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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.

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.

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.

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.

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.

Background During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear. Methodology/Principal Findings We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype. Conclusions Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.

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.

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.