Rhombic Cells Research Articles

Cerebellar cortical lamination and foliation require cyclin A2

The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function.

Dasyclad-like problematic fossils from the lower Cambrian of North Greenland

The problematic fossil Kordesphecia nyeboensis gen. et sp. nov. from the lower Cambrian of North Greenland resembles the slightly younger, putative dasycladalean alga Amgaella Korde, 1957 in terms of its spirally arranged rhombic cells and central cavity. It differs in adding new cells through the sequential addition of roofing plates (‘shingles’) to pre-existing cell rows rather than encrusting lateral branches in dasycladalean fashion, in which character it seems comparable to the contemporaneous Lenaella Korde, 1959) from Siberia of unknown affinity. Kordesphecia is classified as incertae sedis but may be a stem-group dasycladalean.

Rhombic Grid Array Antenna

A rhombic grid array antenna (RGAA) is proposed and its frequency-scanning performance is discussed. The RGAA has numerous bent radiation elements, forming rhombic cells and loops. As the frequency is increased, the radiation beam from the RGAA becomes a forward-fire beam. It is found that, as the bend angle of the radiation elements is decreased, the useful antenna characteristics shift to a lower frequency region. It is also found that the RGAA provides a more stable gain within a frequency band bounded by the 3- and 4-guided-wavelength resonances of the loops, compared with a conventional grid array antenna having non-bent radiation elements. Further investigation reveals that the VSWR is small within a frequency band bounded by the 3- and 4-guided wavelength resonances, as desired, and the bend radiation elements of the RGAA contribute to increasing the radiation efficiency.

Transition between triangular and square tiling patterns in liquid-crystalline honeycombs formed by tetrathiophene-based bolaamphiphiles

A series of 5,5′′′-diphenyl tetrathiophenes with polar glycerol groups at each end and two lateral flexible chains self-assemble into a series of liquid-crystalline honeycombs, formed by the π-conjugated rods which enclose polygonal prismatic cells filled by the lateral chains. With increasing chain length a discontinuous transition from triangular to square honeycombs takes place. At this transition a periodic honeycomb composed of a mixture of square and triangular cells in a ratio 1 : 2 was formed at low temperature, whereas at higher temperature a hexagonal columnar phase composed of triangular and randomly distributed rhombic cells, a new kind of cybotactic nematic phase, and also a cybotactic isotropic phase, both composed of square honeycomb fragments, represent the intermediate states. This provides an example of a dynamic self-assembled system where, depending on the molecular mobility, the transition between two periodic structures with different symmetry either leads to an increase of complexity, or to a chaotic regime with reduced order.

The engrailed homeobox genes are required in multiple cell lineages to coordinate sequential formation of fissures and growth of the cerebellum

The layered cortex of the cerebellum is folded along the anterior-posterior axis into lobules separated by fissures, allowing the large number of cells needed for advanced cerebellar functions to be packed into a small volume. During development, the cerebellum begins as a smooth ovoid structure with two progenitor zones, the ventricular zone and upper rhombic lip, which give rise to distinct cell types in the mature cerebellum. Initially, the cerebellar primordium is divided into five cardinal lobes, which are subsequently further subdivided by fissures. The cellular processes and genes that regulate the formation of a normal pattern of fissures are poorly understood. The engrailed genes (En1 and En2) are expressed in all cerebellar cell types and are critical for regulating formation of specific fissures. However, the cerebellar cell types that En1 and En2 act in to control growth and/or patterning of fissures has not been determined. We conditionally eliminated En2 or En1 and En2 either in both progenitor zones and their descendents or in the two complementary sets of cells derived from each progenitor zone. En2 was found to be required only transiently in the progenitor zones and their immediate descendents to regulate formation of three fissures and for general growth of the cerebellum. In contrast, En1 and En2 have overlapping functions in the cells derived from each progenitor zone in regulating formation of additional fissures and for extensive cerebellar growth. Furthermore, En1/2 function in ventricular zone-derived cells plays a more significant role in determining the timing of initiation and positioning of fissures, whereas in upper rhombic lip-derived cells the genes are more important in regulating cerebellar growth. Our studies reveal the complex manner in which the En genes control cerebellar growth and foliation in distinct cell types.

Ubiquitin-mediated regulation of RhoGTPase signalling: IAPs and HACE1 enter the fray

Ubiquitin-mediated regulation of RhoGTPase signalling: IAPs and HACE1 enter the fray

Design methods of rhombic tensegrity structures

As a special type of novel flexible structures, tensegrity holds promise for many potential applications in such fields as materials science, biomechanics, civil and aerospace engineering. Rhombic systems are an important class of tensegrity structures, in which each bar constitutes the longest diagonal of a rhombus of four strings. In this paper, we address the design methods of rhombic structures based on the idea that many tensegrity structures can be constructed by assembling one-bar elementary cells. By analyzing the properties of rhombic cells, we first develop two novel schemes, namely, direct enumeration scheme and cell-substitution scheme. In addition, a facile and efficient method is presented to integrate several rhombic systems into a larger tensegrity structure. To illustrate the applications of these methods, some novel rhombic tensegrity structures are constructed.

The development of nicotinic receptors in the human medulla oblongata: Inter-relationship with the serotonergic system

Maternal cigarette smoking during pregnancy adversely affects fetal development and increases the risk for the sudden infant death syndrome (SIDS). In SIDS we have reported abnormalities in the medullary serotonergic (5-HT) system, which is vital for homeostatic control. In this study we analyzed the inter-relationship between nicotinic receptors (nAChRs), to which nicotine in cigarette smoke bind, and the medullary 5-HT system in the human fetus and infant as a step towards determining the mechanisms whereby smoking increases SIDS risk in infants with 5-HT defects. Immunohistochemistry for the α4 nAChR subunit and 5-HT neurons was applied in fetal and infant medullae (15–92 postconceptional weeks, n = 9). The distribution of different nAChRs was determined from 39–82 postconceptional weeks ( n = 5) using tissue autoradiography for 3H-nicotine, 3H-epibatidine, 3H-cytisine, and 125I-bungarotoxin; the findings were compared to laboratory 5-HT 1A and 5-HT transporter binding data, and 5-HT neuronal density. Alpha4 immunoreactivity was ubiquitously expressed in medullary nuclei related to homeostatic functions from 15 weeks on, including rhombic lip germinal cells. At all ages, α4 co-localized with 5-HT neurons, indicating a potential site of interaction whereby exogenous nicotine may adversely affect 5-HT neuronal development and function. Binding for heteromeric nAChRs was highest in the inferior olive, and for homomeric nAChRs, in the vagal complex. In the paragigantocellularis lateralis, 5-HT 1A receptor binding simultaneously increased as α7 binding decreased across infancy. This study indicates parallel dynamic and complex changes in the medullary nicotinic and 5-HT systems throughout early life, i.e., the period of risk for SIDS.

Mechanism of polarized protrusion formation on neuronal precursors migrating in the developing chicken cerebellum

Directed cell migration results from the polarization of the cellular motile apparatus by integration of extracellular signals, which are presented in a three-dimensional, spatiotemporal manner in living organisms. To investigate the mechanism underlying the highly polarized and directional nature of migration in vivo, we have developed an imaging system for observing rhombic lip cell migration in the developing chicken cerebellum. First, we show that Cdc42 is the central regulator of the overall polarity, morphology and protrusion formation in these cells. However, perturbation of canonical polarity effectors of Cdc42, e.g. the Par6-Par3-aPKC complex, does not disrupt the cell asymmetry, whereas it affects orientation of the tip of the leading process. In contrast to Cdc42, Rac is required for the generation of protrusions but not the overall polarity. Function interference of class IA phosphoinositide 3-kinase abrogates both directional extension and maintenance of the long leading process, whereas PTEN modulates the size of the protrusion. Actomyosin contractility is important for coordinated spreading of the tip of the leading process in situ. Finally, ErbB4 functions in the generation of protrusions on the rhombic lip cells. These results suggest that polarized protrusion formation on neuronal precursors may occur by a more divergent and complex mechanism than that seen in studies of other cell types growing on planar substrates.

Temporal identity transition in the avian cerebellar rhombic lip

The rhombic lip is a discrete strip of neuroepithelium bordering the roofplate of the fourth ventricle, which gives rise to a defined sequence of migratory neuronal derivatives. In rhombomere 1 of the chick, early born cells give rise to post-mitotic hindbrain nuclei, while later derivatives comprise of cerebellar granule cell precursors, a unique proliferative, migratory precursor population that forms the external granule cell layer. We have examined the temporal specification of these two populations using a heterochronic grafting strategy, in ovo. When transplanted into younger neural tube, rhombic lip cells maintain their characteristic molecular markers and migrate into the hindbrain. Granule cell precursor derivatives of late grafts are, in addition, able to exploit neural crest streams to populate the branchial arches. Within the neural tube, derivatives of early and late rhombic lip progenitors display patterns of migration and process extension, characterised by specific trajectories and targets, which are consistent with their temporal origin. However, the normal temporal progression of cell production is disrupted in grafted progenitors: transplanted early rhombic lip fails to subsequently produce granule cell precursors. This indicates that, while the behaviour of derivatives is intrinsically specified at the rhombic lip, the orderly temporal transition in cell type production is dependent on extrinsic cues present only in the later embryo.

Development of the Deep Cerebellar Nuclei: Transcription Factors and Cell Migration from the Rhombic Lip

The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.

A novel role for the choroid plexus in BMP-mediated inhibition of differentiation of cerebellar neural progenitors

Cerebellar granule cells, the most abundant neurons in the mammalian brain, arise in the rhombic lip located at the roof of the brain's fourth ventricle. Bordering the rhombic lip is the choroid plexus, a non-neuronal structure, composed of blood vessels enveloped by epithelial cells. Here, we show a striking decrease in neural differentiation of rhombic lip-derived cells, which failed to extend neuritic processes and attenuate Math1 promoter activity, when co-cultured with choroid plexus cells. Moreover, a blocking antibody against BMP7, a morphogenetic protein expressed in the choroid plexus, blocked the inhibitory effect of the choroid plexus, whereas purified BMP7 mimicked this effect, demonstrating causal involvement of BMP. On the other hand, the BMP antagonist NBL1 promoted neurogenesis in rhombic lip cultures from Math1 null mice displaying arrested differentiation. Our data indicate that besides its secretory and barrier functions, the choroid plexus has a novel role in attenuating the differentiation of adjacent neural progenitors.

Morphogenetic and Cellular Movements that Shape the Mouse Cerebellum

We used the cerebellum as a model to study the morphogenetic and cellular processes underlying the formation of elaborate brain structures from a simple neural tube, using an inducible genetic fate mapping approach in mouse. We demonstrate how a 90 degrees rotation between embryonic days 9 and 12 converts the rostral-caudal axis of dorsal rhombomere 1 into the medial-lateral axis of the wing-like bilateral cerebellar primordium. With the appropriate use of promoters, we marked specific medial-lateral domains of the cerebellar primordium and derived a positional fate map of the murine cerebellum. We show that the adult medial cerebellum is produced by expansion, rather than fusion, of the thin medial primordium. Furthermore, ventricular-derived cells maintain their original medial-lateral coordinates into the adult, whereas rhombic lip-derived granule cells undergo lateral to medial posterior transverse migrations during foliation. Thus, we show that progressive changes in the axes of the cerebellum underlie its genesis.

Lower rhombic lip-derived cells undergo transmedian tangential migration followed by radial migration in the chick embryo brainstem.

Migration behaviour and fate of cells originated from the lower rhombic lip (LRL) was examined in the chick embryo hindbrain. LRL-derived cells tangentially migrate along the pial surface of the brainstem and form a transient subpial migratory stream. In the initial stages of migration, LRL-derived cells appose each other or axon-like processes, which is indicative of mode of homophilic chain migration and/or axophilic migration. Some LRL-derived cells relocate rostroventrally towards the pontine region, although the majority of them migrate circumferentially to the ventral medulla oblongata. Depending on the stage of generation, LRL-derived cells undergo transmedian migration; late-generated LRL-derived cells preferentially colonize the contralateral brainstem compared with early generated cells. Thus, latecomer neuron precursors may migrate past their predecessors in the migratory stream. When LRL-derived cells leave the subpial migratory stream, they change their migratory direction to a radial one and relocate inwardly, with a profile that resembles a tangential-to-radial change seen in cerebellar granule cell precursors. After they enter the parenchymal region of the brainstem, they exhibited morphological differentiation, and some differentiate into excitatory neurons. The present results suggest that LRL-derived cells migrate across boundaries such as midline or rhombomere, which may facilitate to build up cellular and functional architectures of the hindbrain.

Math1 controls cerebellar granule cell differentiation by regulating multiple components of the Notch signaling pathway.

Cerebellar granule cells (CGC) are the most abundant neurons in the mammalian brain, and an important tool for unraveling molecular mechanisms underlying neurogenesis. Math1 is a bHLH transcription activator that is essential for the genesis of CGC. To delineate the effects of Math1 on CGC differentiation, we generated and studied primary cultures of CGC progenitors from Math1/lacZ knockout mice. Rhombic lip precursors appeared properly positioned, expressed CGC-specific markers, and maintained Math1 promoter activity in vivo and in vitro, suggesting that Math1 is not essential for the initial stages of specification or survival of CGC. Moreover, the continuous activity of Math1 promoter in the absence of MATH1, indicated that MATH1 was not necessary for the activation of its own expression. After 6, but not 3, days in culture, Math1 promoter activity was downregulated in control cultures, but not in cells from Math1 null mice, thus implying that Math1 participates in a negative regulatory feedback loop that is dependent on increased levels of MATH1 generated through the positive autoregulatory feedback loop. In addition, Math1 null CGC did not differentiate properly in culture, and were unable to extend processes. All Notch signaling pathway receptors and ligands tested were expressed in the rhombic lip at embryonic date 14, with highest levels of Notch2 and Jag1. However, Math1-null rhombic lip cells presented conspicuous downregulation of Notch4 and Dll1. Moreover, of the two transcriptional repressors known to antagonize Math1, Hes5 (but not Hes1) was downregulated in Math1-null rhombic lip tissue and primary cultures, and was shown to bind MATH1, thus revealing a negative regulatory feedback loop. Taken together, our data demonstrate that CGC differentiation, but not specification, depends on Math1, which acts by regulating the level of multiple components of the Notch signaling pathway.

Expression of the basic Helix-Loop-Helix ME1 E-protein during development and aging of the murine cerebellum

Genesis of cerebellar granule cells is controlled by key transcription factors, such as the lineage-specific basic Helix-Loop-Helix (bHLH) transcription factor MATH-1, whose activity is dependent upon dimerization with bHLH E-proteins. In an effort to understand the molecular mechanisms of bHLH proteins orchestrating cerebellar development, we explored the spatio-temporal expression of the ME1 E-protein. Our results reveal that ME1 expression is first detected in the cerebellar primordium and then in the rhombic lip cells at E12.5. Its expression persists in the emerging external germinal layer during embryonic expansion. In adult cerebellum, prominent ME1 expression is detected in mature granule cells located in the internal granular layer. However, ME1 expression is not sustained in aged cerebellum. A similar declined pattern of expression is also observed in the aging hippocampus.

Expression of the bHLH gene NSCL-1 suggests a role in regulating cerebellar granule cell growth and differentiation

We report that the neuronal-specific basic helix-loop-helix (bHLH) gene NSCL-1 is expressed at multiple and distinct stages of cerebellar granule cell differentiation. During embryonic development, NSCI-1 expression is initially evenly distributed in the cerebellar primordium and then becomes restricted to the ventricular zone. At the early steps of granule cell development, NSCL-1 is not expressed in rhombic lip cells, but instead in migrating granule cell precursors. Its expression culminates during postnatal proliferation of the external germinal layer, and remains only transiently in the newly formed internal granular layer, and at a much lower level. Thus, NSCL-1 expression is linked to the onset of granule cell differentiation, but is not involved in the maintenance of the differentiated state. These findings suggest that NSCL-1 does not behave as a specification factor, but rather as a factor promoting expansion of progenitor external germinal layer (EGL) cells. Gel mobility shift assays show that NSCL-1 only binds DNA as a heterodimeric complex with the ME1a E-protein. We also provide the first evidence that NSCL-1 functions as a transcriptional activator when heterodimerized with the ME1a E-protein. Taken together, these results suggest that NSCL-1 participates in the regulatory network controlling gene expression during cerebellar granule cell differentiation.

Generation of cerebellar granule neurons in vivo by transplantation of BMP-treated neural progenitor cells.

Cerebellar granule neurons, the most abundant class of CNS neurons, have a critical role in cerebellar function. Granule neurons are generated at the dorsal border of the mesencephalon and metencephalon, the rhombic lip. In the mouse embryo, rhombic lip cells express a number of granule neuron markers, notably the bHLH transcription factor Math1. Dorsal midline cells adjacent to the rhombic lip express Bmp6, Bmp7 and Gdf7, three genes encoding peptide growth factors of the bone morphogenetic protein (BMP) family. These BMPs induced the expression of granule neuron markers in cultured neural tissue. Moreover, BMP-treated neural cells formed mature granule neurons after transplantation into the early postnatal cerebellum, suggesting that BMPs initiate the program of granule cell specification.

Embryonic Precursor Cells from the Rhombic Lip Are Specified to a Cerebellar Granule Neuron Identity

The specification of diverse classes of neurons is critical to the development of the cerebellar cortex. Here, we describe the purification of early embryonic precursors of cerebellar granule neurons from the rhombic lip, the dorsal aspect of the midbrain/hindbrain region. Isolation of rhombic lip cells reveals a homogenous population of precursor cells that express general neuronal markers and the granule cell marker RU49, but fail to extend neurites or express differentiation markers. Differentiation is induced by coculture with external germinal layer (EGL) cells, or their membranes, suggesting that a local inducing factor acts after formation of the EGL. Thus, proliferating precursors within the rhombic lip are specified to be granule cells very early, with the availability of an inducing factor increasing over the course of development.

Scanning tunneling microscopy of the conducting transition metal complex, copper bis-(2,5-dimethyl- N, N′-dicyanoquinonediimine)

We have studied the conducting transition metal complex, copper bis-(2,5-dimethyl- N, N′-dicyanoquinonediimine) (Cu(2,5-DM-DCNQI) 2), using scanning tunneling microscopy (STM). The crystal face imaged by STM at molecular resolution shows structural periodicities of 11 and 4 Å along the orthogonal directions. This surface structure agrees with the reported bulk crystal structure of Cu(2,5-DM-DCNQI) 2, with 11 Å periodicity indicative of close-packed planes of Cu(2,5-DM-DCNQI) 2 complexes along the crystallographic c axis and the 4 Å repeat corresponding to the columnar stacking of 2,5-DM-DCNQI ligands along the crystallographic a axis. We have calculated the lowest unoccupied molecular orbital of a 2,5-DM-DCNQI ligand and have shown that the highest electron density resides on the benzene rings, the cyano groups and the imino groups. Guided by these MO calculations, distorted rhombic cells in the STM image could be drawn by connecting four adjacent high contrast sites, possibly the cyano functional groups of surface-terminating 2,5-DM-DCNQI ligands.