Kirthi Reddy et al. (2001) International C. elegans Meeting "Special handling for the X chromosome in meiosis"

Kirthi Reddy, Gillian Stanfield, Monica Colaiacovo, Stuart Kim, Valerie Reinke, Linwah Yip, Anne Villeneuve

[International C. elegans Meeting, 2001]

During prophase of meiosis I, homologous chromosomes must locate their partners, associate tightly along their lengths, and undergo recombination to ensure proper segregation at the first meiotic division. We are interested in the mechanisms that underly these processes, and have used a functional genomics approach to screen for components of the meiotic machinery in C. elegans. Microarray analysis has identified a set of genes whose expression is enriched in the C. elegans germline relative to somatic tissues (1). We used RNAi to inhibit the function of a subset of these genes that had expression profiles similar to those of known meiosis genes. For a high percentage of the genes analyzed, RNAi elicited germline phenotypes including meiotic defects, gonadogenesis defects, and abnormal chromatin in the mitotic zone of the germline. Among the genes for which RNAi caused a defect in meiotic chromosome segregation, one gene, T09E8.2, had a property that set it apart. Specifically, the relative frequencies of inviable embryos and males produced by affected animals suggested that depletion of T09E8.2 activity preferentially affected the X chromosome. It is not altogether surprising that the X chromosome could be more susceptible to perturbations that affect chromosome behavior, since the X has several properties that distinguish it from the autosomes. For example, each of the autosomes has a central gene-rich region in which recombination is suppressed, whereas both physical density of genes and recombination are more uniformly distributed on the X (2). Further, germline-enriched genes are notably absent from the X chromosome (1). X chromosomes also exhibit distinct chromatin properties during meiotic prophase (3,4). T09E8.2 therefore provides an entry point for understanding how general meiotic mechanisms intersect with special properties of sex chromosomes. The T09E8.2 RNAi phenotype was reminiscent of the meiotic defects we had previously observed in him-17 mutants. The meiotic segregation of both the autosomes and the X chromosome are affected in him-17 mutants, but the X chromosomes are much more strongly affected. There is a high frequency of achiasmate chromosomes in oocyte nuclei, presumably reflecting a defect in a mechanism that ensures crossing over. Interestingly, there is an alteration in both the frequency and distribution of crossovers along the X chromosome in him-17 mutants: recombination levels are elevated in a small region at the left end but are reduced along the remainder of the chromosome, suggesting a defect in associations between X chromosomes. Our genetic mapping data had localized him-17 to a 2 Mb interval containing T09E8.2. We therefore sequenced T09E8.2 in three independently isolated him-17 mutants and identified mutations in all three alleles. This demonstrates how the data from our RNAi screen can greatly aid in the cloning of genes identified by forward genetic screens by reducing the need for extensive mapping of mutants. We are currently investigating the role of HIM-17 during meiotic prophase. Initial analysis using fluorescence in situ hybridization indicates that in him-17 mutants, X chromosomes can initially pair but this paired state may not be stabilized. We are examining different chromosome regions for premature loss of pairing, and are exploring whether loss of pairing might reflect premature dissociation of synaptonemal complex proteins. 1. Reinke et al. (2000) Mol Cell 6(3):605-16. 2. Barnes et al. (1995) Genetics 141(1):159-79. 3. Goldstein and Slaton (1982) Chromosoma 84(4):585-97. 4. A. Dernburg and V. Reinke, personal communication.

Desse, V. et al. (2019) International Worm Meeting "SAX-7/L1CAM in the maintenance of neuronal architecture."

Perrat, P., Blanchette, C., Desse, V., Benard, C.

[International Worm Meeting, 2019]

Whereas remarkable advances have been made in understanding neuronal development, the mechanisms that maintain the nervous system architecture lifelong are poorly understood. How does the nervous system established during embryogenesis subsequently maintain its integrity throughout life, despite the animal's growth, the addition of new neurons, maturation processes, body movements, and aging? The evolutionarily conserved gene sax-7, homologous to mammalian L1CAM, is one of the factors required for maintaining the structural integrity of ganglia and fascicles in C. elegans1. Thus, sax-7 not only contributes to nervous system development (roles in guidance and fasciculation of a subset of developing neurites)2, but is also required for maintaining the organization of neural architecture after its initial development. Indeed, some neural structures that initially develop normally become disorganized in animals lacking the function of sax-7. With heat shock assays, we show that expression of sax-7S(+) during larval stages is sufficient to rescue the maintenance defects of sax-7 mutants that were devoid of functional sax-7 during nervous system development. We are characterizing a true null allele (generated using CRISPR/Cas9), as well as the expression pattern of a functional SAX-7S::sfGFP (insertion in the endogenous locus using CRISPR/Cas9), and we find that SAX-7S is present in neurons, at the plasma membrane. We are exploring the structure-function relationship of SAX-7S, a transmembrane protein harboring extracellular Ig and FnIII domains, in the context of maintenance of the nervous system architecture. A conditional knockout of L1CAM in the adult mouse brain results in behavioral and learning defects, indicating that L1CAM plays a post-developmental role in mammals as well. Our studies are expected to help understand the role of SAX-7/L1CAM in conserved molecular mechanisms ensuring neuronal maintenance, which may go awry in neurodegenerative conditions. 1 Pocock, Benard et al 2008; Sasakura et al 2005; Wang, Chen et al 2005; Zallen et al 1999; Ramirez-Suarez et al 2019 2 Diaz-Balzac et al 2015; Salzberg et al 2013; Yip and Heiman 2018, Dong et al 2013; Zhu et al 2017

Gattegno, Tamar et al. (2013) International Worm Meeting "Menorah-menorah auto-fusion as a mechanism of PVD response to injury."

Gattegno, Tamar, Podbilewicz, Benjamin, Oren, Meital

[International Worm Meeting, 2013]

The PVDs are two nociceptors localized along C. elegans lateral sides, enabling them to sense strong mechanical stimuli such as pain, temperature and posture. The PVDs develop through a dynamic process which involves extension and retraction of dendrite branches. Though we now better understand C. elegans PVD development, very little is known about PVD regeneration and degeneration following injury. In order to study the genetic pathway guiding PVD response to injury, we use laser microsurgery to severe the PVD and live imaging of GFP reporters. We found that when the PVDs are injured at the 1ry branch, AFF-1 mediated fusion occurs between the terminal branches to bypass the site of injury, followed by EFF-1 mediated pruning and arbor simplification. Degeneration of the distal stump occurred in animals that failed to undergo reconnection of the 1ry severed branch or menorah-menorah fusion. We used Kaede photo-convertible fluorescent protein expressed in the PVD to support the observation that injured PVD has reconnected through auto-fusion. We severed the PVD 1ry branch, recovered the worms and detected reconnection after 6-12 hours. The PVD cell body of recovered animals was than photo-converted from green to red and we analyzed the transfer of the Red-Kaede through the reconnected neuron. We found that PVD reconnection occurs through fusion. We then performed a similar experiment in lin-22 animals which have 2 to 5 extra PVD cell bodies (kindly provided by M. Heiman and C. Yip) to study whether adjacent PVDs connect by fusion. We found that following injury different PVDs can fuse as a response to injury. These experiments show that PVD neurons response to dendrotomy involves four steps: (1) Loss of self-avoidance or tiling. (2) Menorah-menorah fusion mediated by AFF-1 and/or reconnection of injured 1ry branches by auto-fusion. (3) Ectopic sprouting of dendrites from the PVD. (4) Simplification by branch retraction mediated by EFF-1. The outcome of the regeneration process is the recovery of the PVD morphology and if regeneration by auto-fusion fails the distal part degenerates.