RanBP1 plays an essential role in directed migration of neural crest cells during development

Collective cell migration is essential for embryonic development, tissue regeneration and repair, and has been implicated in pathological conditions such as cancer metastasis. It is, in part, directed by external cues that promote front-to-rear polarity in individual cells. However, our understanding of the pathways that underpin the directional movement of cells in response to external cues remains incomplete. To examine this issue we made use of neural crest cells (NC), which migrate as a collective during development to generate vital structures including bones and cartilage. Using a candidate approach, we found an essential role for Ran-binding protein 1 (RanBP1), a key effector of the nucleocytoplasmic transport pathway, in enabling directed migration of these cells. Our results indicate that RanBP1 is required for establishing front-to-rear polarity, so that NCs are able to chemotax. Moreover, our work suggests that RanBP1 function in chemotaxis involves the polarity kinase LKB1/PAR4. We envisage that regulated nuclear export of LKB1 through Ran/RanBP1 is a key regulatory step required for establishing front-to-rear polarity and thus chemotaxis, during NC collective migration

The ultrastructural organization of endoplasmic reticulum-plasma membrane contacts is conserved in epithelial cells

Contacts between the endoplasmic reticulum and plasma membrane (ER-PM contacts) have important roles in membrane lipid and calcium dynamics. Yet, their organization in polarized epithelial cells has not been thoroughly described. Here, we examine ER-PM contacts in hepatocytes in mouse liver using electron microscopy, providing the first comprehensive ultrastructural study of ER-PM contacts in a mammalian epithelial tissue. Our quantitative analyses reveal strikingly distinct ER-PM contact architectures spatially linked to apical, lateral, and basal PM domains. Notably, we find that an extensive network of ER-PM contacts exists at lateral PM domains that form inter-cellular junctions between hepatocytes. Moreover, the spatial organization of ER-PM contacts is conserved in epithelial spheroids, suggesting that ER-PM contacts may serve conserved roles in epithelial cell architecture. Consistent with this notion, we show that ORP5 activity at ER-PM contacts modulates apical-basolateral aspect ratio in HepG2 cells. Thus, ER-PM contacts have a conserved distribution and crucial roles in PM domain architecture across epithelial cell types. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Cζ and regulates cell polarity

Lgl (lethal giant larvae) plays an important role in cell polarity. Atypical protein kinase C (aPKC) binds to and phosphorylates Lgl, and the phosphorylation negatively regulates Lgl activity. In this study, we identify p32 as a novel Lgl binding protein that directly binds to a domain on mammalian Lgl2 (mLgl2), which contains the aPKC phosphorylation site. p32 also binds to PKCζ, and the three proteins form a transient ternary complex. When p32 is bound, PKCζ is stimulated to phosphorylate mLgl2 more efficiently. p32 overexpression in Madin–Darby canine kidney cells cultured in a 3D matrix induces an expansion of the actin-enriched apical membrane domain and disrupts cell polarity. Addition of PKCζ inhibitor blocks apical actin accumulation, which is rescued by p32 overexpression. p32 knockdown by short hairpin RNA also induces cell polarity defects. Collectively, our data indicate that p32 is a novel regulator of cell polarity that forms a complex with mLgl2 and aPKC and enhances aPKC activity

Iroquois Complex Genes Induce Co-Expression of rhodopsins in Drosophila

The Drosophila eye is a mosaic that results from the stochastic distribution of two ommatidial subtypes. Pale and yellow ommatidia can be distinguished by the expression of distinct rhodopsins and other pigments in their inner photoreceptors (R7 and R8), which are implicated in color vision. The pale subtype contains ultraviolet (UV)-absorbing Rh3 in R7 and blue-absorbing Rh5 in R8. The yellow subtype contains UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8. The exclusive expression of one rhodopsin per photoreceptor is a widespread phenomenon, although exceptions exist. The mechanisms leading to the exclusive expression or to co-expression of sensory receptors are currently not known. We describe a new class of ommatidia that co-express rh3 and rh4 in R7, but maintain normal exclusion between rh5 and rh6 in R8. These ommatidia, which are localized in the dorsal eye, result from the expansion of rh3 into the yellow-R7 subtype. Genes from the Iroquois Complex (Iro-C) are necessary and sufficient to induce co-expression in yR7. Iro-C genes allow photoreceptors to break the “one receptor–one neuron” rule, leading to a novel subtype of broad-spectrum UV- and green-sensitive ommatidia

Notch inhibits Yorkie activity in Drosophila wing discs.

During development, tissues and organs must coordinate growth and patterning so they reach the right size and shape. During larval stages, a dramatic increase in size and cell number of Drosophila wing imaginal discs is controlled by the action of several signaling pathways. Complex cross-talk between these pathways also pattern these discs to specify different regions with different fates and growth potentials. We show that the Notch signaling pathway is both required and sufficient to inhibit the activity of Yorkie (Yki), the Salvador/Warts/Hippo (SWH) pathway terminal transcription activator, but only in the central regions of the wing disc, where the TEAD factor and Yki partner Scalloped (Sd) is expressed. We show that this cross-talk between the Notch and SWH pathways is mediated, at least in part, by the Notch target and Sd partner Vestigial (Vg). We propose that, by altering the ratios between Yki, Sd and Vg, Notch pathway activation restricts the effects of Yki mediated transcription, therefore contributing to define a zone of low proliferation in the central wing discs

Evidence of Distinct Tumour-Propagating Cell Populations with Different Properties in Primary Human Hepatocellular Carcinoma

Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution

Coe Genes Are Expressed in Differentiating Neurons in the Central Nervous System of Protostomes

Genes of the coe (collier/olfactory/early B-cell factor) family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS) development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians

PAR-Complex and Crumbs Function During Photoreceptor Morphogenesis and Retinal Degeneration

The fly photoreceptor has long been used as a model to study sensory neuron morphogenesis and retinal degeneration. In particular, elucidating how these cells are built continues to help further our understanding of the mechanisms of polarized cell morphogenesis, intracellular trafficking and the causes of human retinal pathologies. The conserved PAR complex, which in flies consists of Cdc42-PAR6-aPKC-Bazooka, and the transmembrane protein Crumbs (Crb) are key players during photoreceptor morphogenesis. While the PAR complex regulates polarity in many cell types, Crb function in polarity is relatively specific to epithelial cells. Together Cdc42-PAR6-aPKC-Bazooka and Crb orchestrate the differentiation of the photoreceptor apical membrane (AM) and zonula adherens (ZA), thus allowing these cells to assemble into a neuro-epithelial lattice. In addition to its function in epithelial polarity, Crb has also been shown to protect fly photoreceptors from light-induced degeneration, a process linked to Rhodopsin expression and trafficking. Remarkably, mutations in the human Crumbs1 (CRB1) gene lead to retinal degeneration, making the fly photoreceptor a powerful disease model system

Shaping an optical dome: The size and shape of the insect compound eye

The insect compound eye is the most abundant eye architecture on earth. It comes in a wide variety of shapes and sizes, which are exquisitely adapted to specific ecosystems. Here, we explore the organisational principles and pathways, from molecular to tissular, that underpin the building of this organ and highlight why it is an excellent model system to investigate the relationship between genes and tissue form. The compound eye offers wide fields of view, high sensitivity in motion detection and infinite depth of field. It is made of an array of visual units called ommatidia, which are precisely tiled in 3D to shape the retinal tissue as a dome-like structure. The eye starts off as a 2D epithelium, and it acquires its 3D organisation as ommatidia get into shape. Each ommatidium is made of a complement of retinal cells, including light-detecting photoreceptors and lens-secreting cells. The lens cells generate the typical hexagonal facet lens that lies atop the photoreceptors so that the eye surface consists of a quasi-crystalline array of these hexagonal facet-lenses. This array is curved to various degree, depending on the size and shape of the eye, and on the region of the retina. This curvature sets the resolution and visual field of the eye and is determined by i) the number and size of the facet lens – large ommatidial lenses can be used to generate flat, higher resolution areas, while smaller facets allow for stronger curvature of the eye, and ii) precise control of the inter facet-lens angle, which determines the optical axis of the each ommatidium. In this review we discuss how combinatorial variation in eye primordium shape, ommatidial number, facet lens size and inter facet-lens angle underpins the wide variety of insect eye shapes, and we explore what is known about the mechanisms that might control these parameters.Work in the Pichaud lab is funded by grants for the MRC (MC_UU_12018/3), the BBSRC (BB/R000697) and the Royal Society grant (Award #181274), and benefited from MRC core funding to the LMCB, covering access to microscopy. (MC_12266B). Work in the Casares lab is funded by PGC2018-093704-B-I00, BFU2016-81887-REDT, and MDM-2016-0687, from MINECO (Ministerio de Ciencia e Innovación (Spain)) and FEDER (European Regional Development Fund (Spain))

<i>otd</i> and <i>sens</i> are required for R8 synaptic layer targeting.

<p>All panels (<b>A-F</b>) show photoreceptor cell projections stained with 24B10 (red). (<b>A</b>) Expression of <i>UAS-sens</i>^<i>RNAi</i> (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. <i>UAS-sens</i>^<i>RNAi</i> expressing cells show a clear reduction in Sens protein levels (blue). (<b>B</b>) Expression of <i>UAS-sens</i>^<i>RNAi</i> transgene (GFP-negative ommatidia, encircled by a dotted line) in wild-type tissue (GFP positive) 48 h after clone induction using the tub>GFP>Gal4 system. Otd expression (red) is unaffected in <i>UAS-sens</i>^<i>RNAi</i> expressing R8 cells (indicated by asterisks). (<b>C</b>) Rh6-lacZ-positive R8 axons in <i>sens</i>^<i>RNAi</i> (R8-specific driver<i>-Gal4</i>^{<i>109–68</i>};<i>UAS</i>-<i>sens</i>^<i>RNAi</i>) and in <i>otd</i>^<i>uvi</i> mutant combined with <i>sens</i>^<i>RNAi</i> retina (<b>D</b>), stained with anti-β-galactosidase (green). <i>sens</i> knockdown in the retina leads to a few defects in R8 axon projection, however the combination of the <i>sens</i> knockdown and <i>otd</i>^<i>uvi</i> mutant leads to a complete failure of R8 layer-specific targeting, indicating that <i>sens</i> and <i>otd</i> act in parallel. (<b>E,F</b>) Layer-specific targeting of the R7 photoreceptors is assessed using the R7 specific transgene <i>Rh4-lacZ</i>. As in wild-type (<b>E</b>), Rh4-lacZ positive R7 terminals (green) correctly target to the M6 layer in <i>otd</i>^<i>uvi</i> mutants flies (<b>F</b>). (<b>G</b>) Quantification of the misprojections of <i>Rh6-lacZ</i>-positive R8 axons in wild-type, <i>otd</i>^<i>uvi</i> mutant and <i>otd</i>^<i>uvi</i> mutant flies where either <i>caps</i>, <i>fmi</i> or <i>gogo</i> expression has been restored. Since Caps is present only in R8, <i>UAS</i>-<i>caps</i> is expressed under the control of an R8-specific Gal4 driver, while <i>UAS</i>-<i>gogo</i> and <i>UAS</i>-<i>fmi are</i> expressed using the pan-photoreceptor <i>GMR-Gal4</i> driver. Restoring <i>caps</i> expression in <i>otd</i>-mutant R8 photoreceptors partially rescues the <i>otd</i>^<i>uvi</i> mutant R8 mis-targeting phenotype (from 59% R8 misprojection in <i>otd</i>^<i>uvi</i> to 25% in <i>otd</i>^<i>uvi</i><i>+ UAS-caps</i>). Similarly, expression of <i>fmi</i> and <i>gogo</i> in <i>otd</i>^<i>uvi</i><i>mutant</i> photoreceptors partially suppress the R8 misprojection phenotype (36% R8 axons misprojecting in <i>otd</i>^<i>uvi</i><i>+ UAS-fmi</i>, n = 374 of 1039; 40% R8 axons misprojecting in <i>otd</i>^<i>uvi</i><i>+ UAS-gogo</i>, n = 436 of 1090). Percentages indicate quantitative assessment of R8 axons as detected in M3 and M6 layers only. In wild-type, all R8 neurons target to the M3 layer, thus the mistargeting percentage is zero.</p