University of Heidelberg

Department for Cell and Molecular Biology

 

 

 

Wnt signaling in Zebrafish

Dr. Matthias Carl

 

Wnt-signalling is involved in a variety of processes, which include the emergence of diseases like cancer as well as early embryonic development.

We are particularly interested in the development of the vertebrate brain and we are using the zebrafish (Danio rerio) as our model organism to study the genetic pathways, which underlie its development. The vertebrate brain is an immensely complex structure, which exhibits numerous asymmetries, both morphological and functional. How these asymmetries are genetically established has remained a key open question, but failure in their proper development in humans can have devastating consequences like schizophrenia or depression.

 

 

Wnt and neural development

At least two structures in the zebrafish forebrain located in the dorsal diencephalon exhibit asymmetric features: The pineal complex and the habenulae. The habenulae connect fore- and ventral midbrain and are formed by a bilateral set of nuclei that are more pronounced on the left side in many vertebrates. The pineal complex consists of the more caudally located pineal and the left sided parapineal organ, which shows an asymmetric ipsilateral fiber projection towards the left habenula. Efferent projections from both habenulae innervate the interpeduncular nucleus in the ventral midbrain. The laterotopic segregation of these projections results in the transformation of the left right asymmetry in the dorsal diencephalon into a dorsal ventral asymmetry in the midbrain.

 


Two photon confocal in vivo time lapse movie using a novel transgenic line highlighting the habenula neural network; dorsal view, anterior to the left (Carlo Beretta with help of the Nikon Imaging Center Heidelberg, Nicolas Dross, Pete Bankhead and Ulrike Engel).

 

 

We could recently show that the Axin1 gene, which is a known inhibitor of the Wnt/b-catenin signalling pathway, is crucially involved in the establishment of brain asymmetries. We are combining molecular biology, genetics and advanced imaging to elucidate where, when and how Axin1/Wnt/b-catenin signalling contributes to the establishment of brain asymmetries. Our main tools are the analysis of zebrafish mutated in genes known to act in this pathway. Furthermore timely and spatially regulated misexpression of genes in combinations of wild type, mutant and transgenic animals complement our studies. Furthermore we are investigating new genes of the Wnt pathway and their potential role in the establishment of brain asymmetries.

 


Confocal in vivo time lapse movie on early habenula development combining GFP fluorescence and DIC; dorsal view, anterior to the upper left (collaboration with Soojin Ryu).


3-D reconstruction of the developing habenula at two days of development.

 

 

Wnt and axis formation

Within the past 350 million years about 25.000 fish species have emerged. This makes fish the most diverse group of animals amongst all vertebrates. The two fish species mainly used in the laboratory are the zebrafish (danio rerio) and the medakafish (oryzias latipes), which are only distantly related. This provides us with the unique opportunity to perform comparative studies in morphologically similar species that are essential if we are to understand the conserved and species specific mechanisms that underlie vertebrate development.
One commonly used method to identify genes which play essential roles during embryonic development is to introduce random mutations into the fish genome. The resulting morphological alterations will then be analysed and the gene affected isolated subsequently. These mutagenesis screens have been frequently and successfully used in the zebrafish. In medaka, a number of mutants have been isolated in recent screens, which show unique phenotypes, which have not been found in any other vertebrate.
We are particularly interested in a group of mutants, which do not develop any trunk- or tail structures, while the head forms apparently normal.

 


The stummelschwanz mutant (upper embryo) lacks trunk- and tail structures.

 

In a collaborative effort one of these mutants has been isolated recently in the group of Hiroyuki Takeda. In this mutant a point mutation in the FGF-receptor1 causes the lack of trunk and tail structures. In contrast, the loss of FGF-receptor1 function in zebrafish causes the loss of a small area in the brain. These phenotypes are consistent with the different regions of FGF-receptor1 gene expression in medaka and zebrafish. This is a nice example of evolutionary divergence in the requirement of a specific receptor, most likely due to differential spatially regulated expression in the two species.
Currently we are analysing four other mutants using genetic, molecular and imaging techniques to identify the nature of the genes responsible for the lack of posterior embryonic structures and to understand the exact function of these genes during embryonic development in medaka. Subsequently, we will knock down the function of the zebrafish homologe and compare the function in these two evolutionary distantly related vertebrate species. We have already identified the gene affected in one of the mutants as being involved in the Wnt/b-catenin signalling cascade. This comparative approach will allow us to discriminate between species specific and conserved function of potentially new genes involved in posterior axis formation in vertebrates.

 

Job offers:

Interested scientists at any level can apply. Salary costs have to be raised beforehand (fellowships etc.).

 

Group members:

Dr. Matthias Carl, Group leader

Luca Guglielmi, Ph.D. Student

Sara Asgharpour, Ph.D. Student

 

Former Group Members:

Nesrin Mwafi, Ph.D. Student

Alessio Paolini, Ph.D. Student

Tillmann Rusch, Practical Student

Aysu Kök, Practical Student

Raquel Jacinto, Ph.D. Student

Krystyna Zawieja, Technician

Carlo Beretta, Ph.D. Student

Ulrike Hüsken, Ph.D. Student

Kirsten Seufert, Fish Facility Assistant

Christian Altbürger, Practical Student

Elke von Ochsenstein, Technician

Shiqi Zhang, Practical Student

Christina Keiner, Internship Student

Ann-Christin Mentzer, Internship Student

Jacquline Siu, Internship Student

Griffin Hartmann, Practical Student

Natalja Mamaeva, Practical Student

Irena Brinkmann, Master Student

 

 

Selected references:

Hüsken, U., Stickney, H.L., Gestri, G., Bianco, I.H., Faro, A., Young, R.M., Roussigne, M., Hawkins, T.A., Beretta, C.A., Brinkmann, I., Paolini A., Jacinto, R., Albadri, S., Dreosti, E., Tsalavouta, M., Schwarz, Q., Cavodeassi, F., Barth, A.K., Wen, L., Zhang, B., Blader, P., Yaksi, E., Poggi, L., Zigman, M., Lin, S., Wilson, S.W., and Carl, M. (2014). Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons. Current Biology 24(19):2217-2227.

Dross, N., Beretta, C.A., Bankhead, P., Carl, M., and Engel, U. (2014). Zebrafish Brain Development Monitored by Long-Term in Vivo Microscopy: A Comparison Between Laser Scanning Confocal and 2-Photon Microscopy. Book chapter in: Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue, Springer Science.

Dreosti, E., Llopis, N.V., Carl, M., Yaksi E., Wilson, S.W. (2014). Left/right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli. Current Biology 24(4):440-5.

Mwafi, N., Beretta, C.A., Paolini, A., Carl, M. (2014). Divergent Wnt8a gene expression in teleosts. Plos One 9(1):e85303.

Beretta, C.A., Dross, N., Bankhead, P., Carl, M. (2013). The ventral habenulae in zebrafish develop in prosomere 2 dependent on Tcf7l2 function. Neural Development 8:19.

Demir, K., Kirsch, N., Beretta, C.A., Erdmann, G., Ingelfinger, D., Moro, E., Argenton, F., Carl, M., Niehrs, C, Boutros, M. (2013). RAB8B is required for activity and caveolar endocytosis of LRP6. Cell Reports 4:1224-34.

Hüsken, U., Carl, M. (2013). The Wnt/beta-catenin signaling pathway establishes neuroanatomical asymmetries and their laterality. Mechanisms of Development 130: 330-335.

McNabb, A., Scott, K., von Ochsenstein, E., Seufert, K., Carl, M. (2012). Donít be afraid to set up your fish facility. Zebrafish 9:120-5.

Beretta, C.A., Dross, N., Gutierrez-Triana, J.A., Ryu, S., Carl, M. (2012). Habenula circuit development: past, present and future. Frontiers in Neuroscience 6:51.

Berns, N., Woichansky, I., Kraft, N., Hüsken, U., Carl, M., Riechmann, V. (2012). “Vacuum-assisted staining”: a simple and efficient method for screening in Drosophila. Development Genes and Evolution 222:113-118.

Beretta, C.A., Brinkmann, I., Carl, M. (2011). All four zebrafish Wnt7 genes are expressed during early brain development. Gene Expression Patterns 11, 277-284.

Wilkinson, C.J., Carl, M. and Harris, W.A. (2009). Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos. BMC Cell Biology 10:17.

Bianco, I.H., Carl, M., Russell, C., Clark, J. and Wilson, S.W. (2008). Brain asymmetry is encoded at the level of axon terminal morphology. Neural Development 3:9.

Carl, M., Bianco, I.H., Bajoghli, B., Aghaallaei, N., Czerny, T., and Wilson, S.W. (2007). Wnt/Axin1/b-catenin signalling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries. Neuron 55: 393-405.

Yokoi, H., Shimada, A., Carl, M., Takashima, S., Kobayashi, D., Narita, T., Jindo, T., Kimura, T., Kitagawa, T., Kage, T., Sawada, A., Naruse, K., Asakawa, S., Shimizu, N., Mitani, H., Shima, A., Tsutsumi, M., Hori, H., Wittbrodt, J., Saga, Y., Ishikawa, Y., Araki, K., and Takeda, H. (2007). Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships. Dev. Biol. 304: 326-337.

 

 

Collaborations:

Francesco Argenton, University Padova, Italy

Patrick Blader and Myriam Roussigne, Université de Toulouse, France

Michael Boutros, DKFZ, Heidelberg, Germany

Filippo Del Bene, Curie Institute, Paris, France

Ulrike Engel, Nikon Imaging Center Heidelberg, Germany

Marika Kapsimali, Ecole Normale Superieure Paris, France

Cheol-Hee Kim, Korea

Suat ÷zbek, Heidelberg University, Germany

Soojin Ryu, MPI Heidelberg, Germany

Veit Riechmann, Heidelberg University, Germany

Sven Sauer, Heidelberg University, Germany

Steffen Scholpp, KIT, Karlsruhe

Christian Schultz, Heidelberg University, Germany

Herbert Steinbeisser laboratory, Heidelberg University, Germany

Rolf-Detlef Treede, Heidelberg University, Germany

Steve Wilson, UCL London, UK

Jochen Wittbrodt, Heidelberg University, Germany

 

 

 

 

Contact:
Last updated: 03.07.2015