The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

Summary The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade

Development of Environmental Management at JSC Valmieras stikla šķiedra

Maģistra darbā " AS „Valmieras stikla šķiedra” vides pārvaldības attīstība" ir veikts pētījums, izmantojot literatūras analīzi un socioloģiskās pētījuma metodes – intervēšanu, anketēšanu. Ir izstrādāts AS „Valmieras stikla šķiedra” vides pārvaldības attīstības plānojums, balstoties uz literatūrā gūtajām atziņām, AS „Valmieras stikla šķiedra” un citu Latvijas un Eiropas uzņēmumu vides pārvaldības praksi un intervēto personu viedokļiem. Tas ietver galvenos ilgtspējīgu attīstību veicinošus rīcības virzienus, konkrētas rīcības, to realizēšanā iesaistāmās mērķgrupas un izmantojamos vides politikas instrumentus visiem korporatīvās vides pārvaldības cikla posmiem. Darbs sastāv no satura rādītāja, ievada, 8 nodaļām, nobeiguma, literatūras saraksta un pielikumiem, iekļauti 38 attēli, 10 tabulas un 10 pielikumi. Atslēgas vārdi: Vides pārvaldība, ilgtspējīga attīstība, ilgtspējīga ražošana, vides pārvaldības instrumenti.The master’s thesis Development of Environmental Management at JSC Valmieras stikla šķiedra represents a study based on literature analysis and sociological research methods (interviews, questionnaires). On the basis of literature konwledge, environmental management practices of JSC Valmieras stikla šķiedra and other Latvian and European businesses and opinions of interviewed persons an environmental management development plan for JSC Valmieras stikla šķiedra has been developed. It covers the main areas of action promoting sustainable development, specific steps, the target groups to be involved in implementing such steps and the environmental policy tools to be deployed, for all stages of the corporative environmental management cycle. The master’s thesis consists of a table of contents, an introduction, eight chapters, 23 sub-chapters, a conclusion, a bibliography and annexes; 38 figures, ten tables and ten annexes have been included. Keywords: environmental management, sustainable development, sustainable manufacturing, environmental management

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: BFU2015-64220-P and BFU2018-094476. We would also like to acknowledge the support from the Drosophila transgenesis and confocal microscopy CBMSO scientific services. The CBMSO enjoys institutional support from the Ramón Areces and Santander Foundatio

Best candidates Salm/Salr Target Genes (STG OK).

<p>“Array” indicates the experiment which the genes were selected, experiment 1 (24/48) or experiment 2 (Df//UAS). “Ph” indicates the phenotype of <i>nub-Gal4/UAS-RNA-i</i> and <i>sal</i>^<i>EPv</i><i>-Gal4/UAS-RNA-i</i> separated by double dash: + (WT); S (wing size); P (vein pattern); S-P (wing size and vein pattern); V+ and V- (extra-veins and loss of veins); Bs (dorso-ventral wing surface adhesion); EPL (early pupal lethal). “IS” indicates the mRNA expression patterns in wild type and <i>salm-i/salr-i</i> discs (N: expression not detected; U: generalised expression and P: patterned expression). We indicated by “+” or “-”that the expression appears ectopic or is reduced in <i>salmi</i>/<i>salri</i> discs, respectively. “Molecular” indicates the molecular nature and simplified molecular class (MC), respectively: D (genes related with the biology of DNA), P (genes related with proteins metabolism), CG (genes without known functional domains or orthology), CS (genes encoding components of signalling pathways), CGh (genes with a known functional domain but not clear orthology relationships), M (genes encoding proteins related to the metabolism of lipids or glucids), CA (genes related with cell adhesion or the cytoskeleton), Cut (genes encoding proteins related with structural constituent of cuticle), Tra (genes related with the transport of metabolites across cellular membranes), RedOx (genes encoding proteins related with oxidation-reduction process), CD (genes related with cell death) and CR (non protein coding gene).</p><p>Best candidates Spalt-repressed genes and Best candidates Spalt-activated genes with a Spalt-related phenotype.</p

Gene regulatory network triggered by Dpp.

<p>(A-D) Schematic representation of the wing pouch (ovals and circles) showing the expected (green fill in A-B) and observed (green fill in C-D) expression in wild type discs (ovals) and in <i>salmi/salri</i> mutant discs (circles). The behaviour of genes repressed by Sal is shown in A (expected) and C (observed), and for genes activated by Sal in B (expected) and D (observed). (E) The expression of Salm/Salr (SAL) is regulated by the Dpp pathway components Brinker (BRK) and Mad (MAD). Dpp, using Mad or Mad and Brk, also activate the expression of additional genes (X and Y) which in turn can be subject to further regulation by Salm/Salr (X) or independent of them (Y). Finally, the Salm/Salr transcription factors regulate by activation (Z) or repression (Z’) the expression of their direct target genes, contributing to the set of active genes in the centre of the wing blade that confer this territory its particular characteristics.</p

Examples of mRNA expression of selected genes (STG+) with increased expression in <i>salm-i/salr-i</i> compared to wild type wing discs.

<p>(A) Frequency of expression patterns encountered in wild type discs and observed changes in <i>UAS-dicer2/+; sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+</i> wing discs. Expression not detected in the wing disc (N), generalised expression (U) and patterned expression (P). We indicated by “=“, “+” and “-”that the corresponding expression does not change, appears ectopic or is reduced in <i>salm-i/salr-i</i> wing discs, respectively. (B-I) Representative examples of <i>in situ</i> hybridizations in late third instar wing discs with probes against the genes <i>CG43676</i> (N =; B-B’), <i>CG43144</i> (N =; C-C’), <i>CG9214</i> (U =; D-D’), <i>CG15784</i> (U+; E-E’), <i>CG13937</i> (U-; F-F’), <i>CG5966</i> (P =; G-G’), <i>CG15009</i> (P+; H-H’) and <i>CG1058</i> (P-; I-I’). In each pair of panels, B-I corresponds to wild type discs and B’-I’ to UAS-<i>dicer2/+; sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP/UAS-salm-i; UAS-salr-i/+</i> wing discs. The expression patterns class and logFC values at 24h and 48 h from experiment 1 or the microarray condition from experiment 2 are indicated at the top and to the bottom corner, respectively, of each panel in B’ to L’ images.</p

Global results of the genome-wide transcriptional changes observed in wing discs with reduced expression of <i>salm</i> and <i>salr</i>.

<p>(A-A’) Expression of Salm (red in A and A’) and <i>sal</i>^<i>EPv</i><i>-Gal4</i> (green in A) in <i>sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP; tub-Gal80</i>^<i>ts</i><i>/UAS-GFP</i> wing discs raised at 29°C. (B-B’) Expression of Salm (red in B) and <i>sal</i>^<i>EPv</i><i>-Gal4</i> (green in B’) in <i>sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP; tub-Gal80</i>^<i>ts</i><i>/UAS-GFP</i> wing discs raised at 25°C. (C-C’’) Imaginal disc of <i>sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP /UAS-salm-i; tub-Gal80</i>^<i>ts</i><i>/UAS-salr-i</i> genotype (<i>salm-i/salr-i</i> 24h) raised at 29°C 24–28 hours before dissection, showing the expression of GFP (green in C and C’) and Salm (red in C and C’’). (D-D’’) Imaginal disc of <i>sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP /UAS-salm-i; tub-Gal80</i>^<i>ts</i><i>/UAS-salr-i</i> genotype (<i>salm-i/salr-i</i> 48h) raised at 29°C 44–48 hours before dissection showing the expression of GFP (green in D and D’) and Salm (red in D and D’’). (E) Number of genes which expression level changes with an adjusted p-value lower than 0.05 that were identified in the comparisons with controls of <i>salm-i/salr-i</i> at 48h (T48); <i>salm-i/salr-i</i> at 24h (T24) and at both time intervals (T24/T48). The blue and red sections of each column correspond to genes showing reduced (sal-; blue) and increassed (sal+; red) expression in <i>salmi/salri</i> compared to their corresponding controls. (F) Number of genes identified at T24 and T48 grouped by logFoldChange values. Blue represents genes which expression is reduced and red those genes which expression is increased. 1: logFC lower than 0.5; 2: logFC between 0.5 and 1 and 3: logFC major than 1. (G) Graphical representation of the logFoldChange values for those genes that were identified both in <i>salm-i/salr-i</i> 48h and <i>salm-i/salr-i</i> 24h. (H) Total number of identified genes from experiment 1 (T) with increased (sal+; red) and reduced (sal-; blue) expression in <i>salm-i/salr-i</i> discs compared to controls. Number of selected genes coming from experiments 1 and 2 (STG) with increased (STG+) and reduced (STG-) expression. Genes selected from experiment 2 are indicated by stripped columns. Number of best candidate sal repressed (STG+OK) and activated (STG-OK) genes. (I-J) Number of genes grouped in GO categories enriched for STG- and STG+ genes (I) and for STG-OK and STG+OK genes (J).</p

Phenotypic analysis of genes with increased expression in <i>sal</i> knockdown discs.

<p>(A) Fraction of genes showing a wing mutant phenotype (Y; green) and no phenotype (N; red) from a total of 126 <i>UAS-dicer2/+; nub-Gal4/ UAS-RNA-i</i> or <i>UAS-dicer2/+; sal</i>^<i>EPv</i><i>-Gal4/ UAS-RNA-i</i> combinations. Genes not analysed from the STG+ class are shown in blue (—) (B) Frequency of defects in both wing size and vein pattern (S-P; blue), changes in wing size (S; red), defects in vein formation with minor or no effect in wing size (V; green) and other wing morphology defects (O; purple) from a total of 35 <i>UAS-RNA-i/Gal4</i> combinations. (C) Wild type wing. (E-F) Representative examples of adult wings knockdown for genes over-expressed in <i>salm-i/salr-i</i> mutant discs but activated in response to JNK. <i>UAS-dicer2/+; nub-Gal4/UAS-CG1512-i</i> (E) and UAS-<i>dicer2/+; nub-Gal4/UAS-CG12505-i</i> (F). (G-P) Representative examples of adult wings of <i>UAS-RNA-i/Gal4</i> combinations for genes over-expressed in <i>salm-i/salr-i</i> knockdown discs independently of JNK activity: <i>sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP/UAS-CG17533-i</i> (G), <i>UAS-dicer2/+; nub-Gal4/UAS-CG10965-i</i> (I), <i>UAS-dicer2/+; nub-Gal4/UAS-CG32021-i</i> (J), <i>UAS-dicer2/+; sal</i>^<i>EPv</i><i>-Gal4/UAS-CG1925-i</i> (K), <i>/+;UAS-dicer2/+; sal</i>^<i>EPv</i><i>-Gal4/ UAS-CG5247-i</i> (L), <i>UAS-dicer2/UAS-CG15784-i; sal</i>^<i>EPv</i><i>-Gal4/+</i> (M), <i>UAS-CG15784-i; sal</i>^<i>EPv</i><i>-Gal4 UAS-GFP/+</i> (N), <i>UAS-dicer2/+; sal</i>^<i>EPv</i><i>-Gal4/UAS-CG18455-i</i> (O) and <i>UAS-dicer2/+; nub-Gal4/UAS-CG3008-i</i> (P). Moderate and strong <i>salm-i/salr-i</i> knockdown phenotypes are shown for comparison in (D) and (H), in wings of <i>sal</i>^<i>EPv</i><i>-Gal4/ UAS-salm-i; UAS-salr-i/+</i> raised at 25°C and 29°C, respectively.</p