'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.

Ndfip1 is required for the development of pyramidal neuron dendrites and spines in the neocortex.

Ubiquitin ligases of the Nedd4 family are important for axon and dendrite development, but little is known about their adaptor, Nedd4 family-interacting protein 1 (Ndfip1), that is responsible for their enzymatic activation. To study the function of Ndfip1 in cortical development, we generated a conditional knock-out (conditional KO) in neurons. The Ndfip1 conditional KO mice were viable; however, cortical neurons in the adult brain exhibited atrophic characteristics, including stunted dendritic arbors, blebbing of dendrites, and fewer dendritic spines. In electron micrographs, these neurons appeared shrunken with compacted somata and involutions of the nuclear membrane. In culture, Ndfip1 KO neurons exhibited exuberant sprouting suggesting loss of developmental control. Biochemical analysis of postsynaptic density (PSD) fractions from Ndfip1 KO cortical and hippocampal neurons showed that the postsynaptic proteins (Arc and PSD-95) were reduced compared with wild-type controls. In addition, the PI3 kinase/Akt signaling pathway was altered. These results indicate that Ndfip1, through its Nedd4 effectors, is important for the development of dendrites and dendritic spines in the cortex.

Altered speeds and trajectories of neurons migrating in the ventricular and subventricular zones of the reeler neocortex.

The Reelin signaling pathway is essential for proper cortical development, but it is unclear to whether Reelin function is primarily important for cortical layering or neuron migration. It has been proposed that Reelin is perhaps required only for somal translocation but not glial-dependent locomotion. This implies that the location of neurons responding to Reelin is restricted to the outer regions of the cortical plate (CP). To determine whether Reelin is required for migration outside of the CP, we used time-lapse imaging to track the behavior of cells undergoing locomotion in the germinal zones. We focused on the migratory activity in the ventricular/subventricular zones where the first transition of bipolar to multipolar migration occurs and where functional Reelin receptors are known to be expressed. Despite Reelin loss, neurons had no difficulty in undergoing radial migration and indeed displayed greater migratory speed. Additionally, compared with the wild-type, reeler neurons displayed altered trajectories with greater deviation from a radial path. These results suggest that Reelin loss has early consequences for migration in the germinal zones that are portrayed as defective radial trajectories and migratory speeds. Together, these abnormalities can give rise to the increased cell dispersion observed in the reeler cortex.

Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons.

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.

Cortical layer development and orientation is modulated by relative contributions of reelin-negative and -positive neurons in mouse chimeras.

Reelin is an important protein that is indispensable for cortical lamination. In the absence of Reelin, cortical layers fail to form due to inappropriate neuron migration and positioning. The inversion of cortical layers is attributed to failure of neurons to migrate past earlier-generated neurons although how Reelin-insufficiency causes this is unclear. The issue is complicated by recent studies showing that very little Reelin is required for cortical layering. To test how variation in the number of Reelin-producing cells is linked to cortical lamination, we have employed Reelin(+/+) <--> Reelin(-/-) chimeras in which the number of Reelin-expressing neurons is adjusted. We found that the Reeler phenotype was rescued in chimeras with a large contribution of Reelin(+/+) neurons; conversely in chimeras with a weak contribution by Reelin(+/+) neurons, the mutant phenotype remained. However, increasing the number of Reelin(+/+) neurons beyond an unknown threshold resulted in partial rescue, with the formation of a correctly layered secondary cortex lying on top of an inverted mutant cortex. Therefore, the development of cortical layers in the correct order requires a minimal level of Reelin protein to be present although paradoxically, this is insufficient to prevent the simultaneous formation of inverted cortical layers in the same hemisphere.

Oligodendrocyte positioning in cerebral cortex is independent of projection neuron layering.

The factors affecting normal oligodendrocyte positioning in the cerebral cortex are unknown. Apart from the white matter, the highest numbers of oligodendrocytes in the rodent cortex are found in Layers V/VI, where the infragranular neurons normally reside. Few, if any, oligodendrocytes are normally found in the superficial cortical layers. To test whether or not this asymmetric positioning of oligodendrocytes is linked to the lamina positions of Layer V/VI projection neurons, mutant mice that cause neuronal layer inversion were examined. In three lines of mutant mice (Reeler, disabled-1, and p35) examined, representing two different genetic signaling pathways, the oligodendrocyte distribution was altered from an asymmetric to a symmetric distribution pattern. Unlike cortical neurons that are inverted in these mutant mice, the lack of oligodendrocyte inversion suggests a decoupling of the genetic mechanisms governing neuronal versus oligodendrocyte patterning. We conclude that oligodendrocyte positioning is not linked to the layer positions of V/VI projection neurons.

Generation and analysis of Siah2 mutant mice.

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

Initial characterization of PTH-related protein gene-driven lacZ expression in the mouse.

UNLABELLED: The PTHrP gene generates low-abundance mRNA and protein products that are not easily localized by in situ hybridization histochemistry or immunohistochemistry. We report here a PTHrP-lacZ knockin mouse in which beta-gal activity seems to provide a simple and sensitive read-out of PTHrP gene expression. INTRODUCTION: PTH-related protein (PTHrP) is widely expressed in fetal and adult tissues, typically as low-abundance mRNA and protein products that maybe difficult to localize by conventional methods. We created a PTHrP-lacZ knockin mouse as a means of surveying PTHrP gene expression in general and of identifying previously unrecognized sites of PTHrP expression. MATERIALS AND METHODS: We created a lacZ reporter construct under the control of endogenous PTHrP gene regulatory sequences. The AU-rich instability sequences in the PTHrP 3' untranslated region (UTR) were replaced with SV40 sequences, generating products with lacZ/beta gal kinetics rather than those of PTHrP. A nuclear localization sequence was not present in the construct. RESULTS: We characterized beta-galactosidase (beta-gal) activity in embryonic whole mounts and in the skeleton in young and adult animals. In embryos, we confirmed widespread PTHrP expression in many known sites and in several novel epidermal appendages (nail beds and footpads). In costal cartilage, beta-gal activity localized to the perichondrium but not the underlying chondrocytes. In the cartilaginous molds of forming long bones, beta-gal activity was first evident at the proximal and distal ends. Shortly after birth, the developing secondary ossification center formed in the center of this PTHrP-rich chondrocyte population. As the secondary ossification center developed, it segregated this population into two distinct PTHrP beta-gal+ subpopulations: a subarticular subpopulation immediately subjacent to articular chondrocytes and a proliferative chondrocyte subpopulation proximal to the chondrocyte columns in the growth plate. These discrete populations remained into adulthood. beta-gal activity was not identified in osteoblasts but was present in many periosteal sites. These included simple periosteum as well as fibrous tendon insertion sites of the so-called bony and periosteal types; the beta-gal-expressing cells in these sites were in the outer fibrous layer of the periosteum or its apparent equivalents at tendon insertion sites. Homozygous PTHrP-lacZ knockin mice had the expected chondrodysplastic phenotype and a much expanded region of proximal beta-gal activity in long bones, which appeared to reflect in large part the effects of feedback signaling by Indian hedgehog on proximal cell proliferation and PTHrP gene expression. CONCLUSIONS: The PTHrP-lacZ mouse seems to provide a sensitive reporter system that may prove useful as a means of studying PTHrP gene expression.

Mandibular coronoid process in parathyroid hormone-related protein-deficient mice shows ectopic cartilage formation accompanied by abnormal bone modeling.

Parathyroid hormone-related protein (PTHrP) null mutant mice were analyzed to investigate an additional role for PTHrP in cell differentiation. We found ectopic cartilage formation in the mandibular coronoid process in newborn mice. While many previous studies involving PTHrP gene knockout mouse have shown that the cartilage in various regions becomes smaller, this is the first report showing an "increase" of cartilage volume. Investigations of mandibular growth using normal mice indicated that coronoid secondary cartilage never formed from E 15 to d 4, but small amount of cartilage temporally formed at d 7, and this also applies to PTHrP-wild type mice. Therefore, PTHrP deficiency consequently advanced the secondary cartilage formation, which is a novel role of PTHrP in chondrocyte differentiation. In situ hybridization of matrix proteins showed that this coronoid cartilage had characteristics of the lower hypertrophic cell zone usually present at the site of endochondral bone formation and/or "chondroid bone" occasionally found in distraction osteogenesis. In addition, the coronoid process in the PTHrP-deficient mouse also showed abnormal expansion of bone marrow and an increase in the number of multinucleated osteoclasts, an indication of abnormal bone modeling. These results indicate that PTHrP is involved in bone modeling as well as in chondrocyte differentiation. In situ hybridization of matrix protein mRNAs in the abnormal mandibular condylar cartilage revealed that this cartilage was proportionally smaller, supporting previous immunohistochemical results.

The influence of parathyroid hormone-related protein (PTHrP) on tooth-germ development and osteoclastogenesis in alveolar bone of PTHrP-knock out and wild-type mice in vitro.

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.

Seizure-related gene 6 (Sez-6) in amacrine cells of the rodent retina and the consequence of gene deletion.

BACKGROUND: Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development. METHODOLOGY/PRINCIPAL FINDINGS: The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear "bright spot" similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG). CONCLUSIONS: In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.