Michael A. Herman

The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously

Mutations in the C. elegans gene lin-44 lead to reversals in the polarity of certain asymmetric cell divisions. We have discovered that lin-44 is a member of the Wnt family of genes, which encode secretory glycoproteins implicated in intercellular signaling. Both in situ hybridization experiments using lin-44 transcripts and experiments using reporter constructs designed to mimic patterns of lin-44 expression indicate that lin-44 is expressed in hypodermal cells at the tip of the tail and posterior to the cells with polarities affected by lin-44 mutations. Our mosaic analysis indicates that lin-44 acts cell nonautonomously. We propose that LIN-44 protein is secreted by tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail.

Distinct β-catenins mediate adhesion and signalling functions in C. elegans.

In flies and vertebrates, Armadillo/β-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signalling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative β-catenin homologues have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homologue POP-1 mediate Wnt signalling by a mechanism that has challenged current views of the Wnt pathway. Here we show that BAR-1 is the only β-catenin homologue that interacts directly with POP-1. BAR-1 mediates Wnt signalling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only β-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. We conclude that a canonical Wnt pathway exists in C. elegans. Furthermore, our analysis shows that the functions of C. elegans β-catenins in adhesion and in signalling are performed by separate proteins.

Caenorhabditis elegans genomic response to soil bacteria predicts environment-specific genetic effects on life history traits.

With the post-genomic era came a dramatic increase in high-throughput technologies, of which transcriptional profiling by microarrays was one of the most popular. One application of this technology is to identify genes that are differentially expressed in response to different environmental conditions. These experiments are constructed under the assumption that the differentially expressed genes are functionally important in the environment where they are induced. However, whether differential expression is predictive of functional importance has yet to be tested. Here we have addressed this expectation by employing Caenorhabditis elegans as a model for the interaction of native soil nematode taxa and soil bacteria. Using transcriptional profiling, we identified candidate genes regulated in response to different bacteria isolated in association with grassland nematodes or from grassland soils. Many of the regulated candidate genes are predicted to affect metabolism and innate immunity suggesting similar genes could influence nematode community dynamics in natural systems. Using mutations that inactivate 21 of the identified genes, we showed that most contribute to lifespan and/or fitness in a given bacterial environment. Although these bacteria may not be natural food sources for C. elegans, we show that changes in food source, as can occur in environmental disturbance, can have a large effect on gene expression, with important consequences for fitness. Moreover, we used regression analysis to demonstrate that for many genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life history traits in those environments.

Control of cell polarity by noncanonical Wnt signaling in C. elegans

The three Caenorhabditis elegans beta-catenin each function in distinct processes: BAR-1 in canonical Wnt signaling that controls cell fates and cell migrations, HMP-2 in cell adhesion and WRM-1 in Wnt signaling pathways that function in conjunction with a mitogen-activated kinase (MAPK) pathway to control the orientations, or cell polarities, of cells that undergo asymmetric cell divisions. In addition, WRM-1 does not interact with the canonical beta-catenin binding site in POP-1/Tcf. Thus, Wnt signaling through WRM-1 is noncanonical and, except for one division that might not include any of the three C. elegans beta-catenin, controls cell polarity in C. elegans.

Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie.

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland

A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial‐feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial‐feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial‐feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.

High-throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure

Nematodes are abundant consumers in grassland soils, but more sensitive and specific methods of enumeration are needed to improve our understanding of how different nematode species affect, and are affected by, ecosystem processes. High‐throughput amplicon sequencing is used to enumerate microbial and invertebrate communities at a high level of taxonomic resolution, but the method requires validation against traditional specimen‐based morphological identifications. To investigate the consistency between these approaches, we enumerated nematodes from a 25‐year field experiment using both morphological and molecular identification techniques in order to determine the long‐term effects of annual burning and nitrogen enrichment on soil nematode communities. Family‐level frequencies based on amplicon sequencing were not initially consistent with specimen‐based counts, but correction for differences in rRNA gene copy number using a genetic algorithm improved quantitative accuracy. Multivariate analysis of corrected sequence‐based abundances of nematode families was consistent with, but not identical to, analysis of specimen‐based counts. In both cases, herbivores, fungivores and predator/omnivores generally were more abundant in burned than nonburned plots, while bacterivores generally were more abundant in nonburned or nitrogen‐enriched plots. Discriminate analysis of sequence‐based abundances identified putative indicator species representing each trophic group. We conclude that high‐throughput amplicon sequencing can be a valuable method for characterizing nematode communities at high taxonomic resolution as long as rRNA gene copy number variation is accounted for and accurate sequence databases are available.

Wnt and EGF pathways act together to induce C. elegans male hook development

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1 degree, 2 degrees, 3 degrees), and 2 degrees HCG fate specification is mediated by LIN-12/Notch. We show that 2 degrees HCG specification depends on the presence of a cell with the 1 degree fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1 degree and 2 degrees HCG fate. A requirement for EGF signaling during 1 degree fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1 degree lineage, and LIN-12/NOTCH induces a 2 degrees lineage. Wnt signaling is also required for execution of the 1 degree and 2 degrees HCG lineages. lin-17 and bar-1/beta-catenin are preferentially expressed in the presumptive 1 degree cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.

Conversion of the LIN-1 ETS Protein of Caenorhabditis elegans from a SUMOylated Transcriptional Repressor to a Phosphorylated Transcriptional Activator

The LIN-1 ETS transcription factor plays a pivotal role in controlling cell fate decisions during development of the Caenorhabditis elegans vulva. Prior to activation of the RTK/Ras/ERK-signaling pathway, LIN-1 functions as a SUMOylated transcriptional repressor that inhibits vulval cell fate. Here we demonstrate using the yeast two-hybrid system that SUMOylation of LIN-1 mediates interactions with a protein predicted to be involved in transcriptional repression: the RAD-26 Mi-2β/CHD4 component of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies indicated that rad-26 functions to inhibit vulval cell fates in worms. Using the yeast two-hybrid system, we showed that the EGL-27/MTA1 component of the NuRD complex binds the carboxy-terminus of LIN-1 independently of LIN-1 SUMOylation. EGL-27 also binds UBC-9, an enzyme involved in SUMOylation, and MEP-1, a zinc-finger protein previously shown to bind LIN-1. Genetic studies indicate that egl-27 inhibits vulval cell fates in worms. These results suggest that LIN-1 recruits multiple proteins that repress transcription via both the SUMOylated amino-terminus and the unSUMOylated carboxy-terminus. Assays in cultured cells showed that the carboxy-terminus of LIN-1 was converted to a potent transcriptional activator in response to active ERK. We propose a model in which LIN-1 recruits multiple transcriptional repressors to inhibit the 1° vulval cell fate, and phosphorylation by ERK converts LIN-1 to a transcriptional activator that promotes the 1° vulval cell fate.

The nuclear receptor NHR-25 cooperates with the Wnt/β-catenin asymmetry pathway to control differentiation of the T seam cell in C. elegans

Asymmetric cell divisions produce new cell types during animal development. Studies in Caenorhabditis elegans have identified major signal-transduction pathways that determine the polarity of cell divisions. How these relatively few conserved pathways interact and what modulates them to ensure the diversity of multiple tissue types is an open question. The Wnt/β-catenin asymmetry pathway governs polarity of the epidermal T seam cell in the C. elegans tail. Here, we show that the asymmetry of T-seam-cell division and morphogenesis of the male sensory rays require NHR-25, an evolutionarily conserved nuclear receptor. NHR-25 ensures the neural fate of the T-seam-cell descendants in cooperation with the Wnt/β-catenin asymmetry pathway. Loss of NHR-25 enhances the impact of mutated nuclear effectors of this pathway, POP-1 (TCF) and SYS-1 (β-catenin), on T-seam-cell polarity, whereas it suppresses the effect of the same mutations on asymmetric division of the somatic gonad precursor cells. Therefore, NHR-25 can either synergize with or antagonize the Wnt/β-catenin asymmetry pathway depending on the tissue context. Our findings define NHR-25 as a versatile modulator of Wnt/β-catenin-dependent cell-fate decisions.