Patrice S. Albert

Genetic and Environmental Regulation of Dauer Larva Development

I. INTRODUCTION Dauer larvae were first identified as a special larval stage of insect-parasitic nematodes. These larvae, which differed structurally from all other stages of the same species, were termed “dauerlarven” by Fuchs (1915). The dauer (enduring) stage of Caenorhabditis elegans is formed when environmental conditions are inadequate for successful reproduction. In abundant food, the animal develops continuously through the four larval stages (L1–L4) to the adult. Coincident with increased population density and limited food supply, development is arrested at the second molt, and the third-stage larva that is formed is structurally and behaviorally specialized for dispersal and long-term survival (Cassada and Russell 1975). Dauer larvae do not feed, but they can survive at least four to eight times the normal 2-week life span of C. elegans (Klass and Hirsh 1976). When favorable conditions are encountered, the dauer larva begins to feed and resumes development to the adult. Both entry into and exit from the dauer stage are developmental responses to specific chemosensory cues. These cues inform the larva whether there will be sufficient food available to support its reproduction. The environmental cues are first assessed and integrated throughout the L1 stage (Golden and Riddle 1984b). The primary cue is a Caenorhabditis-specific pheromone constitutively released by the nematodes (Golden and Riddle 1984c). The pheromone is very stable and hydrophobic and has chromatographic properties similar to those of hydroxylated fatty acids and bile acids. The concentration of pheromone reflects nematode population density. Temperature and food modulate the response to pheromone...

Control of C. elegans Larval Development by Neuronal Expression of a TGF-β Homolog

The Caenorhabditis elegans dauer larva is specialized for dispersal without growth and is formed under conditions of overcrowding and limited food. The daf-7 gene, required for transducing environmental cues that support continuous development with plentiful food, encodes a transforming growth factor-β (TGF-β) superfamily member. A daf-7 reporter construct is expressed in the ASI chemosensory neurons. Dauer-inducing pheromone inhibits daf-7 expression and promotes dauer formation, whereas food reactivates daf-7 expression and promotes recovery from the dauer state. When the food/pheromone ratio is high, the level of daf-7 mRNA peaks during the L1 larval stage, when commitment to non-dauer development is made.

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase.

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.

Fine structure of the Caenorhabditis elegans secretory-excretory system.

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.

Aluminum tolerance in maize is associated with higher MATE1 gene copy number

Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments.

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.

Systematic Interactome Mapping and Genetic Perturbation Analysis of a C. elegans TGF-β Signaling Network

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.

Diversity of Chromosomal Karyotypes in Maize and Its Relatives

Maize is a highly diverse species on the gene sequence level. With the recent development of methods to distinguish each of the 10 pairs of homologues in somatic root tip spreads, a wide collection of maize lines was subjected to karyotype analysis to serve as a reference for the community and to examine the spectrum of chromosomal features in the species. The core nested association mapping progenitor collection and additional selections of diversity lines were examined. Commonly used inbred lines were included in the analysis. The centromere 4 specific repeat and ribosomal RNA loci were invariant. The CentC centromere repeat exhibited extensive differences in quantity on any particular chromosome across lines. Knob heterochromatin was highly variable with locations at many sites in the genome. Lastly, representative examples from other species in the genus Zea (teosintes) were examined, which provide information on the evolution of chromosomal features.

Intragenomic Conflict Between the Two Major Knob Repeats of Maize

Examples of meiotic drive, the non-Mendelian segregation of a specific genomic region, have been identified in several eukaryotic species. Maize contains the abnormal chromosome 10 (Ab10) drive system that transforms typically inert heterochromatic knobs into centromere-like domains (neocentromeres) that move rapidly poleward along the spindle during meiosis. Knobs can be made of two different tandem repeat sequences (TR-1 and 180-bp repeat), and both repeats have become widespread in Zea species. Here we describe detailed studies of a large knob on chromosome 10 called K10L2. We show that the knob is composed entirely of the TR-1 repeat and is linked to a strong activator of TR-1 neocentromere activity. K10L2 shows weak meiotic drive when paired with N10 but significantly reduces the meiotic drive exhibited by Ab10 (types I or II) in Ab10/K10L2 heterozygotes. These and other data confirm that (1) there are two separate and independent neocentromere activities in maize, (2) that both the TR-1 and knob 180 repeats exhibit meiotic drive (in the presence of other drive genes), and (3) that the two repeats can operate in competition with each other. Our results support the general concept that tandem repeat arrays can engage in arms-race-like struggles and proliferate as an outcome.