Lab
Jontes Lab
|
|
Statement of Research Interest
RESEARCH AREA:
* development of the vertebrate central nervous system, with a particular emphasis on the mechanisms involved in synapse and circuit formation.
RESEARCH DESCRIPTION:
The vertebrate brain is immensely complex, yet is wired with an equally impressive precision. A fundamental goal in neuroscience is to understand the mechanisms that balance the huge number of cells with the precision and intricacy with which they are connected. My lab is interested in the development of the vertebrate central nervous system, with a particular emphasis on the mechanisms involved in synapse and circuit formation. We study the roles played by cell-adhesion molecules in synapse formation and assembly, both in terms of potential roles in specificity and cell-cell recognition and in the detailed roles these molecules play in assembling synaptic junctions. Our present focus is on members of the cadherin superfamily, including the classical cadherins and the protocadherins.
Much of our work relies on in vivo 2-photon laser-scanning time-lapse microscopy in living zebrafish embryos. 2-photon microscopy offers several advantages over conventional confocal imaging: 1) reduced photodamage and photobleaching, 2) increased fluorescence collection efficiency and 3) reduced light scattering of IR light allows deeper imaging in tissue. Zebrafish embryos are well-suited for time-lapse imaging, as they are transparent and develop rapidly, allowing the dynamics of early developmental events to be imaged with high spatial and temporal resolution.
Clustered Protocadherins:
The protocadherins are a large group of neuronal cell-surface receptors (~100 in zebrafish) that have been proposed to play a role in selecting appropriate synaptic partners. They are characterized both by the large number of isoforms, as well as the diversity of their extracellular domains. In zebrafish, there are four protocadherin clusters: an a and g cluster present on chromosome 10 and a second pair of a and g clusters present on chromosome 14. Each cluster consists of a tandem array of variable exons, each encoding an entire extracellular domain and a single-pass transmembrane domain. These are each spliced to three constant exons that, together, encode a short, common cytoplasmic domain. We are using BAC (Bacterial Artificial Chromosome) engineering technology (recombineering) to investigate the roles of these genes in neural development and synapse formation.
Classical Cadherins:
1) N-cadherin. We are investigating the role of N-cadherin in early synapse assembly, both in pre- and postsynaptic cells. Using two-photon time-lapse microscopy in conjunction with the expression of gene fusions of N-cadherin with genetically-encoded fluorescent proteins, we are characterizing the dynamics of N-cadherin within developing neurons, as well as its involvement in synapse formation and stabilization.
2) Type II classical cadherins. We have begun to look at type II cadherins, and their roles in CNS circuit formation, using BAC engineering and time-lapse microscopy
TECHNIQUES AND MODELS:
* Imaging - Two-photon laser-scanning time-lapse microscopy, image processing, and image analysis
* Electron Microscopy - Ultra-thin sectioning, ultrastructural analysis, and retrospective localization of recombinant transgenes
* Cell Biology - Transfection of cultured cells, immunocytochemistry, adhesion assays, Western blotting, and antibody production
* Molecular Biology - Cloning of genes, GFP-tagging, expression, site-directed mutagenesis, isolation of promoters, BAC engineering
* Zebrafish model system - transient gene expression, production of transgenics, antisense morpholino knockdown of gene expression
* development of the vertebrate central nervous system, with a particular emphasis on the mechanisms involved in synapse and circuit formation.
RESEARCH DESCRIPTION:
The vertebrate brain is immensely complex, yet is wired with an equally impressive precision. A fundamental goal in neuroscience is to understand the mechanisms that balance the huge number of cells with the precision and intricacy with which they are connected. My lab is interested in the development of the vertebrate central nervous system, with a particular emphasis on the mechanisms involved in synapse and circuit formation. We study the roles played by cell-adhesion molecules in synapse formation and assembly, both in terms of potential roles in specificity and cell-cell recognition and in the detailed roles these molecules play in assembling synaptic junctions. Our present focus is on members of the cadherin superfamily, including the classical cadherins and the protocadherins.
Much of our work relies on in vivo 2-photon laser-scanning time-lapse microscopy in living zebrafish embryos. 2-photon microscopy offers several advantages over conventional confocal imaging: 1) reduced photodamage and photobleaching, 2) increased fluorescence collection efficiency and 3) reduced light scattering of IR light allows deeper imaging in tissue. Zebrafish embryos are well-suited for time-lapse imaging, as they are transparent and develop rapidly, allowing the dynamics of early developmental events to be imaged with high spatial and temporal resolution.
Clustered Protocadherins:
The protocadherins are a large group of neuronal cell-surface receptors (~100 in zebrafish) that have been proposed to play a role in selecting appropriate synaptic partners. They are characterized both by the large number of isoforms, as well as the diversity of their extracellular domains. In zebrafish, there are four protocadherin clusters: an a and g cluster present on chromosome 10 and a second pair of a and g clusters present on chromosome 14. Each cluster consists of a tandem array of variable exons, each encoding an entire extracellular domain and a single-pass transmembrane domain. These are each spliced to three constant exons that, together, encode a short, common cytoplasmic domain. We are using BAC (Bacterial Artificial Chromosome) engineering technology (recombineering) to investigate the roles of these genes in neural development and synapse formation.
Classical Cadherins:
1) N-cadherin. We are investigating the role of N-cadherin in early synapse assembly, both in pre- and postsynaptic cells. Using two-photon time-lapse microscopy in conjunction with the expression of gene fusions of N-cadherin with genetically-encoded fluorescent proteins, we are characterizing the dynamics of N-cadherin within developing neurons, as well as its involvement in synapse formation and stabilization.
2) Type II classical cadherins. We have begun to look at type II cadherins, and their roles in CNS circuit formation, using BAC engineering and time-lapse microscopy
TECHNIQUES AND MODELS:
* Imaging - Two-photon laser-scanning time-lapse microscopy, image processing, and image analysis
* Electron Microscopy - Ultra-thin sectioning, ultrastructural analysis, and retrospective localization of recombinant transgenes
* Cell Biology - Transfection of cultured cells, immunocytochemistry, adhesion assays, Western blotting, and antibody production
* Molecular Biology - Cloning of genes, GFP-tagging, expression, site-directed mutagenesis, isolation of promoters, BAC engineering
* Zebrafish model system - transient gene expression, production of transgenics, antisense morpholino knockdown of gene expression
Lab Members