Crumbs 2 prevents cortical abnormalities in mouse dorsal telencephalon

AbstractThe formation of a functionally integrated nervous system is dependent on a highly organized sequence of events that includes timely division and differentiation of progenitors. Several apical polarity proteins have been shown to play crucial roles during neurogenesis, however, the role of Crumbs 2 (CRB2) in cortical development has not previously been reported.Here, we show that conditional ablation of Crb2 in the murine dorsal telencephalon leads to defects in the maintenance of the apical complex. Furthermore, within the mutant dorsal telencephalon there is premature expression of differentiation proteins. We examined the physiological function of Crb2 on wild type genetic background as well as on background lacking Crb1. Telencephalon lacking CRB2 resulted in reduced levels of PALS1 and CRB3 from the apical complex, an increased number of mitotic cells and expanded neuronal domain. These defects are transient and therefore only result in rather mild cortical abnormalities. We show that CRB2 is required for maintenance of the apical polarity complex during development of the cortex and regulation of cell division, and that loss of CRB2 results in cortical abnormalities

The role of Crumbs2 in neural development

The formation of a functionally integrated nervous system is dependent on a highly organized sequence of events that includes timely division and differentiation of progenitors. Three evolutionarily conserved polarity protein complexes are crucial for defining the apical and basolateral boundaries of cells. In my thesis, I demonstrate that one of the vertebrate homologs of Crumbs- Crumbs2 (Crb2) plays context dependent roles in the developing nervous system using two model systems: the chick embryonic hindbrain and the mouse embryonic telencephalon. In the developing telencephalon, conditional ablation of Crb2 leads to defects in recruitment of apical polarity proteins, cell junction proteins, positioning of mitotic cells and cortical neurogenesis. In the chick embryonic hindbrain, misexpression of Crb2 affects morphology of the neural tube and also affects the apical localization of cell polarity proteins, mitotic cell divisions and neural differentiation. In addition to this, I demonstrate that a novel secreted splice variant of Crb2 plays an important role in regulating neural crest cell migration. Taken together my analyses show that both loss and misexpression of Crb2 have similar effects on the apical domain and in confining mitotic cell divisions to the apical domain. This implies that the level of Crb2 is crucial for its various biological roles in the developing nervous system.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Loss of Usp9x disrupts cell adhesion, and components of the Wnt and Notch signaling pathways in neural progenitors

Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants. Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of β-catenin protein, especially S33/S37/T41 phospho-β-catenin, were markedly increased in Usp9x embryonic cortices. Loss of Usp9x altered composition of the β-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-β-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9x embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors

A Grand Challenge: Unbiased Phenotypic Function of Metabolites from <i>Jaspis splendens</i> against Parkinson’s Disease

A grand challenge in natural product chemistry is to determine the biological effects of all natural products. A phenotypic approach is frequently used for determining the activity of a compound and its potential impact on a disease state. Chemical investigation of a specimen of <i>Jaspis splendens</i> collected from the Great Barrier Reef resulted in the isolation of a new pterin derivative, jaspterin (<b>1</b>), a new bisindole alkaloid, splendamide (<b>2</b>), and a new imidazole alkaloid, jaspnin A (<b>3</b>) TFA salt. Jaspamycin (<b>8</b>) and 6-bromo-1<i>H</i>-indole-3-carboximidamide (<b>16</b>) are reported for the first time as naturally occurring metabolites. Known nucleosides (<b>4</b>–<b>7</b>, <b>9</b>, <b>10</b>), aglycones (<b>11</b>–<b>13</b>), indole alkaloids (<b>14</b>, <b>15</b>, <b>17</b>), and jaspamide peptides (<b>18</b>–<b>22</b>) were also isolated. The structures of the three new compounds <b>1</b>–<b>3</b> were unambiguously elucidated based on NMR and mass spectroscopic data. Jaspnin A (<b>3</b>) contained a rare thiomethylated imidazolinium unit. Coupling an unbiased phenotypic assay using a human olfactory neurosphere-derived cell model of Parkinson’s disease to all of the natural products from the species <i>J. splendens</i> allowed the phenotypic profiles of the metabolites to be investigated

Will Robots Take Your Job?

A chemoinformatic method was developed to extract nonflat scaffolds embedded in natural products within the Dictionary of Natural Products (DNP). The cedrane scaffold was then chosen as an example of a nonflat scaffold that directs substituents in three-dimensional (3D) space. A cedrane scaffold that has three orthogonal handles to allow generation of 1D, 2D, and 3D libraries was synthesized on a large scale. These libraries would cover more than 50% of the natural diversity of natural products with an embedded cedrane scaffold. Synthesis of three focused natural product-like libraries based on the 3D cedrane scaffold was achieved. A phenotypic assay was used to test the biological profile of synthesized compounds against normal and Parkinson’s patient-derived cells. The cytological profiles of the synthesized analogues based on the cedrane scaffold revealed that this 3D scaffold, prevalidated by nature, can interact with biological systems as it displayed various effects against normal and Parkinson’s patient-derived cell lines

Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/- mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S-CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination.</p

Loss of CRB2 in the mouse retina mimics human retinitis pigmentosa due to mutations in the CRB1 gene.

International audienceIn humans, the Crumbs homolog-1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, we generated and analysed conditional knockout mice lacking CRB2 in the developing retina. Progressive disorganization was detected during late retinal development. Progressive thinning of the photoreceptor layer and sites of cellular mislocalization was detected throughout the CRB2-deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Under scotopic conditions using electroretinography, the attenuation of the a-wave was relatively stronger than that of the b-wave, suggesting progressive degeneration of photoreceptors in adult animals. Histological analysis of newborn mice showed abnormal lamination of immature rod photoreceptors and disruption of adherens junctions between photoreceptors, Müller glia and progenitor cells. The number of late-born progenitor cells, rod photoreceptors and Müller glia cells was increased, concomitant with programmed cell death of rod photoreceptors. The data suggest an essential role for CRB2 in proper lamination of the photoreceptor layer and suppression of proliferation of late-born retinal progenitor cells