occur even when lentiviral vectors express low levels of shRNAs. Taken together, these results may explain why neurite outgrowth was not enhanced on an inhibitory substrate following transduction with lentiviral vectors encoding an shRNA targeting LINGO-1. Conclusions These findings highlight the importance of including appropriate controls to verify silencing specificity and the requirement to check for an interferon response when conducting RNA interference experiments. However, the potential benefits that RNA interference and viral vectors offer to gene-based therapies to CNS injuries cannot be overlooked and demand further investigation. Copyright © 2012 John Wiley & Sons, Ltd.

Retinal development is dependent on an accurately functioning network of transcriptional and translational regulators. Among the diverse classes of molecules involved, non-coding RNAs (ncRNAs) play a significant role. Members of this family are present in the cell as transcripts, but are not translated into proteins. MicroRNAs (miRNAs) are small ncRNAs that act as post-transcriptional regulators. During the last decade, they have been implicated in a variety of biological processes, including the development of the nervous system. On the other hand, long-ncRNAs (lncRNAs) represent a different class of ncRNAs that act mainly through processes involving chromatin remodeling and epigenetic mechanisms. The visual system is a prominent model to investigate the molecular mechanisms underlying neurogenesis or circuit formation and function, including the differentiation of retinal progenitor cells to generate the seven principal cell classes in the retina, pathfinding decisions of retinal ganglion cell axons in order to establish the correct connectivity from the eye to the brain proper, and activity-dependent mechanisms for the functionality of visual circuits. Recent findings have associated ncRNAs in several of these processes and uncovered a new level of complexity for the existing regulatory mechanisms. This review summarizes and highlights the impact of ncRNAs during the development of the vertebrate visual system, with a specific focus on the role of miRNAs and a synopsis regarding recent findings on lncRNAs in the retina.

Neuronal migration and axon growth, key events during neuronal development, require distinct changes in the cytoskeleton. Although many molecular regulators of polarity have been identified and characterized, relatively little is known about their physiological role in this process. To study the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in whichRac1is ablated in the whole brain.Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation bothin vivoandin vitro. In addition,Rac1ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott–Aldrich syndrome protein (WASP) family verprolin-homologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth inRac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex to the plasma membrane to enable actin remodeling necessary for axon growth.

The tumour suppressor PTEN can inhibit cell proliferation and migration as well as control cell growth, in different cell types. PTEN functions predominately as a lipid phosphatase, converting PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), thereby antagonizing PI(3)K (phosphoinositide 3-kinase) and its established downstream effector pathways. However, much is unclear concerning the mechanisms that regulate PTEN movement to the cell membrane, which is necessary for its activity towards PtdIns(3,4,5)P(3) (Refs 3, 4, 5). Here we show a requirement for functional motor proteins in the control of PI3K signalling, involving a previously unknown association between PTEN and myosinV. FRET (Förster resonance energy transfer) measurements revealed that PTEN interacts directly with myosinV, which is dependent on PTEN phosphorylation mediated by CK2 and/or GSK3. Inactivation of myosinV-transport function in neurons increased cell size, which, in line with known attributes of PTEN-loss, required PI(3)K and mTor. Our data demonstrate a myosin-based transport mechanism that regulates PTEN function, providing new insights into the signalling networks regulating cell growth.

The study of normal retinal physiology and its perturbations in disease is fundamental in the development of novel therapeutic strategies to treat pathological conditions that affect normal sight. Recent findings suggest an emerging role for microRNAs (miRNAs) during development of the retina and the functionality of visual circuits. This chapter describes the impact and roles of miRNAs during retinal development and the establishment of visual connectivity and function. It focuses on their potential roles in regenerative medicine, highlighting recent findings in retinal gene therapy and retinal cell transplantation.

Pathfinding of retinal ganglion cell (RGC) axons at the midline optic chiasm determines whether RGCs project to ipsilateral or contralateral brain visual centers, critical for binocular vision. Using Isl2tau-lacZ knockin mice, we show that the LIM-homeodomain transcription factor Isl2 marks only contralaterally projecting RGCs. The transcription factor Zic2 and guidance receptor EphB1, required by RGCs to project ipsilaterally, colocalize in RGCs distinct from Isl2 RGCs in the ventral-temporal crescent (VTC), the source of ipsilateral projections. Isl2 knockout mice have an increased ipsilateral projection originating from significantly more RGCs limited to the VTC. Isl2 knockouts also have increased Zic2 and EphB1 expression and significantly more Zic2 RGCs in the VTC. We conclude that Isl2 specifies RGC laterality by repressing an ipsilateral pathfinding program unique to VTC RGCs and involving Zic2 and EphB1. This genetic hierarchy controls binocular vision by regulating the magnitude and source of ipsilateral projections and reveals unique retinal domains.

The vertebrate retina is highly ordered along both its dorsoventral (DV) and nasotemporal (NT) axes, and this order is topographically maintained in its axonal connections to the superior colliculus of the midbrain. Although the graded axon guidance cues that mediate the topographic mapping of retinocollicular connections are increasingly well understood, the transcriptional regulators that set the DV and NT gradients of these cues are not. We now provide genetic evidence that Vax2, a homeodomain protein expressed in the ventral retina, is one such regulator. We demonstrate that in Vax2 mutant mice, retinocollicular projections from the ventral temporal retina are dorsalized relative to wild type. Remarkably, however, this dorsalization becomes systematically less severe in progressively more nasal regions of the ventral retina. Vax2 mutants also exhibit flattened DV and NT gradients of the EphA5, EphB2, EphB3, ephrin-B1 and ephrin-B2 axon guidance cues. Together, these data identify Vax2 as a fundamental regulator of axial polarization in the mammalian retina.

The human brain is capable of generating new functional neurons throughout life, a phenomenon known as adult neurogenesis. The generation of new neurons is sustained throughout adulthood due to the proliferation and differentiation of adult neural stem cells. This process in humans is uniquely located in the subgranular zone of the dentate gyrus in the hippocampus. Adult hippocampal neurogenesis (AHN) is thought to play a major role in hippocampus-dependent functions, such as spatial awareness, long-term mem- ory, emotionality, and mood. The overall aim of current treatments for cancer (such as radiotherapy and chemotherapy) is to prevent aberrant cell division of cell populations associated with malignancy. However, the treatments in question are absolutist in nature and hence inhibit all cell division. An unintended consequence of this cessation of cell division is the impairment of adult neural stem cell proliferation and AHN. Patients undergoing treatment for cancerous malignancies often display specific forms of memory deficits, as well as depressive symptoms. This review aims to discuss the effects of cancer treatments on AHN and propose a link between the inhibition of the neurogenetic process in the hippocampus and the advent of the cognitive and mood-based deficits observed in patients and animal models undergoing cancer therapies. Possible evidence for coadjuvant interventions aiming to protect neural cells, and subsequently the mood and cognitive functions they regulate, from the ablative effects of cancer treatment are discussed as potential clinical tools to improve mental health among cancer patients.

A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore,in vitrogain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculusin vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.

The function of proliferating cell nuclear antigen (PCNA) in DNA replication and repair is to form a sliding clamp with replication factor C (RF-C) tethering DNA polymerase delta or epsilon to DNA. In addition, PCNA has been found to interact directly with various proteins involved in cell cycle regulation. The crystal structure of yeast PCNA shows that the protein forms a homotrimeric ring lining a hole through which double-stranded DNA can thread, thus forming a moving platform for DNA synthesis. Human and yeast PCNA are highly conserved at a structural and functional level. We determined the solution structure of functionally active human PCNA by small-angle neutron scattering. Our measurements strongly support a trimeric ring-like structure of functionally active PCNA in solution, and the data are in good agreement with model calculations based on the crystal structure from yeast PCNA. The human PCNA used in the small-angle neutron scattering experiments was active before and after the measurements in a RF-C independent and a RF-C dependent assay suggesting that the trimeric structure is the in vivo functional form.

DNA polymerase delta core is a heterodimeric enzyme with a catalytic subunit of 125 kDa and a second subunit of 50 kDa with an as yet unknown function. It is an essential enzyme for DNA replication and DNA repair. We cloned the full-length cDNA encoding the DNA polymerase delta small subunit from mouse cells. The sequence of the predicted polypeptide of 51,336 Da is, like the catalytic subunit, highly conserved not only among mammals (93% identity and 96% similarity), but also between yeast and mammals (34% identity and 57% similarity). Fluorescence in situ hybridization experiments indicated that the gene for the small DNA polymerase delta of mouse is localized on chromosome 11, band A2. By using the yeast two-hybrid system we found that the mouse 125-kDa DNA polymerase catalytic subunit is able to interact with the 50-kDa subunit of the human enzyme, suggesting an in vivo interspecies interaction between the two subunits of DNA polymerase delta.

The formation of the retinotopic map depends on the action of axon guidance molecules, activity-dependent mechanisms and axonal competition. However, little is known about the plasticity potential of the system and the effects on the remodelling of retinocollicular connections upon retinal insults. Here we create a mouse model in which retinal ganglion cells that project to anterior and posterior superior colliculus undergo cell death during topographic map formation. We show that the remaining retinal ganglion cells expand the targeted area in the superior colliculus and at the same time increase their spatial coverage in the retina in a correlated fashion. The resulting contralateral topographic map is overall maintained but less precise, while ipsilateral retinal ganglion cell axons are abnormally distributed in anterior and posterior superficial superior colliculus. These results suggest the presence of plastic mechanisms in the developing mammalian visual system to adjust retinal space and its target coverage and ensure a uniform map., Development of the visual system involves remodelling of retinal ganglion cell axons. Maiorano and Hindges create a mouse model where specific retinal portions are genetically eliminated, and find that both the retina and its projections reorganize to maintain uniform visual space coverage.

The entire cDNA encoding the large subunit of mouse DNA polymerase delta (mPol delta; EC 2.7.7.7) has been cloned and expressed in various bacterial expression systems. A soluble protein could only be obtained when mPol delta was produced as a glutathione S-transferase (GST) fusion protein and the incubation temperature of the expression strain was reduced to 30 degrees C. After purification over a glutathione-Sepharose column, the fractions containing the recombinant (re-) fusion protein showed both DNA Pol and 3'-->5' Exo activities. In situ activity gel analysis indicated that the Pol activity resides in the re-protein. This activity, however, was not stimulated by proliferating cell nuclear antigen (PCNA). Our data are discussed in the view of the findings of Goulian et al. [J. Biol. Chem., 265 (1990) 16402-16411] that the second mPol delta subunit, the 48-kDa protein, might play an important role in DNA Pol delta-PCNA interaction.

The oral mucosa plays critical roles in protection, sensation, and secretion and can be classified into masticatory, lining, and specialized mucosa that are known to be functionally, histologically, and clinically distinct. Each type of oral mucosa is believed to develop through discrete molecular mechanisms, which remain unclear. MicroRNAs (miRNAs) are 19 to 25nt non-coding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs. miRNAs are crucial for fine-tuning of molecular mechanisms. To investigate the role of miRNAs in oral mucosa development, we examined mice with mesenchymal ( Wnt1Cre;Dicerfl/fl) conditional deletion of Dicer. Wnt1Cre;Dicerfl/fl mice showed trans-differentiation of lining mucosa into an epithelium with masticatory mucosa/ skin-specific characteristics. Up-regulation of Fgf signaling was found in mutant lining mucosal epithelium that was accompanied by an increase in Fgf7 expression in mutant mesenchyme. Mesenchyme miRNAs thus have an indirect effect on lining mucosal epithelial cell growth/differentiation.

Background: Tooth development is known to be mediated by the cross-talk between signaling pathways, including Shh, Fgf, Bmp, and Wnt. MicroRNAs (miRNAs) are 19- to 25-nt noncoding small single-stranded RNAs that negatively regulate gene expression by binding target mRNAs, which is believed to be important for the fine-tuning signaling pathways in development. To investigate the role of miRNAs in tooth development, we examined mice with either mesenchymal (Wnt1Cre/Dicerfl/fl) or epithelial (ShhCre/Dicerfl/fl) conditional deletion of Dicer, which is essential for miRNA processing. Results: By using a CD1 genetic background for Wnt1Cre/Dicerfl/fl, we were able to examine tooth development, because the mutants retained mandible and maxilla primordia. Wnt1Cre/Dicerfl/fl mice showed an arrest or absence of teeth development, which varied in frequency between incisors and molars. Extra incisor tooth formation was found in ShhCre/Dicerfl/fl mice, whereas molars showed no significant anomalies. Microarray and in situ hybridization analysis identified several miRNAs that showed differential expression between incisors and molars. Conclusion: In tooth development, miRNAs thus play different roles in epithelium and mesenchyme, and in incisors and molars. Developmental Dynamics 241:1465–1472, 2012. © 2012 Wiley Periodicals, Inc.

The Eph tyrosine kinase receptors (a receptor family named for the expression of Eph in an erythropoietin-producing human hepatocellular carcinoma cell line) make up the largest family of receptor tyrosine kinases. In vertebrates, 14 Eph receptor members have been identified, divided in two sub-groups, the EphAs (EphA1 to A8) and EphBs (EphB1 to B6). Their nine membrane-bound ligands, the ephrins, are also subdivided into the ephrin-As (ephrin-A1 to A6) and ephrin-Bs (ephrin-B1 to B3). The first Eph receptor (EphA1) was identified in 1987, whereas the ephrin ligands were cloned in the mid-90s 1. Eph receptors and ephrins have been found in all animal species analyzed so far, from C. elegans to humans, and are highly conserved through evolution 2. Ephs and ephrins are involved in numerous developmental processes, such as boundary formation, angiogenesis and cell migration. Within the nervous system, Eph signaling regulates the migration pattern of neural crest cells, the boundary formation between hindbrain segments (rhombomeres), the proper formation of the corticospinal tract, the establishment of neural topographic maps and the formation and functional properties of neuronal synapses 1, 3, 4, 5, 6. It is therefore not surprising that nature built a complicated and detailed network of proteins interacting with each other to fine tune each of these important processes. The identity of the receptor or ligand molecule is as important as the structure of the receptor-ligand complex to activate a specific signaling pathway and ultimately elicit the right cell decision.

Toward understanding topographically specific branching of retinal axons in their target area, we have studied the interaction between neurotrophin receptors and members of the Eph family. TrkB and its ligand BDNF are uniformly expressed in the retina and tectum, respectively, and exert a branch-promoting activity, whereas EphAs and ephrinAs are expressed in gradients in retina andtectum and can mediate a suppression of axonal branching.We have identified a novelcisinteraction between ephrinA5 and TrkB on retinal ganglion cell axons. TrkB interacts with ephrinA5 via its second cysteine-rich domain (CC2), which is necessary and sufficientfor bindingto ephrinA5. Their functional interaction is twofold: ephrinA5 augments BDNF-promoted retinal axon branching in the absence of its activator EphA7–Fc, whereas EphA7–Fc application abolishes branching in a local and concentration-dependent manner. The importance of TrkB in this process is shown by the fact that overexpression of an isolated TrkB–CC2 domain interfering with the ephrinA/TrkB interaction abolishes this regulatory interplay, whereas knockdown of TrkB via RNA interference diminishes the ephrinA5-evoked increase in branching. The ephrinA/Trk interaction is neurotrophin induced and specifically augments the PI-3 kinase/Akt pathway generally known to be involved in the promotion of branching. In addition, ephrinAs/TrkB modulate axon branching and also synapse formation of hippocampal neurons. Our findings uncover molecular mechanisms of how spatially restricted axon branching can be achieved by linking globally expressed branch-promoting with differentially expressed branch-suppressing activities. In addition, our data suggest that growth factors and the EphA– ephrinA system interact in a way that affects axon branching and synapse development. This work was supported by the Wellcome Trust, Biotechnology and Biological Sciences Research Council, and the Medical Research Council, as well as Ministerio de Educación y Ciencia (MEC) Grant SAF 2007-60287 and Generalitat de Catalunya to (J.C.), MEC Grant SAF2005-0171 (E.S.), Instituto de Salud Carlos III Grants PI04/2433 (A.M.) and P04/2537 (M.L.). M.L. holds a Ramon y Cajal research contract from the Spanish Ministry of Education and Science.

We report that the EphB receptor ligand, ephrin-B1, may act bifunctionally as both a branch repellent and attractant to control the unique mechanisms in mapping the dorsal-ventral (DV) retinal axis along the lateral-medial (LM)axis of the optic tectum. EphB receptors are expressed in a low to high DV gradient by retinal ganglion cells (RGCs), and ephrin-B1 is expressed in a low to high LM gradient in the tectum. RGC axons lack DV ordering along the LM tectal axis, but directionally extend interstitial branches that establish retinotopically ordered arbors. Recent studies show that ephrin-B1 acts as an attractant in DV mapping and in controlling directional branch extension. Modeling indicates that proper DV mapping requires that this attractant activity cooperates with a repellent activity in a gradient that mimics ephrin-B1. We show that ectopic domains of high, graded ephrin-B1 expression created by retroviral transfection repel interstitial branches of RGC axons and redirect their extension along the LM tectal axis, away from their proper termination zones (TZs). In contrast, the primary RGC axons are unaffected and extend through the ectopic domains of ephrin-B1 and arborize at the topographically correct site. However, when the location of a TZ is coincident with ectopic domains of ephrin-B1, the domains appear to inhibit arborization and shape the distribution of arbors. Our findings indicate that ephrin-B1 selectively controls, through either attraction or repulsion, the directional extension and arborization of interstitial branches extended by RGC axons arising from the same DV position: branches that arise from axons positioned lateral to the correct TZ are attracted up the gradient of ephrin-B1 and branches that arise from axons positioned medial to the same TZ are repelled down the ephrin-B1 gradient. Alternatively, EphB receptor signaling may act as a `ligand-density sensor' and titrate signaling pathways that promote branch extension toward an optimal ephrin-B1 concentration found at the TZ; branches located either medial or lateral to the TZ would encounter a gradient of increasingly favored attachment in the direction of the TZ.

Topographic maps are a fundamental organizational feature of axonal connections in the brain. A prominent model for studying axial polarity and topographic map development is the vertebrate retina and its projection to the optic tectum (or superior colliculus). Linked processes are controlled by molecules that are graded along the axes of the retina and its target fields. Recent studies indicate that ephrin-As control the temporal–nasal mapping of the retina in the optic tectum/superior colliculus by regulating the topographically-specific interstitial branching of retinal axons along the anterior–posterior tectal axis. This branching is mediated by relative levels of EphA receptor repellent signaling. A major recent advance is the demonstration that EphB receptor forward signaling and ephrin-B reverse signaling mediate axon attraction to control dorsal–ventral retinal mapping along the lateral–medial tectal axis. In addition, several classes of regulatory proteins have been implicated in the control of the axial patterning of the retina, and its ultimate readout of topographic mapping.

The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA–RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.

The homeodomain protein Vax1 is expressed in a highly circumscribed set of cells at the ventral anterior midline of the embryonic CNS. These cells populate the choroid fissure of the optic disk, the body of the optic stalk and nerve, the optic chiasm and ventral diencephalon, and the anterior midline zones that abut developing commissural tracts. We have generated mutant mice that lack Vax1. In these mice (1) the optic disks fail to close, leading to coloboma and loss of the eye-nerve boundary; (2) optic nerve glia fail to associate with and appear to repulse ingrowing retinal axons, resulting in a fascicle of axons that are completely segregated from optic nerve astrocytes; (3) retinal axons fail to penetrate the brain in significant numbers and fail to form an optic chiasm; and (4) axons in multiple commissural tracts of the anterior CNS, including the corpus callosum and the hippocampal and anterior commissures, fail to cross the midline. These axon guidance defects do not result from the death of normally Vax1(+) midline cells but, instead, correlate with markedly diminished expression of attractive guidance cues in these cells. Vax1 therefore regulates the guidance properties of a set of anterior midline cells that orchestrate axon trajectories in the developing mammalian forebrain.

Many DNA transactions, such as replication, repair and recombination involve DNA synthesis and consequently require the action of DNA synthesizing enzymes called DNA polymerases (Pol). Eukaryotic cells contain at least six different Pols, named alpha, beta, gamma, delta, epsilon, and zeta. Among them Pol delta occupies important roles in DNA replication, nucleotide excision repair, base excision repair and VDJ recombination. Pol a has been extremely conserved in evolution from yeast to man. The function of Pol delta must be considered in the context of two other factors, called proliferating cell nuclear antigen and replication factor C, two protein complexes that build together the moving platform for Pol delta. This moving platform provides an important framework for dynamic properties of an accurate Pol delta such as its recruitment when its function is needed, the facilitation of Pol delta binding to the primer terminus, the increase in Pol delta processivity, the prevention of non-productive binding of the Pol delta to single-stranded DNA, the release of Pol delta after DNA synthesis and the bridging of Pol delta interactions to other replication proteins. In this review we summarize the current knowledge of Pol delta and will focus in particular to its structural conservation, its functional tasks in the cell and its interactions with other proteins.

A mouse DNA polymerase delta (Pol delta)-encoding cDNA (pol delta) was isolated by PCR amplification and cDNA library screening. The sequenced cDNA has a length of 3386 bp and the open reading frame (ORF) encodes a protein of 1105 amino acids (aa) with an M(r) of 123,743. The aa identity to the proteins encoded by the corresponding cDNA from Bos taurus (93%) and Homo sapiens (92%) is very high. The identity to the Pol delta from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Plasmodium falciparum is around 50%. An aa comparison between all available Pol delta sequences reveals several common structural motifs. Polyclonal antibodies raised against a 31-aa synthetic peptide deduced from the ORF specifically recognize Pol delta polymerases from human cells and calf thymus in an immunoblot.

Nature Cell Biol. 11, 1191–1196 (2009); published online 20 September 2009; corrected after print 5 October 2009 In the version of this letter initially published online and in print, the directions of the horizontal arrows in Fig. 4b were reversed. The correct version of this figure is shown below.This error has been corrected in both the HTML and PDF versions of the article.