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Conditions for dye-filling of sensory neurons in Caenorhabditis elegans
Södertörn University, School of Life Sciences, Molecular biology.
Södertörn University, School of Life Sciences, Molecular biology.
2010 (English)In: Journal of Neuroscience Methods, ISSN 0165-0270, E-ISSN 1872-678X, Vol. 188, no 1, 58-61 p.Article in journal (Refereed) Published
Abstract [en]

Dye-filling is a common method used to stain Caenorhabditis elegans ensory neurons in vivo. While the amphids and phasmids are easy to tain, a subset of sensory neurons, the IL2 neurons, are difficult to tain reproducibly. Here we examined the conditions under which the IL2 eurons take up the lipophilic fluorescent dye DiI. We find that IL2 ye-filling depends on salt concentration, but not osmolarity. Low salt rior and during incubation is important for efficient dye uptake. dditional parameters that affect dye-filling are the speed of shaking uring incubation and the addition of detergents. Our modified ye-filling procedure provides a reliable method to distinguish mutant lleles that stain amphids and phasmids, IL2 neurons, or both. An dditional benefit is that it can also stain the excretory duct. The ethod allows genetic screens to be performed to identify mutants that electively affect only one of the sensory structures or the excretory uct.

Place, publisher, year, edition, pages
2010. Vol. 188, no 1, 58-61 p.
National Category
Neurology
Identifiers
URN: urn:nbn:se:sh:diva-13709DOI: 10.1016/j.jneumeth.2010.02.003ISI: 000276866400009PubMedID: 20149821ScopusID: 2-s2.0-77950460160OAI: oai:DiVA.org:sh-13709DiVA: diva2:462203
Available from: 2011-12-06 Created: 2011-12-06 Last updated: 2016-03-14Bibliographically approved
In thesis
1. Regulatory function of homeobox genes in the development of Caenorhabditis elegans
Open this publication in new window or tab >>Regulatory function of homeobox genes in the development of Caenorhabditis elegans
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The nematode worm Caenorhabditis elegans has been widely used as a genetic model for over 40 years to investigate developmental control genes. In this thesis, I studied the roles of several homeobox genes and a novel RNA binding protein (RBD) in the development of C. elegans to understand the function of these genes in higher organisms. Homeobox genes are transcriptional regulators that are highly conserved in evolution and play important domains in eukaryotes, and genes encoding this domain play roles in a wide variety of post-transcriptional gene regulation processes. In paper I, we characterized a novel protein, RNA binding domain-1 (RBD-1), which is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and subcellular localization from yeast to human. RBD-1 is essential for the development of C. elegans. The RNAi experiments using the cDNA of RBD-1 demonstrated various abnormalities in the C. elegans development, such as defects in morphology (dumpy), incomplete molting, and defective gonadal and vulval development. Animals depleted for RBD-1 arrested mainly at the L1 larval stage. In the course of studying the homeobox genes, we often used the dye-filling assay. It is the simplest method presently used to assay the structural integrity of sensory cilia. In paper II, we optimized conditions, in which reliable staining of the inner labial (IL2) neurons could be obtained, namely in low salt conditions, in the presence of determethod to distinguish mutant alleles that stain amphids and phasmids, and IL2 neurons. Using this assay, we found that a mutation in the POU homeobox gene unc-86 abolished dye-filling in IL2 neurons but not amphids and phasmids. mids. In Paper III, we showed that the LIM homeobox gene ceh-14 was expressed in other sensory neurons and interneurons, including the phasmid neurons and the ALA interneuron, while previously it was shown that ceh-14 is expressed in the AFD neurons and required for thermotaxis behavior in C. elegans. ceh-14 mutant animals displayed defects in dendrite outgrowth of the phasmid neurons, while the ALA interneuron and some tail neurons showed ceh-14 and the paired-like homeobox gene ceh-17 act in the separate pathway to control normal axonal outgrowth of ALA neuron. Overexpression of CEH-14 in the nervous system may titrate out interacting factors, such as LDB-1, which caused developmental defects In paper IV, we investigated the function of four homeobox genes, ceh-6, ceh-26, ttx-1 and ceh-37, in the excretory cell development. We showed that the POU-III class homeobox gene ceh-6, the Prospero class homeobox gene ceh-26, and two otd/Otx family homeobox genes, ceh-37 and ttx-1 formed a regulatory hierarchy required for the development and function of the excretory cell in C. elegans. The excretory cell is required for maintaining osmotic balance and excreting waste products. While ceh-6 has previously been demonstrated to play a role in the excretory cell patterning, we showed here that ceh-26 and ceh-37 are expressed in the excretory cell. ceh-26 mutants arrested in early larval development with defects characteristic for a lack of excretory cell function. Double mutant of the otd/Otx genes ceh-37 and ttx-1 was displaying larval arrest, consistent with the excretory cell dysfunction, which indicates that there is functional redundancy between these two genes. Using mutant alleles and RNAi, we showed that ceh-26::GFP and ceh-37::GFP was down-regulated in ceh-6 mutants. Further, we found that ceh-37::GFP was down-regulated in the ceh-26 genes, such as channel proteins (the target genes ) that are expressed in the excretory cell and found that only a subset of the genes regulated by ceh-6 was also regulated by ceh-37/ttx-1. We mapped the promoter regions of ceh-26 and of the target gene clh-4 to identify putative homeodomain proteins binding sites. Given that these homeobox genes are well conserved in evolution, we may expect that parts of this cascade are also conserved in other organisms.

Place, publisher, year, edition, pages
Stockholm: Karolinska Institutet, 2010. 47 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-29738 (URN)978-91-7457-018-2 (ISBN)
Available from: 2016-03-14 Created: 2016-03-14 Last updated: 2016-03-14Bibliographically approved

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