Systems level circuit model of C. elegans undulatory locomotion: mathematical modeling and molecular genetics

To establish the relationship between locomotory behavior and dynamics of neural circuits in the nematode C. elegans we combined molecular and theoretical approaches. In particular, we quantitatively analyzed the motion of C. elegans with defective synaptic GABA and acetylcholine transmission, defective muscle calcium signaling, and defective muscles and cuticle structures, and compared the data with our systems level circuit model. The major experimental findings are: (i) anterior-to-posterior gradients of body bending flex for almost all strains both for forward and backward motion, and for neuronal mutants, also analogous weak gradients of undulatory frequency, (ii) existence of some form of neuromuscular (stretch receptor) feedback, (iii) invariance of neuromuscular wavelength, (iv) biphasic dependence of frequency on synaptic signaling, and (v) decrease of frequency with increase of the muscle time constant. Based on (i) we hypothesize that the Central Pattern Generator (CPG) is located in the head both for forward and backward motion. Points (i) and (ii) are the starting assumptions for our theoretical model, whose dynamical patterns are qualitatively insensitive to the details of the CPG design if stretch receptor feedback is sufficiently strong and slow. The model reveals that stretch receptor coupling in the body wall is critical for generation of the neuromuscular wave. Our model agrees with our behavioral data(iii), (iv), and (v), and with other pertinent published data, e.g., that frequency is an increasing function of muscle gap-junction coupling.Comment: Neural control of C. elegans motion with genetic perturbation

Re-programming of C. elegans male epidermal precursor fates by Wnt, Hox, and LIN-12/Notch activities

In Caenorhabditis elegans males, different subsets of ventral epidermal precursor (Pn.p) cells adopt distinct fates in a position-specific manner: three posterior cells, P(9–11).p, comprise the hook sensillum competence group (HCG) with three potential fates (1°, 2°, or 3°), while eight anterior cells, P(1–8).p, fuse with the hyp7 epidermal syncytium. Here we show that activation of the canonical BAR-1 β-catenin pathway of Wnt signaling alters the competence of P(3–8).p and specifies ectopic HCG-like fates. This fate transformation requires the Hox gene mab-5. In addition, misexpression of mab-5 in P(1–8).p is sufficient to establish HCG competence among these cells, as well as to generate ectopic HCG fates in combination with LIN-12 or EGF signaling. While increased Wnt signaling induces predominantly 1° HCG fates, increased LIN-12 or EGF signaling in combination with MAB-5 overexpression promotes 2° HCG fates in anterior Pn.p cells, suggesting distinctive functions of Wnt, LIN-12, and EGF signaling in specification of HCG fates. Lastly, wild-type mab-5 function is necessary for normal P(9–11).p fate specification, indicating that regulation of ectopic HCG fate formation revealed in anterior Pn.p cells reflect mechanisms of pattern formation during normal hook development

Wnt and EGF Pathways Act Together to Induce \u3ci\u3eC. elegans\u3c/i\u3e 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°, 2°, 3°), and 2° HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° 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° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2° HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1–GFP in P11.p is concordant with the timing of HCG fate determination

Developmental validation of Oxford Nanopore Technology MinION sequence data and the NGSpeciesID bioinformatic pipeline for forensic genetic species identification

Species identification of non-human biological evidence through DNA nucleotide sequencing is routinely used for forensic genetic analysis to support law enforcement. The gold standard for forensic genetics is conventional Sanger sequencing; however, this is gradually being replaced by high-throughput sequencing (HTS) approaches which can generate millions of individual reads in a single experiment. HTS sequencing, which now dominates molecular biology research, has already been demonstrated for use in a number of forensic genetic analysis applications, including species identification. However, the generation of HTS data to date requires expensive equipment and is cost-effective only when large numbers of samples are analysed simultaneously. The Oxford Nanopore Technologies (ONT) MinION™ is an affordable and small footprint DNA sequencing device with the potential to quickly deliver reliable and cost effective data. However, there has been no formal validation of forensic species identification using high-throughput (deep read) sequence data from the MinION making it currently impractical for many wildlife forensic end-users. Here, we present a MinION deep read sequence data validation study for species identification. First, we tested whether the clustering-based bioinformatics pipeline NGSpeciesID can be used to generate an accurate consensus sequence for species identification. Second, we systematically evaluated the read variation distribution around the generated consensus sequences to understand what confidence we have in the accuracy of the resulting consensus sequence and to determine how to interpret individual sample results. Finally, we investigated the impact of differences between the MinION consensus and Sanger control sequences on correct species identification to understand the ability and accuracy of the MinION consensus sequence to differentiate the true species from the next most similar species. This validation study establishes that ONT MinION sequence data used in conjunction with the NGSpeciesID pipeline can produce consensus DNA sequences of sufficient accuracy for forensic genetic species identification

EGF, WNT & HOX Interactions during Patterning of Caenorhabditis elegans Equivalence Groups

During development, as a single-cell zygote divides multiple times to generate a complete organism, previously undifferentiated cells somehow acquire the correct fates. A group of cells that shares the same developmental potential is called an equivalence group. In Caenorhabditis elegans, the most well-characterized equivalence group is the hermaphroditic Vulval Precursor Cell (VPC) group. Epidermal Growth Factor (EGF) signaling specifies VPC fate partly by upregulation of lin-39/SexcombsReduced/Hox5, while Wnt signaling plays a minor role in vulval induction. EGF and Wnt signaling also act together to pattern the P11/12 equivalence group, present in both C. elegans hermaphrodites and males, by upregulating a different Hox gene, egl-5/Antennapedia/Ultrabithorax/Hox6/8, to specify P12 fate. Previous observations suggest that EGF or Wnt signaling may act through Hox genes to specify fate in two other C. elegans equivalence groups: the Hook Competence Group (HCG) and γ/δ pair. I characterized the roles of EGF and Wnt signaling in the HCG and γ/δ pair and found that upregulation of Hox genes is controlled by either pathway in each group. I showed that the major hook inductive pathway involves the Wnt ligands and LIN-17/Fz, which specify the 1° and 2° HCG fates. Also, I identified a role for EGF signaling in specifying the 1° fate, although its role is only revealed when Wnt activity is compromised. I provided a link between mab-5/Hox6/8 and Wnt signaling during normal hook development by determining that LIN-17 is required for mab-5/Hox6/8 expression in P11.p. In the γ/δ pair, I demonstrated that EGF signaling (through the LIN-31/Forkhead and LIN-1/ETS transcription factors) controls ceh-13/Hox1 expression in γ. I did not find any evidence that Wnt signaling specifies the γ fate. Instead, I observed that lin-44/Wnt, mom-2/Wnt and lin-17/Fz are required to orient the γ mitotic spindle. In addition, TGF-β signaling (by dbl-1/Dpp) was previously reported to control γ expression of ceh-13/Hox1. I showed that dbl-1 acts either downstream or in parallel to EGF signaling to specify the γ fate. I also found that dbl-1/Dpp does not appear to specify fates in the VPC and P11/12 equivalence groups, in which EGF signaling plays an important role, suggesting that TGF-β signaling contributes to the specificity of the γ fate.</p

Wnt and EGF Pathways Act Together to Induce \u3ci\u3eC. elegans\u3c/i\u3e 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°, 2°, 3°), and 2° HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° 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° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2° HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1–GFP in P11.p is concordant with the timing of HCG fate determination

MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples

The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples. We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols. For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29%&ndash;100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies. We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification