Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. Here, we have investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. We describe that Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a longrange gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Iglike and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. We also show that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium.This work was supported by Grants BFU2005-04183 and BFU2008-03320/BMC and by Consolider Program CDS 2007-00008 from the Spanish MICINN, by Marie Curie RTN FP6 (RTN 035528-2) and FP7 (ITN 238186) projects, and by an institutional grant from the Fundación Areces to I.G. It was also financially supported by fellowships awarded by the Junta para la Ampliación de Estudios-Consejo Superior de Investigaciones Cientificas program (to N.G. and A.C.), a Juan de la Cierva fellowship from the Spanish MICINN (to A.B.), a Marie Curie RTN 035528-2 FP6 contract (to E.M.), a contract from the Spanish MICINN (to L.D.), and the senior researcher Programa Amarouto from Severo Ochoa Fondation program of the Comunidad Autónoma de Madrid (G.A.)Peer Reviewe

The Vaccinia Virus I3L Gene Product Is Localized to a Complex Endoplasmic Reticulum-Associated Structure That Contains the Viral Parental DNA

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogenous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER

The Vaccinia Virus E8R Gene Product: a Viral Membrane Protein That Is Made Early in Infection and Packaged into the Virions' Core

Vaccinia virus (VV), a member of the poxvirus family, is unique among most other DNA viruses in that both transcription and DNA replication occur in the cytoplasm of the host cell. It was recently shown by electron microscopy (EM) that soon after viral DNA synthesis is initiated in HeLa cells, the replication sites become enwrapped by the membrane of the endoplasmic reticulum (ER). In the same study, a novel VV membrane protein, the E8R gene product, that may play a role in the ER wrapping process was identified (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001). In the present study, the gene product of E8R was characterized both biochemically and morphologically. We show that E8R is made predominantly early in infection but is packaged into the virion. On two-dimensional gel electrophoresis, the protein appeared as a single spot throughout the VV life cycle; however, in the assembled virion, the protein underwent several modifications which resulted in a change in its molecular weight and its isoelectric point. EM of labeled cryosections of infected HeLa cells showed that the protein localized to the ER and to membranes located on one side of the Golgi complex as early as 1 h postinfection. Late in infection, E8R was additionally associated with membranes of immature virions and with intracellular mature viruses. Although E8R is predominantly associated with membranes, we show that the protein is associated with viral cores; the protein is present in cores made with NP-40-dithiothreitol as well as in incoming cores, the result of the viral entry process, early in infection. Finally, we show that E8R can be phosphorylated in vitro by the viral kinase F10L. It is able to bind DNA in vitro, and this binding may be modulated by phosphorylation by F10L. A putative role of the E8R gene product throughout the VV life cycle is discussed

A Simple Predictive Enhancer Syntax for Hindbrain Patterning Is Conserved in Vertebrate Genomes

<div><p>Background</p><p>Determining the function of regulatory elements is fundamental for our understanding of development, disease and evolution. However, the sequence features that mediate these functions are often unclear and the prediction of tissue-specific expression patterns from sequence alone is non-trivial. Previous functional studies have demonstrated a link between PBX-HOX and MEIS/PREP binding interactions and hindbrain enhancer activity, but the defining grammar of these sites, if any exists, has remained elusive.</p><p>Results</p><p>Here, we identify a shared sequence signature (syntax) within a heterogeneous set of conserved vertebrate hindbrain enhancers composed of spatially co-occurring PBX-HOX and MEIS/PREP transcription factor binding motifs. We use this syntax to accurately predict hindbrain enhancers in 89% of cases (67/75 predicted elements) from a set of conserved non-coding elements (CNEs). Furthermore, mutagenesis of the sites abolishes activity or generates ectopic expression, demonstrating their requirement for segmentally restricted enhancer activity in the hindbrain. We refine and use our syntax to predict over 3,000 hindbrain enhancers across the human genome. These sequences tend to be located near developmental transcription factors and are enriched in known hindbrain activating elements, demonstrating the predictive power of this simple model.</p><p>Conclusion</p><p>Our findings support the theory that hundreds of CNEs, and perhaps thousands of regions across the human genome, function to coordinate gene expression in the developing hindbrain. We speculate that deeply conserved sequences of this kind contributed to the co-option of new genes into the hindbrain gene regulatory network during early vertebrate evolution by linking patterns of <i>hox</i> expression to downstream genes involved in segmentation and patterning, and evolutionarily newer instances may have continued to contribute to lineage-specific elaboration of the hindbrain.</p></div

Relationship between Vaccinia Virus Intracellular Cores, Early mRNAs, and DNA Replication Sites

Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed by the process of DNA replication. In the present study the quantitative and structural relationships between these distinct steps of VV morphogenesis were investigated. We show that viral RNA and DNA synthesis increases linearly with increasing amounts of incoming cores. Moreover, at multiplicities of infection that result in 10 to 40 cores per cell, an approximately 1:1 ratio between cores and sites of DNA replication exists, suggesting that each core is infectious. We have shown previously that VV early mRNAs collect in distinct granular structures that recruit components of the host cell translation machinery. Strikingly, these structures appeared to form some distance away from intracellular cores (M. Mallardo, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:3875-3891, 2001). In the present study the intracellular locations of the sites of early mRNA accumulation and those of the subsequent process of DNA replication were compared. We show that these are distinct structures that have different intracellular locations. Finally, we study the fate of the parental DNA after core uncoating. By electron microscopy, cores were found close to membranes of the endoplasmic reticulum (ER) and the parental DNA, once it had left the core, appeared to associate preferentially with the cytosolic side of those membranes. Since we have previously shown that the process of DNA replication occurs in an ER-enclosed cytosolic “subcompartment” (N. Tolonen, L. Doglio, S. Schleich, and J. Krijnse Locker, Mol. Biol. Cell 12:2031-2046, 2001), the present data suggest that the parental DNA is released into the cytosol and associates with the same membranes where DNA replication is subsequently initiated. The combined data are discussed with respect to the cytosolic organization of VV morphogenesis

The PBX-HOX and MEIS/PREP motifs of four enhancers are essential for their function.

<p>Histograms for four elements, <i>pax2</i>.174 (A), <i>meis2a</i>.1042 (B), <i>meis1</i>.1705 (C) and <i>foxd3</i>.327 (D), showing the number of embryos with GFP positive cells in forebrain (fb), midbrain (mb), hindbrain (hb) and spinal cord (sc) when expressing wild-type (green), MEIS/PREP site mutant (blue) or PBX-HOX site mutant (red) constructs. Annotation displays p values for one-tailed paired t tests. All mutant constructs show a significant (student's t test p = <0.05) reduction in the number of embryos positive for hindbrain. Wild-type <i>pax2</i>.174 (E) drives expression in hindbrain and lens (green), whereas mutant constructs do not drive this pattern. Wild-type <i>meis2a</i>.1042 (F) drives expression in the central nervous system particularly the anterior hindbrain. Mutant constructs fail to recapitulate this expression. Wild-type <i>meis1</i>.1705 (G) drives expression in the hindbrain and spinal cord, but mutant constructs drive expression only in spinal cord. Wild-type <i>foxd3</i>.327 (H) drives expression in posterior hindbrain (green), but expression driven by constructs where either the MEIS/PREP (blue) or the PBX-HOX (red) motif is mutated is frequently ectopic in midbrain, anterior hindbrain and spinal cord.</p

Distances between PBX-HOX and MEIS/PREP motifs across the whole human genome.

<p>Graph shows the distribution of gaps between PBX-HOX and MEIS/PREP motifs across the whole genome (black) and human conserved regions defined by GERP (blue).</p

Number of hb_40 elements per Mb in different types of sequence.

<p>GERP regions, CNEs, enhancers, positive enhancers, positive hindbrain enhancers.</p><p>* This figure is artificially low compared with the CNEs, as the Vista enhancers have long flanking regions (an average size of 1,779bp compared with 116bp for CNEs). In terms of numbers, 21/238 hb+ Vista enhancers (11.34%) have hb_40 syntax compared with 93/6691 CNEs (1.4%).</p><p>Number of hb_40 elements per Mb in different types of sequence.</p

CNEs containing PBX-HOX motifs driving expression in hindbrain of transgenic zebrafish.

<p>Images show expression driven during transient transgenic assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130413#sec017" target="_blank">methods</a>) between 2 and 3 dpf. Insets show comparison with RFP in rhombomeres 3 and 5. <i>hoxd</i>.10479 (A) at ~42 hpf in ventral r5r6; hoxd.10482 lateral (B) and dorsal (C) views at ~56 hpf in lateral r2, r4, r6, pectoral fin and spinal cord; <i>bnc2</i>.8642 (D) at ~60 hpf in hindbrain; <i>hmx2</i>.9713 (E) at ~60 hpf in hindbrain and spinal cord; <i>dachd</i>.11206 (F) at ~72hpf in hindbrain and spinal cord; <i>foxd3</i>.327 (G) at ~72hpf in ventral r5 and r6; <i>foxd3</i>.365 throughout the central nervous system, most strongly in the ventral hindbrain, and cranial ganglia. fb: forebrain; mb: midbrain; hb: hindbrain; sc: spinal cord; pf: pectoral fin; pa: pharyngeal arches/neural crest; cg: cranial ganglia; r3 r5: rhombomeres 3 and 5.</p