Final Activity Report Summary - FISH ING (Identification and in vivo functional analysis of novel cell surface receptor-ligand pairs)
The establishment of a complex vertebrate body plan during embryonic development requires intricate cell-cell communication events. This complexity and the need for coordination between cells are reflected by the human brain, which contains more than 1 011 nerve cells, each of them connecting with an average of 103 partners.
During the development of an animal, intercellular communication is fundamental for processes such as the specification and migration of cells, the path finding of the long and thin projections of nerve cells, namely axons, their target recognition and for their connection to other nerve cells, i.e. for synapse formation. Therefore, an understanding of these communication events between cells emerged as a central question in developmental biology. In addition, when these communication events are erroneous, they can lead to common human diseases like cancer.
Inter-cellular signals are received and sent by proteins at the cell-surface and are often referred to as receptor-ligand pairs. Analysis of the human genome indicates that approximately 20 % of our genes encode such cell surface and secreted proteins; however, because of the properties of these proteins, only a fraction of them have known binding partners.
We set out to identify such new cell-surface receptor-ligand pairs by using a new systematic approach involving an avidity-based extracellular interaction screen assay (AVEXIS), which was developed in the laboratory. This assay allowed for the detection of transient physical binary interactions of extracellular proteins.
We took a genome-wide family-based approach to this problem and constructed a library consisting of 102 proteins of the immunoglobulin superfamily (IgSF) and 67 proteins belonging to the leucine rich repeat superfamily (LRR-SF). These two protein families were selected because they contained examples of interacting pairs not only within each family, but also between the two families. In addition, both families had many members which were expressed in the central nervous system. Famous examples of cross-talk between these two families included receptor / ligand pairs involved in guidance of axons (e.g. Slit / Robo) and inhibition of neuronal regeneration (NgR:MAG).
Our ultimate aim was to understand the function of identified interactions in vivo, in the context of a living organism. To achieve this goal, we used zebrafish proteins for the interaction assay and screened 11 584 possible unique binding events. We identified 111 interactions involving 47 proteins. The great majority of interactions in the resulting network were novel.
We determined the expression patterns of the corresponding genes in zebrafish embryos as a first step to understand the in vivo functions of these interactions. The IgSF/LRR cell-surface receptor interaction network we identified was highly enriched for proteins expressed in the brain and contained several proteins suggested to be linked to neurological disorders and diseases like schizophrenia and Alzheimer's disease. We identified new ligands for known receptors which were involved in the navigation of axons and cell migration and entirely novel receptor-ligand pairs whose complementary expression patterns suggested function in neuronal development and wiring of the brain.
By applying a gene-knockdown approach in zebrafish, for both members of a novel receptor-ligand pair, we obtained a strong indication that this interaction was required for nerve-cell migration and axonal pathfinding.
In addition, in collaboration with Derek Stemple's laboratory at the Wellcome Trust Sanger Institute, we started to generate zebrafish which lack functional copies (TILLING mutants) for several genes for which we identified novel interactions. The functional analysis of these TILLING mutants will help to gain insight in developmental processes and will give us a better understanding of the molecular causes of human neurological disorders and diseases.
During the development of an animal, intercellular communication is fundamental for processes such as the specification and migration of cells, the path finding of the long and thin projections of nerve cells, namely axons, their target recognition and for their connection to other nerve cells, i.e. for synapse formation. Therefore, an understanding of these communication events between cells emerged as a central question in developmental biology. In addition, when these communication events are erroneous, they can lead to common human diseases like cancer.
Inter-cellular signals are received and sent by proteins at the cell-surface and are often referred to as receptor-ligand pairs. Analysis of the human genome indicates that approximately 20 % of our genes encode such cell surface and secreted proteins; however, because of the properties of these proteins, only a fraction of them have known binding partners.
We set out to identify such new cell-surface receptor-ligand pairs by using a new systematic approach involving an avidity-based extracellular interaction screen assay (AVEXIS), which was developed in the laboratory. This assay allowed for the detection of transient physical binary interactions of extracellular proteins.
We took a genome-wide family-based approach to this problem and constructed a library consisting of 102 proteins of the immunoglobulin superfamily (IgSF) and 67 proteins belonging to the leucine rich repeat superfamily (LRR-SF). These two protein families were selected because they contained examples of interacting pairs not only within each family, but also between the two families. In addition, both families had many members which were expressed in the central nervous system. Famous examples of cross-talk between these two families included receptor / ligand pairs involved in guidance of axons (e.g. Slit / Robo) and inhibition of neuronal regeneration (NgR:MAG).
Our ultimate aim was to understand the function of identified interactions in vivo, in the context of a living organism. To achieve this goal, we used zebrafish proteins for the interaction assay and screened 11 584 possible unique binding events. We identified 111 interactions involving 47 proteins. The great majority of interactions in the resulting network were novel.
We determined the expression patterns of the corresponding genes in zebrafish embryos as a first step to understand the in vivo functions of these interactions. The IgSF/LRR cell-surface receptor interaction network we identified was highly enriched for proteins expressed in the brain and contained several proteins suggested to be linked to neurological disorders and diseases like schizophrenia and Alzheimer's disease. We identified new ligands for known receptors which were involved in the navigation of axons and cell migration and entirely novel receptor-ligand pairs whose complementary expression patterns suggested function in neuronal development and wiring of the brain.
By applying a gene-knockdown approach in zebrafish, for both members of a novel receptor-ligand pair, we obtained a strong indication that this interaction was required for nerve-cell migration and axonal pathfinding.
In addition, in collaboration with Derek Stemple's laboratory at the Wellcome Trust Sanger Institute, we started to generate zebrafish which lack functional copies (TILLING mutants) for several genes for which we identified novel interactions. The functional analysis of these TILLING mutants will help to gain insight in developmental processes and will give us a better understanding of the molecular causes of human neurological disorders and diseases.