The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear

Background The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. Results We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. Conclusions The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data

ARTEFACTS: How do we want to deal with the future of our one and only planet?

The European Commission’s Science and Knowledge Service, the Joint Research Centre (JRC), decided to try working hand-in-hand with leading European science centres and museums. Behind this decision was the idea that the JRC could better support EU Institutions in engaging with the European public. The fact that European Union policies are firmly based on scientific evidence is a strong message which the JRC is uniquely able to illustrate. Such a collaboration would not only provide a platform to explain the benefits of EU policies to our daily lives but also provide an opportunity for European citizens to engage by taking a more active part in the EU policy making process for the future. A PILOT PROGRAMME To test the idea, the JRC launched an experimental programme to work with science museums: a perfect partner for three compelling reasons. Firstly, they attract a large and growing number of visitors. Leading science museums in Europe have typically 500 000 visitors per year. Furthermore, they are based in large European cities and attract local visitors as well as tourists from across Europe and beyond. The second reason for working with museums is that they have mastered the art of how to communicate key elements of sophisticated arguments across to the public and making complex topics of public interest readily accessible. That is a high-value added skill and a crucial part of the valorisation of public-funded research, never to be underestimated. Finally museums are, at present, undergoing something of a renaissance. Museums today are vibrant environments offering new techniques and technologies to both inform and entertain, and attract visitors of all demographics.JRC.H.2-Knowledge Management Methodologies, Communities and Disseminatio

Aminerge Kontrolle des Verhaltens von Drosophila

The biogenic amines tyramine and octopamine are important neuromodulators involved in many physiological processes in invertebrates. They enable the modulation of behavioral output in response to internal and environmental changes. In this study, the role of octopamine and tyramine in sugar response and locomotion was investigated by genetic modifications affecting amine levels in adult Drosophila melanogaster. The synthesis-mutant tßhnM18 (tyramine-ß-hydroxylase) lacking octopamine and accumulating tyramine was analyzed in two behavioral paradigms: in the proboscis extension assay to test sugar response after starvation and in the Buridan’s assay to test walking speed and stripe deviation. In the proboscis extension assay, starved tßhnM18 mutants showed a reduced sugar response. Their hemolymph carbohydrate concentration was elevated compared to control flies and when starved to death, they survived longer. Temporally controlled rescue experiments revealed an action of the OA/TA system during sugar response, while spatially controlled rescue experiments suggested OA/TA actions inside and outside of the nervous system. Additionally, the analysis of OA- and TA-receptor mutants exhibited an involvement of both amines in the animals' physiological response to starvation. In the Buridan’s paradigm, tßhnM18 mutants walked slower and more directed to the presented stripes. The analysis of tßh overexpression in mutant and wild type flies as well as in OA- and TA-receptor mutants revealed a complex interaction of both aminergic systems in the control of locomotion. The null allele tßhnM18 showed differential dominance effects on the three behavioral traits ranging from recessive to overdominant. The data suggested that the different neuronal networks responsible for the three phenotypes are differentially sensitive to tßh gene dosage. In conclusion, the here presented data indicate a cooperation of biogenic amines on behavioral control, not only in the nervous system but also in non-neuronal tissues. The modulation of the different behavioral aspects is tßh dosage-dependent. The activity of the tßh- enzyme enables the alteration of the relative concentrations of octopamine and tyramine leading to behavioral modulation. Thus, this work provides a mechanism for behavioral plasticity achieved by amine level alterations.Die biogenen Amine Tyramin und Octopamin sind in Invertebraten als wichtige Neuromodulatoren an unzähligen physiologischen Prozessen beteiligt. Sie ermöglichen die Modulation von Verhaltensantworten in Reaktion auf interne und externe Veränderungen. In dieser Arbeit wurde die Rolle von Octopamin und Tyramin in der Zuckerantwort und im Laufverhalten am Modellorganismus Drosophila melanogaster mit Hilfe genetischer Manipulation untersucht, in Folge derer sich die relative Menge der Amine verändert. Die Synthesemutante tßhnM18 (tyramine-ß-hydroxylase), die kein Octopamin aber einen Überschuss an Tyramin aufweist, wurde in zwei Verhaltensparadigmen getestet: im Proboscisextensionsassay (Hunger-abhängige Antwort auf Zucker) und in Buridan’s Assay (Laufgeschwindigkeit und visuelle Fixation). Im Proboscisextensionsassay zeigten gehungerte tßhnM18 Mutanten eine reduzierte Antwort auf Zucker, während die Konzentration der Kohlenhydrate in ihrer Hämolymphe im Vergleich zu Kontrollfliegen erhöht war. Des Weiteren wiesen sie eine längere Überlebensdauer ohne Futter auf. Zeitlich kontrollierte Rettungsversuche zeigten eine Aktivität des octopaminergen und tyraminergen Systems während der Antwort auf Zucker, während räumlich kontrollierte Rettungsversuche eine Aktivität des octopaminergen und tyraminergen Systems innerhalb und außerhalb des Nervensystems zeigten. Eine Analyse von Octopamin- und Tyraminrezeptoren ergab eine Beteiligung beider Amine in der physiologischen und neuronalen Reaktion auf Futterentzug. Im Buridanparadigma zeigten tßhnM18 Mutanten eine geringere Laufgeschwindigkeit und mehr Bewegung in Richtung der präsentierten Streifen. Die Ergebnisse der tßh-Überexpression und der Rezeptormutanten legten eine komplexe Interaktion zwischen den aminergen Systemen zur Bewegungskontrolle dar. Das Nullallel tßhnM18 wies differentielle Dominanzeffekte auf die drei getesteten Verhalten auf, die von rezessiv bis überdominant reichten. Die Daten lassen den Schluss zu, dass verschiedene neuronale Netzwerke für die drei Verhalten verantwortlich sind. Die Modulation von Zuckerantwort und visuelle Fixation korrelierten mit der tßh-Gendosis. Zusammenfassend zeigen die hier präsentierten Daten die Zusammenarbeit zweier biogener Amine bei der Kontrolle von Verhaltensmustern, die nicht nur im Gehirn, sondern auch nicht neuronal stattfindet. Die Veränderung der relativen Mengen von Octopamin und Tyramin durch tßh-Induktion ist ein möglicher Mechanismus für die Verhaltensmodulation, der durch Stress ausgelöst werden könnte. Diese Modulation wird von der tßh-Gendosis beeinflusst

Haemolymph extraction of adult Drosophila

The figure shows the very simple protocol of how to extract haemolymph from adult Drosophila melanogaster

tßh mutant flies walk slower and deviate less from stripes in Buridan's paradigm

A. Buridan’s paradigm. The platform in the middle of the arena is surrounded by water and illuminated with bright white light from behind. Two black opposite stripes are fixed to the arena wall. The centroid position of the fly was recorded by custom tracking software. All data are obtained from 5 min of uninterrupted walk. Data were analyzed using CeTrAn v.4. B. Flies. tßhnM18 mutant and their genetic background control w+ (Schwaerzel et al., 2003) are crossed with CantonS wild type (P). One to two days old male flies were CO2-anesthetized (F1) and their wings were clipped at two thirds of their length. The flies recovered in the food vials overnight. Individuals were captured using a fly aspirator and transferred into the experimental setup on the center of the platform. The platform was cleaned with 70% ethanol after each experiment to minimize nonspecific effects. C. Mean of walking speed. A median of walking speed (traveled distance over time) was calculated for each fly. The mean of all flies is depicted in the graphs ± standard error of the mean. The tßh mutant males walk significantly slower than the wild type males (Welch Two-Sample t-Test, p = 2*10-8). D. Stripe deviation quantifies fixation behavior. It corresponds to the angle between the velocity vector and a vector pointing from the fly position towards the center of the stripe in front of the fly. Data are depicted as medians (bar), the 75%- and 25%-quartiles (box), and total data range (whiskers) excluding outliers outside the 1.5 times the interquartile range (points). Stripe deviation is significantly smaller in tßh mutant males compared to controls (Wilcoxon rank sum test, p = 4*10-10), meaning they fixated the stripes better than control flies. Numbers inside the graphs indicate the sample size. For methodological details see Colomb et al. (2012). References: Colomb, J., Reiter, L., Blaszkiewicz, J., Wessnitzer, J., & Brembs, B. (2012). Open source tracking and analysis of adult Drosophila locomotion in Buridan’s paradigm with and without visual targets. PloS One, 7(8), e42247. doi:10.1371/journal.pone.0042247 Schwaerzel, M., Monastirioti, M., Scholz, H., Friggi-Grelin, F., Birman, S., & Heisenberg, M. (2003). Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 23(33), 10495–502

Drosophila sugar response is increased by starvation duration and sucrose concentration in a locomotion-independent proboscis extension test

A. Proboscis extension response assay. One to two days old CantonS flies of mixed gender were starved for the indicated time with ad libitum access to water. Four hours before testing, they were glued to a copper hook between head and thorax. For the test, the hook was fixed to clamp which was attached to a rack. In this locomotion-independent assay, all tarsi can be stimulated with no stress or pressure on abdomen by a serial dilution of seven increasing sucrose concentrations (0%, 0.1%, 0.3%, 0.6%, 1%, 3%, 10%, 30%, 30% to labellum). B. The proportion of flies that extended proboscis after being stimulated at all the tarsi. Flies either responding to the first (water) or not responding to the last 30% to the labellum were excluded from the data analysis. The proportion of responding flies increases not only with increasing sucrose concentration but also with increasing starvation time of 21 hours compared to 14 hours. C. The response index is significantly higher after 21 hours compared to 14 hours of starvation indicated by asterisk (Wilcoxon rank sum test, p < 0.05) and was calculated by summing the positive proboscis extension responses to all the seven stimulations and are illustrated as medians (bar), the 75%- and 25%-quartiles (box) and the total data range (whiskers). Numbers inside the boxes indicate sample sizes

Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tβh gene in behavioral phenotypes

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-β-hydroxylase (tβh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tβhnM18 allele in 2 behavioral measures in Buridan’s paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tβh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tβh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics

Data and code for behavioral analysis of: Structural and Molecular Properties of Insect Type II Motor Axon Terminals.

<p>Data and code for behavioral analysis of: Structural and Molecular Properties of Insect Type II Motor Axon Terminals.</p> <p>v1.2: typos corrected and all files available in a single .zip file for download</p