Noncanonical function of Capicua as a growth termination signal in Drosophila oogenesis.

Capicua (Cic) proteins are conserved HMG-box transcriptional repressors that control receptor tyrosine kinase (RTK) signaling responses and are implicated in human neurological syndromes and cancer. While Cic is known to exist as short (Cic-S) and long (Cic-L) isoforms with identical HMG-box and associated core regions but distinct N termini, most previous studies have focused on Cic-S, leaving the function of Cic-L unexplored. Here we show that Cic-L acts in two capacities during Drosophila oogenesis: 1) as a canonical sensor of RTK signaling in somatic follicle cells, and 2) as a regulator of postmitotic growth in germline nurse cells. In these latter cells, Cic-L behaves as a temporal signal that terminates endoreplicative growth before they dump their contents into the oocyte. We show that Cic-L is necessary and sufficient for nurse cell endoreplication arrest and induces both stabilization of CycE and down-regulation of Myc. Surprisingly, this function depends mainly on the Cic-L-specific N-terminal module, which is capable of acting independently of the Cic HMG-box-containing core. Mirroring these observations, basal metazoans possess truncated Cic-like proteins composed only of Cic-L N-terminal sequences, suggesting that this module plays unique, ancient roles unrelated to the canonical function of Cic.

Capicua controls Toll/IL-1 signaling targets independently of RTK regulation.

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.

A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer.

HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.

Origins of context-dependent gene repression by capicua.

Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.

Mirror represses pipe expression in follicle cells to initiate dorsoventral axis formation in Drosophila.

Dorsoventral (DV) axis formation in Drosophila begins with selective activation of EGFR, a receptor tyrosine kinase (RTK), in dorsal-anterior (DA) ovarian follicle cells. A critical event regulated by EGFR signaling is the repression of the sulfotransferase-encoding gene pipe in dorsal follicle cells, but how this occurs remains unclear. Here we show that Mirror (Mirr), a homeodomain transcription factor induced by EGFR signaling in DA follicle cells, directly represses pipe expression by binding to a conserved element in the pipe regulatory region. In addition, we find that the HMG-box protein Capicua (Cic) supports pipe expression in ventral follicle cells by repressing Mirr in this region. Interestingly, this role of Cic resembles its function in regulating anteroposterior (AP) body patterning, where Cic supports gap gene expression in central regions of the embryo by repressing Tailless, a repressor induced by RTK signaling at the embryonic poles. Thus, related RTK-Cic repressor circuits regulate the early stages of Drosophila DV and AP body axis formation.

Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.

RTK/Ras/MAPK signaling pathways play key functions in metazoan development, but how they control expression of downstream genes is not well understood. In Drosophila, it is generally assumed that most transcriptional responses to RTK signal activation depend on binding of Ets-family proteins to specific cis-acting sites in target enhancers. Here, we show that several Drosophila RTK pathways control expression of downstream genes through common octameric elements that are binding sites for the HMG-box factor Capicua, a transcriptional repressor that is downregulated by RTK signaling in different contexts. We show that Torso RTK-dependent regulation of terminal gap gene expression in the early embryo critically depends on Capicua octameric sites, and that binding of Capicua to these sites is essential for recruitment of the Groucho co-repressor to the huckebein enhancer in vivo. We then show that subsequent activation of the EGFR RTK pathway in the neuroectodermal region of the embryo controls dorsal-ventral gene expression by downregulating the Capicua protein, and that this control also depends on Capicua octameric motifs. Thus, a similar mechanism of RTK regulation operates during subdivision of the anterior-posterior and dorsal-ventral embryonic axes. We also find that identical DNA octamers mediate Capicua-dependent regulation of another EGFR target in the developing wing. Remarkably, a simple combination of activator-binding sites and Capicua motifs is sufficient to establish complex patterns of gene expression in response to both Torso and EGFR activation in different tissues. We conclude that Capicua octamers are general response elements for RTK signaling in Drosophila.

Polarization gating spectroscopy of normal-appearing duodenal mucosa to detect pancreatic cancer.

BACKGROUND: According to the field effect theory, by detecting microvasculature changes such as early increase in blood supply (EIBS) in the surrounding tissue, neoplastic lesions can be identified from a distance. OBJECTIVE: To determine the feasibility and efficacy of a fiberoptic probe containing novel polarization gating spectroscopy technology to identify patients with pancreatic adenocarcinoma (PAC) by the field effect theory. DESIGN: Prospective cohort (pilot) study. SETTING: Outpatient tertiary care center. PATIENTS: Adult (≥ 18 years) patients undergoing EGD-EUS were screened. Patients with PAC were included in the "cancer" group and patients without PAC were included in the "control" group. We excluded patients with other known malignancies and gastroduodenal premalignant lesions. INTERVENTIONS AND MAIN OUTCOME MEASURES: Spectroscopic measurements of EIBS variables, such as deoxyhemoglobin concentration (DHb) and mean blood vessel radius (BVR), were obtained from 5 periampullary locations. The Mann-Whitney rank sum test was used for the statistical analysis (P ≤ .05). RESULTS: Fourteen patients (mean age 72 years, 79% male) in the cancer group and 15 patients (mean age 63 years, 60% male) in the control group were included in the final analysis. At the ampullary site, both DHb (P = .001) and BVR (P = .03) were higher in PAC patients than in the control subjects. The DHb alone (92% sensitivity, 86% specificity) or in combination with BVR (92% sensitivity, 79% specificity) can differentiate PAC from control subjects with high accuracy. LIMITATIONS: Small sample size, unmatched control subjects. CONCLUSIONS: Spectroscopic measurements of EIBS by fiberoptic probes are feasible. Preliminary evidence suggests that in vivo measurement of normal-appearing duodenal tissue can differentiate PAC patients from a distance with high accuracy.

Non-invasive assessment of liver fibrosis using magnetic resonance elastography in liver transplant recipients with hepatitis C.

BACKGROUND: Liver biopsy has been the reference standard when evaluating fibrosis due to recurrent hepatitis after liver transplantation. Magnetic resonance elastography estimates liver stiffness, correlating to fibrosis. AIM: To investigate the utility of elastography in staging liver fibrosis in transplant recipients with hepatitis C. METHODS: Fifty-four patients, ≥12 months post-transplant, underwent elastography within three months of biopsy. Discriminatory capability for METAVIR fibrosis stages F0-2 vs. F3-4 and receiver operating characteristic curve (ROC) analysis were determined. RESULTS: On biopsy, 27 patients had METAVIR fibrosis score 0-1; 12 had a 3 or 4. There was significant correlation between histologic fibrosis and shear stiffness (R² = 0.588, p < 0.0001). Using a cutoff value of 3.5 kPa, elastography was 91% sensitive and 72% specific in differentiating fibrosis scores of ≥3 from 0 to 1. The AUC of elastography in predicting a fibrosis score of ≥3 was 0.92. Multivariate analysis revealed no correlation between the grade of histologic inflammation and liver stiffness measured by magnetic resonance elastography (R² = 0.265, p = 0.47). CONCLUSION: Magnetic resonance elastography is an accurate non-invasive technique for excluding stage ≥3 graft in recipients with hepatitis C.

Interobserver agreement on the endosonographic features of lymph nodes in aerodigestive malignancies.

BACKGROUND: Endoscopic ultrasound (EUS) is used to locally stage aerodigestive malignancies. Endosonographic features are used to predict malignant lymph nodes (MLN). Interobserver agreement on the endosonographic features of MLN has not been described. AIMS: To evaluate the interobserver agreement among endosonographers on the EUS features of MLN in aerodigestive malignancies along with the specific feature used to make this distinction. METHODS: A total of 760 procedures of suspected LN were reviewed. Cases were selected based on cytology-proven malignant or benign LN involvement by FNA with correspondent photodocumentation. Images of each LN were de-identified and distributed to three expert endosonographers. The experts recorded the LN's echogenicity (hypoechoic or other), shape (round or other), border (sharp or fuzzy) and subjective diagnosis (benign or malignant). The relationship between the endosonographers' subjective as well as the pathological diagnosis and LN's endosonographic features were analyzed using logistic regression analysis. Pair-wise comparison between endoscopist and interobserver agreement (kappa statistics) were performed. RESULTS: Images of 41 malignant and 35 benign LN were evaluated. There was fair agreement on shape, Κ = 0.35 (95% CI 0.2-0.5), and moderate agreement on echogenicity and borders, Κ = 0.46 (95% CI 0.31-0.61) and 0.43 (95% CI 0.27-0.58) respectively. The agreement on malignant LN was good, Κ = 0.65 (95% CI 0.5-0.8). The overall diagnostic predictive accuracy ranged from 70 to 77% among the three endoscopists.Two of the three endoscopists assessed shape as the most predictive feature of malignancy (OR 39.4, 95% CI 3.29-470.96). CONCLUSION: The inter-observer agreement on the individual lymph node features as obtained by EUS is moderate with good overall agreement on the diagnosis. Round shape was the feature most strongly associated with a diagnosis of MLN.

Characterizing extravascular neutrophil migration in vivo in the iris.

Extravascular neutrophil migration is poorly characterized in vivo. To test the hypothesis that this migration is a non-random process, we used videomicroscopy to monitor neutrophils in irises of living mice with endotoxin-induced uveitis (EIU). Paths of individual cells were analyzed. Nearly all of these cells were moving in divergent directions, and mean displacement plots indicated that the predominant movement was random. The paths of some cells were fit to bivariate autoregressive integrated moving average models that revealed at least two modes of movement: random search and linear trend. Cell speed was significantly reduced by the actin inhibitor, cytochalasin D. The pattern of migration for neutrophils is in marked contrast to what we previously described for antigen-presenting cells in the iris, but somewhat resembles recent descriptions for T cells within a lymph node. Characterization of extravascular migration of neutrophils has important implications for understanding infection and immunity.

Posterior expression of nanos orthologs during embryonic and larval development of the anthozoan Nematostella vectensis.

Cnidarians are primitive animals located in a basal position in the phylogenetic tree of the Animal Kingdom, as an outgroup of the Bilaterians. Therefore, studies on cnidarian developmental biology may illustrate how fundamental developmental processes have originated and changed during animal evolution. A particular example of this is the establishment of polarity along the body axes, which is under the control of a number of developmental genes, most of them conserved in evolution and playing similar roles in diverged species. Concerning the anterior-posterior axis, genetic and molecular studies on Drosophila have shown that the nanos gene plays an essential role in defining posterior structures during early embryonic development. Here we report the isolation of two nanos orthologs in the anthozoan Nematostella vectensis. We show that nanos mRNA is asymmetrically distributed in the fertilized egg and this asymmetry is maintained during embryonic development. At gastrula and planula larva stages, nanos expression is permanently associated with posterior body regions. These results, together with our previous analysis in the hydrozoan Podocoryne carnea, indicate that posterior nanos expression during development is a conserved feature among cnidarians. Therefore, the potential role of cnidarian nanos in defining axial polarity as a posterior determinant would represent an ancestral trait in the Animal Kingdom.

Immunohistology of antigen-presenting cells in vivo: a novel method for serial observation of fluorescently labeled cells.

PURPOSE: Dendritic cells and macrophages are phagocytic antigen-presenting cells that bridge the innate and acquired immune systems. The coexistence of subtypes of dendritic cells and macrophages with overlapping properties complicates resolution of their precise roles in an immune response within a given tissue. This report documents a method to identify and observe these cells over time in a living animal and thereby to visualize them during a dynamic immune response. METHODS: To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection. RESULTS: The pattern and density (116-148 cells/mm(2)) of cells labeled in vivo by fluorescent ovalbumin or F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c(+)) and 24 hours (24% CD11c(+)) after injection. CONCLUSIONS: This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.

EGFR-dependent downregulation of Capicua and the establishment of Drosophila dorsoventral polarity.

Dorsoventral (DV) axis formation in Drosophila begins during oogenesis through the graded activation of the EGF receptor (EGFR)-Ras-MAPK signaling pathway in the follicle cell layer of the egg chamber. EGFR signaling, which is higher in dorsal follicle cells, represses expression of the sulfotransferase-encoding gene pipe, thereby delimiting a ventral domain of Pipe activity that is critical for the subsequent induction of ventral embryonic fates. We have characterized the transcriptional circuit that links EGFR signaling to pipe repression: in dorsal follicle cells, the homeodomain transcription factor Mirror (Mirr), which is induced by EGFR signaling, directly represses pipe transcription, whereas in ventral follicle cells, the HMG-box protein Capicua (Cic) supports pipe expression by repressing mirr. Although Cic is under negative post-transcriptional regulation by Ras-MAPK signaling in different contexts, the relevance of this mechanism for the interpretation of the EGFR signal during DV pattern formation remains unclear. Here, we consider a model where EGFR-mediated downregulation of Cic modulates the spatial distribution of Mirr protein in lateral follicle cells, thereby contributing to define the position at which the pipe expression border is formed.

Antigen-specific accumulation of naïve, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.

Uveitis is an immune-mediated ocular disease and a leading cause of blindness. We characterized a novel model of uveitis with intravital microscopy. Transfer of ovalbumin-specific T cells from DO11.10 spleen to BALB/c recipients and subsequent challenge with ovalbumin in the anterior chamber of the eye resulted in anterior uveitis. Antigen-specificity was verified by injection of irrelevant antigen and transfer of T cells with a different specificity. Subsets of CD4 T cells, including naive (DO11.10 RAG(-/-)) and in vitro-activated Th2 effector CD4 T cells, infiltrated anterior segment tissues early in the inflammation. Memory-like CD44(high) CD4 T cells from unprimed transgenic mice and in vitro-activated Th1 effector CD4 T cells accumulated to larger numbers than naive or Th2 effector cells at 48 and 72 h. Of these, the alpha(2)-integrin+CD4 unprimed T cells entered the eye more efficiently, and antibody to alpha(2)-integrin markedly inhibited the inflammatory response. Intravital microscopy revealed the early arrival and antigen-specific accumulation of CD4 T cells in inflamed tissue and should be helpful in understanding T cell migration to other organs.

nanos expression at the embryonic posterior pole and the medusa phase in the hydrozoan Podocoryne carnea.

Summary The distinction between soma and germline is an important process in the development of animals with sexual reproduction. It is regulated by a number of germline-specific genes, most of which appear conserved in evolution and therefore can be used to study the formation of the germline in diverged animal groups. Here we report the isolation of two orthologs of one such gene, nanos (nos), in the cnidarian Podocoryne carnea, a species with representative zoological features among the hydrozoans. By studying nos gene expression throughout the Podocoryne biphasic life cycle, we find that the germline differentiates exclusively during medusa development, whereas the polyp does not contribute to the process. An early widespread nos expression in developing medusae progressively refines into a mainly germline-specific pattern at terminal stages of medusa formation. Thus, the distinction between germline and soma is a late event in hydrozoan development. Also, we show that the formation of the medusa is a de novo process that relies on active local cell proliferation and differentiation of novel cell and tissue types not present in the polyp, including nos-expressing cells. Finally, we find nos expression at the posterior pole of Podocoryne developing embryos, not related to germline formation. This second aspect of nos expression is also found in Drosophila, where nos functions as a posterior determinant essential for the formation of the fly abdomen. This raises the possibility that nos embryonic expression could play a role in establishing axial polarity in cnidarians.

APCs in the anterior uveal tract do not migrate to draining lymph nodes.

The migration of APCs from sites of infection and their maturation are critical elements in the generation of immune responses. However, the paths by which intraocular Ags migrate to draining lymph nodes are not known because the eye has limited lymphatic vessels. To date, only dendritic cells from the cornea and conjunctiva have been shown to emigrate. We demonstrate that phagocytic APCs in the anterior uveal tissues of the murine eye that ingest fluorescent latex beads do not migrate to regional lymph nodes. The beads are ingested in the uveal tract by cells expressing MHC class II, CD11c, or F4/80. Using intravital time-lapse videomicroscopy to monitor iris APC migration after anterior chamber injection of fluorescent Ag, fluorescently labeled APCs fail to move at multiple observation times, even in the presence of Ag and LPS. Whereas an as yet unidentified ocular nonphagocytic APC subset might migrate from the anterior uveal tissues, it is more probable that immune responses in the draining lymph nodes are engendered by soluble Ag escaping the eye through interstitial spaces. The inability of anterior uveal tissue APCs to migrate to lymph nodes may contribute to deviant immune responses that dominate after Ags are introduced into the anterior chamber.