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About Development
Concentric zones, cell migration and neuronal circuits in the Drosophila visual center
Eri Hasegawa, Yusuke Kitada, Masako Kaido, Rie Takayama, Takeshi Awasaki, Tetsuya Tabata, Makoto Sato
Development 2011 138: 983-993; doi: 10.1242/dev.058370
Eri Hasegawa
Yusuke Kitada
Masako Kaido
Rie Takayama
Takeshi Awasaki
Tetsuya Tabata
Makoto Sato
For correspondence: makotos@staff.kanazawa-u.ac.jp
The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. Here, we describe the key features of the developing medulla and report novel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.
In many animals, visual information is processed by visual centers in the brain that are composed of laminar structures and columnar units with topographic input from the retina. The neuronal circuits in the visual centers process various types of visual information such as motion, color and shape. As noted by Ramón y Cajal, the fly optic lobe shares structural characteristics with mammalian visual systems (Sanes and Zipursky, 2010), and Drosophila genetics allow high resolution genetic manipulations (Gao et al., 2008; Rister et al., 2007). The Drosophila visual system may provide a powerful model to investigate the mechanisms underlying the formation of visual processing circuits. However, little is known regarding the developmental processes that create neuronal diversity and organize these circuits in the primary region of the optic lobe, the medulla.
The Drosophila retina is composed of ~800 ommatidial units, each containing eight types of photoreceptor neurons (R1-8). R1-6 detect moving stimuli (Katsov and Clandinin, 2008; Rister et al., 2007; Zhu et al., 2009), while R7-8 are involved in color vision (Gao et al., 2008; Morante and Desplan, 2008). Visual information received in the retina is transmitted to the optic lobe composed of lamina, medulla, lobula and lobula plate. The medulla, lobula and lobula plate are subdivided into ten (M1-10), six (Lo1-6) and four (Lop1-4) strata, respectively.
The medulla is the largest component of the Drosophila optic lobe, containing ~40,000 neurons forming 10 layers. In Golgi studies, 60 types of medulla neurons with different morphological characteristics have been identified (Fischbach and Dittrich, 1989; Hofbauer and Campos-Ortega, 1990). Because R7-8 axons terminate in the medulla and the visual information carried by R1-6 axons is sent to the medulla through a relay by lamina neurons, the medulla might process all of the visual information received in the retina (Takemura et al., 2008). In spite of the importance of the medulla, the developmental mechanisms that produce neuronal diversity and organize medulla circuits remain largely unknown. In the larval brain, the primary source of medulla neurons is thought to be neuroblasts (NBs) situated in the outer proliferation center (OPC) (Hofbauer and Campos-Ortega, 1990; Yasugi et al., 2008). The differentiation of NBs from neuroepithelial cells (NEs) progresses from the medial to the lateral region of the OPC, and each NB produces ganglion mother cells (GMCs) that subsequently divide to become neurons (Egger et al., 2007; Toriya et al., 2006). A key goal in developmental neurobiology is understanding the molecular mechanisms by which each of 40,000 neurons in the medulla establishes an appropriate identity among 60 different possibilities and connects to appropriate targets to form neuronal circuits.
Here, we describe the key features of the developing medulla and report novel observations that could accelerate our understanding of the Drosophila visual system. During larval development, the medulla is subdivided into concentric zones that are characterized by the expression of four conserved transcription factors: Drifter (Drf), Runt (Run), Homothorax (Hth) and Brain-specific homeobox (Bsh) (Anderson et al., 1995; Gergen and Butler, 1988; Jones and McGinnis, 1993; Kurant et al., 1998; Pai et al., 1998; Rieckhof et al., 1997). The expression patterns of these factors correlate with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly, and the concentric zones are disrupted early in pupal development owing to the extensive migration of cell bodies. The projection patterns of medulla neurons are established through a series of steps – axonal projection, cell body migration and dendritic arborization – that are stereotypically defined according to the neuronal type most likely governed by genes that show the concentric zones.
Generation of Gal4 strains
Following DNA fragments were PCR amplified using primers listed in Table S1 in the supplementary material.
drf-Gal4
The drfL, Gal4 and drfR fragments were inserted to pW25 to have drfGal/W25, which was used for homologous recombination (Gong and Golic, 2003).
Although drfR was precisely inserted into the drf locus, as revealed by PCR, drfL was not. As Drf expression was detected in drf-Gal4 homozygous clones, it does not eliminate the drf ORF (not shown).
hth-Gal4
The hthL, Gal4 and hthR fragments were inserted to pW25 to have hthGal/W25, which was introduced to the hth locus by converting to the hth-lacZ insertion using Δ2-3 (Sepp and Auld, 1999). hth-Gal4 was inserted between exons 9 and 10. Hth expression was significantly reduced in hth-Gal4 homozygous clones (not shown).
bsh-Gal4
The bshLz, Gal4, Kmr and bshRz fragments were inserted to pW25 to have bshLRzGal4Kmr, which was inserted to a BAC containing the bsh locus (Kimura et al., 2006; Venken et al., 2006). The bshLB and bshRB fragments at the boundary of the bsh locus were inserted to attB-Pacman-ApR. The bsh locus was retrieved from the BAC to obtain bshGalBAC/Pacman, which was introduced to the fly genome by P-element mediated transformation (third chromosome).
Fly strains
Fly strains used were: repo-Gal4, AyGal4, Actin>stop>lacZ, UAS>CD2>CD8GFP, UAS-flp, hs-flp, elav-Gal4, UAS-CD8GFP, tub-Gal80, FRT40A, FRT2A, FRT82B, M(3R)w, UAS-GFP, UAS-nlsGFP, UAS-SytHA, UAS-tau-myc, drfE82, drfH76, UAS-drf, hthP2, UAS-hth, hth-lacZ, NCadM19, NCadomb405, NP2646 (CG11873) and NP6013 (amnesiac). dpn-Gal4 was provided by Ethan Bier (UC San Diego, La Jolla, CA, USA).
Clonal analysis
Following genetic crosses and heat shock conditions were used: Fig. 2, hs-flp; UAS-CD8GFP was crossed to AyGal4 (32°C, 15 minutes at early 3rd instar, dissected 36 hours after heat shock); Fig. 3, elav-Gal4 UAS-CD8GFP hs-flp; tub-Gal80 FRT2A was crossed to FRT2A (37°C, 60 minutes at 2nd instar); Fig. 4, hs-flp; UAS>CD2>CD8GFP was crossed to hth-Gal4, NP6013 and bsh-Gal4 flies. hs-flp; FRT82B tub-Gal80 was crossed to UAS-SytHA; FRT82B bsh-Gal4 UAS-CD8GFP (32-33°C, 60 minutes at 3rd instar); Fig. 5, y+ FRT40A; hth-Gal4 UAS-CD8GFP was crossed to hs-flp; tub-Gal80 FRT40A; UAS-CD8GFP. hs-flp; FRT82B tub-Gal80 was crossed to FRT82B hth-Gal4 UAS-CD8GFP. hs-flp; UAS-hth; FRT82B tub-Gal80 was crossed to FRT82B hth-Gal4 UAS-CD8GFP (32-33°C, 60 minutes at 3rd instar). hs-flp; FRT82B tub-Gal80 was crossed to FRT82B bsh-Gal4 UAS-CD8GFP or FRT82B bsh-Gal4 UAS-CD8GFP hthP2.
hs-flp; UAS-hth; FRT82B tub-Gal80 was crossed to FRT82B bsh-Gal4 UAS-CD8GFP hthP2 (32-35°C, 60 minutes at 3rd instar). hs-flp; tub-Gal80 FRT40A; UAS-CD8GFP was crossed to NP6013; NCadM19 or NCadomb405 FRT40A; UAS-CD8GFP or NP6013; y+ FRT40A; UAS-CD8GFP (32°C, 60 minutes at 2nd to early 3rd instar). hs-flp; tub-Gal80 FRT40A; UAS-CD8GFP was crossed to NCadM19 or NCadomb405 FRT40A; bsh-Gal4 UAS-CD8GFP or y+ FRT40A; bsh-Gal4 UAS-CD8GFP (32-33°C, 60 minutes at 3rd instar). hs-flp; AyGal4 UAS-GFP was crossed to UAS-hth (34°C, 30-60 minutes at early 3rd instar). hs-flp; FRT82B hthP2 was crossed to FRT82B ubi-GFP M(3R)w flies (37°C, 60 minutes at 1st instar).
In Fig. 6, hs-flp; tub-Gal80 FRT40A; drf-Gal4 UAS-CD8GFP was crossed to UAS-SytHA; FRT40A flies (31°C, 30 minutes at 3rd instar); in Fig. 7, UAS-SytHA; tub-Gal80 FRT2A was crossed to hs-flp; FRT2A UAS-CD8GFP NP2646 or hs-flp; drfE82 or drfH76 FRT2A UAS-CD8GFP NP2646. hs-flp; UAS-drf; tub-Gal80 FRT2A was crossed to hs-flp; drfE82 FRT2A UAS-CD8GFP NP2646 (33°C, 20 minutes at 3rd instar).
Histochemistry
Rabbit anti-Hth [Adi Salzberg (Israel Institute of Technology, Haifa, Israel); 1:1000], guinea pig anti-Bsh (Chi-Hon Lee, NIH Bethesda, MD, USA; 1:500), guinea pig anti-Run (Asian Distribution Center, 1:500), rat anti-Drf [Sarah Certel (University of Montana, Missoula, MT, USA); 1:3000], guinea pig anti-Dpn [James Skeath (Washington University, St Louis, MO, USA); 1:1000), mouse anti-HA (Covance, 1:1000), mouse anti-lacZ (Promega, 1:250), rat anti-CD8 Alexa488-conjugated (Caltag, 1:100) and rabbit anti-GFP Alexa488-conjugated (Invitrogen, 1:2000) were used. Mouse anti-Chaoptin (1:10), mouse anti-ChAT (1:300), mouse anti-Cut (1:20), mouse anti-Dac (1:20), rat anti-Elav (1:100), mouse anti-Myc (1:20), rat anti-NCad (1:20), mouse anti-Pros (1:10) and mouse anti-Repo (1:10) were from DSHB. Custom-made antibodies (rabbit anti-Hth, guinea pig and rat anti-Bsh, Run, Drf antibodies) were raised as described previously (Jones and McGinnis, 1993; Kosman et al., 1998; Kurant et al., 1998; Llimargas and Casanova, 1997). Details are available upon request. In situ hybridization was performed as described previously (Sato et al., 2006). Images were processed using Zeiss LSM image browser, Adobe Photoshop and Bitplane Imaris.
The larval medulla is subdivided into concentric zones expressing conserved transcription factors
To identify genetic subdivisions in the medulla primordium of the larval brain, the expression of conserved transcription factors was examined. We identified concentric zones characterized by the expression of four conserved genes that encode transcription factors: Drifter (Drf) from the Brn family, Runt (Run) from the Runx family, Homothorax (Hth) from the Meis family and Brain-specific homeobox (Bsh) from the Bsx family (Fig. 1D,E) (Anderson et al., 1995; Gergen and Butler, 1988; Jones and McGinnis, 1993; Rieckhof et al., 1997). All of these genes, which we collectively call 'concentric genes', were expressed in differentiated neurons (Fig. 1F); however, weak Hth signals were also found in NBs and NEs (Fig. 1D′,E′). The concentric zones expressing Drf, Run and Hth were adjacent but did not overlap (Fig. 1D). Bsh expression was found in the outer subdomain of the Hth domain (Fig. 1E). Medulla neurons projected axon-like structures toward the center of the brain hemisphere to form concentric neuropiles (Fig. 1A,B,F). Accumulations of tau-Myc suggest that the axon-like structures are indeed axons (Fig. 1J) (Thor et al., 1999). Large glial cells were found on the surface of the medulla, while small glial cells were in the innermost area of the medulla cortex adjacent to the Hth domain (Fig. 1G), which most likely become the medulla neuropile glia (MNG; see Fig. 3B) (Tix et al., 1997). The processes of glial cells were found in the larval medulla cortex (Fig. 1G).
The birth order of medulla neurons correlates with concentric gene expression
The NBs are formed from NEs in the OPC during early third larval instar. NB produces GMCs that subsequently divide to produce medulla neurons (Colonques et al., 2007; Egger et al., 2007; Nassif et al., 2003; Toriya et al., 2006; Yasugi et al., 2008). Many neurons are produced from a single NB with a linear and radial orientation towards the center of the developing medulla (Fig. 1A,B). A weak expression of concentric genes appeared around mid third instar (not shown).
Larval medulla primordium is subdivided into concentric zones. (A-C) Schematics of the Drosophila visual system in third instar larva (A,B) and adult (C). Lateral (A) and horizontal (B,C) views. (D-G′) The larval medulla in lateral (D-G) and horizontal (D′-G′) views. (D,D′) Hth (green), Run (magenta) and Drf (blue) expression. (E,E′) Hth (green) and Bsh (magenta) expression and NBs (Dpn; blue). Weak Hth expression in NE and NB cells (arrowhead in D′ and arrows in D′ and E′, respectively). Bsh expression in lamina neurons (arrowhead in E′). (F,F′) Hth (green) and Drf (blue) expression and neuronal projections (elav-Gal4 UAS-CD8GFP; white). Only GFP signal is shown in the right half (F). Primordia for medulla and lobula complex (arrows and arrowheads, respectively). (G,G′) hth-lacZ (green), glial cell bodies (Repo; magenta) and glial processes (repo-Gal4 UAS-CD8GFP; white). Large and small glial cells in medulla cortex (arrows and arrowheads). Putative MNGs (large arrowheads). (H,I) Schematics of the concentric zones in larval medulla in lateral (H) and horizontal views (I). (J) Clones of larval medulla neurons expressing CD8GFP (green) and tau-Myc (magenta) under the control of AyGal4. Putative axons are indicated by arrows. Lo, lobula; Lop, lobula plate; M, medulla.
Each clone of neurons produced from a single NB formed a line that intersected the concentric zones (Fig. 1A,B,H,I); thus, the expression of concentric genes may not be lineage dependent. Each NB may produce several types of medulla neurons that are distinguished by the expression of concentric genes. To address this possibility, a small group of cells were labeled with constitutive expression of UAS-CD8GFP (Fig. 2A,B) (Ito et al., 1997). Mild induction of FLPase triggered GFP expression in a single NB, as identified by Deadpan (Dpn) expression, and in daughter cells of the NB (Fig. 2B) (Bier et al., 1992). The GFP-positive neurons are thus thought to derive from a single NB. Our observations suggest that various combinations of neurons can be produced from a single NB (Drf/Bsh, Drf/Run, Bsh/Hth, Run/Hth; Fig. 2B and not shown). Because we could not restrict the timing of clone induction in newborn NBs, a subset of daughter cells produced from a NB could be labeled with GFP. However, a single NB clone tended to contain two or three Drf-positive, one Run-positive and one Bsh-positive neurons (Fig. 2C), suggesting that many of the NBs have essentially the same potential to produce a series of medulla neurons (Fig. 2D).
Because NBs produce neurons directed towards the center of the medulla, it is likely that neurons born early in development are located in inner concentric zones, while late-born neurons are found in outer zones. To confirm this theory, we used a neuroblast specific Gal4 driver (dpn-Gal4) that specifically expresses Gal4 mRNA in NB cells (Fig. 2G,H). Differentiated neurons inherit the Gal4 protein, even after Gal4 transcription is terminated; thus, a strong GFP signal was detected in neurons in the outer concentric zones (Fig. 2E). However, the GFP signal was decreased in neurons distant from NBs and was not detectable in the innermost zones. A prominent GFP signal was observed in the innermost, non-neuronal glial cells (Fig. 2F), a result supporting the hypothesis that NBs produce glial cells that subsequently migrate toward the medulla neuropile (Colonques et al., 2007). The signal intensity of GFP roughly correlated with concentric gene expression; strong GFP expression was found in Drf-positive neurons but gradually decreased in Run and Bsh-positive zones (Fig. 1D′, Fig. 2E,I). These results are consistent with the idea that the birth order of medulla neurons may control neuronal type via concentric gene expression.
Birth order of medulla neurons correlates with concentric gene expression. (A,B) Clones of cells expressing CD8GFP (green). Drf (blue; arrowheads) and Bsh (magenta; arrowheads) expression and NBs (Dpn; magenta; arrows). Clones containing multiple NBs (A) or a single NB (B). (C) Number of cells expressing Drf, Bsh and Run in single NB clones that intersect the Drf-Bsh or Drf-Run domains. Clones in the posterior edge of the OPC are not considered. (D) A schematic of NBs producing multiple types of medulla neurons. (E,F) Neuronal birth order visualized using dpn-Gal4 UAS-GFP (green). Later-born neurons show stronger GFP signals. (E) Drf (blue) and Bsh (magenta) expression. (F) Glial cells (Repo; magenta; arrowheads). (G,H) Gal4 mRNA (magenta) expression in NBs (Dpn; blue) in dpn-Gal4 flies. Lateral (G) and horizontal (H) views. (I) A schematic of the GFP gradient produced by NB-specific Gal4 expression.
Medulla neurons migrate extensively and the concentric zones are disrupted during pupal development
The expression of concentric genes in the larval medulla primordium may regulate neuronal identities and contribute to the formation of neuronal circuits in the adult medulla. We thus examined concentric gene expression during pupal development (Fig. 3A). The concentric zones found in the larval medulla were essentially conserved during early pupal development until 8 hours after puparium formation (APF; Fig. 3A, part a). However, the concentric zones began to collapse at 12 hours APF (Fig. 3A, part b). The distribution of neurons continued to change until 24 hours APF, becoming completely disorganized (Fig. 3A, parts c-e). The same disorganized pattern was found in the adult brain (Fig. 3A, part f). Because the expression of concentric transcription factors appeared stable and strong, it is likely that cell bodies migrate extensively between 12 and 24 hours APF without changing gene expression. Hth- and Drf-positive cells were found in the innermost and outer zones of the larval medulla, respectively. However, both Hth- and Drf-positive cells were found in the inner and outer zones in adults (Fig. 3A, part f). This result suggests that the cell bodies of medulla neurons may be migrating in a radial pattern.
In order to address whether they are indeed migrating, clones of medulla neurons were constitutively labeled with GFP (Lee and Luo, 1999). The relative distributions of GFP-positive cells before and after migration were compared (Fig. 3C). Because the NBs situated in the outermost region of the larval medulla primordium produce neurons with a radial orientation, GFP-positive neurons were linearly arranged in the larval brain (Fig. 3C, part a). Similar distributions of GFP-positive cells were found at the beginning of migration at 12 hours APF (Fig. 3C, part b). However, after the completion of migration at 24 hours APF, GFP-positive cells were stochastically distributed (Fig. 3C, part c). Therefore, medulla neurons changed their relative locations between 12 and 24 hours APF. Because a radial migration would not disrupt the radial arrangement of cells, medulla neurons may also migrate with a tangential orientation, perpendicular to the linear arrangement of GFP-positive cells in the larval brain (Fig. 3C, part a).
To determine whether the expression of concentric genes persists during metamorphosis, Drf-positive neurons were constitutively labeled with lacZ (see Fig. S1 in the supplementary material). A Gal4 driver that accurately mimics drf expression (drf-Gal4) was used to express FLPase to constitutively label Drf-positive cells with lacZ (see Fig. S1 in the supplementary material) (McGuire et al., 2003; Struhl and Basler, 1993). If drf expression persists during metamorphosis, all of the lacZ positive neurons in the adult brain should express GFP, which is under the control of drf-Gal4. Indeed, our data suggest that drf expression persists during metamorphosis.
Changes in distributions of Hth-, Run- and Drf-positive neurons were seemingly random. However, Bsh/Hth double positive neurons displayed a clear pattern of migration (Fig. 3B). Because Bsh is expressed in a subset of Hth-positive neurons, Bsh/Hth-positive cells were initially situated in the inner area of the medulla primordium (Fig. 1E, Fig. 3B, part a). However, they always moved outward during metamorphosis and were eventually situated in the outermost area of the adult medulla (Fig. 3B, parts b-f). This finding suggests that the migration of medulla neurons is indeed an organized and patterned process, but that labeling several types of neurons may obscure the migration pattern. Because the projection pattern of Bsh/Hth-positive neurons could be continuously traced from the larval to adult medulla (Fig. 5), we assume that the expression of Bsh and Hth in these neurons does not change throughout development. The locations of Drf-positive neurons in the adult brain could also be classified according to neuronal subtypes (see Fig. S4 in the supplementary material), again suggesting that each type of medulla neuron exhibits a stereotyped migration pattern.
Medulla neurons migrate during pupal development. (A) Hth- (green), Run- (magenta) and Drf- (blue) positive neurons in 8, 12, 16, 20, 24 hours APF pupal (a-e) and adult (f) brains. Run-positive cells in the outer zone (arrowhead in b). Concentric zones are disrupted in 24 hours APF and adult brains (e,f). (B) Hth- (green) and Bsh- (magenta) positive neurons and glial cells (Repo; blue) in 8, 12, 16, 20, 24 hours APF pupal (a-e) and adult (f) brains. Hth/Bsh double-positive neurons migrate outwards (arrows). Putative MNGs are indicated (arrowheads). (C) Clones of medulla neurons visualized by elav-Gal4 UAS-CD8GFP (white; arrows) at late third instar (a), 12 hours APF (b) and 24 hours APF (c), showing tangential migration between 12 and 24 hours APF. Lateral views.
Hth is expressed in four types of medulla neurons, while co-expression of Hth and Bsh is found in a single subtype
To investigate projection patterns of Hth-positive neurons, a Gal4 driver that mimics hth expression was generated. The resulting hth-Gal4 allele widely induced UAS-GFP expression in the larval medulla probably due to residual expression of Gal4 produced in NBs. In the adult brain, hth-Gal4 largely recapitulated Hth expression (Fig. 4A). We also searched for Gal4 drivers that mimic Hth expression. NP6013 induced UAS-CD8GFP expression in a subset of Hth-positive neurons in the larval brain (Fig. 4B). The projections of Hth-positive medulla neurons were already present in the larval brain; their terminals were restricted to the medulla neuropile and were not found in the lobula complex (lobula and lobula plate; Fig. 4B).
Generating single cell clones expressing CD8GFP under the control of hth-Gal4 and NP6013 (Wong et al., 2002), we identified at least four types of Hth-positive neurons in the adult brain (Fig. 4C-F; see Fig. S2B in the supplementary material). As observed in the larval medulla, none of these neurons projected to the lobula complex (Fig. 4B). Neuronal types were classified according to the combinations of layers to which they project, as confirmed by N-Cadherin expression (NCad; Fig. 6E, part c) (Fischbach and Dittrich, 1989; Morante and Desplan, 2008). The neuronal type most frequently observed was Mi1, which shows small arborizations at M1, M5 and M9-10 (Fig. 4C; see Fig. S2B in the supplementary material). Neurons that show stratifications at M1 and M4 in the medulla and projections to the lamina were temporarily named lamina wide field-like1 (Lawfl1; Fig. 4D) (Fischbach and Dittrich, 1989) as arborizations in the lamina were barely detectable owing to weak GFP expression. The other Lawf-like neuron, Lawfl2, showed arborizations at M1 and M8-10 in the medulla and projections to the lamina (Fig. 4E). Lawfl2 has not been described in the literature. Pm3 are local neurons restricted to the proximal medulla (Fig. 4F).
Bsh is expressed in a subset of Hth-positive neurons from larval to adult stages (Fig. 1E, Fig. 3B). We found that Bsh was only expressed in Mi1 type neurons in the adult medulla (see Fig. S2B in the supplementary material). We thus generated a bsh-Gal4 strain by inserting the Gal4-coding sequence into the genomic fragment of the bsh gene that contains the entire bsh locus. The expression of bsh-Gal4 was similar to Bsh in adults (Fig. 4G). Indeed, mosaic analyses using bsh-Gal4 specifically indicated the presence of Mi1 neurons in the medulla. Presynaptic sites were revealed using synaptotagmin-HA (SytHA) (Robinson et al., 2002). The SytHA signal was very strong at M9-10 but weak or hardly detectable at M1/5, suggesting that Mi1 neurons have postsynaptic sites at M1/5 and a presynaptic site at M9-10 shaped like a fish hook (Fig. 4H).
Hth is expressed in four types of medulla neurons. (A) hth-Gal4 UAS-nlsGFP (green) overlaps Hth (red) in adult medulla. (B) NP6013 UAS-CD8GFP (white) overlaps Hth (green) in larval medulla. NCad (blue). Hth-positive neurons project to medulla (M; arrow) but not to lobula complex (Lo/Lop; arrowhead). (C-F) Clones of Hth-positive neurons in adult medulla. NCad (blue) and UAS>CD2>CD8GFP (green) under the control of hth-Gal4 (C,D,F) and NP6013 (E). (C) Mi1, (D) Lawfl1, (E) Lawfl2 and (F) Pm3. A cell body expressing a low level of Hth (arrow). Lamina projections (arrowheads). (G) bsh-Gal4 UAS-CD8GFP (green) overlaps Bsh (red) in adult medulla. NCad (blue). (H) Mi1: a clone expressing UAS-CD8GFP (green) and UAS-SytHA (red) under the control of bsh-Gal4. (I) Co-expression of ChAT (white) and Bsh (magenta) in adult medulla cortex. Lo, lobula; Lop, lobula plate; M, medulla; M1-M10, layers of the medulla.
In addition to the morphological features, neurotransmitters are important characteristics of neurons. We found that choline acetyltransferase (ChAT) was always upregulated in Bsh-positive neurons in adults (Fig. 4I). Therefore, Bsh expression alone can identify a specific type of medulla neuron, Mi1, which stereotypically migrates outward during metamorphosis and is cholinergic.
Hth regulates Bsh expression and neuronal identities
The roles of hth in medulla neuron formation were examined by generating hth-Gal4-homozygous mutant clones and visualizing their neuronal projection patterns (Fig. 5). The loss of hth function completely changed the morphology of hth-Gal4-positive medulla neurons, making cells apparently switch neuronal types (Fig. 5; Fig. S3A in the supplementary material). In a hth-Gal4-heterozygous background, control clones exhibited the normal morphology of Hth-positive neurons, primarily Mi1 (Fig. 5D). In hth-Gal4-homozygous clones, normal Mi1 neurons were not found; instead, we frequently observed neurons very different from Mi1, neurons that were Hth-negative on a wild-type background such as Tm13-like and other Tm-type lobula projection neurons (Fig. 5B,D; see Fig. S3A in the supplementary material). Mi1-like abnormal neurons, which were similar to Mi1 but exhibited irregular arborizations, were occasionally found (Fig. 5A,D). The fish hook-like shape at M9-10 was occasionally disrupted in the Mi1-like abnormal neurons. The phenotype could partially be rescued by introducing exogenous hth (Fig. 5D; see Fig. S3A in the supplementary material). Thus, the loss of hth function transforms Hth-positive neurons into diverse types of medulla neurons that are probably specified by genes other than hth.
Using bsh-Gal4, consistent phenotypes were observed in clones homozygous for a strong hypomorphic allele, hthP2. In control experiments, bsh-Gal4 exclusively labeled Mi1 neurons (Fig. 5D). However, Tm1-like rather than Mi1 neurons were found in hthP2-homozygous clones (Fig. 5C,D; see Fig. S3A in the supplementary material). The formation of Mi1 was partially rescued by introducing exogenous hth (Fig. 5D; see Fig. S3A in the supplementary material). In contrast to the distal localization of Mi1 cell bodies in the wild-type medulla cortex, the cell bodies of hth-mutant Tm-like neurons were found in the proximal to intermediate cortical zone (n=11/16; Fig. 5C). Therefore, hth is essential to determining the identity of Mi1 neurons, including the location of cell bodies in the adult brain. Cell bodies of non-Mi1 type Hth-positive neurons tended to be found in more proximal cortical zones in the wild-type medulla cortex (Fig. 3Bf). Thus, hth and bsh may cooperate to determine the location of Mi1 cell bodies.
Because Hth was weakly expressed in NE and NB cells in the larval brain (Fig. 1D′,E′), the hth-mutant phenotypes shown above may be caused by general defects in neurogenesis resulting from abnormalities in neuronal precursor cells. However, we did not detect such defects in production of GMCs and neurons, as determined by Prospero and Elav expression in hth-mutant clones in the larval medulla (see Fig. S3B in the supplementary material).
Hth may regulate expression of downstream target genes to control specification and formation of Hth-positive neurons. bsh is a potential candidate of Hth-target gene because Bsh expression was frequently reduced in hth mutant clones (Fig. 5L). However, residual Bsh expression was detected (Fig. 5L). This may be due to hypomorphic nature of hthP2, or hth function may not be essential for Bsh expression (e.g. hth and an unidentified gene may act partially redundantly). Interestingly, the transformation of Mi1 neurons to Tm1-like neurons, as revealed by bsh-Gal4, was observed in both Bsh-positive (n=4/65) and Bsh-negative (n=61/65) clones that were hth mutants. In most cases, Mi1-like abnormal neurons were positive for Bsh (n=7/8), suggesting that Bsh expression alone cannot determine the fate and morphology of Mi1 neurons.
hth regulates Bsh and NCad expression and neuronal identities. (A-C) Projections of hth mutant neurons labeled with UAS-CD8GFP (green) under the control of hth-Gal4 (A,B; hth-Gal4 clones) and bsh-Gal4 (C; hthP2 clone) in adults. NCad (blue). Bsh (blue in A,B). (A) Irregular arborizations of Mi1-like neuron (arrowheads). (B,C) Tm13-like and Tm1-like neurons presumably transformed from Hth- and Bsh-positive neurons, respectively. Putative glial cells are indicated (arrowhead in B). (D) Catalogue of neurons in control, hth mutant and hth rescue clones. (E-I) Mi1 neurons of control (E-H) and NCadM19 (I) clones at 0 (E,I), 20 (F), 24 hours APF (G) and in adult (H) brains. Clones expressing UAS-CD8GFP (green) under the control of NP6013 (E,I) or bsh-Gal4 (F-H). NCad, magenta; Bsh, red in E,I. Arrows in E,I indicate Bsh-positive cell bodies. (E,F) Two arborization sites at 'Distal' (D) and 'Proximal' (P) NCad domains (arrowheads). (G) The arborizations of M1 are formed at 24 hours APF. 'Distal' and 'Proximal' arborizations in E,F correspond to M5 and M9-10, respectively. (I) Proximal arborization is missing in NcadM19 clones. (J) NCad upregulation (white; arrow) in Hth domain (magenta) in larval medulla. Only NCad is shown in the right half. (K) Ectopic NCad upregulation (white) in clones expressing hth (GFP; green; arrows). (L) Downregulation (arrows) of NCad (white) and Bsh expression (magenta) in hth mutant clones (dotted lines). Lo1 and 4, lobula layers 1 and 4; M, medulla; M1-M10, layers of the medulla.
NCad may act downstream of Hth to regulate projection of Mi1 neurons
The development of Mi1 neurons was traced from the larval to adult brain using NP6013 and bsh-Gal4 (Fig. 5E-H). At the end of larval development (0 hours APF), Bsh-positive neurons form two arborization sites along the neuropile structures strongly expressing NCad (proximal and distal NCad domains; Fig. 5E). Found in the proximal NCad domain, the arborization at the tip of axonal shafts most likely corresponds to the terminal at M9-10 in adults (Fig. 5E,H). In the distal NCad domain, the third arborization site, which most likely becomes M1 arborization in adults, was occasionally found immediately distal to the pre-existing M5 arborization at 24 hours APF (Fig. 5G). At 20 hours APF, typical Mi1 neurons lacked the third arborization while cell bodies were located in the distal cortical area, suggesting that the M1 arborization is formed around 20-24 hours APF coincidently with the completion of cell body migration (Fig. 5F,G).
Although NCad was widely expressed in the cell bodies of larval medulla neurons, its expression was upregulated in the Hth-positive innermost concentric zone (Fig. 5J). Because NCad was ectopically upregulated in hth-expressing clones and was autonomously reduced to the basal level in hth mutant clones (Fig. 5K,L), NCad upregulation is most likely controlled by hth in the larval brain. NCad is a homophilic cell adhesion molecule; therefore, neurites of Mi1 neurons that strongly express NCad may innervate targets also expressing high levels of NCad (Iwai et al., 1997). NCad is known to regulate the target layer specificity of R-axons and lamina neurons (Lee et al., 2001; Nern et al., 2008; Ting et al., 2005). Thus, NCad upregulation in Mi1 may influence the arborization sites of Mi1 neurons located in the proximal and distal NCad domains in the larval medulla neuropile (Fig. 5E,J). Only the proximal arborizations were occasionally eliminated in clones homozygous for NCadM19 (n=6/10; Fig. 5I) and NCadomb405 (n=3/6; not shown). The defects found in the adult brain were diverse and may not specifically derive from larval defects. However, the loss of or disorganized M9-10 terminals can be explained by a lack of NCad upregulation in Mi1 neurons during larval development (Fig. 5I and not shown).
Drf is expressed in nine types of medulla neurons. (A) drf-Gal4 UAS-CD8GFP (white) overlaps Drf (red) in larval medulla. Medulla (M, arrows) and lobula complex (Lo/Lop, arrowheads). NCad is in blue. (B) drf-Gal4 UAS-nlsGFP (white) overlaps Drf (red) in adult medulla. Drf expression in non-neuronal lamina cells (arrowheads). (C-E) drf-Gal4 UAS-CD8GFP (white), NCad (blue) and R-axons (Chaoptin; red) in larva (C), 24 hours APF pupa (D) and adult (Ea). Drf-positive neurons project to larval medulla and lobula complex (C). Distribution of Drf-neurons changes by 24 hours APF (D). Elongated cell bodies are indicated by arrowheads in D. Definition of layers (Eb,c). Lateral (A) and horizontal (B-E) views. (F-M) Nine types of Drf-positive neurons. Clones expressing UAS-CD8GFP (green) and UAS-SytHA (red) under the control of drf-Gal4. NCad (blue). (F) TmY3, (G) Tm27, (H) Tm27Y, (I) Tm9, (J) Tm3a/b, (K) Mi10b, (L) Dm8a and (M) Dm8b. Three-dimensional reconstructed images (F-M, part a) and single confocal sections (F-M, part b; F-J, part c; J, part d). Lo1 and 4, lobula layers 1 and 4; M1-M10, layers of the medulla.
Drf is expressed in nine types of medulla neurons
To compare neuronal types of Drf- and Hth-positive population, we generated drf-Gal4 strains that accurately mimic drf expression. The drf-Gal4 induced GFP expression in a manner identical to Drf distribution throughout development (Fig. 6A,B). In the adult medulla, restricted subsets of medulla strata were densely labeled with GFP (Fig. 6E). In the lobula, axonal terminals were exclusively found in layers Lo1 and Lo4. Drf-positive neurons thus project to common target layers in the adult lobula.
To identify neuronal types showing Drf expression, we performed MARCM analyses using drf-Gal4 to catalog Drf-positive medulla neurons in adults and identified nine subtypes of Drf-positive neurons (Fig. 6F-M), the majority of which were lobula projection neurons that projected to the lobula complex (TmY3, Tm27(Y), Tm9 and Tm3; Fig. 6F-J). In the distal medulla, SytHA was weak or hardly detectable, but was prominent in the lobula complex. Thus, the arborizations in the distal medulla were primarily postsynaptic, while terminals in the lobula complex were presynaptic. We also identified three types of local neurons (Mi10b, Dm8a and Dm8b; Dm8a is similar to Dm8 in the literature) the innervations of which were restricted to the medulla (Fig. 6K-M). A stochastic distribution of SytHA was found along arborizations of these local neurons. Tm27 and Tm27Y neurons have not been previously described. Tm3b neurons are similar to Tm3 neurons (hereafter Tm3a) with the exception of a small branch at M10 projecting back towards M8 (Fig. 6J).
The Bsh/Hth-positive Mi1 neurons were shown to stereotypically migrate from the inner to outer areas of the medulla cortex (Fig. 3B). To determine whether a similar migration pattern occurs in Drf-positive neurons, locations of cell bodies in the medulla cortex were categorized into distal, intermediate and proximal zones (see Fig. S4 in the supplementary material). For each subtype of Drf-positive neurons, locations of cell bodies were examined. As a result, we found that four subtypes of Drf neurons have cell bodies in the distal zone; three subtypes in the intermediate zone and two subtypes in the proximal to intermediate zones. Thus, cell body location correlated with the neuronal subtype of Drf-positive neuron, suggesting that each subtype shows a stereotyped pattern of migration during pupal development.
Intriguingly, many of Drf-positive neurons already projected to the lobula primordium in the larval brain (Fig. 6A,C). After projecting to the lobula, cell bodies began to migrate between 12 and 24 hours APF (Fig. 3A). At 24 hours APF, Drf-positive cell bodies were found in the inner area of the medulla cortex (Fig. 6D). Cell bodies were occasionally elongated with a radial orientation at 24 hours APF (arrowheads in Fig. 6D), which may imply these cells are migrating in radial orientation.
During pupal development, the medulla and lobula complex rotated 90° around the vertical axis, and the medulla cortex was eventually placed between the lamina and the medulla neuropile (Fig. 6D,E) (Meinertzhagen and Hanson, 1993). The migration of medulla neurons may be a secondary effect of medulla rotation. However, the relative locations of the medulla cortex and the lamina at 24 hours APF were relatively unchanged when compared with the larval brain (Fig. 6C,D), suggesting that the medulla neurons independently migrate before the rotation that occurs after 24 hours APF.
Drf regulates axonal projections and dendritic arborizations
Because the drf-Gal4 we used is not a strong loss-of-function allele, it is not suitable for an analysis of drf mutants. We thus searched for Gal4 drivers that could label Drf-positive neurons independently of drf gene expression. NP2646 expressed Gal4 in some types of medulla neurons, including Tm27(Y) neurons (see Fig. S5 in the supplementary material). Tm27 neurons were rare, so we focused our analyses on Tm27Y. We created drf mutant MARCM clones and examined their morphology (Fig. 7). Although wild-type Tm27Y neurons showed extensive arborizations at M4-5 and M8-10 in the medulla and targeted Lo4 in the lobula, loss of drf function caused reduced arborization at M8-10, ectopic arborization at M2-3 and axonal targeting defects in the lobula (Fig. 7 and see Fig. S5C-F in the supplementary material). These defects are most likely caused by drf mutation because similar phenotypes were observed after using two independent drf alleles. Moreover, the defects were partially rescued by exogenous drf expression (see Fig. S5C-F in the supplementary material). The localization of Tm27Y cell bodies in the proximal to intermediate cortical zone was not noticeably altered in drf mutant clones (see Fig. S5G in the supplementary material). Essentially the same results were observed in Tm27 neurons (not shown). In other types of Drf-positive neurons, minor morphological defects were observed by knocking down drf under the control of drf-Gal4 (not shown).
Neuronal identities are pre-determined in the larval medulla
Concentric genes are expressed in a defined subset of medulla neurons throughout development, suggesting that a part of neuronal identities are pre-determined in the larval medulla primordium. Our data suggest that Drf-positive neurons produce nine types of medulla neurons, including lobula projection and medulla intrinsic neurons, while Hth-positive neurons produce at least four types of neurons, including lamina projection and medulla intrinsic neurons. In Hth-positive neurons, Bsh is exclusively expressed in medulla intrinsic Mi1 neurons. A hth mutation caused the neuron to switch type (Fig. 5), while a drf mutation affected subsets of morphological features of Drf-positive neurons (Fig. 7). Thus, roles of concentric genes may be functionally segregated to form a genetic hierarchy. Apparently, other concentric genes must exist in addition to the four genes reported in this study. Because there are many neurons outside of the Drf domain in the larval medulla, some concentric genes may be expressed in the outer zones. Some transcription factors may have expression patterns that differ from those of concentric genes, and their combined expression may specify restricted subtypes of medulla neurons. For example, apterous (ap) and Cut are widely expressed in medulla neurons (see Fig. S4D in the supplementary material) (Morante and Desplan, 2008). Cut was co-expressed in subsets of Drf-positive neurons, while ap was expressed in all Drf- and Bsh/Hth-positive neurons (see Fig. S4B in the supplementary material and data not shown).
drf regulates axonal targeting and dendritic arborizations. (A,B) Morphology of Tm27Y visualized by NP2646 UAS-CD8GFP (green) in control (A, parts a,b) and drfE82 clones (B, parts a,b) in adults. (a) Ectopic and reduced arborizations at M2-3 (arrows) and M8-10 (arrowheads) in medulla, respectively. (b) Axonal overshooting (arrows) and ectopic branch (arrowhead) in lobula. Lo4 is defined by reduced NCad expression (blue; dotted lines).
Early-born medulla neurons express the inner concentric genes, while late born neurons express the outer ones. Thus, concentric gene expression correlates with neuronal birth order (Fig. 2). However, it is still unknown how concentric gene expression is specified. It would be possible to speculate that genes controlling temporal specification of neurons are expressed in NBs to control the concentric gene expression. However, the genes that are known to control neuronal birth order in the embryonic CNS were not expressed in larval medulla NBs (not shown) (Isshiki et al., 2001). In addition to local temporal mechanisms, such as birth order, global and spatial mechanisms governed by morphogen gradient may also play a role in determining medulla cell type (Jessell, 2000). In addition to birth order or a morphogen gradient, mutual repression among concentric genes may be essential in establishing defined concentric zones. Except for rare occasions, de-repression of other concentric genes was not induced in clones mutant for hth or drf. Additionally, ectopic hth expression did not compromise Drf and Run expression (not shown). These results may suggest that unidentified genes act redundantly with these genes to repress expression of other concentric genes and that weak Hth expression in NBs does not play roles in temporal specification of medulla neurons.
Regulation of migration and its biological significance
Various types of cell migration play important roles during vertebrate neurogenesis (Hatten, 1999; Nobrega-Pereira and Marin, 2009). Although Drosophila has been a powerful model of neural development, extensive neuronal migrations coupled with layer formation found in this study have not been previously reported. Our findings may establish a model to understand molecular mechanisms that govern brain development via neuronal migrations.
It is important to know whether the migration of medulla neurons occurs actively or passively. The distribution of cell bodies in the adult medulla cortex was not random, but organized according to cell type (Fig. 3; see Fig. S4 in the supplementary material). In particular, the Mi1 neurons identified by Bsh expression migrated outwards and were eventually located in the outermost area of the adult medulla cortex (Fig. 3), which was affected in hth mutant clones (Fig. 5). The observation that defined localization of cell bodies is under the control of genetic program may not be explained by passive migration. Repression of apoptosis by expressing p35 under the control of elav-Gal4 did not compromise migration of Bsh- and Drf-positive neurons, suggesting that apoptosis is not a driving force of the migration (not shown). If neurons migrate actively in an organized manner, what regulates the pattern of migration? In many cases, glial cells play important roles in neuronal migration (Hatten, 1999). Indeed, we identified glial cells and their processes in the medulla cortex (Fig. 1G). Glial cells or other cell types could provide cues for neuronal migration.
The medulla neurons project axons near their targets forming subsets of dendrites in the larval brain (Figs 5 and 6); the cell bodies migrated in the presence of preformed neurites during pupal development (Fig. 3). During or following cell body migration, additional dendrites were formed along the axonal shafts (Fig. 5). Therefore, cell body migration may somehow contribute to circuit formation in the medulla. Indeed, similar strategies have been reported in sensory neurons of C. elegans and cerebellar granule cells in mammals (Heiman and Shaham, 2009; Solecki et al., 2006). Thus, cell body migration in the presence of neurites may be a general conserved mechanism of circuit formation. Cell body migration may also allow developing cells to receive inductive cues provided by cells in the vicinity of the medulla cortex. For example, glial cells placed on the surface of the brain may trigger the expression of specific genes (e.g. ChAT) in Mi1 cells that are located in the outermost area of the adult medulla cortex.
Contributions to functional neuronal circuits
In adults, Mi1 neurons have arborization sites at M1 and M5, which coincide with the L1 lamina neuron terminals. In Golgi studies, Mi1 neurons were found in all parts of the retinotopic field (Fischbach and Dittrich, 1989). Indeed, the number of Bsh expressing medulla neurons was about 800, a figure similar to the number of ommatidial units (not shown). Therefore, the Mi1 neurons identified by Bsh expression are most probably columnar neurons with direct inputs from L1 neurons. Because L1 is known to have inputs from R1-6, which processes motion detection, Mi1 may participate in the motion detection circuit (Fischbach and Dittrich, 1989; Joesch et al., 2010; Katsov and Clandinin, 2008; Rister et al., 2007; Zhu et al., 2009).
If the genetic codes that specify each type of neuron are found, it may encourage the functional study of defined neurons. In the medulla, bsh-Gal4 is solely expressed by Mi1 neurons. Although the expression of Bsh is also observed in L4/5 lamina neurons (Zhu et al., 2009), intersectional strategies such as split Gal4 may enable us to specifically manipulate the activity of Mi1 by inducing expression of neurogenetic tools like shibirets (Kitamoto, 2001; Luan et al., 2006). Our findings may thus provide insight into high-resolution functional neurobiology in the Drosophila visual system.
Similarities with vertebrate neurogenesis
Development of the mammalian central nervous system reiteratively establishes cell identity, directs cell migration and assembles neuronal layers (Hatten, 1999), processes similar to the patterns observed during medulla development. In the cerebral cortex, neurons are generated within the ventricular or subventricular zones and migrate outwards, leaving their birthplace along the radial glial fibers. Later-born neurons migrate radially into the cortical plate, past the deep layer neurons and become the upper layers. The layers of the cortex are thus created inside-out (Nobrega-Pereira and Marin, 2009). In the developing spinal cord, neuronal types are specified according to morphogen gradients (Jessell, 2000). Within each domain along the dorsoventral axis, neuronal and glial types are specified according to their birth order (Guillemot, 2007). The spinal cord neurons then migrate extensively along the radial, tangential and rostrocaudal axes (Leber and Sanes, 1995). Therefore, the initial organization of spinal cord neurons is disrupted in the mature system.
The medulla shares intriguing similarities with the mammalian central nervous system. For example, the concentric zones established in the larval medulla resemble the dorsoventral subdivisions of the spinal cord (Jessell, 2000). Extensive migrations of medulla neurons disrupt concentric zones, as observed in the spinal cord. However, we found that the locations of cell bodies were organized according to neuronal type, a distribution that may be similar to the cortical organization of the cerebral cortex (Nobrega-Pereira and Marin, 2009). Thus, the development of the medulla may share characteristics with various forms of neurogenesis found in the mammalian central nervous system. A comprehensive study of important features of neurogenesis will now be possible using the Drosophila visual center and powerful tools of Drosophila genetics. Unveiling all aspects of development in the medulla will not only shed light into the functional neurobiology of the visual system, but also elucidate the developmental neurobiology of vertebrates and invertebrates.
We thank Daiki Umetsu and Miyako Tai for the note that Bsh and Drf are expressed in concentric zones. We thank Karl Fischbach, Kei Ito, Tom Kornberg, Ian Meinertzhagen, Takaki Miyata, Koji Oishi, Michiya Sugimori, Shin-ya Takemura and Larry Zipursky for critical comments. We thank Haruhiro Higashida for use of facilities. We are grateful to Adi Salzberg, Masahide Asano, Ethan Bier, Sarah Certel, Takahiro Chihara, Shin-ichi Higashijima, Takako Isshiki, Kei Ito, Liqun Luo, Richard Mann, Chi-Hon Lee, William McGinnis, Andreas Prokop, James Skeath, Thomas Schwarz, Gary Struhl, Tadashi Uemura and Larry Zipursky for antibodies, strains and plasmids. We thank BestGene for their injection service; Bloomington Stock Center and DGRC, Kyoto for strains; DSHB and Asian Distribution Center for Segmentation Antibodies for antibodies; and DGRC, Indiana for vectors. This work was supported by Program for Improvement of Research Environment for Young Researchers and PRESTO from JST, Takeda Science Foundation, Brain Science Foundation, Mitsubishi Foundation, Sumitomo Foundation, Uehara Memorial Foundation, Nakajima Foundation (to M.S.), Grants-in-Aid from MEXT (to M.S. and T.T.) and JSPS Research fellowship (to Y.K.).
Competing interests statement
Supplementary material for this article is available at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058370/-/DC1
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(1997). Glia in the chiasms and medulla of the Drosophila melanogaster optic lobes. Cell Tissue Res. 289, 397-409.
Toriya M.,
Tokunaga A.,
Sawamoto K.,
Nakao K.,
Okano H.
(2006). Distinct functions of human numb isoforms revealed by misexpression in the neural stem cell lineage in the Drosophila larval brain. Dev. Neurosci. 28, 142-155.
Venken K. J.,
He Y.,
Hoskins R. A.,
Bellen H. J.
(2006). P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314, 1747-1751.
Wong A. M.,
Wang J. W.,
Axel R.
(2002). Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109, 229-241.
(2008). Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT. Development 135, 1471-1480.
Zipursky S. L.,
Frye M. A.
(2009). Peripheral visual circuits functionally segregate motion and phototaxis behaviors in the fly. Curr. Biol. 19, 613-619.
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Developmental geneticist Kathryn Anderson passed away at home on 30 November 2020. Tamara Caspary, a former postdoc and friend, remembers Kathryn and her remarkable contribution to developmental biology.
Zooming into 2021
In a new Editorial, Editor-in-Chief James Briscoe and Executive Editor Katherine Brown reflect on the triumphs and tribulations of the last 12 months, and look towards a hopefully calmer and more predictable year.
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Over 60 institutions in 12 countries are now participating in our Read & Publish initiative. Here, James Briscoe explains what this means for his institution, The Francis Crick Institute. Find out more and view our full list of participating institutions.
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Branch loggers URBAN are able to cut branches into big wood chips with length from 6 to 13cm. You can easily use such big wood chips to heat your house up even in regular solid-fuel stoves, which is more complicated with normal (smaller) wood chips. Using such wood chips for heating is simply clever alternative of cheap and ecological heating, because you can easily use wood from rest branches.
Branch loggers are produced in two versions: the first is designed for connection with the tractor; the second has got its own engine. The chipping device is the same for both versions and works on the same principle: two shafts with chipping knives rotating against each other. These knives are made from high-quality tool steel and you can sharp them repeatedly. After the sharpening, it´s necessary to put washers under the knives so there is only little free space between them. It's possible to grind down knives for 4 mm.
During chipping, branches are automatically fed into the chipping device by chipping knives. Chips fall into bags installed on the branch logger. Bags are filled without any assistance of staff. Please prefer bags with dimensions 57×104 cm. Fifty of them is provided together with the branch logger. You can easily manipulate with bags filled with chips, you can easily stock them and the wood is dried quickly thanks to good ventilation. The weight of 1 bag with wood chips is about 25-30kg and it has got about 0,08 m3 (13 bags = 1m3).


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text:
You are here: Home / Nostalgia / NOSTALGIA: Arsenal visit Boothferry Park for a 'floodlit friendl...
NOSTALGIA: Arsenal visit Boothferry Park for a 'floodlit friendly'
April 18, 2014 /in Nostalgia /by Matt
In January 1953, the first set of floodlights were installed at Boothferry Park, an accessory that was a wild new concept to English football, mistrusted by the Football League. So upon their inauguration, City set about proving they worked with a clutch of night-time friendly games.
Dundee were the slightly left-field visitors for the old place's first ever game by gaslight in January 1953. Over the next two years a number of clubs came to enjoy the new sensation of evening kickoffs, including Wolves, Portsmouth and Falkirk as well as sides from Sweden, Turkey and Austria. Then, on March 23rd 1954, Arsenal kept their appointment to bring a squad up to Hull for a late friendly game.
The novelty of floodlights had yet to wear off, and attendances for most of these friendlies had been excellent. But the arrival of the 1953/4 Arsenal side bumped up the interest back to the levels from when the lights were first connected, and 26,880 people paid to see the game. Arsenal had been in recovery from losing an enormous number of key players to the Second World War, while a number of veterans had to retire by the beginning of the 1950s. Tom Whittaker, a disciple of the great Herbert Chapman in the 20s and 30s, was rebuilding the club with youth, and by 1950 they had started to make inroads again, winning the FA Cup with victory over Liverpool. In 1952, they were one league game and a Cup final collapse away from the Double, but in 1953 they became league champions and Whittaker's reconstruction task was complete. And so it was the glamorous side of the era that was taking an out-of-schedule trip up to East Yorkshire to experience floodlit football for themselves.
"Every match that the Arsenal play is the nature of a cuptie; everyone wants to knock them off their pedestal," declared 'Marksman', the soubriquet used by the Hull Daily Mail on its football writing at the time. The floodlit friendlies, aside from the financial gains and brownie points for innovation, were also proving a decent sideshow for City, as they were having a somewhat featureless season in Division Two.
They had an injury list that clubs wouldn't envy, however; at the time of Arsenal's visit, goalkeeper Billy Bly, utility player Andy Davidson (yet to become a settled right back), half back Denis Durham and winger Brian Bulless were all out, with only Bulless aiming to be fit for the next league game at the weekend. Arsenal brought their regular starting XI and, with a whisky bottle full of shillings next to the meter, the game got underway.
Arsenal started the game slowly, as if their eyes were getting used to this newfangled mixture of light and shade before them, but City weren't in any rush to manipulate their unease, and the word 'friendly' felt decidedly too appropriate for the early tone of the game. Few chances were created, and Bly's stand-in Tommy Forgan got a lot of leaning use out of his posts.
Then, from nowhere, the league champions took the lead. Brian Walsh got past Trevor Porteous on the outside before crossing for the great Tommy Lawton, playing for the Gunners in the twilight of his career, to steer past Forgan from just inside the area. That was with 27 minutes gone, and 13 minutes later Lawton and Peter Goring combined on the wing to feed the ball through the City defence with ease befitting of champions, and the final ball gave a simple scoring opportunity to Don Roper.
Neil Franklin hit the bar for City with a scorching shot just before Leeds ref Arthur Luty blew for half time. Early in the second half, South African inside forward Alf Ackerman though he'd begun City's comeback with a well taken finish but hadn't heard Mr Luty's whistle for offside against Brian Cripsey.
Ackerman was then freed by Ken Horton and hit a shot which struck Arsenal keeper Con Sullivan on the shoulder and flew over. Shortly afterwards, however, Ackerman did reduce the deficit when a "goalfront skirmish" (according to 'Marksman') following a free kick saw Bob Crosbie challenge Sullivan in a typically 1950s "robust" manner and the ball spun out of the keeper's grip and into the air, allowing Ackerman to head in. There were 66 minutes on the watch and City had now scored against the champions.
Franklin then hit the crossbar again as City tried to take advantage of their momentum, but time and energy was at a premium, and Arsenal secured a hard-earned win when Roper and Dave Bowen combined delightfully to split the Tigers' defence and release Goring for a simple finish. Ten minutes remained, and no further goals were scored. Both teams were applauded very heartily from the field by a crowd that generated nearly £4,000 in gate receipts for the club.
THIS ARSENAL WAS PACKED WITH TOO MANY BIG GUNS was how the 'Marksman' match report was headed, though little fuss was made of the game in general, with no after-thoughts of the experience of playing the best side in the country at the time. Attention turned swiftly to the forthcoming home game against Fulham back in the real world of daytime Saturday afternoons. City won it 2-1 but then lost three in a row without scoring before a mixed end to the campaign culminated in a humdrum 15th place finish.
Arsenal were already off their perch in all but name, with their ageing side unable to carry through their status as league champions for another season, and they finished a lowly 12th. Indeed, their 1953 title was their last trophy for 17 years, with the 60s especially proving to be a frustrating and fallow period for the Gunners.
City lined up with Forgan in goal – Bly had been injured two weeks before at Plymouth and was out for the season – with Frank Harrison and Viggo Jensen the overworked full backs. Franklin, using Hull as his rehabilitation spot after his controversial stint in Colombia, was joined in the middle of the park by Porteous and Tom Berry, with the front five consisting of (l-r) Cripsey, Ackerman, Crosbie, Horton and youngster Charlie Atkinson. Mr Luty, the referee, was in charge at Wembley two months later for the FA Cup final.
In competitive games, there are a good number of memorable matches between City and Arsenal without there being a stack of mundane ones. The two have little historic relevance to one another, save for the FA Cup semi-final of 1930, which has been revisited a lot lately. City lost it after a replay. That was only the fourth ever meeting between the two, and there wasn't to be another until the 1977/8 League Cup, when Arsenal pummelled the Tigers 5-1 at Highbury. Another 11 seasons passed when the League Cup again paired the sides, this time in a two-legged affair which Arsenal won 5-1 on aggregate, though the home leg, memorable for Keith Edwards giving City the lead and ex-Tiger Brian Marwood scoring the Gunners' second goal on the night to end the match 2-1, became notable for being Arsenal's only competitive match at Boothferry Park.
City's promotion to the top tier in 2008 brought about the first league games between the two since the First World War, and it started with the season-defining 2-1 win for the Tigers at the Emirates. Arsenal have won every game since – their trips to the KC have yielded 3-1 and 2-1 wins, while they also beat City at the Emirates in a hugely controversial FA Cup quarter final in 2009. This game at the weekend will give City an opportunity to beat Arsenal in Hull for the first time ever, while also (yes, we are going to say it) act as a 'dress rehearsal' (even though it won't, really) for the FA Cup final next month. To put it into some kind of context just how little history exists between these two clubs; there are only five City players who have scored league goals against Arsenal. They are Henry McCorry, Sammy Stevens, Geovanni, Daniel Cousin and Jimmy Bullard.
As for those floodlights, City continued to host friendly games under their flame-fuelled gleam until they became kosher in 1956 when the ever-progressive Football League finally allowed their usage, and the first competitive game under their glare was a 1-1 draw with Doncaster Rovers on April 10th 1956. They were replaced by the famous sextet of electrical free-standers in 1964, funded by the windfall Harold Needler created via shares in his building company that also allowed him to buy three players – Ken Wagstaff, Ken Houghton and Ian Butler – that helped City plunder their way to the Third Division title in 1966.
While the new floodlights illuminated every night-time and winter game at Boothferry Park for almost the next 40 years, the old gas floodlights were bequeathed to Scarborough FC, who used them for more than a decade afterwards.
PODCAST: TWTWT Podcast 33 MATCH REPORT: City 0 Arsenal 3

redpajama_set_name: RedPajamaCommonCrawl


text:
package strtree

import "fmt"

const (
	dirLeft = iota
	dirRight
)

type node struct {
	key   string
	value interface{}

	chld [2]*node
	red  bool
}

func (n *node) dot() string {
	body := ""

	// Iterate all nodes using breadth-first search algorithm.
	i := 0
	queue := []*node{n}
	for len(queue) > 0 {
		n := queue[0]
		body += fmt.Sprintf("N%d %s\n", i, n.dotString())
		if n != nil && (n.chld[0] != nil || n.chld[1] != nil) {
			// Children for current node if any always go to the end of the queue
			// so we can know their indices using current queue length.
			body += fmt.Sprintf("N%d -> { N%d N%d }\n", i, i+len(queue), i+len(queue)+1)
			queue = append(append(queue, n.chld[0]), n.chld[1])
		}

		queue = queue[1:]
		i++
	}

	return body
}

func (n *node) dotString() string {
	if n == nil {
		return "[label=\"nil\" style=filled fontcolor=white fillcolor=black]"
	}

	k := fmt.Sprintf("%q", n.key)
	if n.value != nil {
		v := fmt.Sprintf("%q", fmt.Sprintf("%#v", n.value))
		k = fmt.Sprintf("\"k: \\\"%s\\\" v: \\\"%s\\\"\"", k[1:len(k)-1], v[1:len(v)-1])
	}

	color := "fontcolor=white fillcolor=black"
	if n.red {
		color = "fillcolor=red"
	}

	return fmt.Sprintf("[label=%s style=filled %s]", k, color)
}

func (n *node) insert(key string, value interface{}, compare Compare) *node {
	if n == nil {
		return &node{key: key, value: value}
	}

	// Using fake root to get rid of corner cases with rotation right under the root.
	root := &node{chld: [2]*node{nil, n}}
	dir := dirLeft

	// Nodes down the path to current node. All these nodes are copies of nodes from tree.
	var (
		// Grandparent's parent.
		gp *node

		// Grandparent.
		g *node

		// Parent.
		p *node

		// Childern.
		c [2]*node
	)

	// Start with fake root.
	n = root

	// As real root is right child of fake root - go to the right from start.
	r := -1

	// Continue until keys are equal.
	for r != 0 {
		parentDir := dir
		dir = dirLeft
		if r < 0 {
			// Go to the right if current node is less then given key.
			dir = dirRight
		}

		// Propagate set of nodes.
		gp = g
		g = p
		p = n
		n = n.chld[dir]

		if n == nil {
			// If no child in the direction we go insert new red node.
			n = &node{
				key: key,
				red: true}

			c = [2]*node{nil, nil}
		} else {
			// Make copy of current node or just use copy of child node if it has been made during color flip.
			if n != c[dir] {
				n = n.fullCopy()
			}

			// Color flip case to maintain invariant that the current node is black and has at least one black child.
			if n.chld[dirLeft] != nil && n.chld[dirRight] != nil && n.chld[dirLeft].red && n.chld[dirRight].red {
				n.red = true
				c = [2]*node{
					n.chld[dirLeft].colorCopy(false),
					n.chld[dirRight].colorCopy(false)}
				n.chld = c
			} else {
				c = [2]*node{nil, nil}
			}
		}
		p.chld[dir] = n

		// Fix red violation.
		if n.red && p != nil && p.red {
			// As root is black we can't be here earlier than fake root becomes parent of grandparent.
			grandParentDir := dirLeft
			if gp.chld[dirRight] == g {
				grandParentDir = dirRight
			}

			if n == p.chld[parentDir] {
				// With single rotation if current node goes in the same direction from
				// parent as parent from grandparent.
				gp.chld[grandParentDir] = g.single(parentDir)

				// The rotation changes parent and grandparent so during next iteration
				// grandparent's parent should remain the same. Here we fix grandparent
				// to keep correct gradparent's parent.
				g = gp
			} else {
				// With double rotation if current node goes in the oposite direction.
				gp.chld[grandParentDir] = g.double(parentDir)

				// The rotation puts grandparent and parent as children of current node.
				// The nodes are copied on previous steps so we put them to to children
				// array to prevent additional coping at the next step. Also in the next
				// step grandparent's parent and grandparent iteslf make step back. So we
				// fix parent to keep correct grandparent but there is no information on
				// parent of grandparent's parent to keep corrent grandparent's parent.
				// Luckily after the rotation current node (next parent) becomes black so
				// we can't make red violation on next iteration.
				c = n.chld
				p = gp
			}
		}

		r = compare(n.key, key)
	}

	n.value = value

	n = root.chld[dirRight]
	n.red = false
	return n
}

func (n *node) inplaceInsert(key string, value interface{}, compare Compare) *node {
	if n == nil {
		return &node{key: key, value: value}
	}

	root := &node{chld: [2]*node{nil, n}}
	dir := dirLeft

	var (
		gp *node
		g  *node
		p  *node
	)

	n = root
	r := -1

	for r != 0 {
		parentDir := dir
		dir = dirLeft
		if r < 0 {
			dir = dirRight
		}

		gp = g
		g = p
		p = n
		n = n.chld[dir]

		if n == nil {
			n = &node{
				key: key,
				red: true}

			p.chld[dir] = n
		} else {
			if n.chld[dirLeft] != nil && n.chld[dirRight] != nil && n.chld[dirLeft].red && n.chld[dirRight].red {
				n.red = true
				n.chld[dirLeft].red = false
				n.chld[dirRight].red = false
			}
		}

		if n.red && p != nil && p.red {
			grandParentDir := dirLeft
			if gp.chld[dirRight] == g {
				grandParentDir = dirRight
			}

			if n == p.chld[parentDir] {
				gp.chld[grandParentDir] = g.single(parentDir)
				g = gp
			} else {
				gp.chld[grandParentDir] = g.double(parentDir)
				p = gp
			}
		}

		r = compare(n.key, key)
	}

	n.value = value

	n = root.chld[dirRight]
	n.red = false
	return n
}

func (n *node) fullCopy() *node {
	return &node{
		key:   n.key,
		value: n.value,
		chld:  n.chld,
		red:   n.red}
}

func (n *node) colorCopy(color bool) *node {
	return &node{
		key:   n.key,
		value: n.value,
		chld:  n.chld,
		red:   color}
}

func (n *node) single(dir int) *node {
	nDir := 1 - dir
	s := n.chld[dir]
	n.chld[dir] = s.chld[nDir]
	s.chld[nDir] = n

	n.red = true
	s.red = false

	return s
}

func (n *node) double(dir int) *node {
	n.chld[dir] = n.chld[dir].single(1 - dir)
	return n.single(dir)
}

func (n *node) get(key string, compare Compare) (interface{}, bool) {
	for n != nil {
		r := compare(n.key, key)

		if r == 0 {
			return n.value, true
		}

		dir := dirLeft
		if r < 0 {
			dir = dirRight
		}

		n = n.chld[dir]
	}

	return nil, false
}

func (n *node) enumerate(ch chan Pair) {
	if n == nil {
		return
	}

	n.chld[dirLeft].enumerate(ch)

	ch <- Pair{Key: n.key, Value: n.value}

	n.chld[dirRight].enumerate(ch)
}

func (n *node) del(key string, compare Compare) (*node, bool) {
	// Fake root.
	root := &node{chld: [2]*node{nil, n}}

	// Nodes down the path to current node.
	var (
		// Grandparent.
		g *node

		// Parent.
		p *node

		// Target node.
		t *node
	)

	n = root

	// Direction from current node to next child we need to go.
	dir := dirRight
	for n.chld[dir] != nil {
		// Direction from parent to current node.
		pDir := dir

		g = p
		p = n
		n.chld[dir] = n.chld[dir].fullCopy()
		n = n.chld[dir]

		dir = dirLeft
		r := compare(n.key, key)
		if r < 0 {
			dir = dirRight
		}

		if r == 0 {
			t = n
		}

		if !n.red && (n.chld[dir] == nil || !n.chld[dir].red) {
			nDir := 1 - dir
			if n.chld[nDir] != nil && n.chld[nDir].red {
				n.chld[nDir] = n.chld[nDir].fullCopy()
				p.chld[pDir] = n.single(nDir)
				p = p.chld[pDir]
			} else {
				nPDir := 1 - pDir
				s := p.chld[nPDir]
				if s != nil {
					s = s.fullCopy()
					p.chld[nPDir] = s
					if (s.chld[dirLeft] == nil || !s.chld[dirLeft].red) &&
						(s.chld[dirRight] == nil || !s.chld[dirRight].red) {
						p.red = false
						n.red = true
						s.red = true
					} else {
						// Direction from grandparent to parent.
						gpDir := dirLeft
						if g.chld[dirRight] == p {
							gpDir = dirRight
						}

						if s.chld[pDir] != nil && s.chld[pDir].red {
							s.chld[pDir] = s.chld[pDir].fullCopy()
							g.chld[gpDir] = p.double(nPDir)
						} else {
							s.chld[nPDir] = s.chld[nPDir].fullCopy()
							g.chld[gpDir] = p.single(nPDir)
						}

						n.red = true
						g.chld[gpDir].red = true
						g.chld[gpDir].chld[dirLeft].red = false
						g.chld[gpDir].chld[dirRight].red = false
					}
				}
			}
		}
	}

	if t != nil {
		t.key = n.key
		t.value = n.value

		dir = dirLeft
		if p.chld[dirRight] == n {
			dir = dirRight
		}

		chldDir := dirLeft
		if n.chld[dirLeft] == nil {
			chldDir = dirRight
		}

		p.chld[dir] = n.chld[chldDir]
	}

	n = root.chld[dirRight]
	if n != nil {
		n.red = false
	}
	return n, t != nil
}

redpajama_set_name: RedPajamaGithub


At my 28 week prenatal appointment, Jeramey and I learned our son, Benjamin Lewis, had not grown since 24 weeks. What was going through our minds? Nothing, totally numb. What was going through our hearts? Fear of the unknown and our hearts were in the pits of our stomach. At that point, all hands and trust went to God.
On March 25, 2015, Ben was born at 1 pound 4.8 ounces. He was quickly intubated and rushed to NICU. We saw him for a split second, a tiny little red hand. We spent the next 95 days in NICU, and every day was an eternity. Today, Ben is a healthy 2-year-old who loves to sing songs, play outside, read books and play with "Dada" and "Ma".
We're proud to be representing the March of Dimes as the Ambassador Family and sharing Ben's story. We have set a goal to raise $3250 in honor of Ben's birthday on 3/25. Funds raised through the Signature Chefs Auction are supporting essential research to benefit all babies. Together, we are working towards a day when every baby is born healthy.
Contact Jennifer Torres at (915) 330-9054 or jtorres@marchofdimes.org.


text:
Is a Will Public Information After Death?
By Teo Spengler
Many people keep their last will and testament secret while they are alive, hidden away in a safety deposit box or a personal safe. But unless the will is superseded by another, it loses its private status when the testator dies. Whether the will belongs to a celebrity or your grandmother, it is usually open to public viewing after death.
Wills Initially Private
Courts use the terms "will" and "testament" interchangeably. Both terms refer to a document written and signed by a testator specifying who inherits his property on his death. States authorize different kinds of wills -- holographic, form and international wills, for example -- and prescribe rules for each. No matter what kind of will a testator drafts, it is not a public document during his lifetime. The statutes do not require the testator to show the will contents to anyone, even will witnesses.
Court-Filed Wills Public
A testator executes a will according to state requirements. All states require a testator to sign a form will, for example, before witnesses. A properly executed will remains completely revocable by a living testator. If a will is revoked by an act -- crossing out the will -- or by a superseding testament, it remains private. When the testator dies, his latest will becomes effective and -- once filed in probate court -- becomes a public document.
Probated Wills Public
Probate is the court-supervised process of administering a will. The will usually names an executor; if not, the court appoints one. The executor steers the estate from the testator's death through property distribution -- collecting and appraising assets, locating heirs, paying creditors and liquidating property. The court requires reports from the executor to assure an honest and accurate accounting. Among the first documents the executor files is the will itself. Once the will is in the probate file, it is a public document that's available for viewing.
Viewing Public Wills
Anyone can view a will in probate by appearing at the courthouse with either the probate file number or the name and date of death of the deceased. The court clerk locates the file and permits viewing either at the clerk's window or in a separate file-viewing area. The last will and testament is generally among the first documents in the file. Members of the public can obtain copies of the will by requesting a copy and paying a per-page fee.
Is a Will a Public Record?
American Bar Association: The Probate Process
Inc.com: Executors FAQ
State of Connecticut Judicial Branch: Court Records
Are Wills Open to the Public?
Wills are open to the public after they go into effect, not before. A will is the written description of how the person making the will -- the testator -- wants her property distributed after her death. Since states regulate wills, procedural requirements vary, but every jurisdiction treats wills as private during the life of the testator. Once the testator dies, however, her will moves into the public realm.
How Can I Get a Copy of a Will?
TV shows often portray a lawyer reading the last will and testament to the assembled family. In real life, you usually have to track down a copy of the will yourself. It is easier to get a copy of a will once the maker is dead. You can only see the will of a living person if she agrees to show it you. Once the maker is deceased, however, the will is generally filed with the court for probate. Like most court documents, the vast majority of probated wills are available for public viewing and copying.
How to Read a Last Will & Testament After a Death
TV shows sometimes include scenes in which an attorney for a recently deceased millionaire reads the will to the assembled relatives, causing shock and astonishment. However, no American jurisdiction requires a public will reading. Most people read a will at the court clerk's office. After a testator dies, the probate court reviews his will for validity and supervises its administration until the executor distributes estate property to the heirs. This is termed probate. Anyone can read or copy the will at the courthouse during and after probate.
Can Anyone See Someone's Last Will & Testament?
While a testator remains alive, her will is a private document. She shows it to whom she wishes, and others have no ...
Are Wills Public Information?
State statutes protect a will from the prying eyes of the public until a testator dies. During her lifetime, the ...
How Can I Get a Copy of a Last Will & Testament?
Don't expect to see your grandfather's will while he is alive, unless he decides to show it to you. While the testator ...
Does Probate Make a Will Public?
Many people cloak their wills in mystery, writing them in secret, hiding them in a wall safe or bank vault and refusing ...


text:
import React, { Component, PropTypes } from 'react'
import { I18n } from 'react-redux-i18n'

import { isPublicTeam, getFileUrl } from 'utils'
import { Icon, Dropdown, Modal, MenuSelector } from 'uis'
import { Avatar, Logo } from 'views'
import { TeamCreate } from '../team-create'
import { AccountSettings } from '../account-settings'
import { getWorkspaceBashPath } from '../../index'

import './workspace-header.view.styl'

export class WorkspaceHeader extends Component {

  static propTypes = {
    userMe: PropTypes.object,
    teams: PropTypes.array,
    currentTeam: PropTypes.object,
    actions: PropTypes.object
  }

  saveTeamCreateModalRef = (ref) => {
    this.teamCreateModalRef = ref
  }

  saveAccountSettingsModalRef = (ref) => {
    this.accountSettingsModalRef = ref
  }

  getTeamName (team) {
    if (isPublicTeam(team)) {
      return team.name
    } else {
      return I18n.t('workspace.personal')
    }
  }

  handleNewTeamClick = () => {
    this.teamCreateModalRef.open()
  }

  handleNewTeamSubmitSuccess = () => {
    this.teamCreateModalRef.close()
  }

  handleAccountSettingsClick = () => {
    this.accountSettingsModalRef.open()
  }

  renderTeamCreateModal () {
    return (
      <Modal
        ref={this.saveTeamCreateModalRef}
        title={I18n.t('team.new')}
        size={'small'}
      >
        <TeamCreate
          onSubmitSuccess={this.handleNewTeamSubmitSuccess}
        />
      </Modal>
    )
  }

  renderAccountSettingsModal () {
    return (
      <Modal
        ref={this.saveAccountSettingsModalRef}
        title={I18n.t('account.settings')}
      >
        <AccountSettings />
      </Modal>
    )
  }

  renderWorkspaceInfo () {
    const { currentTeam } = this.props

    return (

        <Dropdown
          className={'workspaceSwitcherDropdown'}
          content={this.getWorkspaceSwitcher()}
        >

              {this.getTeamName(currentTeam)}

            <Icon className={'handlerIcon'} name={'chevron-down'} />

        </Dropdown>

    )
  }

  handleSwitchWorkspace = (teamSelector) => {
    const { teams } = this.props
    const { push } = this.props.actions
    const nextTeam = teams.filter(team => teamSelector.value === team.id)[0]
    push(getWorkspaceBashPath(nextTeam))
  }

  getWorkspaceSwitcher () {
    const { currentTeam, teams } = this.props

    const dataList = teams.map((team) => ({
      className: 'workspaceSwitcherItem',
      value: team.id,
      title: this.getTeamName(team),
      iconName: isPublicTeam(team) ? 'building' : 'user',
      error: team.isFrozen ? I18n.t('team.frozenLabel') : null,
      onClick: this.handleSwitchWorkspace
    }))

    const extraList = [
      {
        className: 'workspaceSwitcherItem',
        iconName: 'plus',
        title: I18n.t('team.new'),
        onClick: this.handleNewTeamClick
      }
    ]

    return (
      <MenuSelector
        dataList={dataList}
        extraList={extraList}
        hasSelected={[currentTeam.id]}
      />
    )
  }

  handleWorkspaceLogoClick = () => {
    const { currentTeam } = this.props
    const { push } = this.props.actions
    push(getWorkspaceBashPath(currentTeam))
  }

  renderWrokspaceLogo () {
    return (

        <Logo className={'defaultLogo'} height={23} />

    )
  }

  renderUserInfo () {
    const { id, avatar } = this.props.userMe

    return (

        <Dropdown
          content={this.getUserInfoDropdownMenu()}
          placement={'bottomRight'}
          offset={[-8, 8]}
        >

            <Avatar className={'infoAvatar'} url={getFileUrl(avatar)} size={'small'} />
            {id}

        </Dropdown>

    )
  }

  handleSignOutClick = () => {
    this.props.actions.signOutUser()
  }

  getUserInfoDropdownMenu () {
    const dataList = [
      { title: I18n.t('account.settings'), onClick: this.handleAccountSettingsClick },
      { type: 'divider' },
      { title: I18n.t('account.signOut'), onClick: this.handleSignOutClick }
    ]

    return (
      <MenuSelector
        dataList={dataList}
      />
    )
  }

  render () {
    return (

        {this.renderWorkspaceInfo()}
        {this.renderWrokspaceLogo()}
        {this.renderUserInfo()}

        {this.renderTeamCreateModal()}
        {this.renderAccountSettingsModal()}

    )
  }

}


text:
Lorem ipsum dolor sit amet, consectetur adipisicing elit. Blanditiis cumque distinctio esse labore maxime sequi voluptatibus. Aut delectus eum ex excepturi in, nemo quae quisquam reiciendis soluta voluptatibus! Cumque, doloremque.

                <?php if($this->session->flashdata('error')):?>

<?php echo $this->session->flashdata('error');?>

                <?php endif;?>

                <?php echo form_open('login',array('class'=>'form-horizontal'));?>

                    Email :

                        <input type="text" name="email" placeholder="Email" value="<?php echo set_value('email');?>">

                    Mot de passse:

                        <input type="password" id="mdp" name="mdp"/>

                <button type="submit" class="btn">Connexion</button>

                <?php echo form_close();?>

        <?php if($this->session->flashdata('success')):?>

<?php echo $this->session->flashdata('success');?>

        <?php endif;?>

        <?php if(validation_errors()):?>

<?php echo validation_errors('
','
');?>

        <?php endif;?>

        <?php echo form_open('login',array('class'=>'form-horizontal'));?>

                Civilité :

                    <input type="checkbox" id="m" value="m" name="civilite" > M.

                    <input type="checkbox" id="mme" value="mme" name="civilite" > Mme

                    <input type="checkbox" id="mlle" value="mlle" name="civilite">Mlle

                Nom :

                    <input type="text" name="lastname" placeholder="Nom" value="<?php echo set_value('lastname');?>">

                Prénom :

                    <input type="text" name="firstname" placeholder="Prénom" value="<?php echo set_value('firstname');?>">

            Mot de passse:

            <input type="password" id="mdp" name="mdp"/>

                Email :

                    <input type="text" name="email" placeholder="Email" value="<?php echo set_value('email');?>">

                Téléphone :

                    <input type="text" name="phone" placeholder="Téléphone" value="<?php echo set_value('phone');?>">

            <button type="submit" class="btn">Poursuivre ma commande</button>

        <?php echo form_close();?>


Bus Users UK, a non-profit organisation that champions the interests of bus and coach passengers, has submitted its response to Somerset County Council's review of local bus services.
Passengers across the county were given a chance to have their say on proposed cuts to services and concessionary fares in a series of Your Bus Matters events run by Bus Users. Their comments and concerns have been included in the consultation response, along with details of the potential impact the proposed funding cuts could have on the people and communities of Somerset.
Bus Use in the South West of England is actually growing with 17 to 20 year olds and those above 60 the most likely to use the bus, and the most likely to be directly affected by the cuts. Passengers at the Your Bus Matters events were particularly concerned that community services and the Slinky Bus will not be able to fill the gap, especially in areas where cuts have already been made. Concerns were also raised over proposals to remove the discretionary element of the Concessionary Bus Pass.


Fantasy Theatre Factory seeks a Bookkeeper to report to Executive Artistic Director while working closely with the Theatre Manager, and Scheduling Director. Must be fast, proactive and highly organized, with extensive working knowledge of Quickbooks and Microsoft Office Suite; and have an impeccable understanding of non-profit accounting procedures. Preference given to applicants who have worked extensively with non-profit theaters. Working with Theater Manager AMS software is a plus.
Please send cover letter and resume to jobs@ftfshows.com with "Bookkeeper" in the subject line or via website at www.ftfshows.com/jobs. Fantasy Theatre Factory values workplace diversity and encourages applicants from diverse backgrounds.


Skako was an ecumenopolis on the Hydian Way in the galactic core and homeworld to the Skakoan species. Though most of Skako was covered by urban sprawl, some examples of the planet's original ecosystem had been preserved in the form of vast fields of sprawling vines.
Discovered during the blazing of the Brentaal–Denon Route of the Hydian Way before the formation of the Galactic Republic, the Skakoans rarely left the world due to their dependence on the methane-based atmosphere and heavy atmospheric pressures. Establishing a major ecumenopolis comparable in terms of population and scale to Coruscant, the planet-wide city lacked the Republic capital's charm and aesthetics. While Skakoans tended to stay out of galactic affairs, war was on their doorstep during the Second Alsakan Conflict which lasted from 16,200 BBY until 15,400 BBY.
In addition to the Skakoan people, the planet was also home to the octuptarras— an eight-eyed creature with a gasbag-shaped head and multiple legs which inhabited the planet's vines. The harsh environment on the planet ensured that the world was rarely visited and left in isolation, much to the contentment of the natives. Developing a religion based on the Book of the Boolmide, the Skakoan people constructed a holy place called the Power Mounds from where the Elders presided over the world's religious affairs.
Attaining membership in the Galactic Republic Senate, the Skakoans also had a leading interest within the Techno Union by the time of the Separatist Crisis which plagued the Republic in its final days. The Skakoan representative Wat Tambor led the Techno Union away from the Republic and to the Confederacy of Independent Systems, for which Skako was expelled from the Republic. When the Clone Wars erupted, Skako was a member world of the CIS, but was placed under the watchful eye of the 1st Sector Army of the Grand Army of the Republic. After Skako fell to the Republic and Foreman Tambor was murdered on Mustafar with the rest of the Separatist Council, the Confederacy collapsed and the Galactic Empire replaced the Republic. Heralding a new era of Human High Culture, the Empire crushed the Techno Union and persecuted the Skakoans for their involvement in the war. Retreating to Skako to resume their isolationist stance, the Skakoans avoided the destruction of the Yuuzhan Vong War by remaining distant from both the New Republic and the Galactic Alliance. By 130 ABY, Skako had been enveloped by the Empire of Darth Krayt.