@article{edens_siegel_beckstead_honaker_hodgson_2024, title={Tissue cytokines in chickens from lines selected for high or low humoral antibody responses, given supplemental <i>Limosilactobacillus reuteri</i> and challenged with <i>Histomonas meleagridis</i>}, volume={14}, ISSN={["1664-042X"]}, DOI={10.3389/fphys.2023.1294560}, abstractNote={Histomonas meleagridis, a protozoan parasite, induces blackhead disease (histomoniasis) in poultry. During hatching, chicks from lines divergently selected for high (HAS) and low (LAS) antibody responses to sheep red blood cells were divided into two groups, each of HAS and LAS, and placed in pens with wood shavings as litter. Feed and water were allowed ad libitum. Half of the chicks from each line had Limosilactobacillus reuteri (L. reuteri) inoculated to their drinking water. On day 18, all chicks were given a transcloacal inoculation of 100,000 H. meleagridis cells. Then, 10 days later, they were euthanized, followed by collection of tissues from the brain, cecal tonsil, ceca, liver, thymus, and spleen for qPCR analyses of cytokines involved in immunological development. Changes in cytokine expressions were most numerous in the cecal tonsil, ceca, and liver. In the absence of a functional medication for control of histomoniasis, L. reuteri and/or its secretory product, reuterin, might serve, in some genetic populations, as a means to reduce the impact of histomoniasis in chickens. The data demonstrate that L. reuteri treatment had tissue specificity between the two genetic lines, in which the effects were targeted primarily toward the cecal tonsil, ceca, and liver, which are the primary tissue targets of the parasite (H. meleagridis), as well as the thymus and spleen. However, interactions among main effects reflect that responses to inflammatory markers observed in tissues for one genetic line may not be observed in another.}, journal={FRONTIERS IN PHYSIOLOGY}, author={Edens, Frank W. and Siegel, Paul B. and Beckstead, Robert B. and Honaker, Christa F. and Hodgson, Dellila}, year={2024}, month={Jan} }
 @article{wei_abraham_chadwick_beckstead_2020, title={Histomonas meleagridis isolates compared by virulence and gene expression}, volume={286}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2020.109233}, abstractNote={Pathology and putative virulence factor expression of three Histomonas meleagridis isolates differing in geographic origin, cell passage number (56 or 100), or cell populations grown from a monoculture were compared. Turkey poults inoculated with the high cell passage number isolates or monoculture isolates varied in gross lesion severity and weight gain (P<0.0001). Screening of a published H. meleagridis cDNA library identified forty- eight cysteine proteinases (CP) and one superoxide dismutase (Fe-SOD) proposed to function in either tissue damage and/or invasion and oxidative defense. The Fe-SOD and eight CPs were analyzed using real time polymerase chain reaction. CP2, CP3, and CP8 showed significant differences in expression among the field isolates (P ≤ 0.05). The high passage isolates had decreased CP2, CP3 and CP4 expression when compared with their field isolate. CP7 did not differ between field isolates or the 56-passaged isolate. The Fe-SOD gene showed significant differences in expression among the various isolates. When exposing cultured H. meleagridis to air, Fe-SOD expression decreased rapidly during the first hour of air exposure but increased progressively through the next 3 h. This study provides information on gross pathology and virulence factors associated with various isolates of Histomonas meleagridis which can aid in determining the pathogenetic mechanisms used by this organism.}, journal={VETERINARY PARASITOLOGY}, author={Wei, Zehui and Abraham, Mathew and Chadwick, Elle V and Beckstead, Robert B.}, year={2020}, month={Oct} }
 @article{beckstead_anderson_mcdougald_2020, title={Oviduct Fluke (Prosthogonimus macrorchis) Found Inside a Chicken Egg in North Carolina}, volume={64}, ISSN={["1938-4351"]}, DOI={10.1637/aviandiseases-D-20-00021.s1}, number={3}, journal={AVIAN DISEASES}, author={Beckstead, R. B. and Anderson, K. and McDougald, L. R.}, year={2020}, month={Sep}, pages={352–353} }
 @article{chadwick_rahimi_grimes_pitts_beckstead_2020, title={Sodium bisulfate feed additive aids broilers in growth and intestinal health during a coccidiosis challenge}, volume={99}, ISSN={["1525-3171"]}, DOI={10.1016/j.psj.2020.07.027}, abstractNote={Sodium bisulfate (SB) was evaluated on its ability to improve broiler growth and intestinal structure with(out) a coccidia challenge. One thousand two hundred Cobb500 day-old males were randomly assigned within 4 experimental groups with a 2 × 2 factorial design, with (out) SB in the diet and with(out) a day 0 coccidia challenge using a 10× dose of a commercial vaccine. At day 7, oocysts per gram of feces were determined. At day 0, 14, 28, and 41, BW and feed consumption were measured. At day 21, 20 birds per treatment were subjectively scored for coccidia lesions, and jejunal histologic samples were collected for villi measurements. Twenty additional birds were given fluorescein isothiocyanate-dextran to determine gut permeability. At day 41, 10 birds per treatment had histologic samples collected. Statistical analysis was conducted in JMP Pro 14 using GLM procedure to compare disease state and diet. Means were separated using Dunnett's test (P ≤ 0.05) with the nonchallenged standard diet treatment that is considered the control. All parameters measured indicated an effect due to the coccidia inoculation. Therefore, effects of diet on (non)challenged treatments were determined using a Student t test (P ≤ 0.05). Limited differences due to diet were seen for the nonchallenged production data. Sodium bisulfate had a thinner villi base width (P = 0.04) on day 21 and greater villi height (P = 0.03), smaller base width (P = 0.04), thicker muscularis (P = 0.03), and lower crypt: height ratio (P = 0.01) on day 41. Challenged SB had similar gut permeability to the nonchallenged control (P = 0.94) on day 21. There was no difference in flock uniformity, feed intake, oocysts per gram of feces, or lesion scores between challenged treatments. Challenged SB had greater BW on day 14 (P < 0.0001), 28 (P < 0.0001), and 41 (P = 0.02). Feed conversion ratio from day 0 to 14 was also lower (P = 0.0002). Challenged SB had smaller crypts (P = 0.02) and therefore a smaller crypt: height ratio (P = 0.03) on day 21. Challenged control had a larger apical width (P = 0.03) and thicker muscularis (P = 0.04) on day 41. Overall, the addition of SB during coccidial enteropathy aided in BW, feed conversion ratio, and villi health with no observed effects on parasite cycling.}, number={11}, journal={POULTRY SCIENCE}, author={Chadwick, Elle and Rahimi, Shaban and Grimes, Jesse and Pitts, John and Beckstead, Robert}, year={2020}, month={Nov}, pages={5324–5330} }
 @article{talghari_behnamifar_rahimi_torshizi_beckstead_grimes_2020, title={The effect of sodium bisulfate and coccidiostat on intestinal lesions and growth performance of Eimeria spp.-challenged broilers}, volume={99}, ISSN={["1525-3171"]}, DOI={10.1016/j.psj.2020.06.060}, abstractNote={Coccidiosis is a high-prevalence disease that annually entails huge costs for the poultry industry. Control of coccidiosis in poultry production is based on the use of coccidiostats and vaccines. However, along with the problem of drug resistance, there is a concern about food safety and drug residues in poultry products. The objective of this study was to evaluate the effect of sodium bisulfate (SBS) in comparison with monensin (M) and their combination (SBSM) effects on controlling coccidiosis in broilers. In a randomized design, 300 chickens (Ross 308) were divided into 5 treatments and 4 replications (15 birds per replicate). All birds, except the negative control (NC), were orally inoculated with 4 Eimeria species on 14 D of age. Treatments included were as follows: NC, an unsupplemented basal diet, nonchallenged; positive control, a basal diet unsupplemented, challenged with Eimeria spp; a basal diet supplemented with 5 g/kg of SBS; a basal diet supplemented with 1 g/kg of M; and a basal diet supplemented with 5 g/kg SBS and 1 g/kg M (SBSM). Oocyst shedding per gram (OPG) of the faecal sample from each experimental unit was counted on 5 to 14 D after inoculation. Two chicks from each experimental unit were euthanized to investigate intestinal lesions on day 5 after inoculation. The NC birds showed the highest BW gain and the lowest feed conversion ratio. The birds in the SBSM group had improved feed consumption compared with the M group in the prechallenge period (P < 0.05). All supplemented treatments resulted in a significant decrease in OPG. The M and SBSM treatments showed more efficacy than the SBS group (P < 0.05) in reducing OPG. There was a significant reduction in cecal lesions owing to supplementation with SBS, but the effect of SBS in the upper part of the intestine was lower than the M and SBSM groups (P < 0.05). Based on the results of this study, SBS has protective effects against coccidiosis in ceca, and the combination of M and SBS (SBSM) did not show any further improvement effect compared with M alone on the control of coccidiosis.}, number={10}, journal={POULTRY SCIENCE}, author={Talghari, Mariam and Behnamifar, Alireza and Rahimi, Shaban and Torshizi, Mohammad Amir Karimi and Beckstead, Robert and Grimes, Jesse L.}, year={2020}, month={Oct}, pages={4769–4775} }
 @article{chadwick_beckstead_2020, title={Two Blackhead Disease Outbreaks in Commercial Turkey Flocks Were Potentially Exacerbated by Poor Poult Quality and Coccidiosis}, volume={64}, ISSN={["1938-4351"]}, DOI={10.1637/aviandiseases-D20-00052}, abstractNote={Field visits at two different farms suggest a correlation between commercial turkey (Meleagridis gallopavo) flocks having increased mortality from blackhead disease (histomoniasis) if they suffer from poor poult quality at placement and coccidiosis (Eimeria spp.) before age 6 wk. In both cases, the flocks were all-in/all-out with curtain-sided houses and received a coccidiosis vaccine on day of hatch. At Farm I 2018, poults from different hatcheries were placed in two houses on the same farm (Houses 1 and 2). House 2 had poults considered poor quality and suffered from mortality associated with coccidiosis at 2 and 4 wk of age. At 8 wk, blackhead disease was diagnosed in both houses by postmortem examination. House 2 had mortality of >2000 poults, and the subpopulation of necropsied poults had gross lesions characteristic of histomoniasis. Gross lesions associated with blackhead disease were only found in eight poults in House 1, which was populated with good-quality poults and did not have a second spike in mortality due to coccidiosis. The Farm II 2020 poults were delivered from the same hatchery onto a three-house farm (Houses A, B, and C). House C had poults that were considered poor quality and had mortality associated with coccidiosis at 3 wk of age. At 8–9 wk, House C had mortality approaching 1000 birds, with all poults examined postmortem having clinical signs of blackhead disease. Houses A and B were populated with good-quality poults and had no diagnosed mortality from coccidiosis or blackhead disease. The similarity of these two cases suggest that poult quality at placement coupled with coccidiosis before 6 wk of age can influence the severity of blackhead disease in commercial turkey flocks.}, number={4}, journal={AVIAN DISEASES}, author={Chadwick, Elle and Beckstead, Robert}, year={2020}, month={Dec}, pages={522–524} }
 @article{sigmon_malheiros_anderson_payne_beckstead_2019, title={Blackhead Disease: Recovery of Layer Flock After Disease Challenge}, volume={28}, ISSN={1056-6171}, url={http://dx.doi.org/10.3382/japr/pfz029}, DOI={10.3382/japr/pfz029}, abstractNote={SUMMARY Blackhead disease, caused by the protozoan Histomonas meleagridis, is commonly found in layer pullets raised on the floor. We examined the effects of blackhead disease during the pullet-rearing period and on subsequent productivity during the first 8 wk of the laying cycle. Treatments were (1) uninfected controls and (2) H. meleagridis -infected pullets, with 4 replicate pens/treatment, 32 pullets/pen (Hy-LineW-36). Pullets in the challenge treatment were infected with H. meleagridis on day 18. Four birds/pen were necropsied on days 23 and 28 for lesion scores and day 176 for detection of H. meleagridis. Hens were moved to individual layer cages on day 120 and observed daily for feed consumption, date of first lay and egg production parameters. Pullets were positive for signs of blackhead disease in 83%–90% of infected birds necropsied on days 23 and 28, with average cecal lesion scores of 2.5 and 2.9. No liver lesions were observed. On day 176, 40% of infected birds were positive for H. meleagridis in the ceca. During the laying cycle, there were no significant differences (P ≤ 0.05) between treatments in terms of date of first lay, hen-day egg production, egg weight, feed conversion, egg mass/hen, or other reproduction measurements. These results showed that while there was no long-term effect of blackhead infection on layer productivity under laboratory conditions, H. meleagridis persisted in the flock, providing a reservoir for infection.}, number={3}, journal={Journal of Applied Poultry Research}, publisher={Elsevier BV}, author={Sigmon, C.S. and Malheiros, R.D. and Anderson, K.E. and Payne, J.A. and Beckstead, R.B.}, year={2019}, month={Sep}, pages={755–760} }
 @article{cupo_beckstead_2019, title={PCR detection of Heterakis gallinarum in environmental samples}, volume={271}, ISSN={["1873-2550"]}, DOI={10.1016/j.vetpar.2019.05.011}, abstractNote={Heterakis gallinarum is a widely distributed cecal nematode that parasitizes gallinaceous birds including chickens and turkeys. H. gallinarum infection poses a problem for the poultry industry as the nematode egg serves as a vector for the protozoan parasite, Histomonas meleagridis, the causative agent of histomonosis. The only means of detecting H. gallinarum in the environment is microscopic identification of the eggs in soil or feces; however, H. gallinarum eggs are often mistaken for those of Ascaridia galli. Three primer sets were designed from sequences cloned from the H. gallinarum genome to develop a diagnostic PCR. Each of these primer sets amplified a single product from H. gallinarum, but were unable to amplify DNA from H. meleagridis, Ascaridia galli, or Cestode sp. H. gallinarum DNA was amplified from Lumbricus sp. (earthworms) and Alphitobius diaperinus (darkling beetles), confirming that the earthworm acts as a paratenic host for H. gallinarum and suggesting that the darkling beetle may be a carrier for this nematode.}, journal={VETERINARY PARASITOLOGY}, author={Cupo, Katherine L. and Beckstead, Robert B.}, year={2019}, month={Jul}, pages={1–6} }
 @article{barrios_kenyon_beckstead_2017, title={Development of a Dry Medium for Isolation of Histomonas meleagridis in the Field}, volume={61}, ISSN={["1938-4351"]}, DOI={10.1637/11530-110816-resnote.1}, abstractNote={SUMMARY Blackhead disease is caused by Histomonas meleagridis, an anaerobic protozoan parasite, and results in mortality rates of up to 100% in turkeys and 30% in chickens. Outbreaks of blackhead disease are unpredictable, and the harvesting of H. meleagridis strains from the field would be a great resource for researchers to study its epidemiology. Therefore, the objective of this study was to develop a dry medium that would allow storage at ambient temperatures until needed. Fifty milliliters of horse serum was dried and then mixed with dry medium M199 with Hanks balanced salts (10.6 g), sodium bicarbonate (0.35 g), and rice powder (0.8 g). To test the ability of reconstituted medium to support growth of H. meleagridis, groups of 10 flasks containing 0.2 g of dry medium were stored for 24 hr at 25 and 60 C before testing. Other groups of flasks containing dry medium were stored at 25, 37, and 42 C for 1, 3, or 6 mo. At each test period, the flasks were reconstituted with 10 ml of water, inoculated with 100 000 H. meleagridis cells, and incubated at 40 C for 48 hr. Fresh liquid medium was used as a control. There were no differences in cell counts in medium stored at 25 or 60 C for 24 hr. After 1 mo, cell counts in reconstituted medium were about half that of fresh liquid medium after 48 hr of incubation. But after 3 and 6 mo, the cell counts were not significantly different in all groups (P < 0.05) after 72 hr of incubation. These results show that dried Dwyer medium can be stored at ambient temperatures for extended times and would be an effective tool for obtaining isolates of H. meleagridis from the field.}, number={2}, journal={AVIAN DISEASES}, author={Barrios, Miguel A. and Kenyon, Anna and Beckstead, Robert}, year={2017}, month={Jun}, pages={242–244} }
 @article{barrios_da costa_kimminau_fuller_clark_pesti_beckstead_2017, title={Relationship Between Broiler Body Weights, Eimeria maxima Gross Lesion Scores, and Microscores in Three Anticoccidial Sensitivity Tests}, volume={61}, ISSN={["1938-4351"]}, DOI={10.1637/11518-102116-reg.1}, abstractNote={SUMMARY Anticoccidial sensitivity tests (ASTs) serve to determine the efficacy of anticoccidial drugs against Eimeria field isolates in a controlled laboratory setting. The most commonly measured parameters are body weight gain, feed conversion ratio, gross intestinal lesion scores, and mortality. Due to the difficulty in reliably scoring gross lesion scores of Eimeria maxima, microscopic analysis of intestinal scrapings (microscores) can be used in the field to indicate the presence of this particular Eimeria. The goal of this study was to determine the relationship between E. maxima microscores and broiler body weights and gross E. maxima lesion scores in three ASTs. Day-old broiler chicks were raised for 12 days on a standard corn-soy diet. On Day 12, chicks were placed in Petersime batteries and treatment diets were provided. There were six birds per pen, four pens per treatment, and 12 treatments, for a total of 288 chicks per AST. The treatments were as follows: 1) nonmedicated, noninfected; 2) nonmedicated, infected; 3) lasalocid, infected; 4) salinomycin, infected; 5) diclazuril, infected; 6) monensin, infected; 7) decoquinate, infected; 8) narasin + nicarbazin, infected; 9) narasin, infected; 10) nicarbazin, infected; 11) robenidine, infected; and 12) zoalene, infected. On Day 14, chicks were challenged with an Eimeria field isolate by oral gavage. On Day 20, broilers were weighed, and gross lesion scores and microscores were classified from 0 to 4 depending on the severity of the gross lesion scores and E. maxima microscores. Data from three trials using different field isolates were statistically analyzed using a logarithmic regression model. There was no relationship (P = 0.1224) between microscores and body weight gain. There was a positive relationship between microscores and gross lesion scores (P = 0.004). However, there was also an interaction between isolate and treatment (P < 0.0001). Lastly, the interaction between isolate and gross lesion scores (P = 0.0041) demonstrates that the significance of the relationship between microscores and gross lesion scores may be dependent on pathogenicity of the challenge Eimeria or the amount of E. maxima in the inoculum.}, number={2}, journal={AVIAN DISEASES}, author={Barrios, Miguel A. and Da Costa, Manuel and Kimminau, Emily and Fuller, Lorraine and Clark, Steven and Pesti, Gene and Beckstead, Robert}, year={2017}, month={Jun}, pages={237–241} }
 @article{venkatesan_rajapaksha_payne_goodfellow_wang_kawabata_tabata_stice_beckstead_liu_2016, title={Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens}, volume={479}, ISSN={0006-291X}, url={http://dx.doi.org/10.1016/J.BBRC.2016.09.064}, DOI={10.1016/J.BBRC.2016.09.064}, abstractNote={The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.}, number={2}, journal={Biochemical and Biophysical Research Communications}, publisher={Elsevier BV}, author={Venkatesan, Nandakumar and Rajapaksha, Prasangi and Payne, Jason and Goodfellow, Forrest and Wang, Zhonghou and Kawabata, Fuminori and Tabata, Shoji and Stice, Steven and Beckstead, Robert and Liu, Hong-Xiang}, year={2016}, month={Oct}, pages={305–311} }
 @article{rajapaksha_wang_venkatesan_tehrani_payne_swetenburg_kawabata_tabata_mortensen_stice_et al._2016, title={Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets}, volume={6}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/SREP37247}, DOI={10.1038/SREP37247}, abstractNote={Abstract}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Rajapaksha, Prasangi and Wang, Zhonghou and Venkatesan, Nandakumar and Tehrani, Kayvan F. and Payne, Jason and Swetenburg, Raymond L. and Kawabata, Fuminori and Tabata, Shoji and Mortensen, Luke J. and Stice, Steven L. and et al.}, year={2016}, month={Nov} }
 @article{goodson_beckstead_payne_singh_mohan_2015, title={Amino acid sequence of Japanese quail (Coturnix japonica) and northern bobwhite (Colinus virginianus) myoglobin}, volume={181}, ISSN={0308-8146}, url={http://dx.doi.org/10.1016/J.FOODCHEM.2015.02.091}, DOI={10.1016/J.FOODCHEM.2015.02.091}, abstractNote={Myoglobin has an important physiological role in vertebrates, and as the primary sarcoplasmic pigment in meat, influences quality perception and consumer acceptability. In this study, the amino acid sequences of Japanese quail and northern bobwhite myoglobin were deduced by cDNA cloning of the coding sequence from mRNA. Japanese quail myoglobin was isolated from quail cardiac muscles, purified using ammonium sulphate precipitation and gel-filtration, and subjected to multiple enzymatic digestions. Mass spectrometry corroborated the deduced protein amino acid sequence at the protein level. Sequence analysis revealed both species’ myoglobin structures consist of 153 amino acids, differing at only three positions. When compared with chicken myoglobin, Japanese quail showed 98% sequence identity, and northern bobwhite 97% sequence identity. The myoglobin in both quail species contained eight histidine residues instead of the nine present in chicken and turkey.}, journal={Food Chemistry}, publisher={Elsevier BV}, author={Goodson, John and Beckstead, Robert B. and Payne, Jason and Singh, Rakesh K. and Mohan, Anand}, year={2015}, month={Aug}, pages={256–262} }
 @article{rumpf_bagley_thompson-peer_zhu_gorczyca_beckstead_jan_jan_2014, title={Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism}, volume={111}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.1406898111}, DOI={10.1073/PNAS.1406898111}, abstractNote={Significance}, number={20}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Rumpf, S. and Bagley, J. A. and Thompson-Peer, K. L. and Zhu, S. and Gorczyca, D. and Beckstead, R. B. and Jan, L. Y. and Jan, Y. N.}, year={2014}, month={May}, pages={7331–7336} }
 @article{jordan_vogel_stark_beckstead_2014, title={Expression of green fluorescent protein in the chicken using in vivo transfection of the piggyBac transposon}, volume={173}, ISSN={0168-1656}, url={http://dx.doi.org/10.1016/J.JBIOTEC.2014.01.016}, DOI={10.1016/J.JBIOTEC.2014.01.016}, abstractNote={The chicken is a well-established model system for studying developmental biology and is recognized as one of the top food production animals in the world. For this reason the chicken is an excellent candidate for transgenic applications, as the technology can be applied to both areas of research. Transgenic technology has not been broadly utilized in the chicken model, however, primarily due to difficulties in targeting germ cells and establishing germ line transmission. Transgenic technologies using non-replicating viral particles have been used in the chick, but are unsuitable for many applications because of size and sequence restraints and low efficiency. To create a more versatile method to target chick germ line stem cells, we utilized the transposable element system piggyBac paired with an in vivo transfection reagent, JetPEI. piggyBac has been previously shown to be highly active in mammalian cells and will transpose into the chicken genome. Here, we show that JetPEI can transfect multiple chick cell types, most notably germline stem cells. We also show that pairing these two reagents is a viable and reproducible method for long-term expression of a transgene in the chicken. Stable expression of the green fluorescent protein (GFP) transgene was seen in multiple tissue types including heart, brain, liver, intestine, kidney and gonad. Combining an in vivo transfection strategy with the PB system provides a simple and flexible method for efficiently producing stable chimeric birds and could be used for production of germ line transgenics.}, journal={Journal of Biotechnology}, publisher={Elsevier BV}, author={Jordan, Brian J. and Vogel, Seth and Stark, Michael R. and Beckstead, Robert B.}, year={2014}, month={Mar}, pages={86–89} }
 @article{ecco_preis_vilela_luppi_malta_beckstead_stimmelmayr_gerhold_2013, title={Corrigendum to “Molecular confirmation of Trichomonas gallinae and other parabasalids from Brazil using the 5.8S and ITS-1 rRNA regions” [Vet. Parasitol. 190 (1–2) (2012) 36–42]}, volume={196}, ISSN={0304-4017}, url={http://dx.doi.org/10.1016/J.VETPAR.2013.04.019}, DOI={10.1016/J.VETPAR.2013.04.019}, abstractNote={a Departamento de Clinica e Cirurgia Veterinarias, Escola de Veterinaria, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b Fundacao Zoo-Botânica de Belo Horizonte, Av. Otacilio Negrao de Lima 8000, 31365-450 Belo Horizonte, MG, Brazil c Department of Poultry Sciences, The University of Georgia, Athens, GA, USA d Department of Wildlife Management, North Slope Borough, P.O. Box 69, Barrow, AK 99723, USA e Center for Wildlife Health, Department of Forestry, Wildlife, and Fisheries, The University of Tennessee Institute of Agriculture, Knoxville, TN, USA}, number={3-4}, journal={Veterinary Parasitology}, publisher={Elsevier BV}, author={Ecco, Roselene and Preis, Ingred S. and Vilela, Daniel A.R. and Luppi, Marcela M. and Malta, Marcelo C.C. and Beckstead, Robert B. and Stimmelmayr, Raphaela and Gerhold, Richard W.}, year={2013}, month={Sep}, pages={552} }
 @article{ecco_preis_vilela_luppi_malta_beckstead_stimmelmayer_gerhold_2012, title={Molecular confirmation of Trichomonas gallinae and other parabasalids from Brazil using the 5.8S and ITS-1 rRNA regions}, volume={190}, ISSN={0304-4017}, url={http://dx.doi.org/10.1016/j.vetpar.2012.05.029}, DOI={10.1016/j.vetpar.2012.05.029}, abstractNote={Clinical, gross, and histopathology lesions and molecular characterization of Trichomonas spp. infection were described in two striped owls (Asio (Rhinoptynx) clamator), one American kestrel (Falco sparverius), two green-winged saltators (Saltator similis), and in a toco toucan (Ramphastos toco) from Brazil. These birds presented clinical signs including emaciation, ruffled feathers, abundant salivation and open mouth breathing presumably due to abundant caseous material. Gross lesions were characterized by multifocal yellow friable plaques on the surface of the tongue, pharynx and/or caseous masses partially occluding the laryngeal entrance. In the owls, the caseous material extended into the mandibular muscles and invaded the sinuses of the skull. Histopathologically, marked necrotic and inflammatory lesions were associated with numerous round to oval, pale eosinophilic structures (6–10 μm) with basophilic nuclei, consistent with trichomonads. Organisms similar to those described above also were found in the liver of the two green-winged saltators. To the authors’ knowledge, this is the first report of trichomonosis in a striped owl and a toco toucan. Sequence analysis of the Trichomonas spp. internal transcribed spacer 1 (ITS-1) region and partial 5.8S of the ribosomal RNA (rRNA) disclosed significant genetic diversity. Two sequences had 100% identity to Trichomonas gallinae, whereas two sequences had a 99% and 92% identity to a Trichomonas vaginalis-like sequence, respectively. One sequence (green-winged saltator 502-08) had a 100% identity to a newly recognized genus Simplicomonas.}, number={1-2}, journal={Veterinary Parasitology}, publisher={Elsevier BV}, author={Ecco, Roselene and Preis, Ingred S. and Vilela, Daniel A.R. and Luppi, Marcela M. and Malta, Marcelo C.C. and Beckstead, Robert B. and Stimmelmayer, Raphaela and Gerhold, Richard W.}, year={2012}, month={Nov}, pages={36–42} }
 @article{ritter_beckstead_2010, title={Sox14 is required for transcriptional and developmental responses to 20-hydroxyecdysone at the onset of drosophila metamorphosis}, volume={239}, ISSN={1058-8388}, url={http://dx.doi.org/10.1002/dvdy.22407}, DOI={10.1002/dvdy.22407}, abstractNote={Abstract}, number={10}, journal={Developmental Dynamics}, publisher={Wiley}, author={Ritter, Amanda R. and Beckstead, Robert B.}, year={2010}, month={Aug}, pages={2685–2694} }
 @article{banerjee_bainton_mayer_beckstead_bhat_2008, title={Septate junctions are required for ommatidial integrity and blood–eye barrier function in Drosophila}, volume={317}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/j.ydbio.2008.03.007}, DOI={10.1016/j.ydbio.2008.03.007}, abstractNote={The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.}, number={2}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Banerjee, Swati and Bainton, Roland J. and Mayer, Nasima and Beckstead, Robert and Bhat, Manzoor A.}, year={2008}, month={May}, pages={585–599} }
 @article{mcbrayer_ono_shimell_parvy_beckstead_warren_thummel_dauphin-villemant_gilbert_o'connor_2007, title={Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila}, volume={13}, ISSN={1534-5807}, url={http://dx.doi.org/10.1016/j.devcel.2007.11.003}, DOI={10.1016/j.devcel.2007.11.003}, abstractNote={In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.}, number={6}, journal={Developmental Cell}, publisher={Elsevier BV}, author={McBrayer, Zofeyah and Ono, Hajime and Shimell, MaryJane and Parvy, Jean-Philippe and Beckstead, Robert B. and Warren, James T. and Thummel, Carl S. and Dauphin-Villemant, Chantal and Gilbert, Lawrence I. and O'Connor, Michael B.}, year={2007}, month={Dec}, pages={857–871} }
 @article{beckstead_lam_thummel_2007, title={Specific transcriptional responses to juvenile hormone and ecdysone in Drosophila}, volume={37}, ISSN={0965-1748}, url={http://dx.doi.org/10.1016/j.ibmb.2007.03.001}, DOI={10.1016/j.ibmb.2007.03.001}, abstractNote={Previous studies have shown that ecdysone (E), and its immediate downstream product 20-hydroxyecdysone (20E), can have different biological functions in insects, suggesting that E acts as a distinct hormone. Here, we use Drosophila larval organ culture in combination with microarray technology to identify genes that are transcriptionally regulated by E, but which show little or no response to 20E. These genes are coordinately expressed for a brief temporal interval at the onset of metamorphosis, suggesting that E acts together with 20E to direct puparium formation. We also show that E74B, pepck, and CG14949 can be induced by juvenile hormone III (JH III) in organ culture, and that CG14949 can be induced by JH independently of protein synthesis. In contrast, E74A and E75A show no response to JH in this system. These studies demonstrate that larval organ culture can be used to identify Drosophila genes that are regulated by hormones other than 20E, and provide a basis for studying crosstalk between multiple hormone signaling pathways.}, number={6}, journal={Insect Biochemistry and Molecular Biology}, publisher={Elsevier BV}, author={Beckstead, Robert B. and Lam, Geanette and Thummel, Carl S.}, year={2007}, month={Jun}, pages={570–578} }
 @article{beckstead_thummel_2006, title={Indicted: Worms Caught using Steroids}, volume={124}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2006.03.001}, DOI={10.1016/j.cell.2006.03.001}, abstractNote={Three recent papers provide new insights into endocrinology in the worm Caenorhabditis elegans. These studies identify natural steroid ligands for the DAF-12 nuclear receptor, define a new enzyme in the hormone biosynthetic pathway, and clarify the role of endocrine signaling in adult longevity. Three recent papers provide new insights into endocrinology in the worm Caenorhabditis elegans. These studies identify natural steroid ligands for the DAF-12 nuclear receptor, define a new enzyme in the hormone biosynthetic pathway, and clarify the role of endocrine signaling in adult longevity. Many animals rely on some form of diapause to cope with difficult environmental circumstances, allowing them to survive until conditions improve and they can return to normal reproductive life. Diapause may seem to be a developmental anomaly in a life cycle; however, detailed genetic studies in the nematode worm Caenorhabditis elegans have shown that this is not the case. Rather, the regulation of C. elegans diapause has provided key insights into the progression of normal postembryonic development—growth, sexual maturation, and senescence—critical steps in the life cycle of higher organisms. Increased temperature, crowding, or nutritional depletion during the early stages of C. elegans development leads to the formation of an alternate third larval stage, the dauer larva, which is specially adapted for long-term survival. Upon a return to favorable conditions, the dauer larva emerges from diapause, resumes feeding, and continues to develop into an adult with a normal life span. Detailed studies have defined a genetic circuit that relays cues from chemosensory neurons to signal-transduction pathways that direct the choice between normal reproductive development and the dauer diapause. These studies arose from screens for mutants defective in their ability to form dauer larvae (Daf-d) or mutants that form constitutive dauers under favorable conditions (Daf-c; Riddle and Albert, 1997Riddle D.L. Albert P.S. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 739-768Google Scholar). Epistasis tests placed these genes in a pathway, with parallel input from TGF-β and insulin/IGF (insulin-like growth factor) signaling. This regulation makes sense as it is critical for the animal to properly assess its nutritional status prior to entering the prolonged period of diapause. During normal development, the DAF-2 insulin receptor acts through an evolutionarily conserved PI 3-kinase/AKT pathway to phosphorylate the transcription factor DAF-16, the worm ortholog of FOXO. This prevents DAF-16 translocation to the nucleus and allows normal growth to proceed (Figure 1A) . In unfavorable environments, insulin/IGF signaling becomes inactive, and dephosphorylated DAF-16 is translocated to the nucleus where it blocks growth and directs dauer formation (Figure 1B). Similarly, in unfavorable conditions, the TGF-β ligand DAF-7 acts through the type II TGF-β receptor DAF-4 to regulate the downstream effectors DAF-3 (SMAD) and DAF-5 (SKI/SNO), promoting dauer formation. These parallel insulin and TGF-β signaling inputs act in a cell nonautonomous manner to control reproductive growth and converge on two genes at the end of the pathway, daf-9 and daf-12, with daf-9 acting upstream from daf-12. The identification of daf-9 as encoding a CYP2 cytochrome P450 enzyme and daf-12 as encoding a nuclear hormone receptor, provided a mechanism for the cell nonautonomous relay of insulin and TGF-β inputs (Antebi et al., 2000Antebi A. Yeh W.H. Tait D. Hedgecock E.M. Riddle D.L. Genes Dev. 2000; 14: 1512-1527PubMed Google Scholar, Gerisch et al., 2001Gerisch B. Weitzel C. Kober-Eisermann C. Rottiers V. Antebi A. Dev. Cell. 2001; 1: 841-851Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Jia et al., 2002Jia K. Albert P.S. Riddle D.L. Development. 2002; 129: 221-231PubMed Google Scholar, Mak and Ruvkun, 2004Mak H.Y. Ruvkun G. Development. 2004; 131: 1777-1786Crossref PubMed Scopus (83) Google Scholar). Based on the ability of mammalian CYP2 enzymes to metabolize steroid hormones and the identification of steroid-hormone ligands for the vertebrate orthologs of DAF-12, an endocrine signaling model was proposed in which a hormone from DAF-9 is received by DAF-12 to direct reproductive growth (Figure 1A). In unfavorable conditions, DAF-9 was proposed to be inactive, leading to a presumed repressive function for the unliganded DAF-12, directing the dauer fate (Figure 1B). Several lines of evidence support this model. First, daf-9 acts nonautonomously and is expressed in endocrine cells or tissues that include the hypodermis, spermatheca, and a pair of cells located in a head ganglion (Gerisch and Antebi, 2004Gerisch B. Antebi A. Development. 2004; 131: 1765-1776Crossref PubMed Scopus (144) Google Scholar, Jia et al., 2002Jia K. Albert P.S. Riddle D.L. Development. 2002; 129: 221-231PubMed Google Scholar, Mak and Ruvkun, 2004Mak H.Y. Ruvkun G. Development. 2004; 131: 1777-1786Crossref PubMed Scopus (83) Google Scholar), whereas DAF-12 is widely expressed, consistent with its role in executing multiple developmental programs (Antebi et al., 2000Antebi A. Yeh W.H. Tait D. Hedgecock E.M. Riddle D.L. Genes Dev. 2000; 14: 1512-1527PubMed Google Scholar). Second, the phenotypes of daf-9 mutants resemble daf-12 mutants that map to critical DAF-12 amino acids that are predicted to act as hormone contact sites (Gerisch et al., 2001Gerisch B. Weitzel C. Kober-Eisermann C. Rottiers V. Antebi A. Dev. Cell. 2001; 1: 841-851Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Jia et al., 2002Jia K. Albert P.S. Riddle D.L. Development. 2002; 129: 221-231PubMed Google Scholar). Third, cholesterol deprivation mimics daf-9 and daf-12 mutations and leads to a few animals that form dauer-like larvae (Gerisch et al., 2001Gerisch B. Weitzel C. Kober-Eisermann C. Rottiers V. Antebi A. Dev. Cell. 2001; 1: 841-851Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Jia et al., 2002Jia K. Albert P.S. Riddle D.L. Development. 2002; 129: 221-231PubMed Google Scholar, Matyash et al., 2004Matyash V. Entchev E.V. Mende F. Wilsch-Brauninger M. Thiele C. Schmidt A.W. Knolker H.J. Ward S. Kurzchalia T.V. PLoS Biol. 2004; 2: e280https://doi.org/10.1371/journal.pbio.0020280Crossref PubMed Scopus (113) Google Scholar). Fourth, mutations in ncr-1 and ncr-2, homologs of the human Niemann-Pick type C gene that are required for proper cholesterol trafficking in C. elegans, form transient dauer larvae that resemble cholesterol-deprived animals (Li et al., 2004Li J. Brown G. Ailion M. Lee S. Thomas J.H. Development. 2004; 131: 5741-5752Crossref PubMed Scopus (64) Google Scholar). Finally, crude lipid extracts can rescue daf-9 mutant phenotypes as well as the dauers that form in the absence of dietary cholesterol (Gill et al., 2004Gill M.S. Held J.M. Fisher A.L. Gibson B.W. Lithgow G.J. Aging Cell. 2004; 3: 413-421Crossref PubMed Scopus (22) Google Scholar, Matyash et al., 2004Matyash V. Entchev E.V. Mende F. Wilsch-Brauninger M. Thiele C. Schmidt A.W. Knolker H.J. Ward S. Kurzchalia T.V. PLoS Biol. 2004; 2: e280https://doi.org/10.1371/journal.pbio.0020280Crossref PubMed Scopus (113) Google Scholar). Taken together, these lines of evidence provide strong support for DAF-9 and DAF-12 acting in an endocrine signaling pathway that regulates postembryonic development. Reproductive growth also involves the proper temporal progression of development in the third and fourth larval stages. This is reflected by the incorrect coordination of developmental programs in some daf-12 mutants, giving phenotypes that are referred to as heterochronic because of their effects on developmental timing (Antebi et al., 2000Antebi A. Yeh W.H. Tait D. Hedgecock E.M. Riddle D.L. Genes Dev. 2000; 14: 1512-1527PubMed Google Scholar). In addition, the DAF-9/DAF-12 pathway regulates longevity during adult stages. Germline ablation of C. elegans results in up to a 60% increase in adult longevity (Hsin and Kenyon, 1999Hsin H. Kenyon C. Nature. 1999; 399: 362-366Crossref PubMed Scopus (686) Google Scholar). This is not due to sterility as removal of both the germline and somatic gonad does not lead to an extended adult life span. In addition, the link between the germline and adult longevity is not unique to worms because similar effects have been seen in Drosophila and mice (Kenyon, 2005Kenyon C. Cell. 2005; 120: 449-460Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar). This coupling could have beneficial effects for survival of the species by allowing the animal to adjust its aging in response to its ability to reproduce. The effect of germline ablation on life span goes through the DAF-16 transcription factor which mediates the output of insulin/IGF and, presumably, other signaling pathways (Hsin and Kenyon, 1999Hsin H. Kenyon C. Nature. 1999; 399: 362-366Crossref PubMed Scopus (686) Google Scholar, Kenyon, 2005Kenyon C. Cell. 2005; 120: 449-460Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, Tatar et al., 2003Tatar M. Bartke A. Antebi A. Science. 2003; 299: 1346-1351Crossref PubMed Scopus (1040) Google Scholar; Figure 1C). One of these pathways is associated with daf-9 and daf-12, which appear to act together with daf-16 to modulate the effects on life span seen in germline-deficient animals. The exact nature of these interactions remains unclear. Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar usher in the age of C. elegans molecular endocrinology by identifying the first steroid hormones in this organism—the much-awaited ligands for the DAF-12 nuclear receptor. They exploit the past studies of DAF-9 and DAF-12, using the enzyme and receptor as tools to identify small chemical compounds that link one with the other. Their initial screen showed that 3-keto-lithocholic acid, but not lithocholic acid, weakly activates DAF-12 in a tissue culture cotransfection assay, independently of coexpressed DAF-9. The identification of a C-3 ketone suggested that 3-keto-sterols may function as DAF-12 ligands. This hypothesis was confirmed by showing that two naturally occurring 3-keto sterols—4-cholesten-3-one (an oxidation product of cholesterol) and lathosterone (a C. elegans cholesterol metabolite)—could activate DAF-12 in the presence, but not the absence, of DAF-9. In addition, either 4-cholesten-3-one or lathosterone that had been incubated with microsomes containing DAF-9 resulted in a complete rescue of daf-9 null mutants. This rescue was dependent upon using 3-keto-sterols as precursors and having DAF-9 in the microsomes. Further analysis of the DAF-9 metabolites of 4-cholesten-3-one and lathosterone identified these compounds as 3-keto-4-cholestenoic acid and 3-keto-7,(5α)-cholestenoic acid, respectively. The observation that these compounds are 3-keto, C-26 oxidized derivatives of cholesterol is consistent with biochemical studies of DAF-9, which showed that it acts as a 3-keto-sterol-26-monooxygenase that modifies 3-keto-sterols through successive side chain oxidation steps to generate DAF-12 ligands. The authors named these ligands Δ4-dafachronic acid (for 3-keto-4-cholestenoic acid) and Δ7-dafachronic acid (for 3-keto-7,(5α)-cholestenoic acid), based on the dauer and heterochronic phenotypes of daf-12 mutants. Chemical synthesis of Δ4-dafachronic acid allowed Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar to show that this compound can effectively activate DAF-12 in cotransfection assays and rescue daf-9 mutant phenotypes at nanomolar concentrations, demonstrating potent biological activity. Intermediate concentrations of Δ4-dafachronic acid resulted in only a partial rescue of daf-9 phenotypes, suggesting that hormone levels are critical for DAF-12 function. In addition, Δ4-dafachronic acid had little effect on daf-12 mutants that carry mutations predicted to affect ligand binding, consistent with the hormone directly regulating receptor activity. Consistent with classic models for nuclear receptor regulation, Δ4-dafachronic acid blocked the interaction between DAF-12 and its corepressor, DIN-1, and significantly enhanced the ability of DAF-12 to recruit the mammalian coactivator SRC-1. This effect of Δ4-dafachronic acid on DAF-12 cofactor interactions also translated to DAF-12 activity, resulting in potent induction of a DAF-12-regulated luciferase reporter gene in cultured cells. Taken together, these studies suggest that Δ4-dafachronic acid directly binds to DAF-12 to influence its interactions with cofactors and thereby regulate its transcriptional activity. Binding to Δ4-dafachronic acid occurs with a high affinity characteristic of natural nuclear receptor ligands. Importantly, Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar showed that 3-keto-cholestenoic acids could be isolated from extracts of wild-type worms, but not daf-9 mutants, and that these molecules can activate DAF-12 and fully rescue daf-9 mutants, defining them as natural hormones for DAF-12. The identification of DAF-12 ligands enabled Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar to determine the biological activity of dafachronic acid in the daf-2/insulin and daf-4/TGF-β pathways. Δ4-dafachronic acid rescued the phenotypes of both daf-7 and weak daf-2 mutants, in agreement with epistasis studies that position these genes upstream from daf-9 and daf-12 in the dauer pathway. Interestingly, Δ4-dafachronic acid was unable to fully rescue a strong daf-2 mutant, suggesting that the insulin pathway can also act downstream of, or parallel to, daf-12 (Figures 1A and 1B), confirming genetic studies which indicate that daf-2 function is difficult to place relative to daf-12 (Riddle and Albert, 1997Riddle D.L. Albert P.S. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 739-768Google Scholar, Vowels and Thomas, 1992Vowels J.J. Thomas J.H. Genetics. 1992; 130: 105-123Crossref PubMed Google Scholar). The requirement of 3-keto-sterols as DAF-9 precursors suggests that their synthesis may be an equally important regulatory step in the production of DAF-12 ligands. The identification of enzymes responsible for converting cholesterol to a 3-keto-sterol thus represents an important future goal for our understanding of how DAF-12 ligands are produced. Rottiers et al., 2006Rottiers V. Motola D.L. Gerisch B. Cummins C.L. Nishiwaki K. Mangelsdorf D.J. Antebi A. Dev. Cell. 2006; PubMed Google Scholar report a first step in this direction by showing that daf-36 encodes a key component of the dafachronic acid biosynthetic pathway. The authors identified this gene in a screen for mutants that display heterochronic and Daf-c phenotypes similar to those of daf-9, reasoning that such genes might represent additional steps in the DAF-9/DAF-12 pathway. Genetic studies positioned daf-36 downstream from TGF-β and insulin signaling and upstream from daf-12. Also, like daf-9, daf-36 mutations block the adult longevity associated with germline ablation, clearly placing this gene in the signaling pathways that control reproductive development and adult life span. daf-36 encodes a protein homologous to Rieske-like oxygenases found in plants, bacteria, and lower vertebrates. Interestingly, a bacterial family member that is most similar in sequence to DAF-36 can catalyze the 9-α hydroxylation of mammalian steroids, suggesting that DAF-36 may participate in the production of a DAF-12 ligand. Consistent with this, Δ4-dafachronic acid can efficiently rescue daf-36 mutant phenotypes at concentrations similar to those required to rescue daf-9 mutants. Lathosterone and 4-cholesten-3-one could also rescue daf-36 mutants—two compounds that have no effect on daf-9 mutants—suggesting that daf-36 acts upstream from daf-9. Moreover, 7-dehydrocholesterol, but not its immediate precursor, cholesterol, could rescue daf-36 mutants, suggesting that DAF-36 acts in the first step of dafachronic acid biosynthesis, modifying cholesterol to form 7-dehydrocholesterol (Figure 1A) or in a parallel pathway. These observations provide the first evidence that Rieske-like oxygenases can function in lipophilic hormone production. This proposal is consistent with the DAF-36 expression pattern, which is most abundant in the intestine—the major tissue for lipid storage in C. elegans. Interestingly, DAF-36 is not detectable in the head ganglion cells that express DAF-9, suggesting that hormonal precursors from the intestine are modified by peripheral cells to produce functional dafachronic acid. DAF-36 expression in the intestine is also consistent with its role in controlling adult life span. daf-36 mutations block the increased life span of germline-deficient animals, suggesting that it contributes to DAF-9 and DAF-12 endocrine signaling in the reproductive pathway. Berman and Kenyon, 2006Berman J.R. Kenyon C. Cell. 2006; 124: 1055-1068Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar provide further insights into how hormone signaling from the reproductive system to the intestine can influence adult longevity. These authors identify a gene, kri-1, that is required for the extended life span due to germline ablation, and characterize the effects of kri-1, daf-9, and daf-12 mutations on this pathway. The kri-1 gene encodes a conserved protein with ankyrin repeats that is constitutively expressed in the pharynx and intestine throughout postembryonic stages. Like daf-9 and daf-12, kri-1 mutations suppress the increase in life span associated with germline loss but do not affect the life span of wild-type animals. In addition, kri-1 mutants have no significant effect on the life span of germline-deprived daf-2 mutants or on their Daf-c phenotype, indicating that kri-1 acts independently of DAF-2 in the reproductive-signaling pathway. Upon germline ablation, DAF-16 in the intestine translocates from the cytoplasm to the nucleus, where its activity accounts for the entire increase in life span (Libina et al., 2003Libina N. Berman J.R. Kenyon C. Cell. 2003; 115: 489-502Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). By using a DAF-16::GFP fusion protein, Berman and Kenyon, 2006Berman J.R. Kenyon C. Cell. 2006; 124: 1055-1068Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar show that kri-1 is required for DAF-16 nuclear localization in the intestinal cells of germline-deficient animals. A less dramatic but significant reduction in nuclear DAF-16::GFP was also seen in daf-9 or daf-12 mutants. The nuclear localization of DAF-16 seen in daf-2 mutants, however, is not affected by kri-1, daf-9, or daf-12 mutations. This indicates that the roles of kri-1 and lipophilic hormone production on DAF-16 nuclear localization are specific to the reproductive pathway and act largely independently of insulin signaling (Figure 1C). The authors use a constitutively nuclear active DAF-16 protein to perform epistasis tests with kri-1, daf-9, and daf-12 mutations to address the long-standing question of how DAF-9 and DAF-12 contribute to the longevity of germline-deficient animals. As expected, nuclear DAF-16 extends the life span of daf-16 mutants that lack a germline. Similarly, nuclear DAF-16 rescues the increase in longevity seen upon germline ablation of kri-1 mutants, demonstrating that a key function for KRI-1 is to facilitate this localization of DAF-16 in the intestine. Interestingly, a daf-12 null mutation strongly blocked the longevity of germline-deficient animals that express nuclear DAF-16, indicating that DAF-12 can control longevity independently of DAF-16. Even more remarkable, a strong daf-9 allele has no effect on the longevity of germline-deficient animals that express nuclear DAF-16, indicating that daf-9 acts upstream from DAF-16 and that DAF-12 has functions in the germline-longevity pathway that are independent of lipophilic hormone signaling (Figure 1C). The identification of dafachronic-acid ligands for DAF-12 provides new directions for these studies. It will be interesting to determine how the hormone affects DAF-16 nuclear localization and adult life span in both wild-type and mutant worms. It is impressive that many of the predictions drawn from in vivo studies of daf-9 and daf-12 mutants are confirmed by the biochemical pathways outlined by Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar and Rottiers et al., 2006Rottiers V. Motola D.L. Gerisch B. Cummins C.L. Nishiwaki K. Mangelsdorf D.J. Antebi A. Dev. Cell. 2006; PubMed Google Scholar—yet more evidence of the elegance of C. elegans. Given the central role of DAF-12 in C. elegans biology, it is likely that the discovery of dafachronic-acid ligands for this receptor will have a major impact on the field. As Motola et al., 2006Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; (this issue)https://doi.org/10.1016/j.cell.2006.01.037Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar mention, the identification of two natural DAF-12 ligands suggests that these hormones may have distinct biological functions. A critical first step for these studies will be the chemical synthesis of Δ7-dafachronic acid, which appears to be more efficacious and abundant in the animal than Δ4-dafachronic acid. These ligands will facilitate more detailed functional characterization of DAF-9 and DAF-12, as well as provide many future directions for studying the critical decision between reproductive growth and dauer diapause and how the germline signals to the intestine to control adult longevity. The development of DAF-12 agonists and antagonists will provide invaluable tools for these studies, as well as a new basis for controlling parasitic nematodes, a major cause of crop damage and human disease in developing countries. In addition, further characterization of DAF-36 and the dafachronic acid biosynthetic pathway will provide critical insights into how hormone production is controlled to coordinate growth, maturation, and aging. Finally, these studies are not just about how nematodes develop but rather have wider implications. Endocrine circuits regulate progression through the life cycle and aging in all higher organisms, from C. elegans up to humans (Pardee et al., 2004Pardee K. Reinking J. Krause H. Sci. Aging Knowledge Environ. 2004; 2004: re8Crossref PubMed Scopus (22) Google Scholar, Tatar et al., 2003Tatar M. Bartke A. Antebi A. Science. 2003; 299: 1346-1351Crossref PubMed Scopus (1040) Google Scholar). Recent studies in Drosophila have shown that both insulin and TGF-β signaling impact the activity of the EcR ecdysteroid receptor, controlling organismal growth and maturation (King-Jones and Thummel, 2005King-Jones K. Thummel C.S. Science. 2005; 310: 630-631Crossref PubMed Scopus (20) Google Scholar, Zheng et al., 2003Zheng X. Wang J. Haerry T.E. Wu A.Y. Martin J. O'Connor M.B. Lee C.H. Lee T. Cell. 2003; 112: 303-315Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Similar signaling pathways play a central role in normal mammalian development and human disease. It is likely that the breakthroughs described in these papers will have a major impact on our broader understanding of postembryonic development, providing a better basis for determining how endocrine signaling helps the animal complete its long and difficult journey through life.}, number={6}, journal={Cell}, publisher={Elsevier BV}, author={Beckstead, Robert B. and Thummel, Carl S.}, year={2006}, month={Mar}, pages={1137–1140} }