dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.MINECO Spain grants: (BFU2012-34324, BFU2015- 66040); Research Foundation—Flanders FWO grants: (G.0640.13, G.0791.14, PhD fellowship); Fundação para a Ciência e Tecnologia grant: (IF/01031/2012)

A Toggle-Switch and a Feed-Forward Loop Engage in the Control of the Drosophila Retinal Determination Gene Network

Dipterans show a striking range of eye sizes, shapes, and functional specializations. Their eye is of the compound type, the most frequent eye architecture in nature. The development of this compound eye has been most studied in Drosophila melanogaster. The early development of the Drosophila eye is under the control of a gene regulatory network of transcription factors and signaling molecules called the retinal determination gene network (RDGN). Nodes in this network have been found to be involved not only in the development of different eye types in invertebrates and vertebrates, but also of other organs. Here we have analyzed the network properties in detail. First, we have generated quantitative expression profiles for a number of the key RDGN transcription factors, at a single-cell resolution. With these profiles, and applying a correlation analysis, we revisited several of the links in the RDGN. Our study uncovers a new link, that we confirm experimentally, between the transcription factors Hth/Meis1 and Optix/Six3 and indicates that, at least during the period of eye differentiation, positive feedback regulation from Eya and Dac on the Pax6 gene Ey is not operating. From this revised RDGN we derive a simplified gene network that we model mathematically. This network integrates three basic motifs: a coherent feedforward loop, a toggle-switch and a positive autoregulation which, together with the input from the Dpp/BMP2 signaling molecule, recapitulate the gene expression profiles obtained experimentally, while ensuring a robust transition from progenitor cells into retinal precursors.MINECOFEDER (BFU2012-34324, BFU2015-66040-P)Agencia Estatal de Investigacion (AEI) of Spai

Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells

This deposit is composed by the main article and supplementary files of the publication.Studies of the role of actin in tumour progression have highlighted its key contribution in cell softening associated with cell invasion. Here, using a human breast cell line with conditional Src induction, we demonstrate that cells undergo a stiffening state prior to acquiring malignant features. This state is characterized by the transient accumulation of stress fibres and upregulation of Ena/VASP-like (EVL). EVL, in turn, organizes stress fibres leading to transient cell stiffening, ERK-dependent cell proliferation, as well as enhancement of Src activation and progression towards a fully transformed state. Accordingly, EVL accumulates predominantly in premalignant breast lesions and is required for Src-induced epithelial overgrowth in Drosophila. While cell softening allows for cancer cell invasion, our work reveals that stress fibre-mediated cell stiffening could drive tumour growth during premalignant stages. A careful consideration of the mechanical properties of tumour cells could therefore offer new avenues of exploration when designing cancer-targeting therapies.Bloomington Drosophila Stock Centre; Vienna Drosophila Research Center (VDRC); Developmental Studies Hybridoma Bank (DSHB); Fundação para a Ciência e Tecnologia (FCT) grant: (IF/01031/2012); Laço Grant in breast cancer 2015; Alexander von Humboldt Foundation grant: (Alexander von Humboldt Professorship); Liga Portuguesa contra o Cancro/Pfizer.info:eu-repo/semantics/publishedVersio

Virtual Physiology: A Tool for the 21st Century

Veterinary physiology is a basic curricular unit for every course within the veterinary field. It is mandatory to understand how the animal body works, and what to expect of a healthy body, in order to recognize any misfunction, and to be able to treat it. Classic physiology teaching involves wet labs, much equipment, many reagents, some animals, and a lot of time. But times are changing. In the 21st century, it is expected that the teaching and learning process can be more active and attractive, motivating students to learn better. It is necessary to understand what students like, and to introduce novelties into the school routine. The use of a game-based learning, using “new” technologies, creating virtual experiences and labs, reducing the costs of reagents, equipment, and especially reducing the use of animals, will be the future for physiology teaching

Composição e abundância de macrófitas num troço do rio Ovelha

As macrófitas fluviais são um grupo relevante para a avaliação ecológica dos rios. Numa amostragem realizada num troço de 100 m do rio Ovelha, localizado a 217 m de altitude, na freguesia de Fornos, Marco de Canaveses, estudou-se a abundância, composição e distribuição das macrófitas. Verificou-se que o troço estudado é pobre em macrófitas, apresentando uma riqueza específica baixa, o que poderá estar relacionado, sobretudo, com o substrato rochoso. Considerando os resultados obtidos é fundamental que, futuramente, sejam estudadas as macrófitas conjuntamente com outros elementos biológicos, no sentido de se proceder a uma correta monitorização do estado ecológico do rio Ovelha.info:eu-repo/semantics/publishedVersio

The role of odd-skipped family genes in the Drosophila head development

Tese de doutoramento em Bioquímica (Biologia Molecular) apresentada à Fac. de Ciências e Tecnologia da Univ. de CoimbraO desenvolvimento de organismos multicelulares requer uma coordenação correcta entre a proliferação e a especificação das células. As mesmas vias de sinalização extracelular que controlam a especificação da identidade das células também regulam a proliferação das mesmas. O principal objectivo desta tese foi compreender o papel da família de genes odd durante a especificação celular e a organogênese do olho e da antena de Drosophila. A família de genes odd é composta por quatro genes: odd-skipped (odd), brother of odd with entrails limited (bowl), drumstick (drm) and sister of odd and bowl (sob), os quais apresentam extensas semelhanças nos domínios de zinco de ligação ao DNA. Diferentes estudos mostraram que estes genes estão implicados na formação do padrão de distintos tecidos, como é o caso do intestino, patas e epiderme embrionária. Nesta tese, demonstrámos que a família de genes odd é expressa ao longo da margem posterior do disco de olho, um centro especializado de sinalização. Este domínio é requerido para o início do desenvolvimento da retina através da produção da molécula de sinalização hedgehog (hh). Nas células da margem, bowl é necessário para a activação de hh e, consequentemente, para o desenvolvimento do olho. Portanto, a família de genes odd é essencial para o desenvolvimento da retina. Além disso, a expressão ectópica de odd e drm nas células indiferenciadas do domínio anterior do olho é suficiente para induzir a expressão de hh com a concomitante formação de olhos ectópicos. Assim sendo, os genes odd são essenciais para definirem o domínio a partir do qual a retina se começa a diferenciar (Chapter I). No seguimento do trabalho, comprovámos que bowl também é necessário durante o desenvolvimento da antena para a repressão da expressão de wg (wingless), na região onde normalmente se expressa a molécula BMP2/4, Dpp (Decapentaplegic). Esta activação de wg no domínio de expressão de dpp origina um novo eixo proximodistal (PD) que, por sua vez, gera o desenvolvimento de antenas extra. Estes resultados podem ser explicados não apenas com base na simples acção repressora da transcrição de wg, mas também se considerarmos que bowl é responsável pela supressão do desenvolvimento de um primórdio cefálico, normalmente ‘silenciado’. Em contraste com o que foi mostrado no desenvolvimento da pata, a família de genes odd parece não ter nenhuma função na segmentação da antena (Chapter II). Mostrámos que a cassete Drm/Lin/Bowl, descrita como funcional durante o desenvolvimento do intestino e da epiderme embrionária, está também em funcionamento durante o desenvolvimento do disco imaginal de olho e antenna. Em ambas as situações, na especificação da margem e do eixo da antenna, Drm, muito provavelmente em associação com Odd, é necessário para aliviar o efeito repressor de Lin sobre a função de Bowl (Chapter I and II). Em colaboração com o Dr. José Luis Gomez-Skarmeta, demonstrámos que a família de genes odd pode estar implicada no normal desenvolvimento dos tubos renais (tubos de Malphigian) de Drosophila, da mesma forma que no desenvolvimento renal de Xenopus e zebrafish, onde ambos os genes odd de vertebrados, Osr1 e Osr2, são suficientes e necessários para o correcto desenvolvimento dos pronefros (Appendix).The development of multicellular organisms requires the correct coordination between proliferation and specification of cells. The same extracellular signaling pathways that control cell fate specifications also regulate proliferation. The main objective of this thesis was to understand the role of odd family genes during specification and organogenesis of the Drosophila eye and antenna. The odd family genes is composed of four genes odd-skipped (odd), brother of odd with entrails limited (bowl), drumstick (drm) and sister of odd and bowl (sob) that display extensive homology in the zinc DNA-binding domains. Different studies have shown that these genes are involved in the patterning of distinct tissues, such as gut, legs and embryonic epidermis. In this thesis, we have shown that odd family genes are expressed along the posterior margin of the eye disc, a specialized signalling center required for the initiation of retinal development by producing the hedgehog (hh) signaling molecule. In the margin cells, bowl is necessary for the activation of hh and therefore for eye development. Thus, odd family genes are essential for retinal development. In addition, misexpression of odd and drm in anterior, undifferentiated eye cells is sufficient to induce hh expression with concomitant formation of ectopic eyes. Therefore, odd genes are essential for defining the retina initiation center (Chapter I). Further investigation revealed that bowl is also required during antennal development for the repression of wg (wingless) expression in territories that normally express the BMP2/4 molecule Dpp (Decapentaplegic). This de-repression of wg in the dpp-expressing domain generates a novel proximo-distal (PD) axis that results in the development of an extranumerary antenna. These results can be explained if rather than simply acting as a block of wg transcription, bowl were suppressing the development of a cephalic primordium that remains normally “silent”. In contrast to what has been shown in leg development, odd family genes do not seem to have any role in antennal segmentation (Chapter II). In addition, we have demonstrated that the Drm/Lin/Bowl cassette, described to be functioning during gut and embryonic epidermis development, is also at work during the development of the eye-antennal imaginal disc. In both situations, margin specification and antennal axis specification, Drm, most likely in association with Odd, is required to relief the repressor effect of Lin on Bowl function (Chapter I and II). In collaboration with Dr. José Luis Gomez-Skarmeta, we have shown that odd family genes may be involved in proper renal (Malphigian) tubules development in Drosophila, like it occurs in Xenopus and zebrafish kidney development, where both vertebrate odd genes, Osr1 and Osr2, are sufficient and required for proper pronephros development (Appendix)

Homeostasis of the Drosophila adult retina by actin-capping protein and the Hippo pathway

The conserved Hippo signaling pathway regulates multiple cellular events, including tissue growth, cell fate decision and neuronal homeostasis. While the core Hippo kinase module appears to mediate all the effects of the pathway, various upstream inputs have been identified depending on tissue context. We have recently shown that, in the Drosophila wing imaginal disc, actin-Capping Protein and Hippo pathway activities inhibit F-actin accumulation. In turn, the reduction in F-actin sustains Hippo pathway activity, preventing Yorkie nuclear translocation and the upregulation of proliferation and survival genes. Here, we investigate the role of Capping Protein in growth-unrelated events controlled by the Hippo pathway. We provide evidence that loss of Capping Protein induces degeneration of the adult Drosophila retina through misregulation of the Hippo pathway. We propose a model by which F-actin dynamics might be involved in all processes that require the activity of the core Hippo kinase module

dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.status: publishe

Loss of <i>dac</i> function affects the dynamics of the MF and proper retinogenesis.

<p>All panels show L3 eye imaginal discs. (A) Cross-section through a wild type disc with anterior to the left and apical side up, revealing that the formation of the MF depends on the coordination of three different cell processes: apical constriction (red arrow), apical-basal contraction (yellow arrow), seen by phalloidin staining, which outlines cell shape (green) and nuclear basal migration (orange arrow), labeled with anti-Elav (blue), which marks PR nuclei and anti-β-Galactosidase to detect <i>hhZ</i> (red). (B,D-J) Standard confocal sections with anterior to the left and dorsal up on this and all subsequent eye disc panels. (C-C´´) Cross section through the wild type disc in B (dashed white line). (B,C-C´´,F) wild type discs stained with (B,C-C´´) anti-Dac (green in B,C and white in C´), anti-β-Galactosidase to detect <i>hhZ</i> (red in B,C and white in C´´) and anti-Ci (blue in B,C) or (F) anti-Ato (red) and anti-Sens (blue). Note that Dac is expressed at high levels in a stripe that comprises the MF (arrows in B,C). (F) Ato shows three distinct expression pattern: ahead of the MF, all cells express Ato (1), posterior to the MF, Ato is restricted to a group of cells that start to co-express Sens (2). More posterior, its expression is only maintained in R8 cell until the 2^nd row of cells (3), while Sens expression persists onwards [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006204#pgen.1006204.ref021" target="_blank">21</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006204#pgen.1006204.ref062" target="_blank">62</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006204#pgen.1006204.ref088" target="_blank">88</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006204#pgen.1006204.ref093" target="_blank">93</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006204#pgen.1006204.ref094" target="_blank">94</a>]. (D-E´ and G-J) <i>dac</i>^<i>3</i> clones marked by the (D) presence or (E-E´ and ´G-J) absence of GFP (green) and outlined by yellow dashed lines in D,E´,G´ and H´. Discs are stained with (D) phalloidin (magenta) or (E-E´) anti-Elav (magenta) or (G-G´) anti-Ato (red) and anti-Sens (blue) or (H-H´) anti-Elav (blue) and anti-β-Galactosidase (red) to reveal <i>hhZ</i> or (I) anti-Elav (blue) and anti-Sal (red) or (J) anti-Pros (magenta), R7 indicates PR7 and C indicates cone cells. Plain white line in E’ marks the MF. Note that in <i>dac</i> mutant clones, the refinement of Ato expression is atypical: its expression is not restricted to the groups of cells that start to co-express Sens (2) and is not singled out properly in R8 cells (3) (1G´, compare green with white annotations). <i>dac</i> mutant ommatidia show irregular number of cells: with only one (white arrow in I) or 2 Sal-expressing PR (yellow arrow in I) and fewer PRs (Elav positive cells, E-E´, H-H´). Scale bars represent 50μm.</p

Expressing <i>dac</i> ectopically in the wing disc is sufficient to enhance Hh signaling.

<p>(A) Schematics of the whole wing disc (upper) and of the distal wing disc (lower) showing the expression domain of <i>ap</i> and of Hh pathway components. <i>ap and hh</i> are expressed in the dorsal (red) and posterior domain, respectively. Hh signals to the anterior domain leading to Ci^FL accumulation and activation of <i>rdx and ptc</i> in the anterior-posterior (AP) compartment boundary (green) and of <i>dpp</i> (blue). (B-C´´,E-G´, I-J´´) Standard confocal sections of L3 wing imaginal discs with anterior to the left and dorsal up. (B-C´´,E-G´) <i>ptc</i>-Gal4 driving the expression of UAS-<i>GFP</i> (B-B´´,E-F´) or UAS-<i>GFP</i> (C-C´´,G-G´) and UAS-<i>HA</i>::<i>dac</i>. (I-J´´) <i>ap</i>-Gal4 driving the expression of (I-I´) UAS-<i>GFP</i> or (J-J´´) UAS-<i>GFP</i> and UAS-<i>HA</i>::<i>dac</i>. Discs are stained with (B-C´´) anti-Ci (magenta in B,C, upper panel in B´´ and C´´; white in B´,C´, bottom panel in B´´ and C´´) or (E-G´) anti-Dac (red in E,F,G) and anti-β-Galactosidase to reveal <i>dppZ</i> (blue in E,F,G and white in F´,G´) or (I-J´´) anti-Ci (blue in I,J and white in J´´) and anti-β-Galactosidase to reveal <i>rdxZ (</i>red in I-J and white in I´,J´). B´´ and C´´ are magnification of B and C, respectively. (D-D´) Profiles of the GFP (green lines) or Ci (magenta lines) intensity signals across the AP axis in B´´ and C´´, respectively. (H-H´) Profiles of the GFP (green lines) and <i>dppZ</i> (blue lines) intensity signals across the AP axis in F and G, respectively. The arrows in D´ and H´ highlight the accumulation of Ci and <i>dppZ</i> in the <i>ptc>dac</i>-expressing domain, respectively. (K-K´) Profiles of the Ci (blue lines) and <i>rdxZ</i> (red lines) intensity signals across the AP axis in the dorsal areal delimited by dashed line in I and J, respectively. Yellow arrows in K and K´ indicate the position of the AP compartment boundary. The plain and dashed yellow lines in F´,G´,I´ and J´-J´´ delimited the AP compartment boundaries. The white dashed line in E outlines the distal wing blade. F-F´ is a magnification of the yellow square in E. The white dashed line in I´,J´-J´´ indicate the dorsal-ventral boundaries. The yellow arrowhead in J´´ indicates the accumulation of Ci in the dorsal, anterior blade. The yellow asterisks in J´,J´´ indicate the upregulation of <i>rdxZ</i> (J´) and the accumulation of Ci (J´´) in the proximal dorsal domain. Scale bars represent 50μm.</p