Cnn3 regulates neural tube morphogenesis and neuronal stem cell properties.

Calponin 3 (Cnn3) is a member of the Cnn family of actin-binding molecules that is highly expressed in the mammalian brain and has been shown to control dendritic spine morphology, density, and plasticity by regulating actin cytoskeletal reorganization and dynamics. However, little is known about the role of Cnn3 during embryonic development. In this study, we analyzed mutant animals deficient in Cnn3 to gain a better understanding of its role in brain morphogenesis. Embryos lacking Cnn3 exhibited massive malformation of the developing brain including exoencephaly, closure defects at the rostral neural tube, and strong enlargement of brain tissue. In wild-type animals, we found Cnn3 being localized to the apical lining of the neuroepithelium in close vicinity to beta-Catenin and N-cadherin. By performing immunohistochemistry on beta-Catenin and p-Smad, and furthermore taking advantage of Wnt-reporter animals, we provide evidence that the loss of Cnn3 during development can affect signaling pathways crucial for correct morphogenesis of the neural tube. In addition, we used embryonic neurosphere cultures to investigate the role of Cnn3 in embryonic neuronal stem cells (NSC). Here, we observed that Cnn3 deficiency in NSCs increased the number of newly formed neurospheres and increased neurosphere size without perturbing their differentiation potential. Together, our study provides evidence for an important role of Cnn3 during development of the embryonic brain and in regulating NSC function.

Trimethylation and Acetylation of ß-Catenin at Lysine 49 Represent Key Elements in ESC Pluripotency.

Wnt/ß-catenin signaling is required for embryonic stem cell (ESC) pluripotency by inducing mesodermal differentiation and inhibiting neuronal differentiation; however, how ß-catenin counter-regulates these differentiation pathways is unknown. Here, we show that lysine 49 (K49) of ß-catenin is trimethylated (ß-catMe3) by Ezh2 or acetylated (ß-catAc) by Cbp. Significantly, ß-catMe3 acts as a transcriptional co-repressor of the neuronal differentiation genes sox1 and sox3, whereas ß-catAc acts as a transcriptional co-activator of the key mesodermal differentiation gene t-brachyury (t-bra). Furthermore, ß-catMe3 and ß-catAc are alternatively enriched on repressed or activated genes, respectively, during ESC and adult stem cell differentiation into neuronal or mesodermal progenitor cell lineages. Importantly, expression of a ß-catenin K49A mutant results in major defects in ESC differentiation. We conclude that ß-catenin K49 trimethylation and acetylation are key elements in regulating ESC pluripotency and differentiation potential.

Presynaptic localization of GluK5 in rod photoreceptors suggests a novel function of high affinity glutamate receptors in the mammalian retina.

Kainate receptors mediate glutamatergic signaling through both pre- and presynaptic receptors. Here, we studied the expression of the high affinity kainate receptor GluK5 in the mouse retina. Double-immunofluoresence labeling and electron microscopic analysis revealed a presynaptic localization of GluK5 in the outer plexiform layer. Unexpectedly, we found GluK5 almost exclusively localized to the presynaptic ribbon of photoreceptor terminals. Moreover, in GluK5-deficient mutant mice the structural integrity of synaptic ribbons was severely altered pointing to a novel function of GluK5 in organizing synaptic ribbons in the presynaptic terminals of rod photoreceptors.

Beta-catenin is vital for the integrity of mouse embryonic stem cells.

ß-Catenin mediated Wnt-signaling is assumed to play a major function in embryonic stem cells in maintaining their stem cell character and the exit from this unique trait. The complexity of ß-catenin action and conflicting results on the role of ß-catenin in maintaining the pluripotent state have made it difficult to understand its precise cellular and molecular functions. To attempt this issue we have generated new genetically modified mouse embryonic stem cell lines allowing for the deletion of ß-catenin in a controlled manner by taking advantage of the Cre-ER-T2 system and analyzed the effects in a narrow time window shortly after ablation. By using this approach, rather then taking long term cultured ß-catenin null cell lines we demonstrate that ß-catenin is dispensable for the maintenance of pluripotency associated genes. In addition we observed that the removal of ß-catenin leads to a strong increase of cell death, the appearance of multiple clustered functional centrosomes most likely due to a mis-regulation of the polo-like-kinase 2 and furthermore, alterations in chromosome segregation. Our study demonstrates the importance of ß-catenin in maintaining correct cellular functions and helps to understand its role in embryonic stem cells.

Reelin together with ApoER2 regulates interneuron migration in the olfactory bulb.

One pathway regulating the migration of neurons during development of the mammalian cortex involves the extracellular matrix protein Reelin. Reelin and components of its signaling cascade, the lipoprotein receptors ApoER2 and Vldlr and the intracellular adapter protein Dab1 are pivotal for a correct layer formation during corticogenesis. The olfactory bulb (OB) as a phylogenetically old cortical region is known to be a prominent site of Reelin expression. Although some aspects of Reelin function in the OB have been described, the influence of Reelin on OB layer formation has so far been poorly analyzed. Here we studied animals deficient for either Reelin, Vldlr, ApoER2 or Dab1 as well as double-null mutants. We performed organotypic migration assays, immunohistochemical marker analysis and BrdU incorporation studies to elucidate roles for the different components of the Reelin signaling cascade in OB neuroblast migration and layer formation. We identified ApoER2 as being the main receptor responsible for Reelin mediated detachment of neuroblasts and correct migration of early generated interneurons within the OB, a prerequisite for correct OB lamination.

Role for Reelin in neurotransmitter release.

The extracellular matrix molecule Reelin is known to control neuronal migration during development. Recent evidence suggests that it also plays a role in the maturation of postsynaptic dendrites and spines as well as in synaptic plasticity. Here, we aimed to address the question whether Reelin plays a role in presynaptic structural organization and function. Quantitative electron microscopic analysis of the number of presynaptic boutons in the stratum radiatum of hippocampal region CA1 did not reveal differences between wild-type animals and Reelin-deficient reeler mutant mice. However, additional detailed analysis showed that the number of presynaptic vesicles was significantly increased in CA1 synapses of reeler mutants. To test the hypothesis that vesicle fusion is altered in reeler, we studied proteins known to control transmitter release. SNAP25, a protein of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was found to be significantly reduced in reeler mutants, whereas other SNARE complex proteins remained unaltered. Addition of recombinant Reelin to organotypic slice cultures of reeler hippocampi substantially rescued not only SNAP25 protein expression levels but also the number of vesicles per bouton area indicating a role for Reelin in presynaptic functions. Next, we analyzed paired-pulse facilitation, a presynaptic mechanism associated with transmitter release, and observed a significant decrease at CA1 synapses of reeler mutants when compared with wild-type animals. Together, these novel findings suggest a role for Reelin in modulating presynaptic release mechanisms.

Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy.

Here, we describe the zebrafish (Danio rerio) as a vertebrate model system to study liver regeneration with the added benefit of its powerful genetics and screening possibilities to uncover the molecular pathways underlying liver regeneration. We developed a partial hepatectomy (PH) protocol in zebrafish and investigated in detail the cellular and morphological changes during the process of liver regeneration. We show that the type of regenerative response is dependent on the size of the injury sustained by the zebrafish liver. Furthermore, we demonstrate for the first time that the mechanisms of liver regeneration in zebrafish after PH are strikingly similar to those of rodents and humans, with 100% recovery of the liver mass after 6-7 d postsurgery. This occurs via compensatory growth mediated by proliferation of hepatocytes throughout the entire liver remnant. By analyzing transgenic fish expressing dominant-negative forms of either bone morphogenetic protein (BMP) receptor or fibroblast growth factor (FGF) receptor 1, we demonstrate that the BMP and FGF signaling pathways are crucial regulators of the early events during liver regeneration after PH. Our study demonstrates that the mechanisms of liver regeneration in zebrafish are highly similar to the processes ongoing during mammalian liver regeneration and make the adult zebrafish a suitable model system to study the mechanisms of liver regeneration.

Postsynaptic and differential localization to neuronal subtypes of protocadherin beta16 in the mammalian central nervous system.

The formation of synapses is dependent on the expression of surface adhesion molecules that facilitate correct recognition, stabilization and function. The more than 60 clustered protocadherins (Pcdhalpha, Pcdhbeta and Pcdhgamma) identified in human and mouse have attracted considerable attention because of their clustered genomic organization and the potential role of alpha- and gamma-Pcdhs in allocating a neuronal surface code specifying synaptic connectivity. Here, we investigated whether beta-Pcdhs also contribute to these processes. By performing RT-PCR, we found a striking parallel onset of expression of many beta-Pcdhs around the onset of neurogenesis and wide expression in the central nervous system. We generated antibodies specific to Pcdhb16 and showed localization of Pcdhb16 protein in the adult mouse cerebellum, hippocampus and cerebral cortex. Analysing the mouse retina in detail revealed localization of Pcdhb16 to specific cell types and, importantly, subsets of synapses. We show that Pcdhb16 localizes predominantly to postsynaptic compartments and the comparison with Pcdhb22 implies differential localization and functions of individual beta-Pcdhs in the mammalian central nervous system. Moreover, we provide evidence for a role of beta-Pcdhs in the outer segments and connecting cilia of photoreceptors. Our data show for the first time that beta-Pcdhs also localize to specific neuronal subpopulations and synapses, providing support for the hypothesis that clustered Pcdhs are candidate genes for the specification of synaptic connectivity and neuronal networks.

Divergent roles of ApoER2 and Vldlr in the migration of cortical neurons.

Reelin, its lipoprotein receptors [very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (ApoER2; also known as Lrp8)], and the cytoplasmic adaptor protein disabled 1 (Dab1) are important for the correct formation of layers in the cerebral cortex. Reeler mice lacking the reelin protein show altered radial neuronal migration resulting in an inversion of cortical layers. ApoER2 Vldlr double-knockout mutants and Dab1 mutants show a reeler-like phenotype, whereas milder phenotypes are found if only one of the two lipoprotein receptors for reelin is absent. However, the precise role of the individual reelin receptors in neuronal migration remained unclear. In the study reported here, we performed fate mapping of newly generated cortical neurons in single and double receptor mutants using bromodeoxyuridine-labeling and layer-specific markers. We present evidence for divergent roles of the two reelin receptors Vldlr and ApoER2, with Vldlr mediating a stop signal for migrating neurons and ApoER2 being essential for the migration of late generated neocortical neurons.

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm.

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.

Endoscope-assisted microsurgical resection of an intraneural ganglion cyst of the hypoglossal nerve.

An unusual case of an intraneural ganglion cyst of the hypoglossal nerve is presented. Only one case of this rare clinical entity has been reported previously. A 51-year-old woman presented with a 6-month history of left-sided hypoglossal nerve palsy. Magnetic resonance imaging revealed a cystic lesion related to the hypoglossal canal. There was no enhancement of the lesion after administration of Gd. A high-resolution computerized tomography scan of the skull base demonstrated an enlargement of the hypoglossal canal. To access the lesion, a far-lateral endoscope-assisted microsurgical approach was used. An intraneural ganglion lesion invading the hypoglossal nerve was found and resected. A histopathological examination confirmed that the lesion was an intraneural ganglion cyst. The occurrence of an intraneural ganglion cyst at the hypoglossal nerve is very rare. This case exemplifies an atypical location of a synovial cyst with cranial nerve involvement.

Mammalian cadherins and protocadherins: about cell death, synapses and processing.

Cadherins have been known for a long time to be key elements in many important biological processes. In particular, the role of classical cadherins in mediating adhesion has been examined in great detail. Over recent years, the accumulation of experimental tools and mice mutants has allowed more refined analysis of cadherin functions, and new aspects such as signaling and synapse dynamics have become the center of interest. In addition, the study of mice lacking the entire protocadherin-gamma cluster shed the first light on a possible novel function of members of this cadherin family in synapse formation and cell survival during development.

Beta-catenin-mediated cell-adhesion is vital for embryonic forebrain development.

Forming a complex structure such as the mammalian brain requires a complex interplay between cells and different signalling cascades during embryonic development. beta-catenin plays pivotal roles in these processes by mediating cadherin-based cell adhesion and Wnt signalling. We show for the first time that beta-catenin functions predominantly as a mediator of cell adhesion during early development of the mammalian telencephalon. Immunohistochemical analysis demonstrates that beta-catenin is localized, together with N-cadherin, to adhesion junctions at the apical lining of the neuroepithelium. The ablation of beta-catenin specifically from the forebrain leads to a disruption of apical adherens junctions and a breakdown of neuroepithelial structures. We show that beta-catenin-deficient neuroepithelial cells delaminate and undergo apoptosis. Newborn beta-catenin mutants lack the entire forebrain and anterior facial structures. Our data also indicate a lack of TCF/LEF-beta-catenin-dependent transcriptional activity in the telencephalon of Wnt reporter embryos. Together with the absence of nuclear beta-catenin, this finding suggests that canonical Wnt signalling is not active during early telencephalic development. In summary, we demonstrate that beta-catenin mediates cell-cell adhesion in the early telencephalon and is vital for maintaining the structural integrity of the neuroepithelium.

The CES-2-related transcription factor E4BP4 is an intrinsic regulator of motoneuron growth and survival.

The regulation of neuronal growth and survival during development requires interplay between extrinsic and intrinsic factors. Among the latter, transcription factors play a key role. In the nematode, the transcription factor CES-2 predisposes neurosecretory motoneurons to death, whereas E4BP4 (NFIL3), one of its vertebrate homologs, regulates survival of pro-B lymphocytes. We show that E4BP4 is expressed by embryonic rat and chicken motoneurons in vivo, with levels being highest in neurons that survive the period of naturally occurring cell death. Overexpression of E4BP4 by electroporation of purified motoneurons in culture protected them almost completely against cell death triggered by removal of neurotrophic factors or activation of death receptors. Moreover, E4BP4 strongly enhanced neuronal cell size and axonal growth. Axons of motoneurons transfected with E4BP4 were 3.5-fold longer than control neurons grown on laminin; this effect required the activity of PI3 kinase. In vivo, overexpression of E4BP4 in chicken embryos reduced the number of dying motoneurons by 45%. Our results define E4BP4 as a novel intrinsic regulator of motoneuron growth and survival. Pathways regulated by E4BP4 are of potential interest both for understanding neuromuscular development and for promoting neuronal survival and regeneration in pathological situations.

The supplementary diagnostic power of selected immunohistochemical, molecular genetic and infective parameters in epithelial hyperplastic laryngeal lesions.

OBJECTIVES: MIB-1 and p53 protein expression, loss of heterozygosity (LOH), microsatellite instability (MSI) of di- and mononucleotide repeats, and HPV status were tested for their potential to characterize different stages of epithelial hyperplastic laryngeal lesions (EHLL). METHODS: Thirty-two EHLL were reclassified according to the Ljubljana classification into simple (SH), abnormal (AbH), atypical hyperplasia (AtH) and carcinoma in situ, and investigated by immunohistochemical methods, PCR and direct sequencing analysis. RESULTS: MIB-1 increased with progressive grades of EHLL, whereas p53 protein expression was distinctive only between SH and AbH. LOH showed increasing frequency with grades of the lesions, but the distribution of altered loci (9p, 9q, 10q, 11q, 17p) was not qualified to differentiate between the stages. MSI was detected in SH, AbH and AtH without clear correlation to histopathological grading. HPV infection occurred mostly in SH and AbH (both: 66.7%). CONCLUSION: MIB-1 labeling and allelic loss could assist histopathological diagnosis in the entire spectrum of EHLL, whereas the MSI results point to a genetic instability of the laryngeal mucosa in general and are therefore not helpful in the distinction of different stages of EHLL. However, future molecular genetic analyses should consider more late events of laryngeal carcinogenesis to improve their diagnostic potential. Furthermore, our results indicate that nonrisky and risky EHLL could probably be caused by different exogenous factors.

Long-term arterial pressure in spontaneously hypertensive rats is set by the kidney.

OBJECTIVES: We investigated whether arterial pressure in spontaneously hypertensive rats (SHR) can be normalized by a kidney graft from normotensive histocompatible donors. In addition, the effect of differential genetic predisposition to hypertension of recipients of an SHR kidney on the development of post-transplantation hypertension was studied. METHODS: SHR were transplanted with a kidney from congenic rats (BB.1K) homozygous for a 2 cM segment of SHR chromosome 20, including the major histocompatibility complex class Ia and class II genes. BB.1K and F1 hybrids (F1H, SHR x Wistar-Kyoto rats) were transplanted with an SHR kidney and the development of renal post-transplantation hypertension was monitored. RESULTS: Thirty days after renal transplantation, mean arterial pressure (MAP) was 116 +/- 4 mmHg in SHR with a BB.1K kidney (n = 8) versus 168 +/- 2 mmHg in sham-operated SHR (n = 10); P < 0.001. Cumulative renal sodium balance (mmol/100 g body weight) over 21 days after bilateral nephrectomy was 6.8 +/- 0.6 in SHR with a BB.1K kidney versus 10.8 +/- 1.6 in sham-operated SHR (P < 0.05). Within 60 days of transplantation, MAP increased in BB.1K and in F1H transplanted with an SHR kidney (n = 7 per group) by 38 +/- 5 mmHg and 43 +/- 8 mmHg, respectively. CONCLUSIONS: In SHR, arterial pressure can be normalized by a kidney graft from normotensive donors. The genetic predisposition of the recipients to hypertension does not modify the rate and the extent of the arterial pressure rise induced by an SHR kidney graft.