α6 integrin subunit regulates cerebellar development

Mutations in genes encoding several basal lamina components as well as their cellular receptors disrupt normal deposition and remodeling of the cortical basement membrane resulting in a disorganized cerebral and cerebellar cortex. The α6 integrin was the first α subunit associated with cortical lamination defects and formation of neural ectopias. In order to understand the precise role of α6 integrin in the central nervous system (CNS), we have generated mutant mice carrying specific deletion of α6 integrin in neuronal and glia precursors by crossing α6 conditional knockout mice with Nestin-Cre line. Cerebral cortex development occurred properly in the resulting α6^{fl/fl;nestin-Cre} mutant animals. Interestingly, however, cerebellum displayed foliation pattern defects although granule cell (GC) proliferation and migration were not affected. Intriguingly, analysis of Bergmann glial (BG) scaffold revealed abnormalities in fibers morphology associated with reduced processes outgrowth and altered actin cytoskeleton. Overall, these data show that α6 integrin receptors are required in BG cells to provide a proper fissure formation during cerebellum morphogenesis.     

A Highlights from MBoC Selection: LPIAT1 regulates arachidonic acid content in phosphatidylinositol and is required for cortical lamination in mice

Dietary arachidonic acid (AA) has roles in growth, neuronal development, and cognitive function in infants. AA is remarkably enriched in phosphatidylinositol (PI), an important constituent of biological membranes in mammals; however, the physiological significance of AA-containing PI remains unknown. In an RNA interference–based genetic screen using Caenorhabditis elegans, we recently cloned mboa-7 as an acyltransferase that selectively incorporates AA into PI. Here we show that lysophosphatidylinositol acyltransferase 1 (LPIAT1, also known as MBOAT7), the closest mammalian homologue, plays a crucial role in brain development in mice. Lpiat1^−/− mice show almost no LPIAT activity with arachidonoyl-CoA as an acyl donor and show reduced AA contents in PI and PI phosphates. Lpiat1^−/− mice die within a month and show atrophy of the cerebral cortex and hippocampus. Immunohistochemical analysis reveals disordered cortical lamination and delayed neuronal migration in the cortex of E18.5 Lpiat1^−/− mice. LPIAT1 deficiency also causes disordered neuronal processes in the cortex and reduced neurite outgrowth in vitro. Taken together, these results demonstrate that AA-containing PI/PI phosphates play an important role in normal cortical lamination during brain development in mice.     

Laminating the hippocampus

Lamination of neurons and fibre projections is a fundamental organizational principle of the mammalian cerebral cortex. A laminated organization is likely to be essential for cortical function, as studies in mutant mice have revealed causal relationships between lamination defects and functional deficits. Unveiling the determinants of the laminated cortical architecture will contribute to our understanding of how cortical functions have evolved in phylogenetic and ontogenetic development. Recently, the hippocampus, with its clearly segregated cell and fibre layers, has become a major subject of studies on cortical lamination.     

Mitotic spindle regulation by Nde1 controls cerebral cortical size

Ablation of the LIS1-interacting protein Nde1 (formerly mNudE) in mouse produces a small brain (microcephaly), with the most dramatic reduction affecting the cerebral cortex. While cortical lamination is mostly preserved, the mutant cortex has fewer neurons and very thin superficial cortical layers (II to IV). BrdU birthdating revealed retarded and modestly disorganized neuronal migration; however, more dramatic defects on mitotic progression, mitotic orientation, and mitotic chromosome localization in cortical progenitors were observed in Nde1 mutant embryos. The small cerebral cortex seems to reflect both reduced progenitor cell division and altered neuronal cell fates. In vitro analysis demonstrated that Nde1 is essential for centrosome duplication and mitotic spindle assembly. Our data show that mitotic spindle function and orientation are essential for normal development of mammalian cerebral cortex.     

Further studies on cortical tangential migration in wild type and Pax-6 mutant mice

In this study we present new data concerning the tangential migration from the medial and lateral ganglionic eminences (MGE and LGE) to the cerebral cortex during development. We have used Calbindin as a useful marker to follow the itinerary of tangential migratory cells during early developmental stages in wild-type and Pax-6 homozygous mutant mice. In the wild-type mice, at early developmental stages, migrating cells advance through the intermediate zone (IZ) and preplate (PP). At more advanced stages, migrating cells were present in the subplate (SP) and cortical plate (CP) to reach the entire developing cerebral cortex. We found that, in the homozygous mutant mice (Pax-6 ^Sey-Neu/Pax-6 ^Sey-Neu), this tangential migration is severely affected at early developmental stages: migrating cells were absent in the IZ, which were only found some days later, suggesting that in the mutant mice, there is a temporal delay in tangential migration. We have also defined some possible mechanisms to explain certain migratory routes from the basal telencephalon to the cerebral cortex. We describe the existence of two factors, which we consider to be essential for the normal migration; the first one is the cell adhesion molecule PSA-NCAM, whose role in other migratory systems is well known. The second factor is Robo-2, whose expression delimits a channel for the passage of migratory cells from the basal telencephalon to the cerebral cortex.     

Fluid compartmentation and electrolytes of cat cerebral cotex in vitro. 3. Ontogenetic and comparative aspects

Studies on swelling and fluid compartmentation have been carried out in vitro on incubated slices of cerebral cortex from kittens 1.5-120 days post-natal age and on incubated sections of corpus callosum and slices of liver and kidney cortex from adult cats. The findings have been compared with analogous data for incubated slices of adult cat cerebral cortex, studied under identical conditions (Bourke and Tower , 1966a, b), in order to identify the probable morphological correlates of fluid and electrolyte distribution. Incubated cortical slices from neonatal (1.5-4-day-old) kittens exhibit none of the relevant characteristics of slices from adult cerebral cortex. By 1 month post-natal age, K⁺-dependent swelling of slices becomes demonstrable, and the K⁺ and Na⁺ contents of slices approximate adult levels. Both these developments coincide with the morphological and physiological maturation of cortical neurons. At 3 months post-natal age, slice swelling accessible to C1^? but not to sucrose becomes observable and the dependence of sucrose space size on time, during incubation, of solute addition becomes demonstrable. Both these developments follow completion of axonal myelination in the cortex but coincide with the period of cortical glial cell proliferation. Incubated sections of corpus callosum from adult cats exhibit none of the relevant characteristics observed for cortical slices under identical conditions. Tissue swelling is minimal and uninfluenced by K⁺ concentrations of incubation media. Tissue fluid spaces accessible to sucrose are approximately twice the size of spaces accessible to inulin. In general, qualitatively similar results have been obtained for incubated slices of cat liver or kidney cortex or for incubated sections of rat diaphragm under the same conditions. A behaviour for glial cells (? astrocytes) in cerebral cortex under such in vitro conditions distinctly different from behaviour of subcortical glial cells is suggested.     

Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex

Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.     

Genetic analysis of Hedgehog signaling in ventral body wall development and the onset of omphalocele formation

Background

An omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear.

Methodology/Principal Findings

To gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4^Lst mutations into the Gli3^Xt/Xt background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3^Xt/+; Alx4^Lst/Lst embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3^Xt/Xt embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles.

Conclusions/Significance

We suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.     

Comparison of Cortical and White Matter Traumatic Brain Injury Models Reveals Differential Effects in the Subventricular Zone and Divergent Sonic Hedgehog Signaling Pathways in Neuroblasts and Oligodendrocyte Progenitors

The regenerative capacity of the central nervous system must be optimized to promote repair following traumatic brain injury (TBI) and may differ with the site and form of damage. Sonic hedgehog (Shh) maintains neural stem cells and promotes oligodendrogenesis. We examined whether Shh signaling contributes to neuroblast (doublecortin) or oligodendrocyte progenitor (neural/glial antigen 2 [NG2]) responses in two distinct TBI models. Shh-responsive cells were heritably labeled in vivo using Gli1-CreER^T2;R26-YFP bitransgenic mice with tamoxifen administration on Days 2 and 3 post-TBI. Injury to the cerebral cortex was produced with mild controlled cortical impact. Yellow fluorescent protein (YFP) cells decreased in cortical lesions. Total YFP cells increased in the subventricular zone (SVZ), indicating Shh pathway activation in SVZ cells, including doublecortin-labeled neuroblasts. The alternate TBI model produced traumatic axonal injury in the corpus callosum. YFP cells decreased within the SVZ and were rarely double labeled as NG2 progenitors. NG2 progenitors increased in the cortex, with a similar pattern in the corpus callosum. To further test the potential of NG2 progenitors to respond through Shh signaling, Smoothened agonist was microinjected into the corpus callosum to activate Shh signaling. YFP cells and NG2 progenitors increased in the SVZ but were not double labeled. This result indicates that either direct Smoothened activation in NG2 progenitors does not signal through Gli1 or that Smoothened agonist acts indirectly to increase NG2 progenitors. Therefore, in all conditions, neuroblasts exhibited differential Shh pathway utilization compared with oligodendrocyte progenitors. Notably, cortical versus white matter damage from TBI produced opposite responses of Shh-activated cells within the SVZ.     

Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination

Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dst^dt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dst^dt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dst^dt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dst^dt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dst^dt-27J optic nerves was normal, as was myelin basic protein expression in both cerebral cortex and spinal cord. Together these data suggest that, unlike Schwann cells, oligodendrocytes do not have an intrinsic requirement for neuronal dystonin for differentiation and myelination.     

Gli1 Deletion Prevents Helicobacter-Induced Gastric Metaplasia and Expansion of Myeloid Cell Subsets

Chronic inflammation in the stomach induces metaplasia, the pre-cancerous lesion that precedes inflammation-driven neoplastic transformation. While Hedgehog signaling contributes to the initiation of some cancers, its role in gastric transformation remains poorly defined. We found that Helicobacter-infected C57BL/6 mice develop extensive mucous cell metaplasia at 6 month but not at 2 months post-infection. Gastric metaplasia coincided with the appearance of CD45⁺MHCII⁺CD11b⁺CD11c⁺ myeloid cells that were normally not present in the chronic gastritis at 2 months. The myeloid regulatory gene Schlafen-4 was identified in a microarray analysis comparing infected WT versus Gli1 null mice and was expressed in the CD11b⁺CD11c⁺ myeloid population. Moreover this same population expressed IL-1β and TNFα pro-inflammatory cytokines. By 6 months, the mucous neck cell metaplasia (SPEM) expressed IL-6, phosphorylated STAT3 and the proliferative marker Ki67. Expression was not observed in Gli1 mutant mice consistent with the requirement of Gli1 to induce this pre-neoplastic phenotype. Ectopic Shh ligand expression alone was not sufficient to induce SPEM, but with Helicobacter infection synergistically increased the histologic severity observed with the inflammation. Therefore Hedgehog signaling is required, but is not sufficient to generate pre-neoplastic changes during chronic gastritis. Gli1-dependent myeloid cell differentiation plays a pivotal role in the appearance of myeloid cell subtypes ostensibly required for SPEM development. Moreover, it suggests that therapies capable of targeting this phenotypic switch might prevent progression to metaplasia, the pre-neoplastic change that develops prior to dysplasia and gastric cancer, which also occurs in other epithelial-derived neoplasias initiated by chronic inflammation.     

Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

Laminins have dramatic and varied actions on neurons in vitro. However, their in vivo function in brain development is not clear. Here we show that knockout of laminin γ1 in the cerebral cortex leads to defects in neuritogenesis and neuronal migration. In the mutant mice, cortical layer structures were disrupted, and axonal pathfinding was impaired. During development, loss of laminin expression impaired phosphorylation of FAK and paxillin, indicating defects in integrin signaling pathways. Moreover, both phosphorylation and protein levels of GSK-3β were significantly decreased, but only phosphorylation of AKT was affected in the mutant cortex. Knockout of laminin γ1 expression in vitro, dramatically inhibited neurite growth. These results indicate that laminin regulates neurite growth and neuronal migration via integrin signaling through the AKT/GSK-3β pathway, and thus reveal a novel mechanism of laminin function in brain development.     

Potassium Transport in Corn Roots : II. The Significance of the Root Periphery

The relative transport capabilities of the cells of the root periphery and cortex were investigated using a variety of experimental techniques. Brief (30 seconds to 1 minute) exposures with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS), dramatically reduced ⁸⁶Rb⁺ (0.2 millimolar RbCl) uptake into 2 centimeter corn (Zea mays [A632 × (C3640 × Oh43)]) root segments. Autoradiographic localization studies with [³H]NEM and [²⁰³Hg]PCMBS demonstrated that, during short term exposures with either reagent, sulfhydryl binding occurred almost exclusively in the cells of the root periphery.

Corn root cortical protoplasts were isolated, and exhibited significant K⁺(⁸⁶Rb⁺) influx. The kinetics for K⁺ uptake were studied; the influx isotherms were smooth, nonsaturating curves that approached linearity at higher K⁺(Rb⁺) concentrations (above 1 millimolar K⁺). These kinetics were identical in shape to the complex kinetics previously observed for K⁺ uptake in corn roots (Kochian, Lucas 1982 Plant Physiol 70: 1723-1731), and could be resolved into a saturable and a first order kinetic component.

The existence of a hypodermal apoplastic barrier was investigated. The apoplastic, cell wall binding dye, Calcofluor White M2R, appeared to be excluded from the cortex by the hypodermis. However, experiments with damaged roots indicated that this result may be an artifact resulting from the binding of dye to the epidermal cell walls. Furthermore, [²⁰³Hg] PCMBS autoradiography demonstrated that the hypodermis was not a barrier to apoplastic movement of PCMBS.

These results suggest that although cortical cells possess the capacity to absorb ions, K⁺ influx at low concentrations is limited to the root periphery. Cortical cell uptake appears to be repressed under these conditions. At higher concentrations, cortical cells may function to absorb K⁺. Such a model may involve regulation of cortical cell ion transport capacity.