Selective expression of hyaluronan and receptor for hyaluronan mediated motility (Rhamm) in the adult mouse subventricular zone and rostral migratory stream and in ischemic cortex.

Hyaluronan is a large glycosaminoglycan, which is abundant in the extracellular matrix of the developing rodent brain. In the adult brain however, levels of hyaluronan are significantly reduced. In this study, we used neurocan-GFP as a histochemical probe to analyze the distribution of hyaluronan in the adult mouse subventricular zone (SVZ), as well as in the rostral migratory stream (RMS). Interestingly, we observed that hyaluronan is generally downregulated in the adult brain, but notably remains at high levels in the SVZ and RMS; areas in which neural stem/progenitor cells (NSPCs) persist, proliferate and migrate throughout life. In addition, we found that the receptor for hyaluronan-mediated motility (Rhamm) was expressed in migrating neuroblasts in these areas, indicating that Rhamm could be involved in regulating hyaluronan-mediated cell migration. Hyaluronan levels are balanced by synthesis through hyaluronan synthases (Has) and degradation by hyaluronidases (Hyal). We found that Has1 and Has2, as well as Hyal1 and Hyal2 were expressed in GFAP positive cells in the adult rodent SVZ and RMS, indicating that astrocytes could be regulating hyaluronan-mediated functions in these areas. We also demonstrate that hyaluronan levels are substantially increased at six weeks following a photothrombotic stroke lesion to the adult mouse cortex. Furthermore, GFAP positive cells in the peri-infarct area express Rhamm. Thus, hyaluronan may be involved in regulating cell migration in the normal SVZ and RMS and could also be responsible for priming the peri-infarct area following an ischemic lesion for cell migration.

NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1.

The balance between self-renewal and differentiation of neural progenitor cells is an absolute requirement for the correct formation of the nervous system. Much is known about both the pathways involved in progenitor cell self-renewal, such as Notch signaling, and the expression of genes that initiate progenitor differentiation. However, whether these fundamental processes are mechanistically linked, and specifically how repression of progenitor self-renewal pathways occurs, is poorly understood. Nuclear factor I A (Nfia), a gene known to regulate spinal cord and neocortical development, has recently been implicated as acting downstream of Notch to initiate the expression of astrocyte-specific genes within the cortex. Here we demonstrate that, in addition to activating the expression of astrocyte-specific genes, Nfia also downregulates the activity of the Notch signaling pathway via repression of the key Notch effector Hes1. These data provide a significant conceptual advance in our understanding of neural progenitor differentiation, revealing that a single transcription factor can control both the activation of differentiation genes and the repression of the self-renewal genes, thereby acting as a pivotal regulator of the balance between progenitor and differentiated cell states.

Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice.

BACKGROUND: Agenesis of the corpus callosum is associated with many human developmental syndromes. Key mechanisms regulating callosal formation include the guidance of axons arising from pioneering neurons in the cingulate cortex and the development of cortical midline glial populations, but their molecular regulation remains poorly characterised. Recent data have shown that mice lacking the transcription factor Nfib exhibit callosal agenesis, yet neocortical callosal neurons express only low levels of Nfib. Therefore, we investigate here how Nfib functions to regulate non-cell-autonomous mechanisms of callosal formation. RESULTS: Our investigations confirmed a reduction in glial cells at the midline in Nfib-/- mice. To determine how this occurs, we examined radial progenitors at the cortical midline and found that they were specified correctly in Nfib mutant mice, but did not differentiate into mature glia. Cellular proliferation and apoptosis occurred normally at the midline of Nfib mutant mice, indicating that the decrease in midline glia observed was due to deficits in differentiation rather than proliferation or apoptosis. Next we investigated the development of callosal pioneering axons in Nfib-/- mice. Using retrograde tracer labelling, we found that Nfib is expressed in cingulate neurons and hence may regulate their development. In Nfib-/- mice, neuropilin 1-positive axons fail to cross the midline and expression of neuropilin 1 is diminished. Tract tracing and immunohistochemistry further revealed that, in late gestation, a minor population of neocortical axons does cross the midline in Nfib mutants on a C57Bl/6J background, forming a rudimentary corpus callosum. Finally, the development of other forebrain commissures in Nfib-deficient mice is also aberrant. CONCLUSION: The formation of the corpus callosum is severely delayed in the absence of Nfib, despite Nfib not being highly expressed in neocortical callosal neurons. Our results indicate that in addition to regulating the development of midline glial populations, Nfib also regulates the expression of neuropilin 1 within the cingulate cortex. Collectively, these data indicate that defects in midline glia and cingulate cortex neurons are associated with the callosal dysgenesis seen in Nfib-deficient mice, and provide insight into how the development of these cellular populations is controlled at a molecular level.

Chapter 28: Future perspective in peripheral nerve reconstruction.

Nerve injuries induce severe disability and suffering for patients. Profound alterations in nerve trunks, neurons, and the central nervous system are induced rapidly after injury. This includes activation of intracellular signal transduction mechanisms aiming at the transfer of the cells into a regenerative state through the induction of the appropriate gene programs. The understanding of the neurobiological mechanisms that occur after injury can be used to design modern strategies for reconstruction after nerve injuries. Signal transduction mechanisms for instance may be targets for pharmacological intervention to stimulate nerve regeneration. Nerve injuries, particularly where there is a defect between the severed nerve trunks like in brachial plexus lesions, remain a challenge for the surgeon. Reconstruction of nerve injuries with a defect requires utilization of graft material, which can be of various designs. Application of autologous nerve grafts and use of nerve transfers are the most common clinical solutions to overcome problems with nerve defects. In this chapter we discuss the future perspective of nerve reconstruction with focus on signal transduction mechanisms and new avenues to bridge nerve defects using nanomodified graft surfaces.

Specific glial populations regulate hippocampal morphogenesis.

The hippocampus plays an integral role in spatial navigation, learning and memory, and is a major site for adult neurogenesis. Critical to these functions is the proper organization of the hippocampus during development. Radial glia are known to regulate hippocampal formation, but their precise function in this process is yet to be defined. We find that in Nuclear Factor I b (Nfib)-deficient mice, a subpopulation of glia from the ammonic neuroepithelium of the hippocampus fail to develop. This results in severe morphological defects, including a failure of the hippocampal fissure, and subsequently the dentate gyrus, to form. As in wild-type mice, immature nestin-positive glia, which encompass all types of radial glia, populate the hippocampus in Nfib-deficient mice at embryonic day 15. However, these fail to mature into GLAST- and GFAP-positive glia, and the supragranular glial bundle is absent. In contrast, the fimbrial glial bundle forms, but alone is insufficient for proper hippocampal morphogenesis. Dentate granule neurons are present in the mutant hippocampus but their migration is aberrant, likely resulting from the lack of the complete radial glial scaffold usually provided by both glial bundles. These data demonstrate a role for Nfib in hippocampal fissure and dentate gyrus formation, and that distinct glial bundles are critical for correct hippocampal morphogenesis.

Nuclear factor I gene expression in the developing forebrain.

Three members of the Nuclear Factor I (Nfi) gene family of transcription factors; Nfia, Nfib, and Nfix are highly expressed in the developing mouse brain. Nfia and Nfib knockout mice display profound defects in the development of midline glial populations and the development of forebrain commissures (das Neves et al. [1999] Proc Natl Acad Sci U S A 96:11946-11951; Shu et al. [2003] J Neurosci 23:203-212; Steele-Perkins et al. [2005] Mol Cell Biol 25:685-698). These findings suggest that Nfi genes may regulate the substrate over which the commissural axons grow to reach targets in the contralateral hemisphere. However, these genes are also expressed in the cerebral cortex and, thus, it is important to assess whether this expression correlates with a cell-autonomous role in cortical development. Here we detail the protein expression of NFIA and NFIB during embryonic and postnatal mouse forebrain development. We find that both NFIA and NFIB are expressed in the deep cortical layers and subplate prenatally and display dynamic expression patterns postnatally. Both genes are also highly expressed in the developing hippocampus and in the diencephalon. We also find that principally neither NFIA nor NFIB are expressed in callosally projecting neurons postnatally, emphasizing the role for midline glial cell populations in commissure formation. However, a large proportion of laterally projecting neurons express both NFIA and NFIB, indicating a possible cell-autonomous role for these transcription factors in corticospinal neuron development. Collectively, these data suggest that, in addition to regulating the formation of axon guidance substrates, these genes also have cell-autonomous roles in cortical development.

Commissure formation in the mammalian forebrain.

Commissural formation in the mammalian brain is highly organised and regulated both by the cell-autonomous expression of transcription factors, and by non-cell-autonomous mechanisms including the formation of midline glial structures and their expression of specific axon guidance molecules. These mechanisms channel axons into the correct path and enable the subsequent connection of specific brain areas to their appropriate targets. Several key findings have been made over the past two years, including the discovery of novel mechanisms of action that 'classical' guidance factors such as the Slits, Netrins, and their receptors have in axon guidance. Moreover, novel guidance factors such as members of the Wnt family, and extracellular matrix components such as heparan sulphate proteoglycans, have been shown to be important for mammalian brain commissure formation. Additionally, there have been significant discoveries regarding the role of FGF signalling in the formation of midline glial structures. In this review, we discuss the most recent advances in the field that have contributed to our current understanding of commissural development in the telencephalon.

Emx and Nfi genes regulate cortical development and axon guidance in the telencephalon.

The Emx and Nuclear Factor One (Nfi) genes encode transcription factors that regulate numerous embryonic developmental processes. The two mammalian Emx genes, Emx1 and Emx2, are expressed in the embryonic cortex and regulate the specification of the cortex into different sensory and motor areas along the rostrocaudal axis. To date, few developmental processes have been attributed specifically to Emx1, with most analyses demonstrating a redundancy of function between Emx1 and Emx2, with Emx2 being most essential for development. Here we provide evidence that Emx1 and Emx2 regulate different developmental processes during corpus callosum formation and review how both genes function in cellular migration and the formation of cortical axon projections. The Nfi gene family is made up of four members, Nfia, Nfib, Nfic and Nfix. Expression analyses show that Nfia, Nfib and Nfix are expressed in the developing telencephalon. They play roles in patterning, glial development, cortical cell migration and axon guidance. We review the role of these genes in cortical cell migration, glial development and the formation of cortical axon projections, and examine the overlapping mutant phenotypes between the Emx and Nfi gene families.

The role of p-c-Jun in survival and outgrowth of developing sensory neurons.

c-Jun activation has been implicated not only in neuronal apoptosis, but also in survival and regeneration. This Janus facet of c-Jun activation could be related to neuronal cell type or to the developmental stage of the neuron. We investigated c-Jun activation in E18 sensory neurons. Cultures of rat dorsal root ganglia neurons were maintained with or without the addition of nerve growth factor or the c-Jun N-terminal kinase inhibitor, (D)-JNKI1. Few dorsal root ganglia neurons survived nerve growth factor deprivation, whereas neurons supplied with nerve growth factor survived and exhibited extensive axonal outgrowth. Activated c-Jun was present in the nuclei of neurons with regenerating axons, but not in apoptotic neurons. c-Jun N-terminal kinase inhibition reduced the number of p-c-Jun immunoreactive and regenerating neurons, and increased cell death. Thus, activation of c-Jun seems to be required for survival and regeneration of developing sensory neurons.

The Janus role of c-Jun: cell death versus survival and regeneration of neonatal sympathetic and sensory neurons.

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.

Retrograde axonal transport of JNK signaling molecules influence injury induced nuclear changes in p-c-Jun and ATF3 in adult rat sensory neurons.

In the present study, we investigated if the previously observed JNK-mediated activation of c-Jun and induction of ATF3 could be ascribed to axonal transport of JNK signaling components, or if axonal transport of the transcription factors themselves contributes to the nuclear changes in injured sensory neurons. We observed retrograde axonal transport of a number of JNK upstream kinases in ligated rat sciatic nerve. In these preparations, axonal transport of JNK/p-JNK, the JNK scaffolding protein JIP, and the transcription factors ATF3 and ATF2/p-ATF2 was also found. No or little retrograde transport of c-Jun and p-c-Jun was found, whereas an anterograde transport of Hsp27, a protein previously reported in the context of p-c-Jun and ATF3, was observed. In separate experiments, we found that in vitro inhibition of axonal transport or axonal inhibition of JNK reduced the number of p-c-Jun- and ATF3-positive neuronal nuclei. The results suggest that retrograde axonal transport of JNK signaling components contributes to the injury induced c-Jun phosphorylation and ATF3 induction.

Inhibition of c-Jun phosphorylation reduces axonal outgrowth of adult rat nodose ganglia and dorsal root ganglia sensory neurons.

The role of c-Jun activation for survival and regeneration of sensory neurons is unclear. Here we report that c-Jun N-terminal kinase (JNK)-mediated c-Jun activation is important for axonal outgrowth of sensory neurons in rat nodose and dorsal root ganglia (DRG). Peripheral severance of the vagus or the sciatic nerve resulted in a massive and rapid, but transient increase of the activated JNK (p-JNK) in neuronal nuclei, followed by c-Jun phosphorylation and activating transcription factor-3 (ATF3) induction. JNK inhibition by the selective JNK inhibitors SP600125 and (D)-JNKI1 did not affect neuronal survival in explanted or dissociated ganglia, but dramatically reduced axonal outgrowth, c-Jun activation, and ATF3 induction. Using retrograde labeling, we demonstrated that activated c-Jun (p-c-Jun) and ATF3 were associated with regenerative neurons. Taken together, our results suggest that JNK-mediated c-Jun activation is one of the first cell body reactions in response to nerve injury and that this activation and subsequent ATF3 induction are associated with axonal outgrowth.