Bruce Wightman et al. (2008) Development & Evolution Meeting "The tailless Ortholog nhr-67 Functions in Uterus, Tail, and Germ-line Development"

Emily Lisco, Bruce Wightman, Sheila Clever, Michael Twardzik, Eliana Verghese

[Development & Evolution Meeting, 2008]

The tailless family of nuclear receptors is highly conserved among animals. In Drosophila, tailless functions in terminal embryonic patterning and central nervous system development. In vertebrates, Tlx functions in the maintenance of neural stem cell identity, but does not play a known role in terminal patterning. The C. elegans tailless ortholog, nhr-67, is expressed in a dynamic pattern in pre-uterine cells: initially in the 4 pre-VU cells during the L2 stage, and subsequently upregulated in the anchor cell (AC), in response to the lin-12/lag-2 reciprocal signaling system. During the L3 stage, nhr-67 expression is maintained at high levels in the AC and at low levels in the six p cells whose twelve progeny form cells of the adult ventral uterus. Development of the p cells also depends on a lin-12 - based signal from the AC. In mutants homozygous for hypomorphic nhr-67 promoter mutations that were identified by Bernard Lakowski's laboratory, the development of the p cells is defective but the AC appears normal by multiple criteria. The expression of nhr-67 is downstream of both lin-12(RNAi) knockdown and lin-12(gf) mutations in AC and p cells, and nhr-67(lf) mutations are epistatic to lin12(gf) mutations in the p cells. These observations indicate that nhr-67 functions downstream of lin-12 in the development of the ventral uterus. Animals that are homozygous for nhr-67 hypomorphic mutations also display a low penetrance sterile phenotype in addition to protruding vulva and egg-laying-defective phenotypes. The germ-line of nhr-67 sterile mutants is under-proliferated, suggesting that the nhr-67 gene may also play a role in maintaining stem cell identity in the germ-line (possibly in the L2 pre-VU cells), an interesting parallel to the role of Tlx in maintaining vertebrate neural stem cell identity. Strong alleles that delete most of nhr-67ligand-binding domain arrest development in the L1 after hatching. The arrested larvae display tail defects in the hyp10 epithelial cell similar to those caused by mutations in the cadherin gene cdh-3. This observation suggests that a function for tailless in terminal development may be conserved among the ecdysozoa. Supported by a grant from the NSF.

Anne Hart (2001) International C. elegans Meeting "Using C elegans to teach genetics: SNP mapping in three weeks!"

Anne Hart

[International C. elegans Meeting, 2001]

C. elegans has been used to the teach fundamentals of genetic mapping for the last three years in the Neurobiology summer course at the Marine Biological Laboratories (MBL). The nine week course is primarily aimed at graduate students and postdoctoral fellows. The molecular section of the course I help teach lasts three weeks including lectures in the morning and lab work in the afternoons and evenings. Two students work with each of the six faculty members in the molecular section of the course. The two students assigned to me spend about 120 hours each working in the lab spread out over a three week period. Consistently, the students have had no experience in C. elegans and, often, little or no prior experience in molecular biology. Given the short duration the molecular section, and the desire to accomplish something of substance, the project has to be chosen with care. The short life cycle of C. elegans makes it an ideal organism to teach the fundamentals of genetic mapping in a limited amount of time. The project chosen last year was recombinant mapping of an interesting mutation, rt70 , using single nucleotide polymorphisms (SNPs) from the Hawaiian strain, CB4856. rt70 causes ASH neuron degeneration and is described in an abstract/poster by Emily Bates, et al. Prior to the start of the course last year, a lon-2(e678) rt70 egl-15(n484) chromosome was generated. All mutations are recessive. Heterozygous lon-2 rt70 egl-15 / CB4856 animals were generated and picked one per plate. These heterozygous animals were transported to MBL along with hundreds of seeded plates. The students practiced picking C. elegans , then singled animals carrying recombinant chromosomes/ lon-2 rt70 egl-15 for the first few days (putative recombinant lines). We attempted to generate homozygous recombinant lines by picking single 9 animals in the next generation and were successful about 80%of the time; the remaining 20% were repicked for analysis at a later date. The homozygous recombinant lines were 1) frozen in a 96 well format using Michael Koelle's protocol and 2) allowed to starve and lysed for PCR analysis using previously defined SNPs identified by Stephen Wicks. To facilitate analysis, only previously characterized SNPs creating a restriction site polymorphism were utilized. Screening more than 100 lines by PCR was not a problem and we were able to refine the rt70 map position. The biggest difficulty we had was scoring egl-15(e484) . In retrospect, I should have chosen marker mutations with very obvious phenotypes so they could be scored unambiguously and easily. PCR reactions were optimized while we were waiting for the recombinant strains to grow. The MBL students also had time to learn how to generate transgenic C. elegans by microinjection. PCR machines for the course were generously loaned by MJ Research, Taq was kindly provided by Fisher and NEB generously donated restriction enzymes. Thanks to all who donated their time, reagents and help- especially MBL students Michael Long and Ryohei Yasuda for their unflagging enthusiasm!