Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression

Robustness of positional specification by the Hedgehog morphogen gradient

AbstractSpatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation

A Hh-driven gene network controls specification, pattern and size of the Drosophila simple eyes

During development, extracellular signaling molecules interact with intracellular gene networks to control the specification, pattern and size of organs. One such signaling molecule is Hedgehog (Hh). Hh is known to act as a morphogen, instructing different fates depending on the distance to its source. However, how Hh, when signaling across a cell field, impacts organ-specific transcriptional networks is still poorly understood. Here, we investigate this issue during the development of the Drosophila ocellar complex. The development of this sensory structure, which is composed of three simple eyes (or ocelli) located at the vertices of a triangular patch of cuticle on the dorsal head, depends on Hh signaling and on the definition of three domains: two areas of eya and so expression - the prospective anterior and posterior ocelli - and the intervening interocellar domain. Our results highlight the role of the homeodomain transcription factor engrailed (en) both as a target and as a transcriptional repressor of hh signaling in the prospective interocellar region. Furthermore, we identify a requirement for the Notch pathway in the establishment of en maintenance in a Hh-independent manner. Therefore, hh signals transiently during the specification of the interocellar domain, with en being required here for hh signaling attenuation. Computational analysis further suggests that this network design confers robustness to signaling noise and constrains phenotypic variation. In summary, using genetics and modeling we have expanded the ocellar gene network to explain how the interaction between the Hh gradient and this gene network results in the generation of stable mutually exclusive gene expression domains. In addition, we discuss some general implications our model may have in some Hh-driven gene networks.Ministerio de Ciencias e Innovacion BFU2009-07044 FIS2008-04120Junta de Andalucía CVI 265

Core regulatory network motif underlies the ocellar complex patterning in Drosophila melanogaster

arXiv:1404.4554v5During organogenesis, developmental programs governed by Gene Regulatory Networks (GRN) define the functionality, size and shape of the different constituents of living organisms. Robustness, thus, is an essential characteristic that GRNs need to fulfill in order to maintain viability and reproducibility in a species. In the present work we analyze the robustness of the patterning for the ocellar complex formation in Drosophila melanogaster fly. We have systematically pruned the GRN that drives the development of this visual system to obtain the minimum pathway able to satisfy this pattern. We found that the mechanism underlying the patterning obeys to the dynamics of a 3-nodes network motif with a double negative feedback loop fed by a morphogenetic gradient that triggers the inhibition in a French flag problem fashion. A Boolean modeling of the GRN confirms robustness in the patterning mechanism showing the same result for different network complexity levels. Interestingly, the network provides a steady state solution in the interocellar part of the patterning and an oscillatory regime in the ocelli. This theoretical result predicts that the ocellar pattern may underlie oscillatory dynamics in its genetic regulation.This work is partially financed by Junta de Andalucía (FQM-122) to A. Córdoba and by the Spanish Ministry for Science and Innovation (MICINN/MINECO) and Feder Funds through grants BFU2012-34324 to F. Casares (CABD, Seville, Spain) and Consolider Ingenio-2010 ‘From Genes to Shape’ (CSD 2007-008), of which F. Casares was a participant researcher.Peer Reviewe

Core regulatory network motif underlies the ocellar complex patterning in Drosophila melanogaster

During organogenesis, developmental programs governed by Gene Regulatory Networks (GRN) define the functionality, size and shape of the different constituents of living organisms. Robustness, thus, is an essential characteristic that GRNs need to fulfill in order to maintain viability and reproducibility in a species. In the present work we analyze the robustness of the patterning for the ocellar complex formation in Drosophila melanogaster fly. We have systematically pruned the GRN that drives the development of this visual system to obtain the minimum pathway able to satisfy this pattern. We found that the mechanism underlying the patterning obeys to the dynamics of a 3-nodes network motif with a double negative feedback loop fed by a morphogenetic gradient that triggers the inhibition in a French flag problem fashion. A Boolean modeling of the GRN confirms robustness in the patterning mechanism showing the same result for different network complexity levels. Interestingly, the network provides a steady state solution in the interocellar part of the patterning and an oscillatory regime in the ocelli. This theoretical result predicts that the ocellar pattern may underlie oscillatory dynamics in its genetic regulation.Junta de Andalucía FQM-122España, Ministerio de Ciencia e Innovación MICINN / MINECOFondos Federales BFU2012-34324Consolider Ingenio-2010 CSD 2007-00

Comparing individual-based approaches to modelling the self-organization of multicellular tissues.

The coordinated behaviour of populations of cells plays a central role in tissue growth and renewal. Cells react to their microenvironment by modulating processes such as movement, growth and proliferation, and signalling. Alongside experimental studies, computational models offer a useful means by which to investigate these processes. To this end a variety of cell-based modelling approaches have been developed, ranging from lattice-based cellular automata to lattice-free models that treat cells as point-like particles or extended shapes. However, it remains unclear how these approaches compare when applied to the same biological problem, and what differences in behaviour are due to different model assumptions and abstractions. Here, we exploit the availability of an implementation of five popular cell-based modelling approaches within a consistent computational framework, Chaste (http://www.cs.ox.ac.uk/chaste). This framework allows one to easily change constitutive assumptions within these models. In each case we provide full details of all technical aspects of our model implementations. We compare model implementations using four case studies, chosen to reflect the key cellular processes of proliferation, adhesion, and short- and long-range signalling. These case studies demonstrate the applicability of each model and provide a guide for model usage

Estudio de la red de regulación génica que controla la especificación y organización del sistema ocelar en "Drosophila melanogaster"

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Biología del DesarrolloClave Programa: DBICódigo Línea: 9Uno de los descubrimientos biológicos más importante de las últimas décadas es que los diferentes grupos de animales multicelulares, no importa cuán divergentes sean, comparten los genes reguladores que controlan los aspectos principales de especificación y diferenciación celular durante el establecimiento del plan corporal. Estos genes reguladores codifican factores de transcripción (FTs) y moléculas de señalización que controlan tanto la expresión de genes efectores, los cuales realizan funciones celulares, como la expresión de otros FTs. Los FTs y vías de señalización forman así redes de regulación que son, finalmente, las que gobiernan la especificación y organización celular durante el progreso del desarrollo y determinan la trayectoria que una célula puede seguir. Conocer, por tanto, cómo operan estas redes de regulación génica (RRGs) es esencial para entender el origen de la diversidad funcional y morfológica observada en los distintos organismos a lo largo de la evolución. Describir una RRG relacionada con un proceso de desarrollo determinado requiere conocer qué FTs y moléculas de señalización están implicados en el sistema, cuándo y dónde se expresan los genes que los codifican y cómo interaccionan unos con otros. En esta tesis hemos explorado estos aspectos en la RRG que controla la especificación y organización del sistema ocelar en Drosophila melanogaster. El sistema o complejo ocelar (CO) es una porción de cutícula triangular, localizada en la cabeza dorsal y en cuyos vértices se encuentran los ocelos. Los ocelos son tres ojos simples [un ocelo anterior (o medial) y dos posteriores (o laterales)] y junto con los ojos compuestos conforman el sistema visual adulto de muchos insectos, donde son esenciales para la estabilización del vuelo y como detectores de movimiento. La cutícula que separa los ocelos y que alberga quetas sensoriales se denomina región interocelar (RIO). La cabeza dorsal se forma a partir de la fusión de los dominios anterior-dorsales del disco de ojo. Este dominio es especificado por la acción dinámica de dos vías de señalización: la vías de Wingless (Wg; el homólogo en Drosophila de Wnt1) y de Hedgehog (Hh); y orthodenticle (otd), un FT miembro de la familia Otx. Trabajos anteriores han descubierto algunas de las relaciones funcionales principales entre estos genes, las cuales resultan en la partición de la cabeza dorsal en tres territorios. De lateral a medial, éstos son: cutícula orbital (COrb), frente (Fr) y CO. Este último es posteriormente divido en tres subdominios, dos dominios precursores de los ocelos, separados por un dominio precursor de la región interocelar (RIO). La formación de estos subdominios requiere de la activación de los FTs eyes absent y sine oculis (eya/so; precursores de los ocelos) y engrailed (en; precursor de la cutícula interocelar) por la vía de Hh, obteniéndose un patrón final aOC-RIO-pOC. En esta tesis, hemos investigado cómo se establecen estos destinos celulares alternativos bajo el control de la vía de Hh. Nuestros resultados resaltan que la expresión temprana de hh a lo largo de todo el CO es necesaria para activar la expresión de los genes diana eya y en; sin embargo, no es suficiente para restringir sus dominios de acción en regiones discretas bien delimitadas. La obtención del patrón final requiere, por tanto, la adición de nuevos factores y/o nuevas interacciones funcionales. Aquí mostramos cómo, por un lado, la acción del FT Optix, el homologo en Drosophila de la subfamilia Six3/6, es requerido como mecanismo anti-represor para restringir la expresión de en en la futura RIO, quien, de lo contrario, actúa como regulador negativo del destino ocelar. Por otro lado, mostramos cómo el establecimiento de bucles de retroalimentación, a lo largo del proceso, estabiliza la expresión de los distintos genes en sus dominios de acción.Universidad Pablo de Olavide. Escuela de Doctorado

Centrosomal kinase Nek2 Cooperates with Oncogenic Pathways to Promote Metastasis

Centrosomal kinase Nek2 is overexpressed in different cancers, yet how it contributes toward tumorigenesis remains poorly understood. dNek2 overexpression in a Drosophila melanogaster model led to upregulation of Drosophila Wnt ortholog wingless (Wg), and alteration of cell migration markers—Rho1, Rac1 and E-cadherin (Ecad)—resulting in changes in cell shape and tissue morphogenesis. dNek2 overexpression cooperated with receptor tyrosine kinase and mitogen-activated protein kinase signaling to upregulate activated Akt, Diap1, Mmp1 and Wg protein to promote local invasion, distant seeding and metastasis. In tumor cell injection assays, dNek2 cooperated with Ras and Src signaling to promote aggressive colonization of tumors into different adult fly tissues. Inhibition of the PI3K pathway suppressed the cooperation of dNek2 with other growth pathways. Consistent with our fly studies, overexpression of human Nek2 in A549 lung adenocarcinoma and HEK293T cells led to activation of the Akt pathway and increase in b-catenin protein levels. Our computational approach identified a class of Nek2-inhibitory compounds and a novel drug-like pharmacophore that reversed the Nek2 overexpression phenotypes in flies and human cells. Our finding posits a novel role for Nek2 in promoting metastasis in addition to its currently defined role in promoting chromosomal instability. It provides a rationale for the selective advantage of centrosome amplification in cancer

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin