Ongoing Projects

The role of slb/wnt11 in regulating convergent extension movements during zebrafish gastrulation
Convergent extension describes the movement of cells within the gastrula to the dorsal side followed by the redistribution of these cells along the future anterior-posterior axis of the embryo. In zebrafish, several mutants have been identified exhibiting defects in convergence and extension movements during gastrulation (Hammerschmidt et al., 1996; Solnica-Krezel et al., 1996; Heisenberg et al., 1996; Marlow et al., 1998). We have recently shown that one of these mutants, silberblick (slb), encodes wnt11, which is expressed within sub-domains of the paraxial mesoderm and neuroectoderm of the gastrula (Heisenberg and Nüsslein-Volhard, 1997; Heisenberg et al., 2000). We further showed that Slb function is required within these paraxial structures in a cell-non-autonomous fashion and that slb/wnt11 signalling shares components with the signalling cascade required for establishment of planar polarity in Drosophila. However, it remains to be established what the precise functions of Slb/Wnt11 are in regulating cell movements during convergent extension and which other genes are required for Slb/Wnt11 function. To address these questions we are undertaking two main sets of experiments:

1. We are analysing in detail the movements and morphologies of single cells within different regions of the gastrula using recently developed cell-tracking software (Concha and Adams, 1998). To follow single cells during the course of gastrulation we are taking high-magnification time-lapse movies of groups of cells within the different germ layers. These movies are then processed by either tracking single cells and subsequently analysing their movements using cell-tracking software or by capturing their morphology at different time points and analysing morphological changes using appropriate software. Initially, we are focussing on wild-type embryos but will subsequently extend these studies to mutant embryos exhibiting defective gastrulation movements such as slb, trilobite (tri) and knypek (kny) (Hammerschmidt et al., 1996; Solnica-Krezel et al., 1996; Heisenberg et al., 1996; Marlow et al., 1998). Eventually, this will allow us to determine how cell movements differ along the anterior-posterior and medial-lateral axis of the gastrula and between the different germ layers (mesendodermal, ectodermal and yolk syncytial cell layers). In the long term, we would like to understand how the regional patterns of cell movements generate global tissue morphogenesis during gastrulation and how these results compare to existing models for cell movements during gastrulation (Keller, 1986).

figure: (A) low magnification image of a shield stage zebrafish embryo showing the region used for image analysis (red square). (B) high magnification timelapse movie (13,1 MB) of paraxial mesodermal cells within the region high lightened in A between 60% and 80% epiboly.

2. We are performing a genetic screen with the aim to identify loci/genes modifying the slb mutant phenotype. By searching for modifiers of the slb mutant phenotype we intend to isolate genes interacting directly or indirectly with the Wnt11 signal transduction pathway. All existing and potential new mutants will be phenotypically analysed as described above (1.). We will also test if the enhancers/suppressors exhibit any phenotype on their own by analysing both potential dominant and recessive phenotypes of the induced mutation in the presence or absence of the slb mutation. Double (and possible triple) mutant embryos between the slb enhancers/suppressors and other known loci involved in convergent extension such as tri and kny will be raised and the mutant phenotype characterised to reveal any potential interaction between these genes.

* Concha, M. L., and Adams, R. J. (1998)
Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis.
Development 125, 983-94

* Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P., Jiang, Y. J., Kelsh, R. N., Odenthal, J., Warga, R. M., and Nusslein-Volhard, C. (1996)
Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.
Development 123, 143-51

* Heisenberg, C. P., Brand, M., Jiang, Y. J., Warga, R. M., Beuchle, D., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J., and Nusslein-Volhard, C. (1996)
Genes involved in forebrain development in the zebrafish, Danio rerio.
Development 123, 191-203

* Heisenberg, C. P., and Nusslein-Volhard, C. (1997)
The function of silberblick in the positioning of the eye anlage in the zebrafish embryo.
Developmental Biology 184, 85-94

* Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000)
Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation.
Nature 405, 76-81

* Keller, R. E. (1986)
The cellular basis of amphibian gastrulation.
Developmental Biology 2, 241-327

* Marlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S. C., Abbas, L., Weaver, M., Driever, W., and Solnica-Krezel, L. (1998)
Functional interactions of genes mediating convergent extension, knypek and trilobite, during the partitioning of the eye primordium in zebrafish.
Developmental Biology 203, 382-99

* Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E., Rangini, Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., and Driever, W. (1996)
Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish.
Development 123, 67-80

Future plans
In the future, we would like to establish methods that allow us to visualise and identify more intracellular components in vivo that are involved in cell polarisation and migration such as cytoskeletal elements, plasma membrane domains and vesicular structures. For doing so, we plan to establish transgenic lines that express proteins fused to GFP or dsRed and to improve our imaging techniques. With the help of those tools we hope to be able to link signalling pathways with a morphogenetic function to specific intracellular events. Eventually this should provide insight into the target mechanisms mediating the morphogenetic activity of those pathways.

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Carl-Philipp Heisenberg research focus group leader projects publications Ongoing Projects The role of slb/wnt11 in regulating convergent extension movements during zebrafish gastrulation Convergent extension describes the movement of cells within the gastrula to the dorsal side followed by the redistribution of these cells along the future anterior-posterior axis of the embryo. In zebrafish, several mutants have been identified exhibiting defects in convergence and extension movements during gastrulation (Hammerschmidt et al., 1996; Solnica-Krezel et al., 1996; Heisenberg et al., 1996; Marlow et al., 1998). We have recently shown that one of these mutants, silberblick (slb), encodes wnt11, which is expressed within sub-domains of the paraxial mesoderm and neuroectoderm of the gastrula (Heisenberg and Nüsslein-Volhard, 1997; Heisenberg et al., 2000). We further showed that Slb function is required within these paraxial structures in a cell-non-autonomous fashion and that slb/wnt11 signalling shares components with the signalling cascade required for establishment of planar polarity in Drosophila. However, it remains to be established what the precise functions of Slb/Wnt11 are in regulating cell movements during convergent extension and which other genes are required for Slb/Wnt11 function. To address these questions we are undertaking two main sets of experiments: 1. We are analysing in detail the movements and morphologies of single cells within different regions of the gastrula using recently developed cell-tracking software (Concha and Adams, 1998). To follow single cells during the course of gastrulation we are taking high-magnification time-lapse movies of groups of cells within the different germ layers. These movies are then processed by either tracking single cells and subsequently analysing their movements using cell-tracking software or by capturing their morphology at different time points and analysing morphological changes using appropriate software. Initially, we are focussing on wild-type embryos but will subsequently extend these studies to mutant embryos exhibiting defective gastrulation movements such as slb, trilobite (tri) and knypek (kny) (Hammerschmidt et al., 1996; Solnica-Krezel et al., 1996; Heisenberg et al., 1996; Marlow et al., 1998). Eventually, this will allow us to determine how cell movements differ along the anterior-posterior and medial-lateral axis of the gastrula and between the different germ layers (mesendodermal, ectodermal and yolk syncytial cell layers). In the long term, we would like to understand how the regional patterns of cell movements generate global tissue morphogenesis during gastrulation and how these results compare to existing models for cell movements during gastrulation (Keller, 1986). figure: (A) low magnification image of a shield stage zebrafish embryo showing the region used for image analysis (red square). (B) high magnification timelapse movie (13,1 MB) of paraxial mesodermal cells within the region high lightened in A between 60% and 80% epiboly. 2. We are performing a genetic screen with the aim to identify loci/genes modifying the slb mutant phenotype. By searching for modifiers of the slb mutant phenotype we intend to isolate genes interacting directly or indirectly with the Wnt11 signal transduction pathway. All existing and potential new mutants will be phenotypically analysed as described above (1.). We will also test if the enhancers/suppressors exhibit any phenotype on their own by analysing both potential dominant and recessive phenotypes of the induced mutation in the presence or absence of the slb mutation. Double (and possible triple) mutant embryos between the slb enhancers/suppressors and other known loci involved in convergent extension such as tri and kny will be raised and the mutant phenotype characterised to reveal any potential interaction between these genes. * Concha, M. L., and Adams, R. J. (1998) Oriented cell divisions and cellular morphogenesis in the zebrafish gastrula and neurula: a time-lapse analysis. Development 125, 983-94 * Hammerschmidt, M., Pelegri, F., Mullins, M. C., Kane, D. A., Brand, M., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Heisenberg, C. P., Jiang, Y. J., Kelsh, R. N., Odenthal, J., Warga, R. M., and Nusslein-Volhard, C. (1996) Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio. Development 123, 143-51 * Heisenberg, C. P., Brand, M., Jiang, Y. J., Warga, R. M., Beuchle, D., van Eeden, F. J., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. A., Kelsh, R. N., Mullins, M. C., Odenthal, J., and Nusslein-Volhard, C. (1996) Genes involved in forebrain development in the zebrafish, Danio rerio. Development 123, 191-203 * Heisenberg, C. P., and Nusslein-Volhard, C. (1997) The function of silberblick in the positioning of the eye anlage in the zebrafish embryo. Developmental Biology 184, 85-94 * Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000) Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81 * Keller, R. E. (1986) The cellular basis of amphibian gastrulation. Developmental Biology 2, 241-327 * Marlow, F., Zwartkruis, F., Malicki, J., Neuhauss, S. C., Abbas, L., Weaver, M., Driever, W., and Solnica-Krezel, L. (1998) Functional interactions of genes mediating convergent extension, knypek and trilobite, during the partitioning of the eye primordium in zebrafish. Developmental Biology 203, 382-99 * Solnica-Krezel, L., Stemple, D. L., Mountcastle-Shah, E., Rangini, Z., Neuhauss, S. C., Malicki, J., Schier, A. F., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., and Driever, W. (1996) Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish. Development 123, 67-80 Future plans In the future, we would like to establish methods that allow us to visualise and identify more intracellular components in vivo that are involved in cell polarisation and migration such as cytoskeletal elements, plasma membrane domains and vesicular structures. For doing so, we plan to establish transgenic lines that express proteins fused to GFP or dsRed and to improve our imaging techniques. With the help of those tools we hope to be able to link signalling pathways with a morphogenetic function to specific intracellular events. Eventually this should provide insight into the target mechanisms mediating the morphogenetic activity of those pathways.
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Carl-Philipp Heisenberg

Ongoing Projects