Ptch2/Gas1 and Ptch1/Boc differentially regulate Hedgehog signalling in murine primordial germ cell migration.

Gas1 and Boc/Cdon act as co-receptors in the vertebrate Hedgehog signalling pathway, but the nature of their interaction with the primary Ptch1/2 receptors remains unclear. Here we demonstrate, using primordial germ cell migration in mouse as a developmental model, that specific hetero-complexes of Ptch2/Gas1 and Ptch1/Boc mediate the process of Smo de-repression with different kinetics, through distinct modes of Hedgehog ligand reception. Moreover, Ptch2-mediated Hedgehog signalling induces the phosphorylation of Creb and Src proteins in parallel to Gli induction, identifying a previously unknown Ptch2-specific signal pathway. We propose that although Ptch1 and Ptch2 functionally overlap in the sequestration of Smo, the spatiotemporal expression of Boc and Gas1 may determine the outcome of Hedgehog signalling through compartmentalisation and modulation of Smo-downstream signalling. Our study identifies the existence of a divergent Hedgehog signal pathway mediated by Ptch2 and provides a mechanism for differential interpretation of Hedgehog signalling in the germ cell niche

Sonic Hedgehog Signaling in Limb Development.

The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs

Regulators of Shh Signaling in Limb Patterning and Development

The embryonic limb is one of the best understood examples of pattern formation in vertebrates. Patterning, along its anterior-posterior (AP) and proximo-distal (PD) axes, has been studied almost exclusively within the framework of positional information - the idea that the fate of individual cells is determined by their positional value in an embryonic field. This value is conferred through signaling molecules, called morphogens, in a concentration dependent manner. This idea is best illustrated in the chick wing whose AP pattern is imposed by an organizing center called the "zone of polarizing activity" (ZPA), through secretion of the morphogen Sonic hedgehog (Shh). Studies over the past 20 years show that Shh regulates patterning and growth through regulation of Gli-family transcription factors. The mouse has been an excellent genetic model for the study of Shh-mediated limb patterning and signal transduction, as well as the pathway's role in development and disease. However, these studies have also revealed critical differences between the mouse and chick models resulting in a need to re-evaluate positional information as a universal framework for vertebrate development. In this thesis, I propose the two-population model as a complimentary framework that allows us to reconcile the discrepancies of existing models. Using genetic models in the mouse, and pharmacological manipulation in the chick, I demonstrate that specific changes in Gli activity produce a defined spectrum of limb malformations and I utilize this spectrum as a tool to study a previously uncharacterized component of the Shh signal cascade, the receptor Ptch2, demonstrating that it is a critical, context-dependent regulator of the Shh signaling receptor Smoothened. This body of work illustrates that the vertebrate limb remains a prescient model for the study of signal transduction, development and disease and opens the door for the study of universal mechanisms of patterning in vertebrate organisms.Ph.D.2016-11-30 00:00:0

Concreto permeable como alternativa de recarga de acuíferos y drenaje urbano

El uso de concreto permeable se está implementando gradualmente en grandes ciudades para mejorar el manejo de la escorrentía de aguas pluviales a nivel regional y local. Al eliminar la necesidad de sumideros, vertederos y otros dispositivos de aguas pluviales, el concreto permeable puede reducir los costos generales del proyecto en un primer costo y hace un uso más eficiente de la tierra. El hormigón permeable tiene un costo de ciclo de vida significativamente más bajo que otras alternativas y permite ahorrar dinero a largo plazo debido a su durabilidad, resistencia y larga vida útil. El concreto permeable permite a los usuarios minimizar el uso del sistema de alcantarillado y evitar las tarifas de mantenimiento de los sistemas de recolección de aguas lluvias. Para el caso del presente trabajo de grado se identificaron las zonas de Santa Marta que son aptas para implementar concretos permeables, además de que fue posible determinar la viabilidad del uso de los mismos. Fue posible diferenciar mediante la realización de un comparativo las características de los pavimentos con drenajes convencionales y los pavimentos con concretos permeables. esto debido a que finalmente se espera establecer el diseño de concreto permeable aplicable en la ciudad de Santa Marta.The use of pervious concrete is gradually being implemented in large cities to improve stormwater runoff management at the regional and local levels. By eliminating the need for sumps, weirs, and other stormwater devices, pervious concrete can reduce overall project costs at first cost and make more efficient use of the land. Pervious concrete has a significantly lower life cycle cost than other alternatives and saves money in the long run due to its durability, strength, and long life. Pervious concrete allows users to minimize sewer system use and avoid maintenance fees for rainwater harvesting systems. In the case of the present degree work, the areas of Santa Marta that are suitable for implementing permeable concretes were identified, in addition to which it was possible to determine the viability of their use. It was possible to differentiate by comparing the characteristics of pavements with conventional drainage and pavements with permeable concrete. This is due to the fact that it is finally expected to establish the applicable permeable concrete design in the city of Santa Marta.Resumen. -- Abstract. -- 0. Introducción. -- 1. Definición del tema a investigar. -- 1.1. Planteamiento del problema. -- 1.2. Formulación del problema. -- 2. Justificación. – 3. Objetivos. -- 3.1. Objetivo general.-- 3.2. Objetivos específicos. -- 4. Delimitación de la investigación. -- 4.1.Línea de investigación. -- 4.2. Delimitación espacial. -- 4.3. Delimitación temporal. -- 5. Metodología. -- 5.1.Tipo de investigación. -- 5.2. Diseño de la investigación. -- 5.3. Técnica para la recolección de información. -- 6. Resultados de la revisión realizada. -- 6.1. Antecedentes. -- 6.2. Bases teóricas. -- 6.2.1. Acuífero. -- 6.2.2. Tipos de acuíferos. -- 6.2.3. Acuífero Manzanares - Gaira, Distrito de Santa Marta. -- 6.2.4. Concreto permeable. -- 6.2.5. Características del Concreto Permeable. -- 7. Análisis y discusión. -- 8. Conclusiones. -- 9. Recomendaciones. -- 10. Referencias Bibliográ[email protected]@campusucc.edu.c

T396I Mutation of Mouse <i>Sufu</i> Reduces the Stability and Activity of Gli3 Repressor

<div><p>Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3^FL) to an N-terminal repressor (Gli3^REP). Recent studies using a <i>Sufu</i> knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, <i>Sufu^T396I</i>, showed that Thr³⁹⁶residue of Sufu played a key role in regulation of Gli3 activity. <i>Sufu^T396I/T396I</i> embryos exhibited severe polydactyly, which is indicative of compromised <i>Gli3</i> activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3^FL) and processed Gli3 (Gli3^REP) were observed <i>in vivo</i> as well as <i>in vitro</i>. Genetic experiments showed that patterning defects in the limb buds of <i>Sufu^T396I/T396I</i> were rescued by a constitutive Gli3^REPallele (<i>Gli3^∆699</i>), strongly suggesting that Sufu^T396I reduced the truncated Gli3 repressor. In contrast, Sufu^T396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr³⁹⁶residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.</p></div

Sufu^T396I can regulate Gli activator function.

<p>(<b>A</b>) Western blotting of Gli2 from embryo lysates at E9.5. Genotypes and antibodies are indicated at the top and left, respectively. The image presented in the Fig. is representative of independent triplicated experiments. The broad gel images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5A Fig</a>. and relative expression of Gli2 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5B Fig</a>. (<b>B, C</b>) Western blotting of immunoprecipitates or lysates from 293T cells transfected with expression constructs as indicated at the top. Antibodies used for immunoprecipitation and western blotting are indicated to the left. The complete gel images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5C and D Fig</a>. Gli1 protein appears to show multiple bands; the reason for this is unknown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref042" target="_blank">42</a>] [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref046" target="_blank">46</a>]. Relative band intensities of lane 5 and 6 (C) are shown. It is note worthy that the expression levels of Gli2 were proportional to the amount of Sufu irrespective of either wild-type or T396I substitutions. (<b>D, E</b>) Luciferase reporter assay in 3T3 transfected with expression constructs as indicated in figures. Error bars indicate standard deviations. In our experimental settings, we observed approximately only 4-fold induction of luciferase reporter activity by Gli1 expression, as had been observed previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref046" target="_blank">46</a>].</p