Comparative analysis of neurogenesis in the branchiopod Daphnia magna and the malacostracan Orchestia cavimana

Lead Research Organisation: Queen Mary, University of London
Department Name: Sch of Biological and Chemical Sciences

Abstract

More than 80 % of the living animal species are arthropods, with over a million modern species described and a fossil record reaching back to the late proterozoic area. Arthropods are common throughout marine, freshwater, terrestrial, and even aerial environments. They include four groups: insects (e.g. flies and beetles), crustaceans (e.g. shrimps and lobsters), chelicerates (e.g. spiders and scorpions) and myriapods (e.g. millipedes and centipedes). There is a long-standing debate on the internal relationships of arthropods. Traditionally, myriapods were thought to be the closest relatives of insects. However, recent comparison of the sequences of similar genes in all arthropod groups resulted in a different hypothesis: insects are more closely related to crustaceans than to myriapods. Interestingly, data on the development of the nervous system in all four groups support this hypothesis and in addition even support a close relationship of chelicerates and myriapods. In contrast to insects and crustaceans, neural stem cells that divide and generate all the cells of the nervous system are absent in chelicerates and myriapods, rather, the area from which the nervous system develops consists of many more cells that directly develop into nerve cells. Despite these differences similar genes control the formation of the nervous system in insects, chelicerates and myriapods. However, their activity and function is adapted to the specific mode of nervous system formation. Crustaceans show the greatest diversity in shape and development among arthropods and therefore it is not clear if all crustacean groups share a common ancestor or if some groups, like the higher crustaceans (e.g. crabs, lobsters), are more closely related to insects while others are not. In addition, the similarities to insects have only been demonstrated in higher crustaceans. Except for two genes, neural development genes have not been identified in crustaceans yet. In the proposed project we will therefore analyse two representatives of crustaceans, the higher crustacean or malacostracan, Orchestia cavimana (a shrimp-like crustacean) and the basal branchiopod, Daphnia magna (a water flea). We will analyse the whole process of nervous system development from the generation of neural stem cells up to the formation of neural networks. On the one hand we will identify genes that are involved in the generation of neural stem cells and study their activity and function. On the other hand, we will verify if neural stem cells are present in the water flea and compare the number and positions of individual stem cells to Orchestia and to insects. Furthermore, we will analyse which nerve cells and support cells are generated by individual stem cells in both crustaceans by single cell labelling of neuroblasts that are adjacent to the ventral midline. In addition, this method will enable us to study the formation of neuronal networks, since the whole cell bodies including the long thin processes that connect individual nerve cells are labelled. The proposed project will (1) contribute substantially to our knowledge on crustacean neurogenesis, (2) contribute to the resolution of arthropod relationships and (3) contribute to the understanding in what way the developmental mechanisms leading to the formation of the nervous system have been modified during evolution in the individual arthropod groups.

Technical Summary

Comparative analysis of neurogenesis in chelicerates, myriapods and insects has revealed that the genetic network controlling the recruitment of neural precursors is conserved in these three arthropod groups. However, the expression pattern and function of these genes is adapted to the distinct morphology of neural precursor formation in each group. Despite of having a similar function in the production of neural cells, insect and crustacean neuroblasts differ among other things in the way they are generated and in their position within the developing neuromeres. Until now, moleculargenetic analyses of neurogenesis in crustaceans are largely missing. In addition, morphological data on neuroblast lineages are only available for higher Crustacea and it is not clear if neuroblasts are present in other major crustacean groups, such as branchiopods. In the course of the proposed project we will analyse the generation of neuroblasts and their identity as well as the establishment of the axonal scaffold in two crustaceans, the branchiopod Daphnia magna and the malacostracan Orchestia cavimana, using both moleculargenetic and morphological approaches. The following techniques will be applied: PCR cloning, in situ hybridisation, cell labelling and functional analysis with RNA interference. We address two main questions: do crustaceans exhibit similar patterns of gene activity during differentiation of neuronal precursors despite apparent differences to insects, and is the situation within crustaceans the same despite differences in the generation of neuronal precursors in the various crustacean groups. We will compare our results to the data on neurogenesis in the remaining arthropod groups to reveal (1) in what way the developmental mechanisms have been modified in the individual arthropod groups and (2) which characters of neurogenesis can be considered as ancestral due to conservation in all groups and thus cannot be used for resolving euarthropod relationships.

Publications

10 25 50
 
Description The freshwater crustacean Daphnia is a model system for ecology, evolution and the environmental sciences. The rapidly growing genomic data for this organism is stimulating interdisciplinary research to understand the complex interplay between genome structure, gene expression, individual fitness, chemical contaminants and environmental change and evolution. We have contributed to the Daphnia pulex and Daphnia magna genome projects by structural and functional annotation of genomic elements. We have identified the open reading frames and gene structures of 22 genes based on homologies to arthropod/vertebrate genes. Furthermore, we have assigned biological functions to 17 of identified genes by expression studies and inactivation of function by a chemical inhibitor (Notch signalling pathway). Nine of the sequences have been deposited in the GenBank data base, the remaining sequences will be submitted publication of the corresponding manuscripts. The expression studies for some of the neural genes (Dam ASH, Dam asense, Dam snail, Dam prospero) have already been published in Developmental Biology (Ungerer, Erkisson & Stollewerk). The Daphnia genome is the only crustacean genome sequenced so far and has therefore the potential to contribute to resolving long-standing debates on arthropod phylogeny. Current views of arthropod phylogenetic relationships are based mainly on two types of datasets -molecular genetic data and morphological characters -and this has led to partly contradictory concepts of arthropod phylogeny. Evolutionary developmental biology joins these two types of data by analysing gene expression and function in a morphological context. Furthermore, it includes an additional factor - the evolutionary comparison of developmental processes. In this project we have contributed to the establishment of Daphnia as a model organism for evolutionary developmental biology. We have developed and/or refined tools for culturing, egg collection, fixation, staging (with particular focus on neurogenesis), in situ hybridisation and immunohistochemistry. These techniques have been published in the materials and methods part of our paper on Daphnia magna neurogenesis (Ungerer, Erkisson & Stollewerk, 2011). We have also designed a general staging system for all embryonic stages that facilitates the comparative analysis of developmental stages in arthropods (Mittmann et al., 2014). Furthermore, we have developed tools for functional analysis of Notch signalling, one of the major developmental signalling pathways which contributes to the formation of all organs. We have established a protocol for non-invasive chemical inhibition of Notch signalling in Daphnia magna embryos by DAPT (2,5-bis[4-dimethylaminophenyl]-1,3,4-thiadiazole). The data are included in the materials and methods part of our manuscript on 'Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna' (Ungerer, Eriksson & Stollewerk, 2012). We have also developed a protocol for RNA interference but similar results have already been published by Kato Y, Shiga Y, Kobayashi K, Tokishita S, Yamagata H, Iguchi T and Watanabe H. in 2011 ('Development of an RNA interference method in the cladoceran crustacean Daphnia magna' Dev Genes Evol. 220:337-45).
In this project we have uncovered a remarkable case of parallel evolution of neural stem cells in crustaceans and vertebrates which does not only involve the cooption of conserved genetic networks but also the independent evolution of similar morphogenetic processes. We show that both in the crustacean Daphnia magna and in vertebrates neural stem cells are maintained in the neuroepithelium and exist in two different states.
Exploitation Route The question how neural stem cells are maintained and how they differentiate into the diverse cell types is a main focus in neural stem cell research since stem cells have been suggested as possible source for cell replacement therapy to treat diseases and conditions like Parkinson and spinal cord injury. However, research is hindered by the complexity of the vertebrate, particularly the mammalian brain. Individual stem cells cannot be identified and thus it is difficult to design experiments that aim to understand how neural stem cells develop into particular cell types in vivo. Furthermore, the abundant presence of duplicated genes impairs with functional analysis. Cell culture experiments have advanced our knowledge; however, in what ways the results can be translated into the in vivo situation remains to be shown. Compared to vertebrates, Daphnia has several advantages that might advance our understanding of the complex interaction of signal transduction pathways in maintenance and differentiation of neural stem cells. Daphnia has a short generation time, it is easy to culture and the maintenance costs are low. Furthermore, we have shown here that the ventral nervous system of Daphnia is generated by a limited number of neural stem cells which can be individually identified and are located at stereotyped positions. Cell lineage studies in another crustacean show that the neural stem cells generate fixed lineages depending on their position in the neuroepithelium. Together with the non-invasive method developed for chemical inhibition of signalling pathways in Daphnia magna, this opens up the possibility of analysing the function and interaction of major signalling pathways in neural stem cell maintenance, differentiation and neural subtype production in vivo.
Sectors Other

 
Description Collegestudentship
Amount £60,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 09/2010 
End 08/2014
 
Description Collegestudentship
Amount £60,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 04/2009 
End 07/2013
 
Title Daphnia magna 
Description Advancement of a crustacean model system for developmental biology and evolution 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact Staging system which is used for collecting samples for gene expression studies (RNAseq) Identification of neural genes which are used for annotating the Daphnia genome Morphological and gene expression data which are used for phylogenies 
 
Title Daphnia genes and expression 
Description in situ gene expression data identification of new neural genes in Daphnia magna 
Type Of Material Database/Collection of data 
Year Produced 2011 
Provided To Others? Yes  
Impact First molecular genetic data on early crustacean neurogenesis - impact on our understanding of crustacean neurogenesis and arthropod nervous system evolution. 
 
Description Daphnia FIREFLEA 
Organisation AZ Delta
Country Belgium 
Sector Hospitals 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation AZ Groenige
Country Belgium 
Sector Hospitals 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation Aquafin
Country Belgium 
Sector Private 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation Norwegian Institute for Water Research (NIVA)
Country Norway 
Sector Public 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation Osaka University
Country Japan 
Sector Academic/University 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation Spanish National Research Council (CSIC)
Country Spain 
Sector Public 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation University of Bayreuth
Department Department of Animal Ecology
Country Germany 
Sector Academic/University 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation University of Ghent
Country Belgium 
Sector Academic/University 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia FIREFLEA 
Organisation bbe Moldaenke GmbH
Country Germany 
Sector Private 
PI Contribution Joint grant application - deputy coordinator
Collaborator Contribution Joint grant application
Impact Innovative Training Networks (ITN) Call: H2020-MSCA-ITN-2016 Title: Development of a Fast, action-mode based Informative scREening tool for pollutants - the water FLEA Daphnia as aquatic biosensor for endocrine disruptors submitted January 2016 Part of the Consortium submitted a joint grant application (same title) to the H2020 FET call in 2015 (rejected)
Start Year 2011
 
Description Daphnia development of phenotypic plasticity 
Organisation University of Bayreuth
Department Department of Animal Ecology
Country Germany 
Sector Academic/University 
PI Contribution Joint grant applications
Collaborator Contribution Joint grant application
Impact Submission of two grant applications (BBSRC, Leverhulme Trust), title: How does prey develop adaptive phenotypes in response to predators?
Start Year 2010
 
Description Daphnia development of phenotypic plasticity 
Organisation University of Birmingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint grant applications
Collaborator Contribution Joint grant application
Impact Submission of two grant applications (BBSRC, Leverhulme Trust), title: How does prey develop adaptive phenotypes in response to predators?
Start Year 2010