Rook, Vicky et al. (2021) International Worm Meeting "Sexually dimorphic glia-neuron reprogramming in Caernorhabditis elegans."

Marti I Sabari , Albert, Rook, Vicky, Sammut , Michele, Bonnington, Rachel, Poole, Richard, Lloret-Fernandez , Carla

[International Worm Meeting, 2021]

How fully differentiated cells can retain their neurogenic potential remains a central question in developmental biology. Previously, we described a glia-to-neuron cell fate switch during sexual maturation in the nervous system of male Caenorhabditis elegans; whereby stably differentiated amphid socket (AMso) glial cells in the head undergo asymmetric cell division to self-renew and give rise to the MCM (Mystery Cells of the Male) neurons1. Using forward genetic screening and candidate gene approaches we have identified a particularly exciting class of mutants (Class III) in which the AMso divides but fails to adopt MCM neuronal identity, instead retaining glial marker expression. We also find that mutations in the zinc finger transcription factor lin-48, an ortholog of vertebrate OVO1 and in the proneural bHLH transcription factor hlh-14, a homolog of vertebrate ASCL1 generate a Class III phenotype. In addition, we have isolated two novel Class III mutants; nom-7 and nom-9 and we are currently performing mapping-by-sequencing to identify the causal locus and will present our most recent findings here. Our analysis of Class III genes will help us to elucidate the molecular mechanisms regulating the plasticity underlying this sexually dimorphic AMso-to-MCM transdifferentiation event. 1Sammut, M. et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature 526, 385-390, doi:10.1038/nature15700 (2015).

Janet Duerr et al. (2005) International Worm Meeting "mRNA localization in vivo"

Nanda Filkin, Janet Duerr

[International Worm Meeting, 2005]

We are developing a method to examine the location of specific tagged mRNAs in living adult C. elegans. An elegant technique has been used to examine the distribution of mRNAs in yeast (Bertrand et al. 1998, Beach et al. 1999) and in vertebrate neurons (Rook et al. 2000) in vivo. In this method, cells are simultaneously transformed with two constructs. One construct encodes a fusion between GFP and a viral RNA binding protein (MS2) with a nuclear localization signal (nls) (GFP-MS2-nls). The second construct includes a reporter gene (lacZ), several MS2 binding sites, and the RNA of interest (lacZ-MS2bs-RNAX). When both constructs are present, the GFP-MS2 fusion protein binds to the MS2 binding sites present on the mRNA of the lacZ-MS2bs-RNAX fusion. When the lacZ-MS2bs-RNAX message is present in the cytoplasm, it will bind the GFP-MS2-nls protein and the fluorescence will be detectable in the cytoplasm. If the tagged mRNA is non-uniformly distributed, then the fluorescence will be similarly non-uniform in distribution. Work in a number of organisms has identified a number of localized mRNAs. In most cases, the sequences necessary for sub-cellular localization are present in the 3 untranslated region (3-UTR). If these mRNAs are also localized in C. elegans, then one would expect conservation of the localization sequences in the 3UTR. We have used published information on subcellular mRNA localization as well as conservation of 3-UTR sequences in C. elegans vs. C. briggsae to identify 3UTRs that may be involved in mRNA localization. Initial constructs include lacZ-MS2bs-RNAX where RNAX is the 3-UTR from actin, myosin (unc-54), Calcium-calmodulin dependent Kinase II (unc-43), or, as a control, let-858. Due to difficulties in expression and localization with our initial GFP constructs, we have also had to vary the location and number of nls sequences in our GFP-MS2-nls constructs. We will report on our progress in identifying and monitoring localized mRNAs using this system.

Janet Duerr (2003) International Worm Meeting "Localization of mRNAs in vivo in C. elegans."

Janet Duerr

[International Worm Meeting, 2003]

We are developing a system to examine the location of specific tagged mRNAs in living adult C. elegans. Many studies in C. elegans and other organisms have used in situ hybridization to examine the subcellular distribution of mRNAs in larger cells or in culture, but detection of localized mRNAs in smaller cells has been problematic (Lyczak et al. 2002). Recently, an elegant technique has been developed to examine the distribution of mRNAs in yeast (Bertrand et al. 1998, Beach et al. 1999) and in vertebrate neurons (Rook et al. 2000) in vivo. In this method, cells are simultaneously transformed with two constructs. One construct encodes a fusion between GFP and a viral RNA binding protein (MS2) with a nuclear localization signal (nls) (GFP-MS2-nls). The second construct includes a reporter gene (lacZ), several MS2 binding sites, and the RNA of interest (lacZ-MS2bs-RNAX). The interactions of the GFP fusion protein with the lacZ fusion mRNA are monitored. Any free GFP-MS2-nls protein should be confined to the nucleus. When the lacZ-MS2bs-RNAX message is present in the cytoplasm, it will bind the GFP-MS2-nls protein and the fluorescence will be detectable in the cytoplasm. If the tagged mRNA is non-uniformly distributed, then the fluorescence will be similarly non-uniform in distribution. We are adapting this technique for use in C. elegans by modifying some of the Andy Fire's transformation constructs to include the GFP-MS2 or lacZ-MS2bs sequences. Initial plans are to test the constructs with RNA sequences that are known to be non-uniformly distributed in early embryos. But, as a neuro lab, our long range goal is to refine these tools so that it is possible to examine mRNA localization in the nervous system. In vertebrate neurons, specific subcellular localization of mRNAs encoding a number of proteins, including cytoskeleton proteins and regulatory proteins such as CaMKII, has been noted. Localized protein synthesis has been implicated in the regulation of neuronal polarity and neuronal plasticity (Rogiers et al. 2003). By developing tools for examining mRNA localization in C. elegans, we hope to be able to identify genes and proteins that are important for regulating mRNA localization in adult (neuronal) cells.