Katharyn Schmitt et al. (2006) Development & Evolution Meeting "Cell Architecture of the Distal Tip Cell"

Katharyn Schmitt, Dana Byrd, Judith Kimble

[Development & Evolution Meeting, 2006]

A stem cell niche is required for maintenance of stem cells. Many niches have been identified in a variety of organisms and tissues (Li and Xie, 2005). For example, the hub cells provide the niche for germline stem cells in Drosophila, and osteoblasts do so for hematopoietic stem cells in mammals. Two typical functions of a cellular niche are signaling to the stem cells and anchoring of the stem cells within the niche. We have focused on analysis of the distal tip cell (DTC), which forms the niche for germline stem cells in C. elegans. The DTC signals to the germline stem cells by Notch signaling, and it appears to anchor them by cellular processes that embrace the distal-most germ cells within the mitotic region. Our goal is to study the relationship between the DTC cellular architecture and its function as a stem cell niche. To visualize DTC architecture, we are building a panel of fluorescent protein markers that localize to major DTC cellular compartments. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. We will present our progress at the meeting.

Dana T. Byrd et al. (2007) International Worm Meeting "Interactions between the germline stem cells and their niche."

Dana T. Byrd, Audra Amasino, Katharyn Schmitt, Judith Kimble

[International Worm Meeting, 2007]

Stem cells are defined by their unlimited or prolonged capacity for self-renewal and ability to produce at least one differentiated cell type. Their decision between self-renewal and differentiation is governed by both intrinsic signals and extrinsic stimuli from the surrounding microenvironment, or niche. The niche for C. elegans germline stem cells is provided by the distal tip cell (DTC) since it is both necessary and sufficient for maintenance of cells in the mitotic region. The DTC controls germline stem cells by Notch signaling (Crittenden et al. 2003). Specifically, germ cells in the mitotic region express the GLP-1 Notch receptor and respond to the LAG-2 DSL ligand expressed by the DTC. However, there may be additional factors required in the DTC for LAG-2 signaling and maintenance of germline stem cells. We have taken several approaches to further dissect the molecular and cellular properties of the DTC that enable it to function as a niche for the germline stem cells. Using a candidate gene approach, we are screening for genes required in the DTC for fertility by tissue-specific RNAi. To both narrow down the list of candidate genes and to determine the gene expression profile of the DTC, we have used a microarray approach, enriching for DTC transcripts by immunoprecipitation of Flag-tagged PAB-1 (poly-A binding protein) with associated RNAs (Roy et al. 2002). To investigate the cellular characteristics of the DTC that may contribute its role as a stem cell niche, we made a panel of fluorescent protein markers to examine the subcellular structure of the DTC in vivo. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. Using a membrane marker, we found that contact between the DTC niche and germ cells in the mitotic region is extensive and dynamic. The DTC extends membrane processes that surround adjacent germ cells. The intimate contact between the niche and germ cells may provide a mechanism to physically anchor the distal-most germ cells within the niche and/or provide more localized signaling to maintain the germline stem cells.

Dana Byrd et al. (2008) Development & Evolution Meeting "Subcellular Architecture of the Germline Stem Cell Niche"

Karla Knobel, Judith Kimble, Katharyn Schmitt, Dana Byrd, Audra Amasino

[Development & Evolution Meeting, 2008]

A stem cell's decision between self-renewal and differentiation is governed by extrinsic stimuli from the surrounding microenvironment, or niche, as well as by intrinsic cues. In C. elegans, the distal tip cell (DTC) provides the niche for germline stem cells and maintains a "mitotic region" by Notch signaling (e.g. Kimble and Crittenden 2007). DTC number and dosage of the LAG-2 DSL ligand, which is expressed by the DTC, can affect the number of germ cells in the mitotic region (Dyan Vogel and Myon-Hee Lee). To investigate DTC niche function, we made a panel of fluorescent protein markers to visualize its subcellular structure in vivo. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. Using membrane markers, we found that contact between the DTC niche and germ cells in the mitotic region is extensive. The DTC extends membrane processes that surround adjacent germ cells. The intimate contact between the niche and germ cells may provide a mechanism to physically anchor the distal-most germ cells within the niche and/or provide more localized signaling to maintain the germline stem cells. With a combination of DTC, sheath cell, and germ cell markers, we are examining the relationship between the extent of niche contact and the region of cells with stem cell potential. We are also using tissue-specific RNAi to test which genes are required in the DTC for regulation of germline stem cells.

Karen Erbguth et al. (2009) European Worm Neurobiology Meeting "Applying and combining optogenetic and imaging tools in the C.elegans nervous system."

Alexander Gottschalk, Karen Erbguth, Martin Brauner

[European Worm Neurobiology Meeting, 2009]

Stimuli are evaluated and an animal responds to them with an appropriate behavior. How behavior is guided by neuronal circuits is one of the most fascinating questions approached by neuroscience today. C. elegans provides a good model system for the investigation of neuronal networks, due to its compact nervous system and rather easy access to genetic manipulation. Most physical connections between neurons have been characterized using EM reconstruction, a big step towards an extensive elucidation of the functional connections between these neurons, e.g. by mutant studies, electrophysiology or calcium imaging, and linking them to the animals. behavior. Here we want to combine the method of calcium imaging with activity-regulating light-activated ion channels. This should provide a basic and fast method in order to facilitate an easy and widespread investigation of neuronal networks, which ideally can also be correlated to behavior. Depolarizing or hyperpolarizing neurons with light-sensitive proteins, namely channelrhodopsin or halorhodopsin, and expressing genetically encoded Ca2+ sensors (GECIs) like G-CaMPs or cameleons postsynaptically to monitor the resulting effect on neuronal activity, could be the easiest and minimally invasive way towards this. Here, different approaches in spatial and spectral separation are presented, to overcome the major limitation in combining these tools, i.e. the overlapping excitation spectra. The challenge of specific expression also is addressed within our group (see posters by Christian Schultheis and Cornelia Schmitt). To be able to test our systems in terms of functionality and to have a behavioral readout as a control, we are currently applying our techniques on testing paradigms like mini-networks consisting of just two neurons.