N-cadherin (Cdh2) Maintains Migration and Postmitotic Survival of Cortical Interneuron Precursors in a Cell-Type-Specific Manner.

The multiplex role of cadherin-based adhesion complexes during development of pallial excitatory neurons has been thoroughly characterized. In contrast, much less is known about their function during interneuron development. Here, we report that conditional removal of N-cadherin (Cdh2) from postmitotic neuroblasts of the subpallium results in a decreased number of Gad65-GFP-positive interneurons in the adult cortex. We also found that interneuron precursor migration into the pallium was already delayed at E14. Using immunohistochemistry and TUNEL assay in the embryonic subpallium, we excluded decreased mitosis and elevated cell death as possible sources of this defect. Moreover, by analyzing the interneuron composition of the adult somatosensory cortex, we uncovered an unexpected interneuron-type-specific defect caused by Cdh2-loss. This was not due to a fate-switch between interneuron populations or altered target selection during migration. Instead, potentially due to the migration delay, part of the precursors failed to enter the cortical plate and consequently got eliminated at early postnatal stages. In summary, our results indicate that Cdh2-mediated interactions are necessary for migration and survival during the postmitotic phase of interneuron development. Furthermore, we also propose that unlike in pallial glutamatergic cells, Cdh2 is not universal, rather a cell type-specific factor during this process.

Acetaminophen Relieves Inflammatory Pain through CB1 Cannabinoid Receptors in the Rostral Ventromedial Medulla.

Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug with only incompletely understood mechanisms of action. Previous work, using models of acute nociceptive pain, indicated that analgesia by acetaminophen involves an indirect activation of CB1 receptors by the acetaminophen metabolite and endocannabinoid reuptake inhibitor AM 404. However, the contribution of the cannabinoid system to antihyperalgesia against inflammatory pain, the main indication of acetaminophen, and the precise site of the relevant CB1 receptors have remained elusive. Here, we analyzed acetaminophen analgesia in mice of either sex with inflammatory pain and found that acetaminophen exerted a dose-dependent antihyperalgesic action, which was mimicked by intrathecally injected AM 404. Both compounds lost their antihyperalgesic activity in CB1-/- mice, confirming the involvement of the cannabinoid system. Consistent with a mechanism downstream of proinflammatory prostaglandin formation, acetaminophen also reversed hyperalgesia induced by intrathecal prostaglandin E2 To distinguish between a peripheral/spinal and a supraspinal action, we administered acetaminophen and AM 404 to hoxB8-CB1-/- mice, which lack CB1 receptors from the peripheral nervous system and the spinal cord. These mice exhibited unchanged antihyperalgesia indicating a supraspinal site of action. Accordingly, local injection of the CB1 receptor antagonist rimonabant into the rostral ventromedial medulla blocked acetaminophen-induced antihyperalgesia, while local rostral ventromedial medulla injection of AM 404 reduced hyperalgesia in wild-type mice but not in CB1-/- mice. Our results indicate that the cannabinoid system contributes not only to acetaminophen analgesia against acute pain but also against inflammatory pain, and suggest that the relevant CB1 receptors reside in the rostral ventromedial medulla.SIGNIFICANCE STATEMENT Acetaminophen is a widely used analgesic drug with multiple but only incompletely understood mechanisms of action, including a facilitation of endogenous cannabinoid signaling via one of its metabolites. Our present data indicate that enhanced cannabinoid signaling is also responsible for the analgesic effects of acetaminophen against inflammatory pain. Local injections of the acetaminophen metabolite AM 404 and of cannabinoid receptor antagonists as well as data from tissue-specific CB1 receptor-deficient mice suggest the rostral ventromedial medulla as an important site of the cannabinoid-mediated analgesia by acetaminophen.

Flying under the radar: CDH2 (N-cadherin), an important hub molecule in neurodevelopmental and neurodegenerative diseases.

CDH2 belongs to the classic cadherin family of Ca2+-dependent cell adhesion molecules with a meticulously described dual role in cell adhesion and ß-catenin signaling. During CNS development, CDH2 is involved in a wide range of processes including maintenance of neuroepithelial integrity, neural tube closure (neurulation), confinement of radial glia progenitor cells (RGPCs) to the ventricular zone and maintaining their proliferation-differentiation balance, postmitotic neural precursor migration, axon guidance, synaptic development and maintenance. In the past few years, direct and indirect evidence linked CDH2 to various neurological diseases, and in this review, we summarize recent developments regarding CDH2 function and its involvement in pathological alterations of the CNS.

Microglial control of neuronal development via somatic purinergic junctions.

Microglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment.

ABHD4-dependent developmental anoikis safeguards the embryonic brain.

A specialized neurogenic niche along the ventricles accumulates millions of progenitor cells in the developing brain. After mitosis, fate-committed daughter cells delaminate from this germinative zone. Considering the high number of cell divisions and delaminations taking place during embryonic development, brain malformations caused by ectopic proliferation of misplaced progenitor cells are relatively rare. Here, we report that a process we term developmental anoikis distinguishes the pathological detachment of progenitor cells from the normal delamination of daughter neuroblasts in the developing mouse neocortex. We identify the endocannabinoid-metabolizing enzyme abhydrolase domain containing 4 (ABHD4) as an essential mediator for the elimination of pathologically detached cells. Consequently, rapid ABHD4 downregulation is necessary for delaminated daughter neuroblasts to escape from anoikis. Moreover, ABHD4 is required for fetal alcohol-induced apoptosis, but not for the well-established form of developmentally controlled programmed cell death. These results suggest that ABHD4-mediated developmental anoikis specifically protects the embryonic brain from the consequences of sporadic delamination errors and teratogenic insults.

Microglia monitor and protect neuronal function through specialized somatic purinergic junctions.

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.

The small molecule Mek1/2 inhibitor U0126 disrupts the chordamesoderm to notochord transition in zebrafish.

BACKGROUND: Key molecules involved in notochord differentiation and function have been identified through genetic analysis in zebrafish and mice, but MEK1 and 2 have so far not been implicated in this process due to early lethality (Mek1-/-) and functional redundancy (Mek2-/-) in the knockout animals. RESULTS: Here, we reveal a potential role for Mek1/2 during notochord development by using the small molecule Mek1/2 inhibitor U0126 which blocks phosphorylation of the Mek1/2 target gene Erk1/2 in vivo. Applying the inhibitor from early gastrulation until the 18-somite stage produces a specific and consistent phenotype with lack of dark pigmentation, shorter tail and an abnormal, undulated notochord. Using morphological analysis, in situ hybridization, immunhistochemistry, TUNEL staining and electron microscopy, we demonstrate that in treated embryos the chordamesoderm to notochord transition is disrupted and identify disorganization in the medial layer of the perinotochordal basement mebrane as the probable cause of the undulations and bulges in the notochord. We also examined and excluded FGF as the upstream signal during this process. CONCLUSION: Using the small chemical U0126, we have established a novel link between MAPK-signaling and notochord differentiation. Our phenotypic analysis suggests a potential connection between the MAPK-pathway, the COPI-mediated intracellular transport and/or the copper-dependent posttranslational regulatory processes during notochord differentiation.

Hedgehog signaling patterns the outgrowth of unpaired skeletal appendages in zebrafish.

BACKGROUND: Little is known about the control of the development of vertebrate unpaired appendages such as the caudal fin, one of the key morphological specializations of fishes. Recent analysis of lamprey and dogshark median fins suggests the co-option of some molecular mechanisms between paired and median in Chondrichthyes. However, the extent to which the molecular mechanisms patterning paired and median fins are shared remains unknown. RESULTS: Here we provide molecular description of the initial ontogeny of the median fins in zebrafish and present several independent lines of evidence that Sonic hedgehog signaling emanating from the embryonic midline is essential for establishment and outgrowth of the caudal fin primordium. However, gene expression analysis shows that the primordium of the adult caudal fin does not harbor a Sonic hedgehog-expressing domain equivalent to the Shh secreting zone of polarizing activity (ZPA) of paired appendages. CONCLUSION: Our results suggest that Hedgehog proteins can regulate skeletal appendage outgrowth independent of a ZPA and demonstrates an unexpected mechanism for mediating Shh signals in a median fin primordium. The median fins evolved before paired fins in early craniates, thus the patterning of the median fins may be an ancestral mechanism that controls the outgrowth of skeletogenic appendages in vertebrates.

Expression of pcp4a in subpopulations of CNS neurons in zebrafish.

The molecular organization of the zebrafish brain and its relation to neuroanatomical divisions are still largely unknown. In this study we have analyzed the expression of a small transcript encoding for the IQ containing polypeptide Pcp4a in developing and juvenile zebrafish. The transcript is exclusively expressed in neural structures with a pattern that is highly specific for restricted domains and cell populations throughout development, and it allows us to follow the development of these structures at different times. The expression of pcp4a characterizes the dorsocaudal telencephalon, dorsal habenula, pretectal nuclei, preglomerular complex, mammillary bodies, and deep layers of the optic tectum and is a hallmark of a subpopulation of reticulospinal neurons. In the telencephalon, comparison of the expression of pcp4a with other pallial markers showed a rostrocaudal gradient in the expression of these genes, which suggests that the dorsal telencephalon of zebrafish may be organized in distinct areas with different molecular natures. Pcp4 has been involved in modulating calcium signals and in binding to calmodulin, but its precise role in neuronal functions is not known. The analysis of pcp4a expression and localization in the zebrafish brain suggests that pcp4a may be a useful marker for sensory and some motor neuronal circuitries and for telencephalic areas processing sensory inputs.

Comparative analysis of type II classic cadherin mRNA distribution patterns in the developing and adult mouse somatosensory cortex and hippocampus suggests significant functional redundancy.

The type II classic cadherin subfamily contains a number of extensively studied genes (cdh6, cdh8, cdh11); however, the expression and function of the other members have only been partially described. Here we employed reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization to characterize cortical and hippocampal expression of all type II cadherins (with the exception of the nonneural Cdh5) in the developing and adult mouse brain. Many of these genes have ubiquitous mRNA distribution patterns throughout development, indicating high functional redundancy, which might be necessary for safe production of the strictly laminated structure of these regions. A few of the genes examined, however, exhibit a unique spatiotemporal pattern of expression, particularly during cortical development, indicating a potentially specific function. In the developing and adult hippocampus, almost all of these genes are strongly expressed in glutamatergic neurons of the CA1-CA3 pyramidal cell layer and the granular layer of the dentate gyrus. In contrast, there are significant expression differences within the GABAergic cells of the adult hippocampus. Our results indicate that selective expression of type II cadherins may generate a flexible cell-adhesion machinery for developing neurons to selectively bind to each other, but can also provide a high level of security due to the multiple overlaps in the expression domains.

Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog.

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.

N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites.

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube.

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.

Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish.

Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development.