s2fieldsofstudy: Biology Medicine

text:
ApoE deficiency exacerbates the development and sustainment of a semi-chronic K/BxN serum transfer-induced arthritis model

Background The risk for developing cardiovascular disease is greater in patients with rheumatoid arthritis (RA) than in the general population. While patients with RA also have dyslipidemia, the impact of dyslipidemia on the severity of inflammatory arthritis and associated cardiovascular disease is unclear. Currently, there are conflicting results regarding arthritis incidence in apolipoprotein E (ApoE) deficient mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis. Here, we utilize a distinct approach to investigate the contribution of a hyperlipidemic environment on the development of arthritis and atherosclerosis in mice lacking ApoE. Methods K/BxN serum transfer-induced arthritis (STIA) was assessed in C57BL/6 (control) and ApoE−/− mice using clinical indices and immunohistochemical staining. Ankle synoviums were processed for flow cytometry. Aortic atherosclerosis was quantitated using Sudan IV staining. Serum cholesterol and cytokine levels were determined via enzymatic and luminex bead-based assays, respectively. Results ApoE−/− mice developed a sustained and enhanced semi-chronic inflammatory arthritis as compared to control mice. ApoE−/− mice had increased numbers of foamy macrophages, enhanced joint inflammation and amplified collagen deposition versus controls. The presence of arthritis did not exacerbate serum cholesterol levels or significantly augment the level of atherosclerosis in ApoE−/− mice. However, arthritic ApoE−/− mice exhibited a marked elevation of IL-6 as compared to non-arthritic ApoE−/− mice and arthritic C57BL/6 mice. Conclusions Loss of ApoE potentiates a semi-chronic inflammatory arthritis. This heightened inflammatory response was associated with an increase in circulating IL-6 and in the number of foamy macrophages within the joint. Moreover, the foamy macrophages within the arthritic joint are reminiscent of those within unstable atherosclerotic lesions and suggest a pathologic role for foamy macrophages in propagating arthritis. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0912-y) contains supplementary material, which is available to authorized users.

Background
Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease associated with significant disability and increased mortality [1]. Life expectancy is reduced in patients with RA, largely due to the development of premature cardiovascular disease [2,3]. Patients with RA have roughly a 50-60 % increase in the risk of death from cardiovascular causes compared to the general population [4]. This elevated cardiovascular risk is independent of traditional risk factors and may be due to increased systemic inflammation in RA [3].
Atherosclerotic disease is a major cause of cardiovascular morbidity and mortality. One of the primary determinants of the propensity of atherosclerotic plaques to rupture and subsequently cause vascular occlusion is the stability of the foamy macrophages within the plaques [5]. Foamy macrophages are formed through the uptake of oxidized LDL through scavenger receptors, such as CD36. When foamy macrophages become saturated with oxidized LDL cell death can occur, which results in the release of proinflammatory cellular contents and formation of a necrotic core within the atherosclerotic plaque [5]. The understanding of this process has been facilitated by the use of ApoE knockout mice [6,7] since functional mutations in ApoE lead to elevations in serum cholesterol levels and premature atherosclerosis in both mice and humans [8,9].
The role that dyslipidemia plays in the development of atherosclerosis in RA is highly controversial. Numerous studies have shown that patients with RA have reduced total cholesterol (TC) levels compared to controls, which may be attributed to the increased inflammatory burden present in RA [10]. Surprisingly, decreases in TC are associated with adverse cardiovascular outcomes in patients with RA [11]. However, the atheroprotective effects of high-density lipoprotein (HDL) may be dampened in RA, as patients with more active disease exhibit reduced HDL-mediated cholesterol efflux as compared to patients with quiescent disease [12].
Conversely, the impact of the presence of hyperlipidemia on the development of inflammatory arthritis is also unclear. The collagen-induced arthritis (CIA) model [13] has previously been utilized to examine the development of arthritis and atherosclerosis in ApoE −/− mice, but has led to conflicting results. One group found that ApoE −/− mice were resistant to CIA [14], while another group showed that ApoE −/− mice developed more severe disease [15]. Thus the uncertainty of the role that dyslipidemia plays in the progression of arthritis in RA and whether the changes in lipid profile associated with inflammatory arthritis can alter the progression of atherosclerosis in a hyperlipidemic setting further underscores the critical need to utilize murine models of altered cholesterol metabolism to better understand these processes.
Here, we examined the development of arthritis in ApoE −/− mice compared to controls using a semi-chronic K/BxN serum transfer-induced arthritis (STIA) model [16]. The STIA model mimics the effector phase of RAlike disease and does not require lymphocytes. ApoE −/− mice developed enhanced semi-chronic inflammatory arthritis as compared to control mice. Elevated foamy macrophage numbers were observed in the synovial lining and extra-articular tissue from ApoE −/− mice, but not in controls. Moreover, arthritic ApoE −/− mice secreted increased levels of circulating pro-inflammatory IL-6. However, the degree of arthritis failed to significantly impact dyslipidemia or atherosclerotic disease burden in ApoE −/− mice on a high fat diet. These data suggest that a dyslipidemic environment can alter the progression of inflammatory arthritis during the effector phase of disease. Further, these data support a potential pathogenic role for foamy macrophages not only in atherosclerotic lesions, but also within the arthritic joint.

Animals
For generation of arthritogenic serum, KRN mice were kindly provided by Drs. Diane Mathis and Christophe Benoist, and non-obese diabetic mice were purchased from Taconic, Germantown, NY. Male and female wildtype control mice (C57BL/6, Jackson Laboratory, Bar Harbor, ME) and ApoE −/− mice, which spontaneously exhibit both hyperlipidemia and atherosclerosis (Jackson Laboratory-B6.129P2-Apoe tm1Unc /J stock number 002052), were purchased from commercial vendors. Genotyping was confirmed by Transnetyx (Memphis, TN). Mice were maintained on chow until 8 weeks of age and then switched to Harlan Teklad Western diet TD.94059 (Harlan Technologies, Houston, TX) containing 15.8 % fat and 1.25 % cholesterol. Animals were bred and maintained in a barrier facility within the Center for Comparative Medicine and were approved by the Northwestern University IACUC.

Scoring and induction of arthritis
For acute K/BxN serum transfer-induced arthritis (STIA), animals received a single injection of 50 μl of K/ BxN serum at 12 weeks of age as previously described [17][18][19]. STIA involves the passive transfer of arthritogenic anti-glucose-6-phosphate isomerase antibodies. The arthritis that ensues in these mice has almost 100 % penetrance and the mice develop maximum arthritis within 1 week after each injection of K/BxN serum. Arthritis severity was determined via the change in thickness of the hind paw ankle joints and a clinical score (total = 12) for all four paws, where 0 = normal, 1 = swollen wrist, 2 = swelling extending to dorsal paw, 3 = swelling extending to the digits. Acute arthritis severity was assessed every other day via clinical score for 2 weeks. Semi-chronic K/BxN STIA was elicited in ApoE −/− and control (C57BL/6) mice using 50 μl of K/ BxN serum every 2-3 weeks as previously described [17,20]. Animals were scored weekly for arthritis severity.

Tissue preparation and flow cytometry
Peripheral blood was harvested by cardiac puncture, followed by ventricular perfusion with 20 ml of PBS. Aortas were excised, fixed in 4 % paraformaldehyde and stained with Sudan IV (Sigma-Aldrich, St. Louis, MO). Ankles were either fixed in 10 % formalin, decalcified and then paraffin embedded prior to immunohistochemistry or processed for flow cytometry as previously described [17]. Data were acquired on BD LSR II flow cytometer. Additional file 1 lists antibodies, clones, fluorochromes and manufacturers used for all flow cytometric studies. The following dyes were utilized: fixable Viability Dye eFluor 506 from eBioscience and BoDIPY 493/503 from Molecular Probes. Compensation and analysis of the flow cytometry data were performed using FlowJo software (TreeStar).

Immunohistochemistry
Paraffin-embedded ankle sections were stained with haematoxylin and eosin, picrosirius red, rat anti-mouse F4/80 (clone BM8, Caltag) antibody or CD45 (Caltag) antibody and were scored for severity of disease by a pathologist (GKH) blinded to the conditions as previously described [20][21][22][23]. Imaging was performed using an Olympus DP40 microscope (Tokyo, Japan) equipped with a DP71 camera or a Zeiss upright AXIO (TissueGnostics GmbH) with a polarizing filter at the Northwestern Cell Imaging Facility (supported by NCI CCSG P30 CA060553 awarded to Robert H Lurie Comprehensive Cancer Center). Acquisition and analysis of picrosirius red staining utilized Tissue FAXS and TissueQuest. The picrosirius red quantification represents the sum of trans mean intensity for each tissue section.

Serum analysis
Serum TC, low-density lipoprotein/very low-density lipoprotein (LDL/vLDL) and HDL cholesterol levels were measured using enzymatic assays in accordance with the manufacturer's instructions (AbCam, Cambridge, MA). Serum cytokine and chemokine levels were assessed using Luminex (Austin, TX) Panomics 16-plex assays (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.

Evaluation of atherosclerosis
Atherosclerosis was quantified by computer-assisted morphometric analysis (Cell sense and ImagePro 6.3 software) of whole aortic (aortic root to iliac bifurcation) specimens. Results were expressed as the percentage of Sudan IV-stained lesion area (calculated by Sudan IV stained area/total aortic area × 100).

Statistical analysis
GraphPad Prism 5.0 Software (GraphPad Software, San Diego CA, USA) was utilized for all statistical analyses. Two-way ANOVA with Bonferroni posttests were used for arthritis analyses. Mann-Whitney U tests were used for histologic scoring, Sudan IV staining, levels of cytokines and chemokines, flow cytometry analysis, TC, LDL/vLDL and HDL levels among groups of mice. Statistical significance was established at p < 0.05.

ApoE −/− mice exhibit greater severity of semi-chronic arthritis than control mice
There are conflicting data concerning the effect of ApoE deficiency on the development of inflammatory arthritis in the CIA model [14,15]. Here we employed a different model of RA-like disease, the STIA model, which mimics the effector phase of RA and does not require the adaptive immune response. When the traditional STIA model was utilized (i.e., single injection of serum), we observed no difference in arthritis severity between ApoE −/− mice and C57BL/6 mice over a 2-week period (Additional file 2). However, this acute model represents a shorter time frame than the CIA model and does not provide adequate time for atherosclerotic plaques to develop. Therefore we employed a modified semi-chronic version of the STIA model, where animals were injected with K/ BxN serum every 2 weeks starting at 8 weeks of age and the development of arthritis was scored weekly over a 2-4 month period [24].
Throughout the development of semi-chronic arthritis ApoE −/− mice demonstrated significantly (p < 0.001) more severe inflammatory arthritis as compared to C57BL/6 mice (Fig. 1). The clinical scores were also associated with the presence of histologically assessed inflammation. Ankle joint histologic severity scores of inflammation were elevated 2.3-fold in arthritic ApoE −/− mice compared to C57BL/6 mice ( Fig. 2a, b). Additionally, extra-articular inflammation was increased 7.7-fold in arthritic ApoE −/− mice compared to C57BL/6 mice ( Fig. 2a, b). The histologic appearance of the extraarticular inflammation resembled that of a vulnerable atherosclerotic plaque, which is unstable and thus has an increased risk of progressing from a non-obstructive lesion to a vascular occlusion [25]. Although there were not significant differences in the total number of F4/80 + macrophages or in the number of F4/80 + macrophages within the synovial lining or pannus (Fig. 3a, b), when we focused on the subset of F4/80 + macrophages that were histologically consistent with foamy macrophages there was a marked increase in this particular population of macrophages in ApoE −/− mice as compared to C57BL/6 mice ( Fig. 3a, b). Moreover, foamy macrophages represented the overwhelming majority of F4/80 + cells within the pannus and synovial lining in ApoE −/− mice. In most of the synoviums, the foamy macrophages resided beneath the synovial lining but in some cases they were mixed within the synovial cell lining. The surface lining cells in ApoE −/− synoviums also showed variable foam cell morphology. In addition to the higher level of inflammation surrounding the foamy macrophages, there was a 1.5-fold increase in the amount of picrosirius red staining, indicative of collagen deposition, in arthritic ApoE −/− mice as compared to C57BL/6 mice ( Fig. 4a, b). This illustrates another parallel between the pathology surrounding foamy macrophages within the synovium and those within atherosclerotic lesions. Taken together, these results indicate that ApoE −/− mice demonstrate enhanced arthritis compared to C57BL/6 mice.

ApoE deficiency alters synovial macrophages characteristics
The development of acute arthritis has been shown to be associated with a progressive change in the cellular composition of immune cells within the joint, particularly macrophages [17]. We previously established our ability to identify at least two different populations of synovial macrophages (MHCII + and MHCII − ) using flow cytometry [17]. Therefore, we analyzed the impact of semi-chronic STIA on synovial macrophage composition in ApoE −/− mice. Total numbers of synovial macrophages did not differ between ApoE −/− and C57BL/6 mice with semi-chronic STIA (Fig. 5a). However, the higher ratio of MHCII + to MHCII − macrophages was more pronounced both at baseline and in the presence of semi-chronic arthritis in ApoE −/− compared to C57BL/6 mice (Fig. 5b). In addition, both MHCII + and MHCII − synovial macrophages in ApoE −/− mice displayed increased expression of CD36, a scavenger receptor that is involved in the uptake of oxidized lipoproteins (Fig. 5c-e). Both MHCII − and MHCII + foamy synovial macrophages exhibited a higher uptake of lipophilic dye in ApoE −/− mice ( Fig. 5f-h) and had higher side scatter ( Fig. 5i-k), consistent with an increased accumulation of lipids within the cytoplasm.

Arthritis does not impact atherosclerosis susceptibility in ApoE −/− mice
We have previously shown that induction of inflammatory arthritis led to the development of atherosclerosis in mice [20]. To determine if inflammatory arthritis exacerbates atherosclerosis in ApoE −/− mice, a known model of hyperlipidemia-induced atherosclerosis, atherosclerotic lesion area via Sudan IV staining was investigated following STIA. Both C57BL/6 ( Fig. 6a, b) and ApoE −/− (Fig. 6c, d) mice exhibited a trend towards increased atherosclerosis in the presence of STIA with a 2-fold and 1.4-fold increase, respectively (Fig. 6e). When ApoE −/− mice were compared to C57BL/6 there was a 5.2-fold increase in area of atherosclerosis in the absence of arthritis and a 3.6-fold increase in the area of atherosclerosis in the presence of arthritis (Fig. 6e). These data demonstrate that inflammatory arthritis has a minimal impact on the high level of atherosclerosis that is already present in ApoE −/− mice on a high fat diet.

Serum cholesterol levels are elevated in ApoE −/− mice
To determine whether induction of arthritis alters serum cholesterol profiles in ApoE −/− and C57BL/6 mice, levels of TC, LDL/vLDL and HDL were measured in serum from semi-chronic STIA animals (Fig. 7). Serum levels of both TC (Fig. 7a) and LDL/vLDL (Fig. 7b) were significantly greater in arthritic ApoE −/− mice (2.5-fold for TC and 3.6-fold for LDL/vLDL) compared to arthritic C57BL/6 mice. HDL levels were 38 % lower in arthritic ApoE −/− mice compared to arthritic C57BL/6 animals (Fig. 7c). However, there were no significant intra-genotype differences in lipid profile upon induction of STIA. These data suggest that although baseline lipid profiles are substantially different between ApoE −/− and C57BL/6 Immunohistopathological scores for F4/80 + total cells, F4/80 + cells within synovial lining and F4/80 + cells within the pannus from control and ApoE −/− mice. F4/80 + foam cells within each of these categories are subsequently quantified from the mice described above. Data are represented as mean ± SEM. Asterisk denotes statistically significant differences: ***p < 0.001. SL synovial lining, P pannus, arrowheads foamy macrophages arthritic ApoE −/− mice compared to arthritic C57BL/6 mice (Fig. 8). As IL-6 is a known mediator of both atherosclerosis and RA, our data suggest a potential role for this pro-inflammatory cytokine in our model.

Discussion
The role that dyslipidemia plays in the development of arthritis in murine models of RA remains controversial. Here, we utilized the semi-chronic STIA model of inflammatory arthritis since this model resembles the effector phase of RA and does not require the adaptive immune response. We show that ApoE −/− mice, which spontaneously develop hyperlipidemia and atherosclerosis, also exhibit a heightened level of inflammatory arthritis when compared to control mice. This heightened inflammation is associated with an increased number of foamy macrophages in the synovium and elevated serum IL-6 levels. Nonetheless, the presence of inflammatory arthritis is not associated with changes in atherosclerosis or hyperlipidemia in ApoE −/− mice. These data suggest that ApoE may play a novel suppressive role in inflammatory arthritis.
Currently, there are a plethora of therapeutics to treat dyslipidemia in RA patients. However, treatment of RA patients with atorvastatin, a cholesterol lowering medication, has been shown to decrease swollen joint count [26]. Further, previous studies using hypercholesterolemic mice to evaluate the relationship between dyslipidemia and inflammatory arthritis have yielded conflicting results. Prior studies utilized the CIA model to assess atherosclerosis and arthritis in ApoE −/− mice [14,15] We utilize an entirely different approach with the acute and semi-chronic STIA model where there is passive transfer of arthritogenic anti-glucose-6-phosphate isomerase antibodies twice a month. The arthritis that ensues in these mice has almost 100 % penetrance and the mice develop maximum arthritis within 1 week after each injection of K/BxN serum. This is in contrast to the CIA model, which has a delayed onset of disease (6-8 weeks after CII immunization) and diminished penetrance (65-75 % of control animals developing disease in the CIA models used by Asquith et al. and Postigo et al.). In addition, the semi-chronic STIA model allows us to bypass the use of adjuvant, which may complicate interpretation of the results. Moreover, this model allows us to assess the impact of semi-chronic arthritis on the development of atherosclerosis, which is a complication of longstanding disease. Here, we demonstrate that deficiency in ApoE potentiates and sustains the presence of inflammatory arthritis.
Global deletion of ApoE results in extreme elevations in serum cholesterol [27]. ApoE deficiency is known to exert effects on hematopoietic stem cell proliferation and mobilization, monocytosis and macrophage accumulation in tissue [28,29]. In our semi-chronic STIA model, loss of ApoE is associated with changes to the properties of synovial macrophages. In our previous study using the acute STIA model (one time passive transfer glucose-6-phosphate antibodies), we demonstrated that MHCII + macrophage subsets predominate during the propagation phase of arthritis while the MHCII − macrophage becomes the primary cell in the resolution phase of arthritis [17]. These data suggest that ApoE −/− mice may be more primed for the induction of arthritis since they exhibit a higher proportion of MHCII + to MHCII − macrophages. However, it is also possible that the increase in inflammation is related to a lack of resolution of inflammation. ApoE −/− mice are also able to amass foamy macrophages within the synovium. Although foamy macrophages are a well-established mediator of atherosclerotic disease, their role in the arthritic joint is not as clear. Immunohistochemical analysis of human synovial membranes reveal that foam cells are present in the synovium of RA patients [30]. In addition, a recent study observed foamy macrophages in arthritic/atherosclerotic rabbit joints [31]. Our data show increases in the number of foamy macrophages in ApoE −/− mice compared to control in the semi-chronic STIA model. Interestingly this increase in foamy macrophages is associated with higher levels of inflammation in ApoE −/− mice (Fig. 2) but not an increase in damage to the joint or erosive disease. However, the presence of foamy macrophages appear to be associated with enhanced collagen deposition (Fig. 4) that is reminiscent of atherosclerotic plaques and further support an extension of the pathogenic role of foamy macrophages from the vasculature to the synovium. Our data show that ApoE −/− mice have increased expression of CD36 on synovial macrophages. This is consistent with previous reports showing elevated expression of CD36 in ApoE −/− peritoneal macrophages [32]. CD36 is a scavenger receptor involved in the uptake of oxLDL and the subsequent development of foamy macrophages. CD36 signaling can inhibit migration and thus promote macrophage trapping within tissue. It can also stimulate the production of inflammatory cytokines [32,33]. It is possible that CD36 is critical for the development of arthritis in our model similar to its role in atherosclerosis where loss of CD36 improves atherosclerosis in ApoE −/− mice by over 60 % [32].
The exact role of the differential expression of CD36 on MHCII − and MHCII + synovial macrophages is unclear. Data from an acute arthritis model suggest that the majority of tissue-resident macrophages are MHCII − and are involved in attenuating arthritis as opposed to the MHCII + cells that are monocyte-derived and of which a specific subpopulation is involved in propagating arthritis [17]. If these data are extrapolated to the semi-chronic arthritis model it would suggest that the potential impact of CD36 up-regulation on enhancing inflammatory arthritis is more critical in MHCII + cells, which can propagate inflammation, than the MHCII − cells which attenuate inflammation. Future studies will be needed to investigate the requirement for CD36 in synovial macrophages and to further characterize this heterogeneous macrophage population. These studies should evaluate not only the expression of surface markers on foamy macrophages isolated from the synovium in order to differentiate them from foamy macrophages within other cellular niches, but also look at gene expression on a single-cell basis.
Transcriptional profiling will also help further investigations into another potential target in our system, IL-6. Polymorphisms in IL-6 and serum levels of IL-6 have been shown to be associated with an increased risk of metabolic syndrome [34,35]. In addition, studies evaluating the chronic inflammation associated with obesity and increased risk of metabolic syndrome have suggested that IL-6 is an important cytokine released by adipose tissue macrophages [36]. Our data reveal large systemic increases in IL-6 during the course of inflammatory arthritis in ApoE −/− mice. This is similar to data from a French cohort showing a correlation between serum IL-6 levels and both swollen joint count and structural damage to the joint in RA patients [37]. It is also consistent with the success of anti-IL-6 receptor therapy, tocilizumab, in treating RA [38]. However, it is likely that IL-6 is a downstream product of the foamy macrophages as it is not required for the acute STIA model [39]. IL-6 may also propagate the production of foamy macrophages, as in vitro experiments have shown that IL-6 plays a role in increasing lipid uptake by macrophages [40]. Future studies will be needed to determine the requirement for IL-6 in the development of foamy macrophages and their infiltration into the joint and the ability of foamy macrophages to produce IL-6.
Although IL-6 is also an important mediator of cardiovascular disease, its augmentation in arthritic ApoE −/− mice is not associated with a pronounced increase in atherosclerosis. It is possible that the systemic increase in IL-6 is not associated with local production of IL-6 in both the joint and the aorta. It may also be difficult to further enhance atherosclerosis in ApoE −/− mice, which is already at a saturated level due to a combination of both genetic and dietary factors. In support of this assertion, CIA studies performed on ApoE −/− mice show no change in atherosclerosis [41,42]. Nonetheless this negative data is important to help understand the interplay between the presence of semichronic inflammatory arthritis and the development of atherosclerosis.
Taken together, these studies demonstrate that ApoE itself has a major effect on the development of arthritis, which may be mediated through the formation of foamy macrophages within the synovium. The identification of foamy macrophages within the inflamed arthritic joint provides a potential mechanistic link between inflammatory arthritis and atherosclerosis. Improved understanding of the connection between arthritis and atherosclerosis is crucial as we evaluate therapies that will not only improve arthritis but also decrease patients' risk of cardiovascular disease.

Conclusions
The present study examines the relationship between inflammatory arthritis and atherosclerosis. Our model shows that ApoE is essential for limiting the development of semi-chronic inflammatory arthritis. In contrast, the combination of inflammatory arthritis and ApoE deficiency does not enhance atherosclerosis beyond the high level observed in non-arthritic ApoE −/− mice. However, our data provide additional support that factors beyond the absolute levels of lipids may be important for the development of inflammatory arthritis in ApoE −/− mice. Our data also support a critical role for foamy macrophages and IL-6 production in the progression of inflammatory arthritis. Future studies may uncover the dual role for monocytes in the development of atherosclerosis and arthritis in ApoE −/− mice and address the effects of ApoE deficiency on joint foam cell formation, IL-6 production and arthritis development in a cell-type specific fashion.

Authors' contributions
AMA carried out arthritis studies, processed and quantified atherosclerosis staining, performed picrosirius red analysis, interpreted data, designed experiments and wrote manuscript. RS carried out arthritis studies, processed atherosclerosis staining, performed cholesterol assays, was involved in experimental design and reviewed manuscript. SR carried out arthritis studies, processed and analyzed atherosclerosis staining, performed data interpretation, was involved in experimental design and wrote manuscript. AS processed atherosclerosis staining, analyzed atherosclerosis data and reviewed the manuscript. AVM was involved in experimental design, data collection, flow analysis and edited the manuscript. GKH quantified pathologic changes in joints and assisted in writing the manuscript. SD was involved in experimental design, data collection and reviewed the manuscript. FT was involved in experimental design, data collection and reviewed the manuscript. ME was involved in experimental design and reviewed the manuscript. DEV was involved in experimental design, data interpretation and reviewed the manuscript. CMC carried out arthritis studies, performed luminex assays, processed and quantified atherosclerosis staining, performed data interpretation, designed experiments and wrote the manuscript. HP was involved in experimental design, data interpretation and wrote the manuscript. All authors read and approved the final manuscript.


extfieldsofstudy: Biology Medicine

text:
A new species of Baenothrips Crawford from China (Thysanoptera, Phlaeothripidae)

Abstract A new urothripine species, Baenothrips cuneatus sp. n., is described from China. This is distinguished from its congeners by the following combination of characteristics: dorsal surface of head having a wedge-shaped reticulation extending from median to the posterior margin; antennal segments VII–VIII is closely joined with a complete suture; the mesoacrotergite strongly constricted in the middle; abdominal tergite I divided into 5 plates; width of membranous gap between ovispan on abdominal sternite IX approximately 1/3 of the apical width of segment IX.

Introduction
The genus Baenothrips Crawford currently comprises 11 species in the world, of which five are distributed in Asia (ThripsWiki 2016). These thrips are considered to be fungusfeeding, with most living in leaf litter, grass tussocks or dead twigs (Stannard 1970;Mound 1972;Okajima 1994). However, some species, such as B. moundi Stannard of Australia, can crawl up above soil level to grass stems, and are likely to be winddispersed (Mound 1972;Ulitzka and Mound 2014). The new species described below has similar dispersive behaviour, and can be collected not only in leaf litter but also on fresh leaves or stems of grass, fern, and dicotyledons. Presumably this species normally inhabits leaf litter, but crawls up fresh plants occasionally and is then dispersed by wind.

Materials and methods
The thrips were extracted by using Tullgren funnels from leaf litter, or collected by beating vegetation over a white plastic tray using a small stick, and then sorted and preserved in 90% alcohol. Specimens were then mounted into Canada balsam on microscope slides. Structural details were examined with a ZEISS Imager A1 microscope, photos were taken by a Photometrics CoolSNAP camera, and the figures were subsequently processed with Adobe Photoshop CS6. All type specimens are deposited in the Insect Collection, South China Agricultural University (SCAU).
Paratypes. 8 females 1 male, taken with holotype; 3 females 7 males, same locality and habitat as holotype, 5.ix.2015, Zhaohong Wang. CHINA, Hunan: 1 female, Yanling Description. Female macroptera ( Fig. 1): Head and prothorax dark brown; pterothorax yellowish white with dark brown anteriorly and laterally; abdominal tergites I-IX yellowish white with brown laterally, of which tergites II-V each with a pair of light brown circular patches on either side; tube yellow with extreme apex dark brown. All coxae, trochanters and apical half of tarsi brown; fore and mid femora yellowish white except for inner base brown, hind femora pale yellowish brown with brown on dorsal margins; fore tibiae yellowish white, mid tibiae yellowish yellow shaded with light brown on outer margins, hind tibiae whitish but brown medially. Antennal segment I pale brown, segments II-VI yellowish white, segments VII and VIII pale brown.
Head (Fig. 3) almost as long as broad or a little shorter; head broadly rounded in front, with three pairs elongate cephalic setae on anterior margin; dorsal surface tuberculate and with a wedge-shape reticulation extending from middle to posterior margin; cheeks almost straight. Eyes with approximately eight facets dorsally and six ventrally, of which three dorso-lateral facets are distinctly larger than the others; three ocelli present, anterior ocellus placed between inner cephalic setae, posterior ocelli behind outer pair of cephalic setae and placed close to eyes. Antenna 8-segmented, arising ventrally ( Fig. 13), segments VII-VIII closely joined with a complete suture; segment III with no sense cones, IV with two sense cones, each approximately two-thirds as long as the segment; segment V with one sense cone, situated outside of apex; segments VI and VII each with one sense cone dorsally. Maxillary stylets retracted to base of compound eyes, approximately one-third of head width apart medially.
Pronotum rectangular (Fig. 5), shorter than head, dorsal surface with irregular sculpture and wart-like tubercles; epimeral setae well developed. Mesoacrotergite strongly constricted medially by a very narrow bridge (Figs 7, 11); mesonotum sculptured with transverse dotted lines on anterior third; meta-epimeron bulging with one well developed seta. Fore wing bulging at base without basal setae; both fore wing and hind wing with a median vein or thickening, and with many, but not closely spaced, fringe cilia. Basantra weakly developed, largely membranous; ferna well developed, strongly narrowed posteromedially (Fig. 6); mesopresternum complete and transverse; mesoeusternum anterior margin entire. Mesosternal furcae fused in the middle; metasternal furcae placed laterally and widely separated (Fig. 8). All tarsi unarmed.
Abdominal tergite I divided into five plates, a slender median longitudinal plate bearing a campaniform sensillum (Fig. 14); tergite II with a pair of expanded wingretaining setae; tergites III-VII with two pairs of wing-retaining setae, inner pair knife-like and outer pair fin-shaped ( Fig. 9; cf. Bhatti 2002: fig 15a); tergites III-VIII posterolateral setae enlarged, each with a transverse row of 12-18 short setae medially; tergite IX 4.4 times as long as distal wide; ovispan slightly reduced, the width of membranous gap between ovispan approximately 1/3 of the apical width of segment IX (Fig. 12). Tube approximately twice as long as head with three pairs of anal setae; anal setae nearly 2.2 times as long as tube, but median dorsal pair half as long as the lateral two pairs. Male aptera (Fig. 2a): Head with only two pairs of elongate cephalic setae on anterior margin; wedge-shape reticulation of head wider than in female (Fig. 4); abdominal tergites III-VII without brown circular patches on either side; tergites II-VII with only one pair of wing-retaining setae (Fig. 10); abdominal tergite IX nearly three times as long as distal wide. Male genitalia as in figure 2b.

Remarks.
Only two species of the genus Baenothrips Crawford are validly recorded from China, B. cuneatus, and B. ryukyuensis Okajima. The record by Kudô (1978), of B. asper (Bournier) from China in Taiwan, was considered by Okajima (1994) to be a misidentification and to actually refer to ryukyuensis. Similarly, Bhatti (2002) suggested that the species B. asper is known only from Africa, and that the Asian records refer to some other species. Recently, B. ryukyuensis was recorded by Dang and Qiao (2014) from Fujian, China. Moreover, during sorting of specimens Baenothrips from China we found in our collections slide-mounted specimens labelled by Wang and Tong (2007) as B. murphyi (Stannard), and recognised that these actually represent B. cuneatus, the new species described above.
Baenothrips cuneatus sp. n. is most closely related to B. asper (Bournier) in colour pattern and several other features, but in the new species, dorsal surface of head having a wedge-shaped reticulation extending from median to the posterior margin; the mesoacrotergite is strongly constricted medially by a very narrow bridge (Figs 7, 11) as in B. moundi (Stannard) (cf. Bhatti 2002: fig 25) and the membranous gap (Fig. 12) between the ovispan on abdominal sternite IX is much wider than those in B. asper (Bournier) (cf. Bhatti 2002: fig 18). In addition, there are other five species of the genus occurring in Asia, some of them are also similar to B. cuneatus sp. n., but this new species can be distinguish from them by the below key. Head with a wedge-shaped reticulation extending from median to the posterior margin; the mesoacrotergite is strongly constricted medially (Figs 7, 11); abdominal tergite I divided into five plates (Fig. 14); the width of membranous gap (Fig. 12)


extfieldsofstudy: Medicine Biology

text:
The mRNA transportome of the BicD/Egl transport machinery

ABSTRACT mRNA (mRNA) transport focuses the expression of encoded proteins to specific regions within cells providing them with the means to assume specific functions and even identities. BicD and the mRNA binding protein Egl interact with the microtubule motor dynein to localize mRNAs in Drosophila. Because relatively few mRNA cargos were known, we isolated and identified Egl::GFP associated mRNAs. The top candidates were validated by qPCR, in situ hybridization and genetically by showing that their localization requires BicD. In young embryos these Egl target mRNAs are preferentially localized apically, between the plasma membrane and the blastoderm nuclei, but also in the pole plasm at the posterior pole. Egl targets expressed in the ovary were mostly enriched in the oocyte and some were apically localized in follicle cells. The identification of a large group of novel mRNAs associated with BicD/Egl points to several novel developmental and physiological functions of this dynein dependent localization machinery. The verified dataset also allowed us to develop a tool that predicts conserved A'-form-like stem loops that serve as localization elements in 3′UTRs.

Introduction mRNA transport is an important mechanism for focusing protein expression to specific regions of cells, to individual cells in a group of interconnected cells, or even to regions of an organism in cases where its nuclei share a common cytoplasm. mRNA localization plays important functions wherever spatial restriction of protein expression is needed and the importance of mRNA localization for normal development and physiology is underlined by the fact that this mechanism is found form bacteria to humans. 1 Drosophila oocytes and embryos are well-established systems to study mRNA localization. Oocyte determination depends on mRNA localization and the spatial localization of bicoid (bcd), oskar (osk) and gurken (grk) transcripts determines the embryonic anterior-posterior axis as well as the dorsalventral axis already during oogenesis. 2 During the syncytial blastoderm stage of embryonic development pair-rule transcripts, such as fushi tarazu (ftz), even-skipped (eve) and hairy (h) become localized via microtubule (MT) minus-end directed transport to the apical cytoplasm, above the layer of nuclei. Their local translation in the apical cytoplasm close to the nuclei allows the local synthesis of the transcription factors encoded by them. This is an efficient way to produce the proteins where they are needed and to restrict diffusion to the surrounding nuclei. Other transcripts that are targeted apically to the nuclei are the mRNA encoding the signaling molecule Wingless (Wg), which is subsequently translated and secreted apically, and the inscuteable (insc) mRNA that becomes apically localized in neuroblasts. 3,4,5 Altogether, 71% of the genes expressed during Drosophila embryonic development show discernable subcellular mRNA localization patterns. 6 In Drosophila, mRNA localization involves a machinery composed of Bicaudal-D (BicD) and Egalitarian (Egl), which interact with dynein/dynactin microtubule motors on the one hand and with the mRNA cargo on the other (reviewed in 1 ). In this way the mRNA cargo is targeted to distinct cellular compartments. While we have learned much about the genes and proteins involved in mRNA transport and localization, until recently we knew only a small number of mRNAs that use the BicD/Egl localization machinery for their transport. Here we describe many novel mRNAs localized by BicD/Egl. The results for the top candidates were validated in independent experiments and in several ways. Aside from quantifying their enrichment during immunopurification with qPCR, we also included genetic analyses combined with in situ mRNA localization experiments to confirm that these mRNAs indeed require BicD to reach their target regions. This approach also revealed novel cellular compartments that are targeted by the BicD/Egl transport machinery in embryos and ovaries. Finally we used the resource we established to develop a search algorithm to identify putative A'-form-like localizing elements in 3 0 UTRs. Testing two of the newly identified putative localization sequences revealed that the program successfully identified novel localizing signals in the 3 0 UTR of novel Egl targets.

RIPseq uncovers novel putative Egl targets
Anti-GFP antibodies were used to immunoprecipitate Eglassociated mRNAs from cytoplasmic extracts prepared from 0-8 hours old Drosophila embryos expressing a functional Egl::GFP fusion protein. 7 As control for unspecific binding we used extracts from wild type embryos lacking the GFP epitope. Immunoprecipitation (IP) conditions were optimized by analyzing the enrichment of known BicD/Egl mRNA targets in the experimental IP compared to the control IP (Fig. 1A, B). This procedure efficiently co-immunoprecipitated Egl::GFP, BicD and endogenous Egl ( Fig. 1A and data not shown). Because BicD forms dimers, 8,9 this likely indicates that a multimeric Egl::GFP/BicD/BicD/Egl complex was pulled down. The apically localizing mRNAs h, ftz and wg 4,10 were strongly enriched in the Egl::GFP precipitates compared to the controls (Fig. 1B). The abundance of 2 mRNAs that get localized through BicD/Egl during oogenesis (osk, bcd) was also elevated in the embryonic Egl::GFP IP. As negative controls we tested the known nonlocalized transcripts from Kr€ uppel (Kr) and string (stg), which are not recruited to the BicD/Egl localization machinery in embryos 10 and we included the house keeping genes Tub67C, RpL32, Act5C and Act42C (Fig. 1B). While we observed residual amplification of all transcripts in the control IPs (wt; probably due to minor amounts of unspecific binding to the beads), only localized mRNAs showed a clear enrichment in the Egl::GFP IP (Fig. 1B). Enrichment was observed when using the same amount of pulled down total RNA (Fig. 1B) and the same amount of initial embryonic material, respectively (Fig. S1).
Immunoprecipitations were performed with 2 independent biological samples of Egl::GFP and controls. Biological replicates were pooled for Illumina sequencing and enrichment factors and P-values were determined and compiled (Table S1). Many of the known BicD/Egl mRNA cargoes such us osk, bcd, insc, nos, orb and Chc mRNAs were found to be highly enriched in the Egl::GFP IPs and they ranked in the top 140 enriched mRNAs, while our non-localizing mRNAs were not in this group ( Fig. 1C; Table S1). Therefore, the preparative purification scheme effectively enriched for Egl mRNA targets. Using the known target osk mRNA as a cut off, a list of the top 50 mRNAs with the highest enrichment scores was produced. Table 1 lists the embryonic expression data for these genes and Table 2 their oogenesis expression data. These predicted targets were selected for further analysis. Interestingly, many top candidate mRNAs show higher enrichment values than the already described Egl targets (Fig. 1D). Because the known BicD/Egl targets had been identified by genetic and not by biochemical experiments and because many of them perform their function primarily in the ovarian germ line, it is not surprising that we identified mRNAs that are more enriched than the already known ones. To our knowledge this was the first biochemical approach taken to identify targets of this machinery and it revealed numerous novel mRNAs that seem to attach significantly and specifically to Egl:GFP.

Validation of top Egl target candidates
We validated the 28 top candidates in independent experiments by qPCR (Fig. 1E), comparing their enrichment again to known BicD/Egl targets as well as to non-localizing transcripts from the same IPs. 27 of these 28 putative novel Egl target candidates turned out to be enriched in anti Egl::GFP IPs compared to the mock IPs. The fold enrichment values from the qPCR analysis cannot be compared directly with the ones from the sequencing experiment because they are normalized and calculated using different parameters (see methods). Nevertheless, both experimental methods revealed a higher enrichment score than the known Egl targets for most of the top hits. The enrichment was also observed when comparing the Egl::GFP IPs to the total input mRNA (not shown). Only the enrichment of RpS29 could not be confirmed. The fact that this mRNA showed also no specific localization pattern by in situ hybridization (see bellow, Tables 1 and 2) suggests that this is a false positive hit. This indicates that our approach to find novel targets gives consistent results even with different analysis tools and that results are reproducible in independent IPs.

Egl target mRNAs are enriched for specific localization patterns
BicD/Egl is needed to transport mRNAs from the nurse cells into the oocyte and to the apical region in embryos. To validate the data set we therefore analyzed the subcellular localization of the top 50 mRNAs in ovaries and embryos. We searched for known embryonic and ovarian localization patterns in the literature and the following databases: Fly-Fish 6 (http://fly-fish. ccbr.utoronto.ca), BDPG (http://insitu.fruitfly.org/cgi-bin/ex/ insitu.pl) and DOT 11 (http://tomancak-srv1.mpi-cbg.de/DOT/ main.html). The different localization patterns found for Egl candidates are summarized in Table 1 and 2. snmRNA:331/ snRNA:7SK and snoRNA:U3:9B were not analyzed and the expression of CG34357 was bellow detection. Because pbl expression was only studied in embryos, the localization of 47 top candidates was studied in embryos and 46 top candidates in ovaries.
Because the purification was performed with embryonic extracts as starting material, we expected the identified mRNAs to show specific localization patterns during embryogenesis. 10 mRNAs localized to the embryonic pole plasm and/or pole cells, 10 localized apically of the syncytial blastoderm nuclei and 4 of them showed both localization patterns (Table 1, examples in Figs. 2, 3). Interestingly, these locations are cellular  compartments where components of the BicD/Egl complex can also be found enriched. 10 Surprisingly however, Egl protein showed a strong posterior localization ( Fig. 2A), while posterior anti BicD staining was only slightly elevated ( Fig. 2B) and sometimes even not recognizably. 12 Examples of mRNAs showing preferential localization to the pole plasm and pole cells are shown in Fig. 2C.
Among the apically localizing mRNAs some showed a continuous apical distribution along the a-p body axis. These are Acf1, lok (chk2/mnk), Dok, Sry-a and Uba1 (Fig. 3A). Others, however, were enriched apically but displayed a non-uniform pattern of expression along one of the body axes ( Fig. 3B-D). arc (a) mRNA was enriched apically in the anterior region of the embryo (Fig. 3B) and consistent with this localization, Arc Oocyte enrichment-Apical in follicle cell epithelia Oocyte enrichment-Apical in follicle cell epithelia @

Tsp39D
Ubiquitous-very weak ND RpL38 Ubiquitous-strong (stronger in nc than oocyte) ND Acf1 Oocyte Oocyte enrichment ND @ Ubiquitous-strong (slightly stronger in nc than oocyte) ND tefu Ubiquitous-weak (oocyte and nc expression equal) ND @ snoRNA:U3:9B NA ND NA fs(1)N oocyte enrichment Oocyte enrichment @ CG17698 Ubiquitous-strong (slightly stronger in nc than oocyte) Nuclear foci somatic cells nc and follicle cells nmdyn-D6 Ubiquitous-very weak ND CG33298 Ubiquitous-strong (oocyte and nc expression equal) ND @ CG6459 Ubiquitous-strong (stronger in nc than in the oocyte in ealry stages). St. 9 more accumulation in the oocyte. protein was also localized apically in several epithelia. 13 Arc has been reported to be a component of adherens junctions and to be required for eye and wing development. 13 T48 mRNA signal was enriched apically, but only in the ventral region. This apical localization remarkably correlates with its protein localization and its function in ventral furrow formation ( Fig. 3C; 14 ). CG8841 is apically enriched at the anterior and posterior poles, in a dorsal-ventral stripe in the middle of the embryo, and it accumulates in the pole cells (Fig. 3D, Fig. 2C). Interestingly, the CG33129 mRNA shows apical localization in invaginating furrows during gastrulation and also in tracheal precursor cells later in embryogenesis ( Fig. 3E-F). The apical enrichment of these mRNAs in cells that are undergoing morphogenetic movements suggests that localization of this mRNA and the protein it encodes might play a role in the apical movement of these cells. in situ hybridization experiments revealed that 28 of the 46 (61%) mRNAs expressed in ovaries showed clear enrichment in the oocyte relative to the nurse cells at some point during oogenesis (Table 2, Figs. 4, 5 and Fig. S2). Eight additional mRNAs (17%) displayed strong oocyte staining even though the oocyte signal was not higher than the nurse cell signal ( Table 2, Fig. 4 and Fig. S2). Because transcription in the female germ line is almost exclusively happening in nurse cell nuclei, this distribution pattern most likely requires active transport as well. Consistently, we also found that 79% of the Egl::GFP target candidates were expressed maternally in embryos (Table 1). Interestingly, many oocyte enriched mRNAs displayed specific subcellular localization patterns. Most of them localized posteriorly up to stage 6 and then relocalized to the anterior cortex ( Table 2, Figs. 4, 5 and Fig. S2). Additionally, 3 of the oocyteenriched candidates were also expressed in the somatic follicle cells of the ovary where they localized apically ( Table 2, Fig. 5A). Work with different mRNAs had shown previously that this localization pattern is also dependent on dynein. 15 In summary, the present knowledge about the function of BicD/ Egl in mRNA localization can explain for about 80% of the top candidates (Table 2) why they have been isolated.
To systematically analyze whether the BicD/Egl targets are enriched for specific gene ontology (GO) groups we also compiled an extended top target list. Sorted according to cellular components, the members of this list were significantly (p < 0.05) enriched for mRNAs that encode cytoplasmic proteins, components of the pole plasm and of ribonucleoprotein granules (Fig. S3A). Among the biological processes, developmental processes such as germ cell development, oocyte and embryonic axis specification, cell fate determination and cell maturation were predominant (Fig. S3B). Interestingly, these processes are well known for their dependence on mRNA localization. The enrichment of molecular functions is, however, less clear (Fig. S3C). While nucleotide and RNA binding proteins might be expected, we presently do not know why calmodulin binding proteins made it to the top of the list.

Localization of Egl target candidates requires BicD
To test whether the localization of the candidate mRNAs indeed depends on the BicD/Egl localization machinery we used the BicD mom flies described previously to turn off BicD expression in the germ line once the oocyte is made. 16,17 Around 4 d after shutting down BicD expression, ovaries contained egg chambers with strongly reduced or undetectable levels of BicD mRNA and BicD protein (Figs. 4, 5B-C and 17 ). Indeed, at the time BicD mRNA became undetectable, oocyte enrichment of the candidate mRNAs was severely impaired (Fig. 4), indicating that the top candidate Egl::GFP targets   . Egl targets are enriched for mRNAs that localize to the oocyte in a BicD dependent manner. 61% of the BicD/Egl targets tested showed accumulation in the oocyte compared to the expected 17% observed in a random set of mRNAs. In situ hybridization to wild-type (OreR) controls and to egg chambers 4 d after turning off BicD expression (BicD mom ). Antisense RNA probes for the candidate mRNAs were labeled with green or red fluorescent signals. Almost no BicD mRNA signal is observed when BicD expression is off (green signal, upper-most panels). During early wild-type oogenesis, clos, fwe, lok, CG43340, nos, T48, and a accumulated in the oocyte where they became enriched at the posterior till stage 6. Subsequently the signal appeared at the anterior cortex by stage 7, showing this pattern until mid to late oogenesis (left panels). These mRNAs failed to efficiently accumulate in BicD mom oocytes when BicD was off and the late localization in the oocyte was also severely impaired in BicD mom oocytes (right panels). CG6459 showed expression in the oocyte cytoplasm at all stages. However, in this case the oocyte signal was not enriched compared to its accumulation in nurse cells. A weak BicD dependent concentration of the mRNA at the dorsal side of the oocyte nucleus was observed by stage 8 (arrowhead, and magnified region in inset picture). By stage 9 a more clear presence of a dotted CG6459 signal was observed in the oocyte, but levels did not seem to exceed nurse cell levels. In BicD mom ovarioles big blobs of CG6459 mRNA signal were seen in the nurse cell cytoplasm, suggesting a problem in transport of this mRNA. Hoechst (blue) visualizes the DNA. Scale bars are 20 mm. Figure 5. BicD is needed for apical mRNA localization in follicle cells. 6.5% of the mRNA tested show apical localization in follicle cells. This mRNAs class is enriched 8-fold among the Egl::GFP targets compared to a random set (6.5% vs 0.8% 11 ) (A) In situ hybridization to wild-type (OreR, wt) controls and to egg chambers 4 d after turning off BicD expression (BicD mom ). Oocyte enrichment (arrows) as well as apical follicle cell localization of CG33129, egl and Uba1 was impaired when BicD was off. High magnification of the stage 10 follicle cell epithelium is shown in the right panels. Note that apical is toward the oocyte (up in the magnified pictures). Scale bars are 20 mm. Uba1 apical enrichment is weaker and less cortical, but still requires BicD. Histograms depicted on the right side of the pictures show the fluorescence intensity along the apical-basal axis of the marked cortical region around the nuclear layer (dotted square). This region is also shown magnified on the right side of the micrographs. (B) BicD mRNA expression (red) is drastically reduced in stage 9-10 BicD mom egg chambers in the germline and also in the somatic follicle cells (high power pictures on the right). Note that BicD mRNA is also apically localized as expected since it is also a target of Egl. (C) BicD protein (red) is also apically enriched in wild-type follicle cells, but is almost undetectable in BicD mom egg chambers, oocytes and follicle cells. Hoechst (blue) visualizes the DNA. Scale bars are 20 mm.
indeed depend on BicD for their localization. mRNAs from egl, CG33129 and Uba1 showed oocyte enrichment as well as apical localization in the somatic follicle cells. Interestingly, their localization was affected in both cell types in BicD mom ovaries (Fig. 5A). Because BicD mRNA (Figs. 4, 5B) and BicD protein (Fig. 5C) levels were not only reduced in the germ line, but also in the follicle cells, it appears that the BicD/Egl machinery also localizes mRNAs apically in the somatic tissue of the female gonad.
To study the involvement of BicD in localizing mRNAs that are enriched in the embryonic pole plasm, we studied transcript localization in embryos from mothers carrying a dominant Bic-D IIIE48 allele. Embryos laid by such mothers lack anterior structures and many of them show a strong double abdomen phenotype called bicaudal. 18 A large proportion of the BicD protein gets mislocalized to the anterior of these embryos, producing ectopic anterior localization of the posterior mRNA determinants osk and nos, 2 BicD/Egl targets. 12,19 lok mRNA is enriched in the pole plasm and pole cells of wild-type embryos 20 (Fig. 2C, Fig. S4). Embryos from BicD IIIE48/r5 mothers showed additional enrichment of lok mRNA at the anterior of the young embryo (Fig. S4) providing good evidence that the lok mRNA is a target of BicD/Egl in embryos as well.
Putative localization elements in Egl target mRNAs mRNA localization motives facilitating the binding to the BicD/ Egl localization machinery could so far not be predicted based on their primary sequence. NMR studies of the K10 TLS localization signal showed that it forms a stem-loop with 2 doublestranded RNA helices adopting an unusual A 0 -form conformation. 21 These helices were associated with runs of 3 or more purines on one side of the stem. Consistently, stem-loops within the mapped localization elements of well known BicD/ Egl targets, such as grk, ftz, h, bcd, wg, osk and the I-factor RNAs, contain 2 or more stretches of at least 3 contiguous purines on the same side of the stem. 21,22 Based on this we set up a bioinformatics approach to predict functionally important helices of this kind in our candidate mRNA data set. We expect such signals to be conserved between different Drosophila species. Accordingly, orthologous sequences from 10 Drosophila species were aligned based on their secondary structure prediction. Conserved stem-loops were then sorted for the presence of 2 or more stretches of at least 3 contiguous purines on the same side of the stem. Using this approach we optimized the program by searching for the known BicD/Egl dependent localization hairpins (Fig. S5). For each hairpin we then plotted the conservation across Drosophila species against the percentage of bases that make pair bonds for each hairpin (this serves as a simplified measurement of the hairpin stability). We found that in the case of h, orb, grk, K10, osk and bcd the best predicted localizing A'-form hairpin (best conserved and with highest frequency of base matches) coincided with the known hairpins responsible for apical or oocyte localization of the transcripts ( 21,22 ; Fig. S5). In the case of wg and ftz the program still predicted the known localization sequences and they were among the best scoring ones, but not at the top. It is possible that in these cases the presence of redundant localization signals or the high conservation intrinsic to open reading frames (ORFs) was distorting the values (see Discussion). We also consider it possible that the 2 best predicted hairpins in ftz and wg are the main localization elements in several other Drosophila species, but have become partially redundant with new ones in melanogaster.
We next analyzed the Egl:GFP targets that showed a clear oocyte, pole plasm or apical enrichment in ovaries or embryos for the presence of conserved predicted A'-form helices (Fig. S6  and Fig. S7). Two mRNAs, pgc and sry-a, showed the presence of only one putative A'-form type helix, and this was identified in their ORF (Fig. S7). lok, Dok, fs(N)1 and T48 have one most likely localization hairpin that shows the best folding energy and is highly conserved (Fig. S6A) The analysis of CG33129, egl, a, mus210/Xpc, insc, dap and fatp/CG7400 revealed that although the best-scoring hairpin might likely be the localizing signal, some other hairpins would probably need to be tested as well (Fig. S6 and Fig. S7). Similarly CG43340, didum, CG12488, Uba1, Acf1, CG8841, fwe and hts have at least 2 putative A'form helices with high scores (Fig. S6 and Fig. S7). We selected Dok and lok to test the localization activity of the predicted hairpins ( Fig. 6) by injecting fluorescently labeled RNAs into the basal cytoplasm of syncytial embryos and following their apical transport. For lok, the most highly conserved A'-form hairpin resides in the ORF, but there is also one in the 3 0 UTR that, although less conserved, has the highest frequency of base matches (Fig. S6A). Only lok sequences containing the 3 0 UTR supported apical localization, while fragments containing the ORF regions did not localize (Fig. 6A, C C',). Furthermore, deletion of the predicted conserved A'-form in the 3 0 UTR greatly impaired apical localization of the 3 0 UTR region (Fig. 6B, D, D'). Similarly, the best predicted A'-form hairpin of Dok was also identified in the ORF (Fig. S6A), but the ORF RNA alone did not support apical transport (Fig. 6E, G, G'). In contrast, the 3 0 UTR localized strongly to the apical side (Fig. 6E, G, G') and the deletion of its predicted 3 0 UTR A'-form stem loop abolished most of the transport (Fig. 6E-G, G'). Egl protein is recruited to its target RNAs injected into the basal region of the embryo and it gets co-transported apically with the RNA. 10 Injecting lok and Dok 3 0 UTRs, but not the corresponding constructs lacking the localizing hairpins, also led to an apical enrichment of Egl above the site of injection (Fig. 7A-D). The bioinformatics predictions can therefore reveal functional localization elements in the 3 0 UTR that serve to localize these mRNAs apically by the BicD/Egl transport machinery.

Discussion
We have identified BicD/Egl mRNA targets in 0-8 hour old Drosophila embryos and found that 80% of the top candidates showed localization patterns that are compatible with their being actively transported during oogenesis and/or during the first 4 hours of embryogenesis by the BicD/Egl dependent localization machinery. At present we do not know for most of the remaining transcripts whether they are transported by Egl during different developmental stages, whether a competing process prevents their accumulation at the expected target site or whether the Egl-mediated transport also leads to yet uncharacterized distribution patterns. For CG33129, however, we observed that the transcripts are also localized apically in later stages in the tracheal precursors and insc is known to be localized in older embryos apically in the neuroblasts. 3,4 Compared to a random group of mRNAs, 6 the BicD/Egl top targets described here show a 6-fold enrichment for apically localizing embryonic mRNAs (10/47; 21% vs 3.4%). Similarly 21% (10/47) of the BicD/Egl targets are also enriched for mRNAs that localize to the pole plasm or pole cells compared to the reported 8.4% of a random set. 6 Compared to a random set of mRNAs 11 we also observed a 3-4-fold increase in oocyte accumulation among the BicD/Egl targets (61% vs 17%) and an 8fold increase in apical follicle cell localization (6.5% vs 0.8%) although our sample size for this phenotype is very small (n D 3). The same localization patterns were also enriched in a statistical analysis that took into account a much larger group of Egl targets (see Fig. S3D, E). In contrast, basally localized transcripts were clearly underrepresented in our data set (Fig. S3D). The enrichment for specific mRNA localization patterns in ovaries and embryos indicates that the BicD/Egl machinery specifically acts for nurse cell to oocyte transport, for apical transport of mRNAs during oogenesis and embryogenesis, and for enrichment in the pole plasm and pole cells. All these processes indeed involve a polarized MT network and the BicD/Egl/Dynein machinery. These results further imply that different subcellular localization patterns identified for other mRNAs are generated by different mechanisms or at least require an additional element. The multitude of different mRNA localization patterns described for instance by the high throughput screens 6,11 are therefore likely to involve additional mechanisms such as mRNA diffusion coupled with entrapment and degradation, and active transport utilizing different transport systems.
The data set of apically localized mRNAs bound to Egl can be grouped into 3 functional groups: 1) Genes with nuclear functions, involved in DNA metabolism, DNA repair, chromosome structure and DNA damage response. 2) Genes coding for proteins that associate directly or indirectly with the plasma membrane. 3) Genes coding for secreted proteins. The first group includes the genes like lok that encodes the Drosophila Chk2 kinase involved in monitoring DNA damage, Mutagen-sensitive 210 (Mus-210, also Xpc), which encodes a protein with DNA binding domains and a predicted DNA repair function, and Acf1 (ATP-dependent chromatin assembly factor 1), which is involved in the assembly and maintenance of heterochromatin. 20,23,24 Also belonging to this group of nuclear proteins are the transcription factors encoded by the segmentation genes h and ftz. At the time the proteins encoded by these mRNAs become active, their target nuclei are also positioned apically. It thus appears that the localization of their mRNAs facilitates an efficient reaching of their target site by the encoded protein. Furthermore, as for the apically localized mRNAs with striped localization patterns like ftz and h (but also wg, CG8841, a, T48), the apical localization of these transcripts might serve to avoid lateral diffusion of their products to the neighboring nuclei and compartments. For example, Acf1 is a subunit of the nucleosome-remodeling complex involved in chromatin assembly and chromatin-mediated gene repression, and in Acf1 mutants heterochromatin assembly is disturbed. 23 Interestingly, when pericentric heterochromatin forms during the blastoderm stages, it is localized to the apical side of the nuclei, adjacent to where we found Acf1 mRNA localized. Because Acf1 mRNA localization depends on BicD activity, 25 it appears that BicD and egl might also play a role in heterochromatin assembly by localizing Acf1 mRNAs in embryos.
The second group contains mRNAs encoding proteins that associate with the plasma membrane. The PDZ domain protein Arc is a component of the adherens junctions. Protein and mRNA reside apically in several epithelia, and in embryos they are found in regions that undergo morphogenetic movements, such us invagination, elongation or convergent extension. 13 Serendipity a (Sry-a) is associated with the plasma membrane and with actin invaginations during cellularization at the blastoderm stage. 26 Dok is a signaling protein required upstream of JNK signaling during dorsal closure. It is found at the cell cortex associating with the plasma membrane where it is ideally positioned to respond to Src signaling. 27 T48 is a transmembrane protein that is needed together with G protein signaling to recruit adherens junctions and cytoskeletal regulators to sites of apical constriction during ventral furrow formation. 14 Remarkably, CG8841, the novel pole plasm component that displays additional apical mRNA localization, encodes a predicted transmembrane domain protein, too.
The localization of most Egl target mRNAs in ovaries depends on BicD. This testifies to the quality of the data presented in the candidate list and it suggests that aside from the top candidates, the list may contain numerous additional targets with lower scores. Indeed, known BicD/Egl targets, like Chc, orb, K10, h and ftz appear further down in the list with lower enrichment scores (Table S1). While validating the BicD/ Egl targets we noticed that BicD is also required for apical localization of many mRNAs in "normal" somatic cells (aside from the syncytial embryo). However, loss-of-function mutations in egl and BicD were initially isolated as female sterile mutants because of their essential function in the female germ line. 28 The fact that we now also found Egl target mRNAs that are apically localized in developing tracheal cells and in salivary glands (data not shown), together with the known role for BicD in localizing insc mRNA in neuroblasts, suggest that this machinery is also working in somatic embryonic and larval cells and tissues where a polarized cytoskeleton is present. This indicates that mRNA localization by BicD/Egl is at work in many different cell types, but that it is more important in large cells, such as the female germ line, the syncytial embryo and the nervous system. This is consistent with the BicD null phenotype, which reveals important zygotic functions for this gene. Most BicD null animals die during larval stages but a very small fraction makes it to adulthood. Such adults are uncoordinated and lethargic and die within 2 d. 29 Taking advantage of the new pool of verified Egl target mRNAs and of the published NMR structure of the K10 localization signal, we also set up a bioinformatics approach to identify A'-form hairpin structures in Egl-bound mRNAs. Interestingly, although most of the conserved A'-form hairpins were predicted to be located in the ORF of the mRNAs (Fig. S6D), we could not validate their functionality in the 2 cases we tested. Instead A'-form hairpins identified in the 3 0 UTR turned out to serve as functional localization elements. This suggests that giving priority to the hairpins found in the 3 0 UTR should improve the chance of finding the correct localization sequences. One reason for this could be that hairpins of ORFs that are in a state of repressed translation -bound to translational repressors or to a repressed translation machinery -may be masked such that the transport machinery cannot bind to them. In contrast, the 3 0 UTR may be free of stalled ribosomes and accessible for recruitment by the transport complex. Another possibility is that additional sequences in the 3 0 UTR serve as binding site for additional factors that may facilitate Egl binding. Similarly, embedding the localization signal in the context of the 3 0 UTR, which is more AT rich than the ORF, may facilitate the folding of the Egl recognition structure. This view is also supported for example by the finding that the efficient localization of h and ftz is context dependent with sequences surrounding the minimal localization signal working non autonomously, but enhancing the activity of the localization element. 30,31 Material and methods

Purification of Egl bound mRNAs
RNA immunoprecipitations were performed essentially as described with some modifications. 32 Briefly, protein G Sepharose beads (Gamma bind Plus agarose, Roche) were washed 3 times with blocking buffer (20 mM HEPES pH 7.9, 150 mM KCl, 20% glycerol containing 0.5% Tween-20, 1mg/ml BSA, 2 mg/ml heparin and EDTA free protease inhibitors, Roche). Beads were then blocked for 3 h at RT with the same buffer. 1 ml of monoclonal anti-GFP antibody supernatant was added per 40 ml of blocked beads and incubated for 2 h at RT with gentle rotation. Coated antibody beads were then washed with non-hypotonic buffer (20 mM HEPES pH 7.9, 2mM MgCl 2 , 150 mM KCL, 1mM DTT, 20% glycerol containing 0.5% Tween and EDTA free protease inhibitors, Roche). For extract preparation, 1 gr of 0-8 h old embryos were dechorionated and homogenized in 2 ml hypotonic buffer (20 mM HEPES pH 7.9, 2 mM MgCl 2 , 10 mM KCL, 1 mM DTT containing 0.5% Tween-20, EDTA free protease inhibitors, Roche, and RNase inhibitor, Biolabs, 100 units/ml). All further steps were performed at 4 C. The homogenized extract was cleared at 10,000 £ g for 20 min. For each IP 323 ml of salt adjusting buffer (20mM HEPES pH 7.9, 57% glycerol, 0.4M KCl, 2mM MgCl 2 , 1mM DTT) and 2 ml of RNase free DNase I (20 U/ml, Roche) were added to 600 ml of cleared supernatant. This salt adjusted extract was added to 40 ml of coupled antibody beads and incubated overnight at 4 C with rotation. The immunoprecipitate was washed 8 times with high salt wash buffer (20 mM HEPES pH 7.9, 2 mM MgCl 2 , 200 mM KCL, 20% Glycerol, 1mM DTT containing 0.5% Tween, EDTA free protease inhibitors and RNase inhibitor (Biolabs, 100 units/ml)), rotating it for 10 min, followed by centrifugation at 2,000 rpm for 2 min. Beads were treated with 30 mg of Proteinase K (Roche) in proteinase K buffer (150 mM NaCl, 12.5 mM EDTA, 10 % SDS, 0.1M Tris-HCL pH 7.5) in a total volume of 100 ml for 30 min at 55 C. RNA was extracted using the Trizol reagent (Invitrogen) following the manufacture's instructions. The integrity and quality of the coimmunoprecipitated RNA was assessed using the Agilent RNA 6000 Nano Kit on an Agilent 2100 bioanalyzer (Agilent Technologies) following the manufacturer's instructions.

Preparation of the library for sequencing
The material of 2 independent IPs was pooled and used for library preparation (15 ml of RNA with 47,7 ng/ml for the control IP and with 73,7 ng/ml for the Egl::GFP IP) following the manufacture's instructions (TruSeq RNA Sample Prep Kit V2, Illumina, La Jolla, USA).

RT-PCR and RT-qPCR analysis
The RNA from each IP was resuspended in 25 ml of DEPC treated water and its quality was analyzed with the Eukaryote Total RNA Nano chip in an Agilent bioanalyzer. RT-PCR assays were performed to optimize the RNA IP protocol. For this, sequences were amplified using the Access RT-PCR system (Promega) and primers that spanned introns. Optimal cycle numbers were determined for each gene.15 ng of IPed RNAs were used as template for each RT-PCR reactions. Using the same amount for the Egl::GFP IPs and the control IPs allows us to normalize the total number of reads from the subsequent sequencing reaction even though it will produce an underestimated enrichment value. The enrichment for localizing mRNAs but not for housekeeping genes was also observed when using the same amount of embryonic material for normalization. No amplification was observed in reactions without RT, indicating that we do not observe amplification from DNA.
For RT-qPCR assays, cDNAs from the different samples were prepared with the SuperScript II Reverse Transcriptase and oligo dT primers (Invitrogen) following the manual's instructions and using 133 ng of IPed mRNAs. Primers used for the RT-qPCR analysis were designed in a way that one of a pair spanned an exon-junction sequence and that the amplicon length ranged from 100 to 150 bp. Sequences were amplified using the QuantiTect SYBR Green PCR Kit (Qiagen) and a Rotor Gene instrument (Qiagen). Calculation of mRNA levels was done using the 2 [-DDC( T )] method. 33,34 Fold enrichment (mRNA levels present in the IP over mRNAs levels in the mock IP) was plotted. C( T ) values used were the means of duplicate technical and biological repeats.

Bioinformatics analysis of Illumina data
The two single-end RNAseq libraries produced 49 M and 52 M reads with a read length of 100bp on an Illumina HiSeq2000 Instrument (Illumina, La Jolla, USA). The quality of the reads was assessed using FastQC (version 0.10.1, www.bioinformat ics.babraham.ac.uk/projects/fastqc) and reads mapping to rRNA genes (»80%) were removed. The remaining reads (9,5 M and 10,8 M reads) were mapped to the fly genome (version 5.45 of the Flybase) using the spliced alignment approach implemented in TopHat2 version 2.0.5. 35 For each annotated gene (annotation version 5.45 of Flybase) we counted the number of reads mapping to it using the program HTSeq-count (www.w-huber.embl.de/users/anders/HTSeq, version 0.5.3). To test for significant differences of gene expression levels between the Egl::GFP and the y w control the RÄpackage DESeq was used. 36 More precisely, raw read counts were normalized to the total number of reads, and the variation of read counts per gene was assumed to follow the variation of counts across genes. The reported p-value is corrected for multiple testing following Benjamini and Hochberg. 37 Fold enrichment represent the ratio between the normalized reads from the Egl IP over the control IP. GO term enrichment analysis was done with the website http://geneontology.org.

Fluorescent in situ hybridization (FISH) to whole mount embryos and ovaries and immunostainings
Plasmids (BDGP resources; 38 ) containing the cDNA of the candidate genes were linearized with the corresponding restriction enzymes (New England Biolabs) and used as templates to generate digoxigenin-or Fluorescein (FITC)-labeled RNA antisense probes. In situ hybridization experiments on ovaries and embryos were performed essentially as described 39,40 but 5% milk powder (Rapilat) in PBT (PBS, 0.1% Tween) was used as blocking reagent. The digoxigenin labeled probes were detected with sheep-anti-digoxigenin antibodies and Cy3-conjugated donkey anti-sheep IgG F(ab')2 fragments (Jackson Immunoresearch). Fluorescein-labeled probes were detected with mouse anti-fluorescein antibodies (Roche) and Cy3-conjugated goat anti-mouse antibodies (Jackson Immunoresearch). For double RNA in situs, Cy3-conjugated anti-mouse antibodies and Cy5conjugated anti-sheep antibodies were used (Jackson Immunoresearch). Immunostainings were done using the following primary antibodies: mouse anti-BicD (1B11, 1: 10 dilution; 41 ) anti-Egl (1:5,000 dilution; 42 ). Where required, nuclei were stained for 20 min with 2.5 mg/ml of Hoechst 33258 during the final wash step. Images were analyzed with a Leica TCS-SP8 or Leica TCS-SP5 confocal microscopes. Different pictures were taken for each egg chamber in an ovariole focusing on the oocyte center. The picture showing the entire ovariole was then assembled in Adobe Photoshop. All patterns described were observed reproducibly and were observed in most of the embryos and ovaries analyzed. Histograms depicting the fluorescence along the apical-basal axis were done using Fiji software.

Fluorescent RNA synthesis and injections
RNAs for injections were synthesized in the presence of Alexa-488-UTP (Molecular probes) as described. 10 Dok and lok RNAs were synthesized using LD32155 and LD27875 plasmids, respectively (BDGP resources). The Dok (1-2271) DNA template was generated by linearizing the corresponding plasmid with BamHI. The lok plasmid was linearized with SphI or BamHI and used as template to generate lok (1-948) and lok (1-1495) transcripts. Deletion of the corresponding localizing hairpins in Dok and lok 3 0 UTR was done on LD32155 and LD27875 by site directed mutagenesis. Wild-type and deleted forms of the 3 0 UTR regions were amplified by PCR and used as templates for transcription. Fluorescently labeled RNAs were used at 0.5-1 mg ml ¡1 . Fixing was done 12 min after injection of the last embryo and immunofluorescent staining of injected embryos was done as described. 10 Images were analyzed with a Leica TCS-SP8 confocal microscope. Strong localization means that most of the signal was in the apical cytoplasm. Weak localization means most signal remained basally, but there were some apical caps of signal; "in very weak localization" apical cap signals were barely above background fluorescence of the basal cytoplasm while no fluorescent puncta were observed in the apical cytoplasm in "no apical localization."

Stem loop predictions
The sequences of the candidate genes were retrieved from Fly-Base (release 5.1). The longest isoform of every gene was chosen. Local secondary structures were explored by means of RNALfold, from the Vienna package 43 (version 2.1.8), with the following options: 25 C temperature and no "lonely pairs." A conservation score for the Drosophila melanogaster genome was available on the UCSC genome browser. 44  Predicted hairpins were further analyzed for the presence of at least 2 A/G stretches, each at least 3 nucleotides in length. Overlapping hairpins were removed, retaining the ones with the lowest energy. Hairpins with a conservation score higher than 0.5 and longer than 40 nt were reported.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.