MiR-9 and the Midbrain-Hindbrain Boundary: A Showcase for the Limited Functional Conservation and Regulatory Complexity of MicroRNAs.

MicroRNAs regulate gene expression at post-transcriptional levels. Some of them appear to regulate brain development and are involved in neurodevelopmental disorders. This has led to the suggestion that the role of microRNAs in neuronal development and function may be more central than previously appreciated. Here, we review the data about miR-9 function to depict the subtlety, complexity, flexibility and limited functional conservation of this essential developmental regulatory system. On this basis we propose that species-specific actions of miR-9 could underlie to a large degree species differences in brain size, shape and function.

The zinc-finger transcription factor GLI3 is a regulator of precerebellar neuronal migration.

Hindbrain precerebellar neurons arise from progenitor pools at the dorsal edge of the embryonic hindbrain: the caudal rhombic lip. These neurons follow distinct migratory routes to establish nuclei that provide climbing or mossy fiber inputs to the cerebellum. Gli3, a zinc-finger transcription factor in the Sonic hedgehog signaling pathway, is an important regulator of dorsal brain development. We demonstrate that in Gli3-null mutant mice, disrupted neuronal migratory streams lead to a disorganization of precerebellar nuclei. Precerebellar progenitors are properly established in Gli3-null embryos and, using conditional gene inactivation, we provide evidence that Gli3 does not play a cell-autonomous role in migrating precerebellar neurons. Thus, GLI3 likely regulates the development of other hindbrain structures, such as non-precerebellar nuclei or cranial ganglia and their respective projections, which may in turn influence precerebellar migration. Although the organization of non-precerebellar hindbrain nuclei appears to be largely unaffected in absence of Gli3, trigeminal ganglia and their central descending tracts are disrupted. We show that rostrally migrating precerebellar neurons are normally in close contact with these tracts, but are detached in Gli3-null embryos.

Glial Cells in the Fish Retinal Nerve Fiber Layer Form Tight Junctions, Separating and Surrounding Axons.

In the retina of teleost fish, cell addition continues throughout life involving proliferation and axonal growth. To study how this is achieved in a fully functioning retina, we investigated the nerve fiber layer (NFL) of the cichlid fish Astatotilapia burtoni for components that might regulate the extracellular environment. We hypothesized that growing axons are surrounded by different cell structures than signal conducting axons. Using immunohistochemistry and freeze fracture electron microscopy we found that the endfeet of Müller cells (MCs) expressed aquaporin-4 but not in high densities as in mammals. The presence of this water channel indicates the involvement of MCs in water homeostasis. Remarkably, we discovered conspicuous tight junctions in the retinal NFL. These tight junctions formed branching strands between myelin-like wrappings of ganglion cell axons that differed morphologically from any known myelin, and also an elaborate meshwork on large membrane faces between axons. We speculated that these tight junctions have additional functions than solely facilitating nerve conductance. Immunostainings against the adaptor protein ZO-1 labeled the NFL as did antibodies against the mammalian claudin-1, 3, and 19. Performing PCR analysis, we showed expression of claudin-1, 3, 5a, 5b, 9, 11, and 19 in the fish retina, claudins that typically occur at brain barriers or myelin. We could show by immunostains for doublecortin, a marker for differentiating neurons, that new axons are not surrounded by the myelin-like wrappings but only by the endfeet of MCs. We hypothesize that the tight junctions in the NFL of fish might contribute to the separation of an extracellular space around axons facilitating conductance, from a growth-promoting environment. For a functional test we applied Evans Blue dye to eye cup preparations which showed a retention of the dye in the NFL. This indicates that these remarkable tight junctions can indeed act as a diffusion barrier.

Expression and function of microRNA-9 in the mid-hindbrain area of embryonic chick.

BACKGROUND: MiR-9 is a small non-coding RNA that is highly conserved between species and primarily expressed in the central nervous system (CNS). It is known to influence proliferation and neuronal differentiation in the brain and spinal cord of different vertebrates. Different studies have pointed to regional and species-specific differences in the response of neural progenitors to miR-9. METHODS: In ovo and ex ovo electroporation was used to overexpress or reduce miR-9 followed by mRNA in situ hybridisation and immunofluorescent stainings to evaluate miR- expression and the effect of changed miR-9 expression. RESULTS: We have investigated the expression and function of miR-9 during early development of the mid-hindbrain region (MH) in chick. Our analysis reveals a closer relationship of chick miR-9 to mammalian miR-9 than to fish and a dynamic expression pattern in the chick neural tube. Early in development, miR-9 is diffusely expressed in the entire brain, bar the forebrain, and it becomes more restricted to specific areas of the CNS at later stages. MiR-9 overexpression at HH9-10 results in a reduction of FGF8 expression and premature neuronal differentiation in the mid-hindbrain boundary (MHB). Within the midbrain miR-9 does not cause premature neuronal differentiation it rather reduces proliferation in the midbrain. CONCLUSION: Our findings indicate that miR-9 has regional specific effects in the developing mid-hindbrain region with a divergence of response of regional progenitors.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.

Differences in the spatiotemporal expression and epistatic gene regulation of the mesodiencephalic dopaminergic precursor marker PITX3 during chicken and mouse development.

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesencephalon and caudal diencephalon of all tetrapod species studied so far. They are the most prominent DA neuronal population and are implicated in control and modulation of motor, cognitive and rewarding/affective behaviors. Their degeneration or dysfunction is intimately linked to several neurological and neuropsychiatric human diseases. To gain further insights into their generation, we studied spatiotemporal expression patterns and epistatic interactions in chick embryos of selected marker genes and signaling pathways associated with mdDA neuron development in mouse. We detected striking differences in the expression patterns of the chick orthologs of the mouse mdDA marker genes Pitx3 and Aldh1a1, which suggests important differences between the species in the generation/generating of these cells. We also discovered that the sonic hedgehog signaling pathway is both necessary and sufficient for the induction of ectopic PITX3 expression in chick mesencephalon downstream of WNT9A-induced LMX1a transcription. These aspects of early chicken development resemble the ontogeny of zebrafish diencephalic DA neuronal populations, and suggest a divergence between birds and mammals during evolution.

Engrailed homeoproteins in visual system development.

Engrailed is a homeoprotein transcription factor. This family of transcription factors is characterized by their DNA-binding homeodomain and some members, including Engrailed, can transfer between cells and regulate protein translation in addition to gene transcription. Engrailed is intimately involved in the development of the vertebrate visual system. Early expression of Engrailed in dorsal mesencephalon contributes to the development and organization of a visual structure, the optic tectum/superior colliculus. This structure is an important target for retinal ganglion cell axons that carry visual information from the retina. Engrailed regulates the expression of Ephrin axon guidance cues in the tectum/superior colliculus. More recently it has been reported that Engrailed itself acts as an axon guidance cue in synergy with the Ephrin system and is proposed to enhance retinal topographic precision.

In ovo expression of microRNA in ventral chick midbrain.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

The tumor suppressor p53 fine-tunes reactive oxygen species levels and neurogenesis via PI3 kinase signaling.

Mounting evidence points to a role for endogenous reactive oxygen species (ROS) in cell signaling, including in the control of cell proliferation, differentiation, and fate. However, the function of ROS and their molecular regulation in embryonic mouse neural progenitor cells (eNPCs) has not yet been clarified. Here, we describe that physiological ROS are required for appropriate timing of neurogenesis in the developing telencephalon in vivo and in cultured NPCs, and that the tumor suppressor p53 plays a key role in the regulation of ROS-dependent neurogenesis. p53 loss of function leads to elevated ROS and early neurogenesis, while restoration of p53 and antioxidant treatment partially reverse the phenotype associated with premature neurogenesis. Furthermore, we describe that the expression of a number of neurogenic and oxidative stress genes relies on p53 and that both p53 and ROS-dependent induction of neurogenesis depend on PI3 kinase/phospho-Akt signaling. Our results suggest that p53 fine-tunes endogenous ROS levels to ensure the appropriate timing of neurogenesis in eNPCs. This may also have implications for the generation of tumors of neurodevelopmental origin.

Genetic and physical interaction of Meis2, Pax3 and Pax7 during dorsal midbrain development.

BACKGROUND: During early stages of brain development, secreted molecules, components of intracellular signaling pathways and transcriptional regulators act in positive and negative feed-back or feed-forward loops at the mid-hindbrain boundary. These genetic interactions are of central importance for the specification and subsequent development of the adjacent mid- and hindbrain. Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced. RESULTS: Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage. Using in ovo electroporation of the mesencephalic vesicle of chick embryos we show that (i) Pax3 and Pax7 mutually regulate each other's expression in the mesencephalic vesicle, (ii) Meis2 acts downstream of Pax3/7 and requires balanced expression levels of both proteins, and (iii) Meis2 physically interacts with Pax3 and Pax7. These results extend our previous observation that Meis2 cooperates with Otx2 in tectal development to include Pax3 and Pax7 as Meis2 interacting proteins in the tectal anlage. CONCLUSION: The results described here suggest a model in which interdependent regulatory loops involving Pax3 and Pax7 in the dorsal mesencephalic vesicle modulate Meis2 expression. Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.

Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones.

Engrailed 1 and engrailed 2 homeoprotein transcription factors (collectively Engrailed) display graded expression in the chick optic tectum where they participate in retino-tectal patterning. In vitro, extracellular Engrailed guides retinal ganglion cell (RGC) axons and synergises with ephrin A5 to provoke the collapse of temporal growth cones. In vivo disruption of endogenous extracellular Engrailed leads to misrouting of RGC axons. Here we characterise the signalling pathway of extracellular Engrailed. Our results show that Engrailed/ephrin A5 synergy in growth cone collapse involves adenosine A1 receptor activation after Engrailed-dependent ATP synthesis, followed by ATP secretion and hydrolysis to adenosine. This is, to our knowledge, the first evidence for a role of the adenosine A1 receptor in axon guidance. Based on these results, together with higher expression of the adenosine A1 receptor in temporal than nasal growth cones, we propose a computational model that illustrates how the interaction between Engrailed, ephrin A5 and adenosine could increase the precision of the retinal projection map.

Notch signalling stabilises boundary formation at the midbrain-hindbrain organiser.

The midbrain-hindbrain interface gives rise to a boundary of particular importance in CNS development as it forms a local signalling centre, the proper functioning of which is essential for the formation of tectum and cerebellum. Positioning of the mid-hindbrain boundary (MHB) within the neuroepithelium is dependent on the interface of Otx2 and Gbx2 expression domains, yet in the absence of either or both of these genes, organiser genes are still expressed, suggesting that other, as yet unknown mechanisms are also involved in MHB establishment. Here, we present evidence for a role for Notch signalling in stabilising cell lineage restriction and regulating organiser gene expression at the MHB. Experimental interference with Notch signalling in the chick embryo disrupts MHB formation, including downregulation of the organiser signal Fgf8. Ectopic activation of Notch signalling in cells of the anterior hindbrain results in an exclusion of those cells from rhombomeres 1 and 2, and in a simultaneous clustering along the anterior and posterior boundaries of this area, suggesting that Notch signalling influences cell sorting. These cells ectopically express the boundary marker Fgf3. In agreement with a role for Notch signalling in cell sorting, anterior hindbrain cells with activated Notch signalling segregate from normal cells in an aggregation assay. Finally, misexpression of the Notch modulator Lfng or the Notch ligand Ser1 across the MHB leads to a shift in boundary position and loss of restriction of Fgf8 to the MHB. We propose that differential Notch signalling stabilises the MHB through regulating cell sorting and specifying boundary cell fate.

Chondroitin sulfates are required for fibroblast growth factor-2-dependent proliferation and maintenance in neural stem cells and for epidermal growth factor-dependent migration of their progeny.

The neural stem cell niche of the embryonic and adult forebrain is rich in chondroitin sulfate glycosaminoglycans (CS-GAGs) that represent complex linear carbohydrate structures on the cell surface of neural stem/progenitor cells or in their intimate environment. We reported earlier that the removal of CS-GAGs with the bacterial enzyme chondroitinase ABC (ChABC) reduced neural stem/progenitor cell proliferation and self-renewal, whereas this treatment favored astroglia formation at the expense of neurogenesis. Here, we studied the consequences of CS-deglycanation further and revealed that CS-GAGs are selectively required for neurosphere formation, proliferation, and self-renewal of embryonic cortical neural stem/progenitor cells in response to fibroblast growth factor (FGF)-2. Consistently, the FGF-2-dependent activation of the MAPKinase in neural stem/progenitor cells was diminished after ChABC treatment, but unaltered after epidermal growth factor (EGF) stimulation. Upon EGF treatment, fewer radial glia were brain lipid-binding protein (BLBP)-positive, whereas more were glutamate aspartate transporter (GLAST)-positive after CS-GAG removal. Only in this latter situation, GLAST-positive radial glia cells extended processes that supported neuronal migration from differentiating neurospheres. CS-deglycanation also selectively increased astrocyte numbers and their migration in response to EGF. Thus, our approach revealed that CS-GAGs are essential for FGF-2-mediated proliferation and maintenance of neuron-generating neural stem/progenitor cells. Simultaneously, CS-GAGs act as a brake on the EGF-dependent maturation, migration, and gliogenesis of neural stem/progenitor cells. We conclude that neural stem/progenitor cell subpopulations reside in neurospheres that are distinguishable by their responsiveness to FGF-2 and EGF which is differentially regulated by CS-carbohydrate structures.

Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo.

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. We show here that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo with antibodies expressed in the tectum causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, we tested whether Engrailed cooperates with EphrinA5 in vitro. We find that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, our results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA.

MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary.

The midbrain-hindbrain boundary (MHB) is a long-lasting organizing center in the vertebrate neural tube that is both necessary and sufficient for the ordered development of midbrain and anterior hindbrain (midbrain-hindbrain domain, MH). The MHB also coincides with a pool of progenitor cells that contributes neurons to the entire MH. Here we show that the organizing activity and progenitor state of the MHB are co-regulated by a single microRNA, miR-9, during late embryonic development in zebrafish. Endogenous miR-9 expression, initiated at late stages, selectively spares the MHB. Gain- and loss-of-function studies, in silico predictions and sensor assays in vivo demonstrate that miR-9 targets several components of the Fgf signaling pathway, thereby delimiting the organizing activity of the MHB. In addition, miR-9 promotes progression of neurogenesis in the MH, defining the MHB progenitor pool. Together, these findings highlight a previously unknown mechanism by which a single microRNA fine-tunes late MHB coherence via its co-regulation of patterning activities and neurogenesis.

Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube.

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells.

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.

Loss- and gain-of-function analyses reveal targets of Pax6 in the developing mouse telencephalon.

Appropriate neurogenesis and patterning of the forebrain requires the transcription factor Pax6, yet it is largely unknown how Pax6 exerts its effects at the molecular level. To characterize Pax6-mediated regulation of gene expression during murine forebrain neurogenesis, we performed microarray analysis with tissue from the dorsal Pax6-dependent telencephalon and the ventral Pax6-negative telencephalon at the onset of neurogenesis (E12) and at mid-neurogenesis (E15) in wild-type and Pax6-deficient mutant littermates. In the Pax6-deficient cortex the expression levels of various transcription factors involved in neurogenesis (like Satb2, Nfia, AP-2gamma, NeuroD6, Ngn2, Tbr2, Bhlhb5) and the retinoic acid signalling molecule Rlbp1 were reduced. Regulation by Pax6 could be confirmed upon electroporation of a Pax6- and a dominant-negative Pax6-containing vector into embryonic cortex. Taken together, our data reveal novel insights into the molecular pathways regulated by Pax6 during cortical neurogenesis. Most intriguingly, this analysis revealed time- and region-specific differences in Pax6-mediated transcription, explaining the specific function of Pax6 at early and later stages of neurogenesis.

The transcription factors Emx1 and Emx2 suppress choroid plexus development and promote neuroepithelial cell fate.

The transcription factors Emx1 and Emx2 exert important functions during development of the cerebral cortex, including its arealization. Here, we addressed their role in development of the derivatives of the midline region in the telencephalon. The center of the midline region differentiates into the choroid plexus, but little is known about its molecular specification. As we noted a lack of Emx1 or 2 expression in the midline region early in development, we interfered by misexpressing Emx1 and/or Emx2 in this region of the chick telencephalon. Ectopic expression of either Emx1 or Emx2 prior to HH 13 instructed a neuroepithelial identity in the previous midline region instead of a choroidal fate. Thus, Gli3 and Lhx2 normally restricted to the neuroepithelium expanded into the Emx misexpressing region. This was accompanied by down-regulation of Otx2 and BMP7, which implicates that these factors are essential for choroid plexus specification and differentiation. Interestingly, the region next to the ectopic Emx-misexpression then acquired a hybrid identity with some choroidal features such as Bmp7, Otx2 and Ttr gene expression, as well as some neuroepithelial features. These observations indicate that the expression levels of Emx1 and/or Emx2 restrict the prospective choroid plexus territory, a novel role of these transcription factors.