Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.     

Dynamic,Non-binary Specification of Sexual State in the C. elegans Nervous System

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The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos.

During C. elegans embryogenesis an 8-cell stage blastomere, called MS, undergoes a reproducible cleavage pattern, producing pharyngeal cells, body wall muscles, and cell deaths. We show here that maternal-effect mutations in the pie-1 and mex-1 genes cause additional 8-cell stage blastomeres to adopt a fate very similar to that of the wild-type MS blastomere. In pie-1 mutants one additional posterior blastomere adopts an MS-like fate, and in mex-1 mutants four additional anterior blastomeres adopt an MS-like fate. We propose that maternally provided pie-1(+) and mex-1(+) gene products may function in the early embryo to localize or regulate factors that determine the fate of the MS blastomere.     

The C. elegans gene lin-36 acts cell autonomously in the lin-35 Rb pathway.

The Caenorhabditis elegans gene lin-36 acts to antagonize Ras-mediated vulval induction in a pathway that includes genes with products similar to the mammalian retinoblastoma (Rb) protein and the Rb-binding protein p48. We report that lin-36 encodes a novel protein of 962 amino acids. We demonstrate that lin-36 functions in and is expressed in the vulval precursor cells, establishing that the lin-36 pathway is involved in intercellular signaling. We also report that the lin-36 pathway and/or another pathway that is functionally redundant with the lin-36 pathway antagonize a ligand-independent activity of the receptor tyrosine kinase/Ras vulval induction pathway.     

Identification of cis-regulatory elements from the C. elegans T-box gene mab-9 reveals a novel role for mab-9 in hypodermal function

We have identified Conserved Non-coding Elements (CNEs) in the regulatory region of Caenorhabditis elegans and Caenorhabditis briggsae mab-9, a T-box gene known to be important for cell fate specification in the developing C. elegans hindgut. Two adjacent CNEs (a region 78 bp in length) are both necessary and sufficient to drive reporter gene expression in posterior hypodermal cells. The failure of a genomic mab-9::gfp construct lacking this region to express in posterior hypodermis correlates with the inability of this construct to completely rescue the mab-9 mutant phenotype. Transgenic males carrying this construct in a mab-9 mutant background exhibit tail abnormalities including morphogenetic defects, altered tail autofluorescence and abnormal lectin-binding properties. Hermaphrodites display reduced susceptibility to the C. elegans pathogen Microbacterium nematophilum. This comparative genomics approach has therefore revealed a previously unknown role for mab-9 in hypodermal function and we suggest that MAB-9 is required for the secretion and/or modification of posterior cuticle.     

Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.     

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.