Spot(ters) the difference: Bringing traditional anatomical examinations online.

The COVID-19 pandemic caused a shift in anatomy education forcing institutions to find innovative ways to teach and assess online. This study details the development of an online spotter across multiple modules that allowed students to sit the examination at home whilst still maintaining the integrity of the assessment. The online spotter consisted of individual, Zoom calls between students and examiners whereby slides with images and questions were screen shared. To examine the viability of this spotter in non-lockdown scenarios several parameters were considered. Mean marks were compared to traditional versions and Pearson's r correlation coefficients were calculated between online and traditional spotters and between online spotters and overall performance in anatomy modules. A survey was carried out to determine the students' view of the assessment. Pearson's r was between 0.33 and 0.49 when comparing online spotters to the traditional format, and between 0.65 and 0.75 (p < 0.01) when compared to a calculated anatomy score. The survey indicated overall student satisfaction as 82.5% reported that it was a fair way to test their knowledge and 55% reported the same or lower levels of anxiety when compared to traditional spotters. However, there was nothing to indicate that the students preferred this format over laboratory-based spotters. These results indicate that this new exam format would be useful for small cohorts who are undertaking online or hybrid courses, or in circumstances when running a full spotter is too costly, and represents a fair and robust way to assess practical anatomical knowledge online.

Metabolic Exploration for Cyhalofop-Butyl Antagonism in Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] Following Pretreatment of Malathion.

The present study investigated the effects of broad-spectrum metabolic inhibitors malathion (cytochrome P450 inhibitor) and/or 4-chloro-7-nitrobenzofurazan (NBD-Cl; glutathione S-transferase inhibitor) on the metabolism of cyhalofop-butyl (CyB) in barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes confirmed previously with multiple resistance to two herbicides CyB and florpyrauxifen-benzyl. The metabolic inhibitors were not effective at recovering the sensitivity of resistant barnyardgrass biotypes to CyB treated at the labeled rate (313 g ai ha-1). Rather, treatment with malathion followed by CyB caused antagonism, reducing the efficacy of CyB and promoting the growth of resistant biotypes. Pretreatment with malathion did not influence absorption/translocation of the applied form CyB and its conversion to the active herbicide form cyhalofop-acid (CyA), in both susceptible and resistant biotypes. In contrast, metabolism of the applied form (CyB) decreased 1.5 to 10.5 times by the malathion pretreatment. Taken together, the maintained CyA production against the reduced CyB metabolism could be the mechanism to account for the cause of CyB antagonism observed in barnyardgrass following malathion pretreatment. Additionally, the evolution of CyB resistance in barnyardgrass might be associated with reduced production of CyA in resistant biotypes, independent of activities of cytochrome P450 or GST enzymes.

Exploratory Analysis on Herbicide Metabolism and Very-Long-Chain Fatty Acid Production in Metolachlor-Resistant Palmer Amaranth (Amaranthus palmeri S. Wats.).

A Palmer amaranth (Amaranthus palmeri S. Wats.) biotype resistant to S-metolachlor was confirmed from crop fields in Arkansas, USA. This study investigated the metabolic effects of malathion (cytochrome P450 inhibitor) and 4-chloro-7-nitrobenzofurazan [NBD-Cl; glutathione S-transferase inhibitor] on the S-metolachlor-resistant A. palmeri biotype. Root elongation of the resistant biotype was 20% more inhibited by treatment of NBD-Cl (50 nM) and S-metolachlor (2 µM) in mixture than by treatment of S-metolachlor alone. Metabolites of S-metolachlor were 1.4-12.1 times greater produced in the resistant biotype for 7 d than in the susceptible standard. Production of cerotic acid, one of the very-long-chain fatty acids containing 26 carbons, was more reduced in the susceptible standard (3.8-fold) than in the resistant biotype (1.8-fold) by S-metolachlor treatment. Conclusively, evolution of S-metolachlor resistance observed in this study was likely associated with improved activity of glutathione S-transferases. Further studies are needed to genetically evaluate plant endogenous enzymes involving cerotic acid production.

Impact of volatility reduction agents on dicamba and glyphosate spray solution pH, droplet dynamics, and weed control.

BACKGROUND: Regulations in 2021 required the addition of a volatility reduction agent (VRA) to dicamba spray mixtures for postemergence weed control. Understanding the impact of VRAs on weed control, droplet dynamics, and spray pH is essential. RESULTS: Adding glyphosate to dicamba decreased the solution pH by 0.63 to 1.85 units. Across locations, potassium carbonate increased the tank-mixture pH by 0.85 to 1.65 units while potassium acetate raised the pH by 0.46 to 0.53 units. Glyphosate and dicamba in tank-mixture reduced Palmer amaranth control by 14 percentage points compared to dicamba alone and decreased barnyardgrass control by 12 percentage points compared to glyphosate alone 4 weeks after application (WAA). VRAs resulted in a 5-percentage point reduction in barnyardgrass control 4 WAA. Common ragweed, common lambsquarters, and giant ragweed control were unaffected by herbicide solution 4 WAA. Dicamba alone produced a larger average droplet size and had the fewest driftable fines (% volume < 200 µm). Potassium acetate produced a larger droplet size than potassium carbonate for Dv0.1 and Dv0.5 . The addition of glyphosate to dicamba decreased droplet size from the entire spray droplet spectrum (Dv0.1 , Dv0.5 , Dv0.9 ). CONCLUSION: A reduction in spray pH, droplet size, and weed control was observed from mixing dicamba and glyphosate. It may be advisable to avoid tank-mixtures of these herbicides and instead, apply them sequentially to maximize effectiveness. VRAs differed in their impacts on spray solution pH and droplet dynamics, but resulted in a minimal negative to no impact on weed control. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Herbicide spray drift from ground and aerial applications: Implications for potential pollinator foraging sources.

A field spray drift experiment using florpyrauxifen-benzyl was conducted to measure drift from commercial ground and aerial applications, evaluate soybean [Glycine max (L.) Merr.] impacts, and compare to United States Environmental Protection Agency (US EPA) drift models. Collected field data were consistent with US EPA model predictions. Generally, with both systems applying a Coarse spray in a 13-kph average wind speed, the aerial application had a 5.0- to 8.6-fold increase in drift compared to the ground application, and subsequently, a 1.7- to 3.6-fold increase in downwind soybean injury. Soybean reproductive structures were severely reduced following herbicide exposure, potentially negatively impacting pollinator foraging sources. Approximately a 25% reduction of reproductive structures up to 30.5-m downwind and nearly a 100% reduction at 61-m downwind were observed for ground and aerial applications, respectively. Aerial applications would require three to five swath width adjustments upwind to reduce drift potential similar to ground applications.

Cross-resistance of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] to aryloxyphenoxypropionate herbicides.

Managing emerged weeds that have evolved resistance to acetyl CoA carboxylase (ACCase)-inhibiting herbicides is a challenging task. A dose-response experiment was conducted on barnyardgrass biotypes resistant (R) and susceptible (S) to three aryloxyphenoxypropionate herbicides cyhalofop-butyl (CyB), fenoxaprop-ethyl (FeE), and quizalofop-ethyl (QuE) along with investigations into the potential resistance mechanism of these biotypes. The tested R barnyardgrass biotypes had strong resistance to CyB and FeE (resistant/susceptible ratio: 7.9-14.4) but weak resistance to QuE (resistant/susceptible ratio: 2.4-3.1). Absorption, translocation, and total metabolism of CyB and QuE were not associated with differences among S and R barnyardgrass biotypes. However, differences between S and R barnyardgrass were observed in production of active acid forms of each herbicide (cyhalofop-acid and quizalofop-acid). Production of cyhalofop-acid was >1.6-fold less in R barnyardgrass (3-8%) for 24 h after herbicide application than in the S barnyardgrass (8-16%). Meanwhile, production of quizalofop-acid was less in R barnyardgrass (< 14%) throughout the study period than in the S barnyardgrass (< 22%). Sequencing results of ACCase gene showed no difference between S and R barnyardgrass. Overall results show that a non-target-site resistance mechanism altering metabolism of CyB and QuE likely contributes to resistance of the barnyardgrass biotypes to these herbicides.

Evaluation of dicamba volatilization when mixed with glyphosate using imazethapyr as a tracer.

Expansion of dicamba-resistant crops increased the frequency of off-target movement issues, especially in the midsouthern United States. Six field trials were conducted over two growing seasons with the purpose to determine the contribution of volatilization and physical suspension of particles to the off-target movement of dicamba when applied with glyphosate and imazethapyr - a non-volatile herbicide used as a tracer for physical off-target movement. Applications included dicamba at 560 g ha-1, glyphosate at 1260 g ha-1, and imazethapyr at 105 g ha-1. Applicators include glyphosate with dicamba to increase the spectrum of weed control from these applications; however, this addition increases potential for dicamba volatilization. Following application of the mixture, air samplers were placed in the field to collect dicamba and imazethapyr. Results showed there was at least 50 times more dicamba than imazethapyr detected even though the dicamba:imazethapyr ratio applied was 5.3:1. Dicamba was detected in the treated area and the off-site locations and all intervals of air sampling, ranging from 126 to 5990 ng. No more than 37.5 ng of imazethapyr was detected during the first 24-h after application (HAA) inside the treated area. Imazethapyr was only detected in 9 of the 20 sampling combinations during these experiments, and most of these detections (6) occurred during the first 24 HAA and inside the treated area. While some movement from the suspension of particles occurred based on the detection of imazethapyr in air samples, results show that most dicamba detection was due to the volatilization of the herbicide.

Large-scale evaluation of physical drift and volatility of 2,4-D choline in cotton: a four-year field study.

BACKGROUND: Cotton with resistance to 2,4-d choline, glufosinate and glyphosate allows over-the-top use of these herbicides for postemergence weed control. Nontransgenic cotton is highly responsive to low rates of 2,4-d, causing concern among farmers when the herbicide is applied near the crop. Injury to nearby cotton following application of 2,4-d choline is sometimes blamed on volatilization of the herbicide. RESULTS: A large-scale experiment was conducted in 2018-2021 to better understand causes of damage to nearby sensitive cotton following an application of 2,4-d choline plus glufosinate-ammonium. The herbicides were applied to 0.4 ha in the center of a 4-ha non-Enlist cotton field. At 30 min after application, air samplers were established in the treated center and outside the treated area in cardinal directions. The 72-h cumulative air concentration of 2,4-d in the swath ranged from 3.3 to 7.1 ng m-3 , and most volatile residues (5.0-25.5 ng m-3 ) were detected in samplers established in the downwind direction directly adjacent to the treated field. Cotton plants in three downwind transects that were covered for 30 min after application were not damaged by 2,4-d, whereas noncovered plants along the downwind transects were injured. No cotton injury occurred outside the treated area, except in the downwind direction during application even though wind direction changed after application. CONCLUSION: 2,4-d choline volatilizes, but findings show that the volatilization is not sufficient to damage cotton in the neighboring area following applications, pointing to the importance of applicators understanding wind direction/shift during the application along with proximity of sensitive crops in the downwind direction. © 2022 Society of Chemical Industry.

Utility of roller wiper applications of dicamba for Palmer amaranth control in soybean.

BACKGROUND: The commercialization of dicamba-resistant soybean has resulted in increased concern for off-target movement of dicamba onto sensitive vegetation. To mitigate the off-target movement through physical drift, one might consider use of rope wicks and other wiper applicators. Although wiper-type application methods have been efficacious in pasture settings, the utility of dicamba using wiper applicators in agronomic crops is not available in scientific literature. To determine the utility of roller wipers for dicamba applications in dicamba-resistant soybean, two separate experiments were conducted in the summer of 2020 and replicated in both Keiser and Fayetteville, AR, USA. RESULTS: Utilizing opposing application directions and a 2:1:1 ratio of water: formulated glyphosate: formulated dicamba were the most efficacious practices for controlling Palmer amaranth. The high herbicide concentrations and wiping in opposing directions increased dicamba-resistant soybean injury when the wiper contacted the crop, but no yield loss was observed because of this injury. Broadcast applications resulted in greater Palmer amaranth mortality than roller wiper applications, and the most effective roller wiper treatments were when two sequential applications were made inside the crop canopy. CONCLUSIONS: Dicamba applications require adequate coverage for optimum weed control. While efforts can be made to increase roller wiper efficacy by optimizing coverage and timing of applications, broadcast applications are superior to roller wiper applicators for weed control. Roller wiper applications did not reduce soybean yield, thus wiper-type applications may be safely used in dicamba-resistant soybean, albeit the likelihood for off-target damage caused by volatilization of these treatments would need to be investigated. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Non-target-site resistance mechanism of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] to florpyrauxifen-benzyl.

BACKGROUND: Florpyrauxifen-benzyl (FPB) is an arylpicolinate herbicide (Group IV) for barnyardgrass control in rice. One susceptible (Sus) and three putative FPB-resistant (R1, R2, and R3) barnyardgrass biotypes were selected based on resistant/susceptible (R/S) ratios obtained from dose-response tests and used to investigate the potential resistance mechanisms. RESULTS: Based on visual control results, the R/S ratios of barnyardgrass biotypes R1, R2, and R3 were 60-, 33-, and 16-fold greater than the Sus standard, respectively. Sequencing results of TIR1 and AFB genes in the tested barnyardgrass revealed no difference between Sus and R barnyardgrass biotypes. Absorption of [14 C]-FPB in Sus barnyardgrass increased over time and reached 90%, which was >10 percentage points greater than that in R biotypes. The [14 C]-FPB absorption in all R barnyardgrass equilibrated after 48 h. For both Sus and R barnyardgrass, most [14 C]-FPB absorbed was present in the treated leaf (79.8-88.8%), followed by untreated aboveground (9.5-18.6%) and belowground tissues (1.3-2.2%). No differences in translocation were observed. Differences between Sus and R barnyardgrass biotypes were found for FPB metabolism. Production of the active metabolite, florpyrauxifen-acid, was greater in Sus barnyardgrass (21.5-52.1%) than in R barnyardgrass (5.5-34.9%). CONCLUSION: In conclusion, reductions in FPB absorption and florpyrauxifen-acid production may contribute to the inability to control barnyardgrass with FPB. © 2021 Society of Chemical Industry.

Absorption, translocation, and metabolism of florpyrauxifen-benzyl and cyhalofop-butyl in cyhalofop-butyl-resistant barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.].

Dose-response experiments were conducted to assess the sensitivity of one susceptible and three putative resistant (R1, R2, and R3) barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes to florpyrauxifen-benzyl and cyhalofop-butyl alone and as a formulated premix. Subsequently, potential resistance mechanisms of the barnyardgrass were evaluated. Based on biomass reduction results, resistant/susceptible ratios were calculated for R1 (7.0-50), R2 (7.0-150), and R3 (18-214) biotypes. Absorption and translocation of [14C]-florpyrauxifen-benzyl decreased in R1 and R3 biotypes, but not for [14C]-cyhalofop-butyl. The metabolism of [14C]-florpyrauxifen-benzyl to [14C]-florpyrauxifen-acid was >2-fold less in resistant biotypes (9-11%) than in the susceptible biotype (23%). Moreover, the production of [14C]-florpyrauxifen-acid in susceptible barnyardgrass (not in the R biotypes) increased 3-fold when florpyrauxifen-benzyl and cyhalofop-butyl were applied in mixture compared to florpyrauxifen-benzyl applied alone. The tested barnyardgrass biotypes had no mutation in the Transport Inhibitor Response1, auxin-signaling F-box, and acetyl coenzyme A carboxylase genes. Although further studies on cyhalofop-butyl resistance with respect to analysis of specific metabolites are needed, our findings in this study demonstrates that the evolution of florpyrauxifen-benzyl resistance in multiple resistant barnyardgrass can be related to non-target-site resistance mechanisms reducing absorption and translocation of the herbicide and causing reduced conversion or rapid degradation of florpyrauxifen-acid.

A machine learning interpretation of the contribution of foliar fungicides to soybean yield in the north-central United States.

Foliar fungicide usage in soybeans in the north-central United States increased steadily over the past two decades. An agronomically-interpretable machine learning framework was used to understand the importance of foliar fungicides relative to other factors associated with realized soybean yields, as reported by growers surveyed from 2014 to 2016. A database of 2738 spatially referenced fields (of which 30% had been sprayed with foliar fungicides) was fit to a random forest model explaining soybean yield. Latitude (a proxy for unmeasured agronomic factors) and sowing date were the two most important factors associated with yield. Foliar fungicides ranked 7th out of 20 factors in terms of relative importance. Pairwise interactions between latitude, sowing date and foliar fungicide use indicated more yield benefit to using foliar fungicides in late-planted fields and in lower latitudes. There was a greater yield response to foliar fungicides in higher-yield environments, but less than a 100 kg/ha yield penalty for not using foliar fungicides in such environments. Except in a few production environments, yield gains due to foliar fungicides sufficiently offset the associated costs of the intervention when soybean prices are near-to-above average but do not negate the importance of disease scouting and fungicide resistance management.

Echinoderms provide missing link in the evolution of PrRP/sNPF-type neuropeptide signalling.

Neuropeptide signalling systems comprising peptide ligands and cognate receptors are evolutionarily ancient regulators of physiology and behaviour. However, there are challenges associated with determination of orthology between neuropeptides in different taxa. Orthologs of vertebrate neuropeptide-Y (NPY) known as neuropeptide-F (NPF) have been identified in protostome invertebrates, whilst prolactin-releasing peptide (PrRP) and short neuropeptide-F (sNPF) have been identified as paralogs of NPY/NPF in vertebrates and protostomes, respectively. Here we investigated the occurrence of NPY/NPF/PrRP/sNPF-related signalling systems in a deuterostome invertebrate phylum - the Echinodermata. Analysis of transcriptome/genome sequence data revealed loss of NPY/NPF-type signalling, but orthologs of PrRP-type neuropeptides and sNPF/PrRP-type receptors were identified in echinoderms. Furthermore, experimental studies revealed that the PrRP-type neuropeptide pQDRSKAMQAERTGQLRRLNPRF-NH2 is a potent ligand for a sNPF/PrRP-type receptor in the starfish Asterias rubens. Our findings indicate that PrRP-type and sNPF-type signalling systems are orthologous and originated as a paralog of NPY/NPF-type signalling in Urbilateria.

Extracellular matrix protein anosmin-1 modulates olfactory ensheathing cell maturation in chick olfactory bulb development.

Olfactory ensheathing cells (OECs) are a specialized class of glia, wrapping around olfactory sensory axons that target the olfactory bulb (OB) and cross the peripheral nervous system/central nervous system boundary during development and continue to do so post-natally. OEC subpopulations perform distinct subtype-specific functions dependent on their maturity status. Disrupted OEC development is thought to be associated with abnormal OB morphogenesis, leading to anosmia, a defining characteristic of Kallmann syndrome. Hence, anosmin-1 encoded by Kallmann syndrome gene (KAL-1) might modulate OEC differentiation/maturation in the OB. We performed in ovo electroporation of shRNA in the olfactory placode to knock-down kal in chick embryos, resulting in abnormal OB morphogenesis and loss of olfactory sensory axonal innervation into OB. BLBP-expressing OECs appeared to form a thinner and poorly organized outmost OB layer where SOX10 expressing OECs were completely absent with emergence of GFAP-expressing OECs. Furthermore, in embryonic day 10 chick OB explant cultures, GFAP expression in OECs accumulating along the OB nerve layers was dramatically reduced by recombinant anosmin-1. We then purified immature OECs from embryonic day 10 chick OB. These cells express GFAP after 7 days in vitro, exhibiting a multipolar morphology. Overexpression of chick anosmin, exogenous anosmin-1 or FGF2 could inhibit GFAP expression with cells presenting elongated morphology, which was blocked by the FGF receptor inhibitor Su5402. These data demonstrate that anosmin-1 functions via FGF signalling in regulating OEC maturation, thereby providing a permissive glial environment for axonal innervation into the OB during development.

Evaluation of spray pattern uniformity using three unique analyses as impacted by nozzle, pressure, and pulse-width modulation duty cycle.

BACKGROUND: The increasing popularity of pulse-width modulation (PWM) sprayers requires that application interaction effects on spray pattern uniformity be completely understood to maintain a uniform overlap of spray, thereby reducing crop injury potential and maximizing coverage on target pests. The objective of this research was to determine the impacts of nozzle type (venturi vs. non-venturi), boom pressure, and PWM duty cycle on spray pattern uniformity. Research was conducted using an indoor spray patternator located at the University of Nebraska-Lincoln in Lincoln, NE, USA. Coefficient of variation (CV), root mean square error (RMSE), and average percent error (APE) were used to characterize spray pattern uniformity. RESULTS: Generally, across nozzles and pressures, the duty cycle minimally impacted the CV of spray patterns. However, across nozzles and duty cycles, increasing pressure decreased CV values, resulting in more uniform spray patterns. The RMSE values typically increased as pressure and duty cycle increased across nozzles. This may be the result of a correlation between RMSE values and flow rate as RMSE values also increased as nozzle orifice size increased. Generally, APE increased as the duty cycle decreased across nozzles and pressures with significant increases (40%) caused by the 20% duty cycle. Within non-venturi nozzles, increasing pressure reduced APE across duty cycles, while venturi nozzles followed no such trend. CONCLUSION: Overall, results suggest PWM duty cycles at or above 40% minimally impact spray pattern uniformity. Further, increased application pressures and the use of non-venturi nozzles on PWM sprayers increase the precision and uniformity of spray applications. © 2019 Society of Chemical Industry.

Spray particle drift mitigation using field corn (Zea mays L.) as a drift barrier.

BACKGROUND: Herbicide particle drift reduces application efficacy and can cause severe impacts on nearby vegetation depending on the herbicide mode of action, exposure level, and tolerance to the herbicide. A particle drift mitigation effort placing windbreaks or barriers on the field boundaries to reduce off-target movement of spray particles has been utilized in the past. The objective of this research was to evaluate the effectiveness of field corn (Zea mays L.) at different heights as a particle drift barrier. RESULTS: Applications with a non-air inclusion flat fan nozzle (ER11004) resulted in greater particle drift when compared with an air inclusion nozzle (TTI11004). Eight rows of corn were used as barriers (0.91, 1.22, and 1.98 m height) which reduced the particle drift for both nozzles, especially at shorter downwind distances. Applications with the ER11004 nozzle without corn barriers had 1% of the applied rate (D99 ) predicted to deposit at 14.8 m downwind, whereas this distance was reduced (up to 7-fold) when applications were performed with corn barriers. The combination of corn drift barriers and nozzle selection (TTI11004) provided satisfactory particle drift reduction when the D99 estimates were compared with those for applications with the ER11004 nozzle without corn barriers (up to 10-fold difference). CONCLUSION: The corn drift barriers were effective in reducing particle drift from applications with the ER11004 and the TTI11004 nozzles (Fine and Ultra Coarse spray classifications, respectively). The corn drift barrier had appropriate porosity and width as the airborne spray was captured within its canopy instead of deflecting up and over it. © 2018 Society of Chemical Industry.

Spray droplet size and carrier volume effect on dicamba and glufosinate efficacy.

BACKGROUND: Pesticide applications using a specific droplet size and carrier volume could maximize herbicide efficacy while mitigating particle drift in a precise and efficient manner. The objectives of this study were to investigate the influence of spray droplet size and carrier volume on dicamba and glufosinate efficacy, and to determine the plausibility of droplet-size based site-specific weed management strategies. RESULTS: Generally, across herbicides and carrier volumes, as droplet size increased, weed control decreased. Increased carrier volume (187 L ha-1 ) buffered this droplet size effect, thus greater droplet sizes could be used to mitigate drift potential while maintaining sufficient levels of weed control. To mitigate drift potential and achieve satisfactory weed control (≥ 90% of maximum observed control), a 900 µm (Ultra Coarse) droplet size paired with 187 L ha-1 carrier volume is recommended for dicamba applications and a 605 µm (Extremely Coarse) droplet size across carrier volumes is recommended for glufosinate applications. Although general droplet size recommendations were created, optimum droplet sizes for weed control varied significantly across site-years. CONCLUSION: Convoluted interactions occur between droplet size, carrier volume, and other application parameters. Recommendations for optimizing herbicide applications based on droplet size should be based on a site-specific management approach to better account for these interactions. © 2018 Society of Chemical Industry.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.