Andrea Wizenmann

Appearance of target-specific guidance information for regenerating axons after CNS lesions

During development of the vertebrate visual system, an orderly projection of ganglion cells from the retina onto the superior colliculus (SC) is established. Mechanisms that might govern this process include the coordinated action of guidance and corresponding receptor molecules that are specifically distributed on the axons and their targets. In birds and mammals, information for axonal guidance and targeting appears to be confined to the time when the retinocollicular projection is being formed. Here we show that putative guidance activities for temporal and nasal retinal axons, which are not detectable in the normal adult SC, appear after optic nerve transection in adult rats. Both embryonic and adult retinal axons are able to respond to these guiding cues, although the guidance activities detectable in the deafferented adult rat SC might be different from those found during development. These findings imply that it might be possible to reestablish an ordered projection after lesions in the adult mammalian visual system.

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.

Selective Adhesion of Cells from Different Telencephalic Regions

We asked whether specifications of different regions of the rodent and avian telencephalon during development involved the acquisition of differential adhesive properties. Cells from different regions were aggregated in a short-term aggregation assay, and their segregation was analyzed. Both neurons and precursor cells from cortex segregate from striatal cells at early, but not later, stages, whereas cells from rodent neocortex and hippocampus segregated only during later stages. Segregation was abolished when Ca2+-dependent but not Ca2+-independent adhesion molecules were selectively removed. Thus, selective adhesion appears to be a conserved mechanism that restricts cellular mixing and might serve to maintain positional information during forebrain development. A candidate for mediating the Ca2+-dependent segregation is the CD15 (Lewis(x)) carbohydrate epitope, which is selectively expressed by mammalian cortex but not striatum.

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.

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. STEM CELLS 2010;28:775–787

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.

Gene Deletion Mutants Reveal a Role for Semaphorin Receptors of the Plexin-B Family in Mechanisms Underlying Corticogenesis

ABSTRACT Semaphorins and their receptors, plexins, are emerging as key regulators of various aspects of neural and nonneural development. Semaphorin 4D (Sema4D) and B-type plexins demonstrate distinct expression patterns over critical time windows during the development of the murine neocortex. Here, analysis of mice genetically lacking plexin-B1 or plexin-B2 revealed the significance of Sema4D-plexin-B signaling in cortical development. Deficiency of plexin-B2 resulted in abnormal cortical layering and defective migration and differentiation of several subtypes of cortical neurons, including Cajal-Retzius cells, GABAergic interneurons, and principal cells in vivo. In contrast, a lack of plexin-B1 did not impact on cortical development in vivo. In various ex vivo assays on embryonic forebrain, Sema4D enhanced the radial and tangential migration of developing neurons in a plexin-B2-dependent manner. These results suggest that Sema4D-plexin-B2 interactions regulate mechanisms underlying cell specification, differentiation, and migration during corticogenesis.

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 sulfate glycosaminoglycans are required for the development of telencephalic neural stem/progenitor cells

Neural stem cells (NSCs) have the ability to self-renew and give rise to neurons, glia and oligodendrocytes; however, the signals that regulate NSCs are not well understood. In order to determine the role of -catenin in neural stem and progenitor cells of the mouse embryonic forebrain, we used Emx1-Cre and Nestin-Cre to restrict inactivation of a floxed β-catenin allele to neural precursor cells in the cerebral cortex and neuroepithelium, respectively. Emx1-Cre-mediated knockout of β-catenin is compatible with viability; however, null mice possess a significantly smaller cerebral cortex, defects in cortical lamination and loss of the hippocampus. Nestin-Cre-mediated knockout of β-catenin also leads to a smaller cerebral cortex and perinatal death. To determine how the neural precursor cell population is affected by loss of β-catenin, we performed an in vitro clonal neurosphere assay on E14.5 striatal germinal zone cells obtained from NestinCre/β-catenin-knockout embryos. Although β-catenin mutant cultures did not develop intact neurospheres, many individual cells were scattered throughout the cultures. This loss of cell adhesion seen in vitro may phenocopy the abnormal cortical morphogenesis that occurs in mutant mice in vivo. We also examined the self-renewal capacity of β-catenin mutant NSCs using a collagen-based, clonal colony forming assay. Although β-catenin mutant cells gave rise to smaller colonies than control cells, the total number of mutant colonies was significantly greater indicating the presence of more NSCs in the β-catenin mutant brain. These mutant colonies were passageable, demonstrating that -catenin is not required for NSC self-renewal. Furthermore, β-catenin mutant colonies retained multipotentiality as demonstrated by their ability to differentiate into neurons, astrocytes and oligodendrocytes. Therefore, we hypothesize that -catenin is required for proper adhesion of neural precursor cells via cadherins, and the proliferation of neural progenitor cells, but is not required for neural progenitor differentiation, and may act to inhibit the symmetric division of NSCs.