Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs  showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of miRNAs biogenesis, reinforcing the role of such layer of miRNA biogenesis in miRNA-based regulation of cell activities. The two forms are associated to different sets of target genes, thus contributing in different ways to the regulation of cell activities; experiments conducted on selected miRNAs pairs demonstrated that they could be both functionally effective According to the conventional model, Dicer cleaves the pre-miRNA hairpin to produce a miRNA duplex (∼22 nt), which is incorporated into the RISC. The RISC recognizes the duplex, unwinds it, selects the guide miRNA strand (while degrading the passenger strand), and mediates recognition and silencing of target RNAs. To date, such asymmetry of the strand selection process is considered determined by differential thermodynamic stability of alternative sister miRNAs  elucidate possible transcriptional and post-transcriptional regulatory circuits and ii) clarify, from a methodological point of view, whether host genes expression data may be profitably used as proxy of intragenic miRNAs expression.Mature miRNAs can cause translation inhibition or mRNA cleavage, by base pairing with the 3′ untranslated region (3′-UTR) of their target mRNAs, depending on the complementarity degree between the miRNA and its target sequence Recently, some evidences have been provided that in specific contexts some miRNAs are co-expressed with their host genes We considered all mature miRNAs in miRBase, where 676 (corresponding to 869 mature sequences) were assigned to unique genomic locations whereas the remaining were discarded, since unmapped or corresponding to more than one different localization per miRNA.Two different mature miRNA sequences (miRNA/miRNA*) are generated from a fraction of precursor hairpins and are associated to different sets of target genes and regulated cell activities. We considered expression profiles of mature miRNAs, obtained by microarray platforms specifically designed to measure mature forms.Part of miRNA hairpin sequences is represented, in each dataset, by two different mature miRNAs, each one associated to an individual expression profile. Identical mature pairs derived from hairpins belonging to different genomic localizations were considered only once, obtaining a set of 237 couples of sister mature miRNAs from the same hairpin represented in the MCN, MM datasets was considered. In ALL, ALZ and PRO datasets we found respectively 32, 37 and 95 sister miRNA pairs.The investigation of expression relationships between sister miRNA pairs provided interesting clues about strand selection bias regulation. We were willing to understand if one miRNA in the pair is more expressed than the other in all tissues/cell types/conditions or if the strand selection bias may be cell/tissue-specific. The cluster analysis of samples and of miRNAs pairs according to standardised per sample log2(ratio) between expression values of 5′ and 3′ sister miRNAs provides a general picture of expression prevalence among sister miRNA pairs. Only five miRNA pairs are represented in all considered datasets . The hea2(ratio) of expression values between two sister miRNAs, is able to fairly well separate different samples/tissue types. Even if a laboratory/study effects can not be excluded, normal and malignant B cells, derived from the same dataset (MM) are correctly separated, suggesting that standardised log2(ratio) of expression values may help distinguish normal and tumour samples. 2(ratio) of expression values close to zero in all considered samples. We can conclude that for the large majority of pairs the strand selection bias may be tissue/cell specific. Indeed, the heatmap shows lines in which positive and negative values are mixed, corresponding to sister miRNA pairs showing a not deterministic strand selection bias. At least two sets of miRNAs seem to be expressed in B cells with inverse ratio respectively to other tissues. For instance, 22 pairs shows mean values of expression log2(ratio) in the two sample sets of opposite sign, and 16 miRNA pairs shows mean values of expression log2(ratio) in MM and in all the other samples differing at least one point in the scale of standardised values.We found that the standardised logMany mature miRNAs are characterized by low expression values, slightly over background, and possibly associated to miRNA cellular concentrations insufficient to guarantee the biological activity. Thus, as explained in Methods, mature miRNAs were tagged as “expressed” in a given sample whenever the expression level was higher than the median of all expression values in the matrix. Then, sister miRNA pairs may be alternatively (i.e. only one out of two sister miRNAs is present) or concurrently expressed (both miRNA and miRNA* are present) in a given sample. Therefore, for each of the five considered datasets, miRNA pairs fall in one of the following categories , Table 2A: alternatively expressed, with concurrent expression never occurring in considered samples;C: concurrently expressed pairs in the same set of samples (expressed concurrently whenever expressed);AC: miRNAs pairs resulting alternatively expressed in some samples and concurrently expressed in others.2(ratio) not exceeds 1. Excluding the PRO dataset, in which only one C pair was recorded, in the remaining datasets in average 17%±13% of miRNA pairs have comparable expression levels. Moreover, the distribution of log2(ratio) among expression levels of AC class miRNA pairs shows that about one third of them (34%±28%), are expressed at comparable level.According to microarray data, the majority of miRNA pairs  belong to the AC class, whereas a more or less small percentage results always concurrently expressed . Pairs showing pure “alternative” behaviour, according to the classical biogenesis model, represent less than one quarter of total expressed pairs, in average (23%±7%). When two mature forms are expressed in the same sample, we considered the comparability between their expression levels, as per sample expression ratios distribution among C pairs expression levels, in those samples showing concurrent expression. We considered that two expression levels are comparable when their absolute value of expression logThe heatmap in −16, A few studies considered miRNAs host genes genomic length/organization and their possible regulatory role. In particular, Golan and colleagues We considered the pair-wise expression correlations of respectively 309, 147, 148 and 170 mature miRNA/host gene pairs in the MM, ALL, ALZ and PRO datasets . In all We reasoned that about 20% considered host genes is associated each to two mature miRNA forms, derived from the same hairpin whereas the remaining host genes are associated to only one mature miRNA. Since sister miRNA expression profiles may not be considered independent, we carried out again, for each of the four expression datasets, the above reported analysis of co-expression between intragenic miRNAs and host genes, but considering, for host genes including two mature miRNAs, only the mature miRNA of the pair with the highest miRNA-host correlation. Considering only the highest miRNA-host gene correlation, when a pre-miRNA hosted in a gene produces two mature forms, may give an overestimation of general miRNA-host co-expression tendency. Anyway, the percentages of miRNA-host correlations being positive, >0.25 and >0.5, for each dataset (data not shown), resulted to be almost equal to that reported in These observations discouraged the usage of host gene expression profiles as a proxy to monitor the expression of its embedded miRNA. Thus, we tested whether such procedure affected the results of an integrated analysis of target prediction with miRNA and target expression profiles, using datasets in which real and not inferred miRNAs expression data are available. In particular, for each of the four miRNA and genes matched datasets, a comparative evaluation of results was obtained, by contrasting two integrated analyses, the first (REAL) was conducted on real miRNA and gene expression profiles, whereas the second one (PROXY) was conducted on host genes expression profiles, used as proxy for miRNAs, and gene expression profiles. For each dataset, different numbers of miRNA and genes were considered for target prediction, using TargetScan A set of 2, 848 validated miRNA-target interactions, resulting from Diana Tarbase In this study we exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection and expression relationships between intragenic miRNAs and host genes.Our observations were based on a comprehensive collection of miRNAs and genes/transcripts whose annotation and localization was integrated with expression profiles computed from five large microarray-based datasets, regarding different biological contexts and including both normal and tumour/disease samples. At least 8 different tissues types are represented  plus different T- and B-lineage blood cells. The high number of samples and the broad coverage of cell types would guarantee both significance and fair generality of the obtained results.The first evidence emerging from our analysis regards the expression behaviour of pairs of sister mature miRNAs produced from the same hairpin. As mentioned above, in the classic model of miRNA biogenesis, the duplex of mature miRNAs is produced by Dicer processing of the hairpin precursor. Then, a following strand selection step determines which mature miRNA is the degraded “passenger” strand and which is the major and stable form that will act as guide for the mature miRISC complex in the post-transcriptional silencing of target genes. It is worth notice that the two mature miRNAs have different sets of target genes and may differently contribute to the regulation of cell activities. Our analysis of expression profiles showed that, considering different samples, representing different tissue types under various conditions, the strand selection is highly regulated. In fact, we observed miRNA pairs in which the same miRNA is the most expressed in the majority of considered samples, as well as miRNA pairs expressed at comparable level in almost all considered samples. Nevertheless, the large majority of miRNA pairs show a not deterministic strand selection bias, which may be highly regulated since it shows tissue-/cell-/condition-specific modulation. This is confirmed by the fact that unsupervised analysis, using for samples classification only information about the direction and the strength of the strand selection bias, is able to distinguish different tissues, and sometimes also different conditions . Moreover, when considering a minimum expression threshold, only a minority of pairs were expressed alternatively in all considered cell types, whereas the majority were concurrently expressed in some cell types and alternatively in others. A significant fraction of concurrently expressed pairs showed highly comparable levels, suggesting that both the major and the minor forms may contribute to post-transcriptional gene silencing, possibly in a coordinate way. Also considering separately alternatively and concurrently expressed pairs, the regulated nature of the strand selection is evident. Our results on genome-wide investigation of co-expression relationships of sister miRNAs showed that different biological contexts share complex mechanisms of strand selection regulation. This observation complements recent findings coming from deep sequencing of short RNAs which recently conducted to identification of new miRNAs The second aim of the study was to clarify to what extent intragenic miRNAs were co-expressed with the corresponding host genes. An intragenic miRNAs and host genes related behaviour has been taken for proven by different Authors and used as a strong assumption for the design of computational methods for miRNA targets identification Results of the last part of our study are relevant mainly from a technical and methodological point of view. As previously said, host genes expression profiles were proposed as possible proxies for the intragenic miRNA expression profile, when the latter is unavailable, to identify most probable miRNA target genes. We conducted two integrated analysis of target prediction and expression profiles, one using real miRNA and gene expression profiles, and the second using host genes expression profiles as proxy for miRNAs, and gene expression profiles. The comparative evaluation of the two methods was based on an independent true solution, represented by a set of validated regulatory interactions. This allowed to measure and compare the effectiveness of the two methods in finding validated regulatory interactions, among the subset of predicted miRNA-target relations supported by negative correlations of expression profiles. Our results support the usefulness of the integrated analysis conducted on real miRNAs expression profiles, when stringency is kept reasonably high. Moreover, as expected from previous observations about intragenic miRNAs and host genes scarce co-expression, we experienced that the use of host genes expression as a proxy for miRNA profiles for the integrated analysis seems not significantly enrich in validated relations.In conclusion, our analysis of miRNA sister pairs expression prevalence across samples from five sizeable and diverse expression datasets showed that the large majority of miRNA pairs show a not deterministic strand selection bias, which may be highly regulated, since it presents tissue-/cell-/condition-specific modulation. Our conclusions strengthened recent evidences about the previously underestimated importance of the strand selection regulation, reinforcing the role of such layer of miRNA biogenesis in miRNA-based control of cell activities. Furthermore, our results showed that most host genes and intragenic miRNAs are scarcely co-expressed. In specific cases, they might be co/expressed but mainly in a cell/tissue-specific way. This actually does not rule out the importance of already documented cooperation of specific intragenic miRNAs and host genes products, but proves that the expression information of corresponding host genes can hardly be used as estimator for actual expression of the co-transcribed miRNA and encourage more detailed studies of transcriptional regulation of miRNAs expression.We obtained 49, 506 human genes and 132, 056 transcripts sequences from ENSEMBL (version 56) each associated to a unique chromosomal position. The complete set of hairpin precursors of human microRNA sequences was downloaded from miRBase version 14, thus obtaining a set of 721 pre-miRNA hairpin sequences and 904 mature miRNAs, 185 of which are tagged as “minor”, according to miRBase annotation (i.e. hsa-miR-30e*). Hairpin miRNA sequences were aligned with the version 37.1 of the human genome to establish their genomic positions as start and end coordinates of the aligned region in a specific chromosome and strand. Alignments associated to at least 95% sequence identities, calculated over the hairpin sequence length, have been considered for miRNA genome position definition. As genomic localization is referred to hairpin sequences whereas miRNA microarray platforms measure expression profiles of mature miRNAs, mature miRNAs to hairpin correspondence info was used for data integration. miRNA hairpins localizations were compared with those of protein-coding genes to identify intragenic miRNAs, putatively transcribed from the coding gene promoter [see affy package for Bioconductor and re-annotated using Manhong Dai custom cdf, HuGene10stv1_Hs_ENSG (available at http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/12.1.0/ensg.asp).MM dataset consists of matched miRNAs and genes expression profiles from purified plasma cells of thirty-nine human samples, including 33 patients with multiple myeloma (MM), 2 with plasma cell leukaemia (PCL) and 4 healthy donors. The miRNA expression was profiled on the Agilent Human miRNA Microarray V2. The human miRNAs data were re-annotated and normalized as suggested in http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/12.1.0/ensg.asp).ALL dataset consists of matched miRNA and genes expression profiles in nineteen adult Acute Lymphoblastic Leukaemia (ALL) cases, including T-lineage and B-lineage cells, harbouring specific molecular lesions ALZ dataset consists of 16 matched miRNA and gene expression experiments, obtained by USC/XJZ Human 0.9 K miRNA-940-v1.0 and Affymetrix Human Genome U133 Plus 2.0 Array, in parietal lobe tissue from 4 Alzheimer Disease patients and 4 age-matched controls (GSE16759) PRO dataset consists of the subset of 140 matched miRNA and gene expression experiments, obtained respectively by Agilent-019118 Human miRNA Microarray 2.0 and Affymetrix Human Exon 1.0 ST, of the prostate data reported in MCN dataset includes miRNAs expression profiles in 32 samples from 14 different patients and 8 different tumour types, with tumour cells and normal cells counterpart for each patient 2(ratio) between expression values of 5′ and 3′ sister miRNAs. Matrix values were standardised and used for cluster analysis of samples and of miRNA pairs, using Euclidean distance and average clustering. Then, for each dataset we considered not expressed in a given sample those miRNAs associated to expression values lower than the median of the dataset expression matrix . We calculated the per sample log2(ratio) between expression values of sister miRNAs as indicated before, but miRNA pairs expressed in alternative way in a given sample were associated to extreme values. When only one out of two sister miRNAs was expressed over the threshold, log2(ratio) values (generating ±∞) were artificially set to maxLog(ratio)+0.1, if only the 5′ miR is expressed, or to minLog(ratio)-0.1 in the opposite case. Values of log2(ratio) of samples in which both miRNAs of the pair are not expressed were not considered for the clustering analyses.Mature miRNA pairs may be generated by both 5′ and 3′ strands of the hairpin stem. For historical reasons, miRNA names are assigned in miRBase according to an inhomogeneous notation. miR-X/miR-X* sister pairs refers to the fact that one miRNA of the pair, the miRNA*, is the minor form. An older convention used miR-142-s and miR-142-as. For other pairs, the strand is explicitly indicated in the mature miRNA name (miR-X-5p/miR-X-3p). The latter naming convention is most informative and unambiguous and it will be used for future miRBase releases. Thus, in the present study, we report mature miRNA according to current miRBase names but the order of mature miRNAs in considered sister pairs was kept consistent with the physical position of mature sequences in the precursor, i.e. miRNAs are given in 5′ to 3′ order. For each sister miRNA pair represented in at least one of the five considered datasets, we calculated the per sample logThe integrated analysis of miRNAs and target genes expression profiles combines target predictions with miRNAs and gene expression data correlation-based analysis to identify, among predicted target genes for each considered miRNA, those regulatory relationships significantly supported by expression data.In details, the procedure comprises the identification of miRNA target genes by computational predictions and compilation of the adjacency matrix of targeting relationships, and the computation of pair-wise relatedness of miRNAs and targets from matched expression matrices, to identify relationships supported by expression data, which could be used for post-transcriptional regulatory networks reconstruction and study. The set of microRNA-target predictions were defined using TargetScan 5.1 considering both conserved and non conserved sites that match the seed region of each miRNA. The pair-wise Pearson correlation coefficient between miRNA and target genes expression profiles in exactly the same samples was calculated. We then selected as reliable and potentially functional the subset of predicted relationships associated to most negative Pearson coefficients. Different percentile-based cutoffs were applied, to define the groups of supported regulatory interactions.Figure S1Variability of strand selection bias across samples among all considered datasets.(TIF)Click here for additional data file.Figure S2Variability of strand selection bias across samples considering miRNA pair classification.2(ratio) and to extreme values derived from observed distribution. Cluster analysis performed with these values, produce an heatmap showing both the regulation of the strand selection bias and alternative expression occurrence in different samples.(TIF)Click here for additional data file.Table S1miRNAs genomic localization.(TXT)Click here for additional data file.


Phagocytosis of invading microorganisms by specialized cells such as macrophages and neutrophils is a key component of the innate immune response. These cells capture and engulf pathogens and subsequently destroy them in intracellular vacuoles—the phagosomes. Pathogen phagocytosis and progression and maturation of pathogen-containing phagosomes, a crucial event to acquire microbicidal features, occurs in parallel with accentuated formation of lipid-rich organelles, termed lipid bodies (LBs), or lipid droplets. Experimental and clinical infections with different pathogens such as bacteria, parasites, and viruses induce LB accumulation in cells from the immune system. Within these cells, LBs synthesize and store inflammatory mediators and are considered structural markers of inflammation. In addition to LB accumulation, interaction of these organelles with pathogen-containing phagosomes has increasingly been recognized in response to infections and may have implications in the outcome or survival of the microorganism within host cells. In this review, we summarize our current knowledge on the LB-phagosome interaction within cells from the immune system, with emphasis on macrophages, and discuss the functional meaning of this event during infectious diseases. Phagocytosis of invading microorganisms by specialized cells such as macrophages and neutrophils is a key component of the immune innate response. Phagocytosis is also a fundamental process for removal of cells undergoing apoptosis. The first stage of the elimination process is the internalization of the pathogens or apoptotic bodies into a plasma membrane-derived vacuole, known as phagosome . Newly fIn cells from the immune system, mainly macrophages, both pathogen phagocytosis and progression of pathogen-containing phagosomes generally occurs in parallel with accentuated formation of lipid-rich organelles, termed lipid bodies (LBs) or lipid droplets LBs are intracellular organelles of all cell types including plants and microorganisms  and therefore have specific molecules linked to this synthesis  associated with pools of phospholipid and/or neutral lipids 4 (LTC4)-synthase 2 (cPLA2) and its activating protein kinases, mitogen-activated protein (MAP) kinases , the upstream involved in AA liberation, have been described within LBs The major enzymes involved in the enzymatic conversion of AA into eicosanoids are also present within LBs from activated cells of the immune system, mainly macrophages, eosinophils, and neutrophils. These enzymes include cyclooxygenases (COX) 2 (PGE2) was found in LBs from mouse macrophages infected with Mycobacterium bovis BCG, which causes bovine tuberculosis Trypanosoma cruzi, the causal agent of Chagas' disease, a debilitating cardiac illness 4 was demonstrated in LBs from human eosinophils and basophils stimulated with the chemokines eotaxin/CCL11 and RANTES/CCL5 4in LBs from neutrophils and macrophages during sepsis Overall, LBs from cells of the immune system compartmentalize the substrate (AA) and the entire enzymatic machinery for eicosanoid synthesis. Because eicoisanoids are non-storable mediators, newly formed and rapidly released upon cell stimulation, the detection of these molecules is not simple. However, by means of a new strategy to cross-link newly formed eicosanoids at its sites of synthesis, the presence of eicosanoids has been directly demonstrated within LBs In summary, there is good evidence that LBs are able to change their composition in concert with cell activation acting as inflammatory organelles with roles in the innate immune response to infections and inflammatory processes.ze and to induce LB ultrastructural alterations. LB biogenesis is a process that happens in vivo and in vitro in response to a variety of pathogens.Pathogens induce several changes in the host cell signaling and trafficking mechanisms. One prominent pathogen-mediated change is the formation of LBs in the host cell cytoplasm. Most cells contain a small number of cytoplasmic LBs, but they can be rapidly stimulated to form new LBs under interaction with pathogens. This interaction is also able to increase LB siT. cruzi (Y strain), a significant increase of the LB numbers in peritoneal macrophages at day 6 and 12 of the infection was found. While control peritoneal macrophages presented ∼2.19±0.4 (mean ± SEM) LBs/cell, peritoneal macrophages from infected animals showed ∼18.09±1.4 LBs/cell at day 12 of infection. At this time, the most intense inflammatory process and parasitism in the heart, a target organ of Chagas' disease, compared to other points during the acute phase in rats, is observed T. cruzi is also capable to induce in vitro LB formation within mouse peritoneal macrophages through a Toll-like receptor -2 (TLR2)-dependent mechanism The first observation of newly formed LBs in response to an in vivo parasite infectious disease dates to 2003 Plasmodium berghei, the causative agent of the disease, induces LB accumulation in host cells Toxoplasma gondii, which causes human toxoplasmosis Leishmania amazonensis, a causal agent of human leishmaniasis Pathological studies of target organs of malaria, such as kidney and liver, found the presence of a high number of LBs in infected mice, indicating that the parasite Mycobacterium tuberculosis is characterized by a tight interplay between M. tuberculosis and host cells within cellular aggregates (granulomas) M. tuberculosis in both humans and experimental settings Mycobacterium bovis bacillus Calmette-Guerin (BCG), it was found that this pathogen is capable of inducing a dose- and time-dependent increase on LB formation within pleural macrophages M. bovis BCG-induced LB formation within 24 h. In parallel, the BCG-induced PGE2 generation was completely abrogated Interaction of pathogenic bacteria with host cells leads to LB biogenesis. Tuberculosis caused by Mycobacterium leprae. Leprosy is characterized by widespread skin lesions in which M. leprae lives and replicates in foamy macrophages. These macrophages are highly positive for ADRP M. leprae infection increases expression of ADRP/perilipin mRNA in THP-1 cells, a human promonocytic cell line M. leprae infection. In fact, the capacity of M. leprae to induce LB formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human monocytes and murine peritoneal macrophages Accumulation of lipid-filled foamy macrophages is also a hallmark of lepromatous leprosy, a chronic disease caused by Chlamydia pneumonia and characterizes the early atherosclerosis in the presence of low-density lipoprotein (LDL) Chlamydia muridarum, which causes genital infection in mice, demonstrated that epithelial host cells accumulate LBs in parallel to bacteria replication LB formation within macrophages is also driven by infection with M. leprae, LB biogenesis is observed in the cytoplasm of both cells bearing bacteria and cells with no bacteria cruziInterestingly, the bacterium uptake does not seem essential for LB formation within macrophages and other cells. In human peripheral blood monocytes and murine macrophages exposed to M. tuberculosisBacterial derivates such as lipopolysaccharide (LPS) present in all Gram-negative bacteria M. bovis BCG Mycobacterium smegmatis or Bacillus subtilisM. leprae-infected Schwann cells Chlamydia pneumoniaDistinct signaling pathways can trigger LB formation within cells from the immune system. Specific bacteria- and receptor-mediated pathways activate intracellular signaling that leads to enhanced LB formation. For instance, LB formation is also induced by infection with viruses such as the hepatitis C virus (HCV), the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma A list of pathogens that induce LB biogenesis within different mammalian cells is shown in ze and ultrastructure. In scoring the diameters of LBs within inflammatory macrophages from rats experimentally infected with T. cruzi, 74% of LBs had a size <0.5 µm in non-infected whereas 54% of LBs from infected animals were >0.5 µm, reaching up to 3 µm. When macrophages from T. cruzi-infected animals were challenged in vivo with higher parasite load, a significant increase of LB sizes compared to LBs induced by the infection alone was observed Newly formed LBs within pathogen-infected macrophages can show changes in siM. bovis BCG show distinct morphology, becoming larger and less dense organelles compared to LBs from non-infected cells T. cruzi infection within inflammatory macrophages also exhibited changes in electron-density compared to uninfected cells One interesting ultrastructural aspect of LBs is their electron-density (osmiophilia), which is dependent on the cell type and can change in response to pathogens. When observed by TEM, macrophages from mice infected with 3H AA by macrophages that were also exposed to zymosan particles for phagocytosis, a striking approximation of 3H AA-incorporated LBs with phagolysosomes was observed  within Hep2 or HeLa cells, penetrate the vacuole membrane, and intimately associate with reticulate bodies, the replicative form of ChlamydiaOther studies have been demonstrating LB translocation into the lumen of bacteria-containing phagosomes using live cell fluorescence microscopy and/or TEM M. leprae also show accumulating LBs in close association with M. leprae-containing phagosomes within Schwann cells, a target cell of this pathogen. These LBs are then promptly recruited to the bacteria-containing phagosomes, a process that depends on cytoskeletal reorganization and phosphatidylinositol 3 kinase (PI3K) signaling Chlamydia muridarum demonstrated the same phenomenon—multiple LBs in contact with the pathogen-containing vacuole and LBs entering into the vacuole within host cells Nerve biopsies of patients infected with Chlamydia trachomatisOverall, infection with different pathogens leads to movement of LBs into phagosomes. It is intriguing how pathogens target LB and how intact and non-membrane-bound organelles such as LBs are translocated across the phagosome membrane. Bacterial proteins seem to be involved in capturing LB into bacteria-containing vacuoles while the translocation process seems to involve displacement of the LB structural protein ADRP from the LB surface to the phagosome membrane, as observed during the in vitro infection with LB-phagosome association has also been observed in other situations. Contact sites between LBs and latex bead-containing phagosomes were identified by high resolution Raman microscopy in neutrophilic granulocytes As noted, the LB-phagosome interaction seems to be a general event found during infections with different pathogens in both humans and experimental models. Although little is known about the functional meaning of this interaction, it raises intriguing possibilities in light of the LB composition and functions.M. tuberculosisM. lepraeChlamydia trachomatisM. tuberculosis bacilli are able to accumulate lipids during dormancy from which it derives both carbon and energy for its own metabolism Much interest has been focused on LBs as conduits for the transport of potential nutrients, especially neutral lipids, to the phagosome. The lipid content of LBs may, therefore, serve as a nutrient source for the pathogen enabling its survival within the cell M. tuberculosis-induced LB formation but also the bacterial viability in Schwann cells Pharmacological inhibition of LB formation within pathogen-infected cells was investigated. Using triacsin C, which prevents LD biogenesis by specifically inhibiting the activity of a subset of long chain acyl-coA synthetases (ACSL) required for triacylglyceride and cholesterol ester biosynthesis, a decrease was observed in the phagosome size and reduction of chlamydial growth within Hep2 cells Escherichia coliThe possibility of LBs to deliver other nutrients into the phagosome for pathogen growth was also raised. Complexes of iron and mycobactins, lipophilic siderophores of mycobacteria, accumulate in LBs within macrophages infected with 2 in macrophages act as a potent inhibitor of Th1 type response Taken together, a picture emerges in which pathogens usurp host, newly-formed LBs to obtain nutrients, mainly lipids, as a survival strategy. Moreover, the enhanced capacity of host cells to generate inflammatory mediators in the course of pathogenic infections due to increased LB formation and compartmentalization of signaling and eicosanoid production within LBs may also be contributing to mechanisms that intracellular pathogens have developed to survive in host cells. For example, high concentration of PGEM. tuberculosis-infected macrophages, selected lipids, including AA, can activate actin assembly, phagosome-lysosome fusion, and phagosome maturation, resulting in bacteria killing On the other hand, lipids have been gaining attention as co-directors of phagocytosis (reviewed in 3), an ER-resident 47 KDa immune-related GTPase involved in phagosomal maturation and phagocytic cross-presentation, was also identified in LBs within dendritic cells, indicating that LBs regulate cross-presentation of phagocytosed antigens in these cells Key molecules, such as Rab 5 and Rab 7, are involved in the sequential interactions of early and late endosomes with phagosomes LBs emerge as key organelles involved in experimental and clinical infections with different pathogens, such as bacteria, parasites, and viruses. Within cells recruited in response to these infections, especially macrophages, LBs contribute to the genesis of inflammatory mediators and are considered as structural markers of inflammation. In addition to LB accumulation, interaction of these organelles with pathogen-containing phagosomes has increasingly been recognized. Recent observations have indicated that this intriguing and intimate association is a pathogen-driven process, evolved as a strategy to survive within the host cells by sequestering mainly host lipids. However, it should be noted that the LB-phagosome interaction may be linked to defense events in which the host cell seeks to kill the pathogen invader. Because phagosomal membrane and luminal contents must undergo remodeling to transform the initially inert environment into a microbicidal one, attention should be paid to LBs as potential determinant organelles in the phagocytic event. While this is more speculative, to date we cannot answer whether LBs have a major and a definitive role in the intracellular survival or destruction of pathogens and/or if these organelles are able to interfere with phagocytosis pathways. Other questions regarding how pathogens target LBs and/or how LBs target pathogen-containing phagosomes and are translocated into these vaccuoles await a lot of more investigations. Novel approaches such as electron tomographic analysis of the LB-phagosome interaction dynamics combined with further refined functional analyses will be required to address these questions. A better understanding of the cell biology of LBs and their potential role during pathogen phagocytosis may be crucial for the application of novel therapies addressing different pathological conditions.


What is the cellular origin of melanoma? What role do melanocyte stem cells (MSC) and other melanocyte precursors play in the development of melanoma? Are MSCs and other latent melanocyte precursors more susceptible to solar radiation? These and many other questions can be very effectively addressed using the zebrafish model. Zebrafish have a robust regenerative capability, permitting the study of how MSCs are regulated and recruited at specific times and places to generate the pigment pattern following fin amputation or melanocyte ablation. They can be used to determine the effects of environmental radiation on the proliferation, survival, repair, and differentiation of MSCs. Our lab is using zebrafish to investigate how UVA- (320–400 nm) and UVB- (290–320 nm) induced damage to MSCs may contribute to the development of melanoma. A review is given of MSCs in zebrafish as well as experimental techniques and drugs for manipulating MSC populations. These techniques can be used to design experiments to help answer many questions regarding the role of MSCs or melanocyte precursors in the formation of melanoma stem cells and tumors following exposure to UVA/UVB radiation. Danio rerio, a small tropical fish known commonly as zebrafish, is becoming a widely used model system for studying many human diseases [diseases . Zebrafidiseases –4. TheirZebrafish has great promise as a model for investigating the earliest origins of melanoma . AlthougTraditionally, it has been assumed that melanoma has its earliest beginnings in a mature melanocyte, but due to the accumulating evidence that early-stage precursors to melanocytes exist in the dermis –17, equaXiphophorus hybrid model of melanoma that removes 50–85% of the CPDs within 15 min and (6-4) PDs within 60 min [in vivo electroporation to introduce a fluorescein-tagged antisense morpholino to block expression of photolyase and prevent PER in the regenerating tail fin [UV light causes damage when DNA and proteins directly absorb the light energy or when it produces free radicals, including reactive oxygen species, which indirectly cause damage to these molecules. UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (PDs). Generation of these photoproducts is wavelength dependent with UVB radiation being 1000-fold more efficient than UVA in their production. These two photoproducts can block DNA replication, transcription and affect protein-DNA interactions. If the photoproducts are improperly repaired or escape repair processes they cause mutations contributing to the development of melanoma –23. In hn 60 min , 27. Thitail fin . Thus, tp53 mutants are deficient in NER and are more sensitive than wild-type fish to UVB radiation [p53 gene expression is developmentally regulated and its expression depends upon the stage of development [p53 may play an important role in DNA damage repair in MSCs similar to what has been shown in other cells [The significance of NER in preventing melanoma is illustrated in the inherited human condition xeroderma pigmentosum (XP). People homozygous for this recessive mutation have defective NER. Unable to repair DNA damage effectively, they develop skin tumors with high frequency in sun-exposed areas of the skin , 30. Ageadiation . Many stelopment –36. Thuser cells . The zebrafish model lends itself well to testing the hypothesis that UV-induced damage to MSCs rather than to mature melanocytes may be how melanoma begins. UVA penetrates deeper in the dermal layer of the skin where MSC niches may reside . UVA finThe zebrafish model offers a variety of strategies to determine if UVA and UVB radiation impairs MSC function simply by activating melanocyte regeneration by either fin amputation or chemical ablation of the adult melanocyte population –45. Any Zebrafish MSCs exhibit almost unlimited self-renewal capacity and share many regulatory signals and pathways with melanoma and cancer stem cells in humans –49. GeneThere are two distinct populations of melanocytes in zebrafish—larval melanocytes and adult melanocytes. During embryonic development neural crest cells differentiate directly into early larval melanocytes. At 3 days after fertilization (dpf) there are approximately 460 larval melanocytes . A “pigmCells of the melanocyte lineage that have not yet differentiated into a melanin-producing melanocyte are called melanoblasts, melanocyte progenitors, or latent melanocyte precursors. They all represent cells in a pre-melanocyte stage, somewhere along the developmental pathway that ends with differentiation into a mature, melanin-producing melanocyte. In contrast, a MSC has both the capacity for producing differentiated melanocyte progeny as well as the ability to self-renew. The existence of self-renewing MSCs in zebrafish is supported by studies showing continued regeneration of the melanocyte pigment pattern in fish following multiple rounds of adult melanocyte ablation or fin amputation , 53, 54.dct), permits the detection of melanoblasts very early in development. Analysis of dct expression suggests that melanoblast specification may be established prior to migration at about 18-19 hpf [The cloning and expression of an early zebrafish melanoblast marker, dopachrome tautomerase  and proliferation (tumors and nevi) over the lifetime of the fish. Exposing the MSCs at specific stages of development for extended periods of time can be more precisely achieved by adding WNT pathway inhibitors to temporarily suspend regeneration while irradiating [The zebrafish model can be used to investigate how adult stem cells may be more susceptible to environmental radiation at different stages of development. In general, when tissues are regenerated, adult stem cells switch from a quiescent stage and proceed through all the intermediate stages leading up to fully differentiated cell types. A major challenge of using stem cells adiating . The amputation of the zebrafish caudal fin is a very facile method to initiate and synchronize the regeneration of melanocytes from the resident MSC population. Amputation of the caudal fin does not affect fish viability. Fin regeneration is completed within two weeks. The caudal fin has an almost unlimited capacity to regenerate; repeated amputations up to 29 times over a period of 11 months do not alter regenerative capacity , 59. DurAlthough the fin amputation offers many advantages to study the mechanisms regulating MSC proliferation and their distribution in regenerating tissue, this method does not offer the opportunity to study the effects of UVA/UVB radiation on MSCs in isolation from other biological mechanisms operating during epimorphic regeneration. This limitation can be overcome by using the conditional drug, neocuproine (NCP), to ablate the entire population of adult melanocytes  or by us KIT, RAS, MITF, and ERBB3 genes and the Wnt/β-catenin signaling pathway are known to play important roles in the development and regulation of MSCs or melanocyte precursors. Zebrafish mutants help to delineate the mechanisms, underlying the establishment, maintenance and recruitment of MSCs or melanocyte precursors during development and regeneration . The KITKIT gene [KIT signaling influences the establishment, recruitment, regulation, and maintenance of both the pre-MSC and the MSC population itself [KIT receptor, and it is believed that SCF guides these cells to their terminal locations. SCF also regulates survival and proliferation of fully differentiated melanocytes in adults [KIT in transgenic larvae leads to recruitment of MSCs, bringing about an increased proliferation of melanocytes. This suggests that KIT signaling is involved in MSC recruitment during regeneration [Tyrosine-protein kinase is encoded by the KIT gene . When thn itself , 64, 65.n adults . MutantsRAS proteins function as binary molecular switches that control intracellular signaling networks. RAS-regulated signaling pathways control such processes as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. RAS proteins are often deregulated in cancers, leading to increased invasion and metastasis and decreased apoptosis. RAS determines the supply of melanocyte precursors, facilitating stripe pattern recovery during zebrafish fin regeneration. During regeneration following amputation of the caudal fin, RAS activity levels control the repopulation and expansion of the MSC or melanocyte precursor cell population that exists prior to amputation, rather than amplifying existing differentiated melanocytes. The level of active RAS in regenerating zebrafish fins controls the density of melanocyte precursors and differentiated progeny within newly forming fin structures. Transgenic increases in active RAS markedly amplify the number of melanocytes present during fin regeneration, hyperpigmenting new fin structures and disrupting the stripe pattern. The amplification of melanocyte precursors occurs through a mechanism that does not depend on intact KIT signaling [HRAS under the KIT promoter induces melanoma development with high efficiency without the need for additional mutations in tumor suppressor genes [ignaling . Expressor genes .MIT gene encodes the microphthalmia-associated transcription factor, a protein of the basic helix-loop-helix leucine zipper family. It is the master regulator of vertebrate melanocyte development [MITF promotes melanoblast survival immediately after migration from the dorsal neural tube and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration [MITF expression is thought to be a key event in triggering MSCs to enter the cell cycle [The elopment –69. It ielopment , 70, 71.igration . Activatll cycle . ERBB3 gene encodes a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. This membrane-bound protein has a neuregulin binding domain but not an active kinase domain. Therefore, it can bind this ligand but not convey the signal into the cell through protein phosphorylation. However, it does form heterodimers with other EGF receptor family members which do have kinase activity. This leads to the activation of cell proliferation or differentiation pathways [ERBB3 is required during embryonic development for establishing the pool of melanocyte precursors responsible for developing adult melanocytes during the transition from the larval to adult stage. This is in contrast to embryonic melanocytes that develop directly, without the need for ERBB3 signaling [ERBB3-deficient fish [The pathways , 75. ERBignaling , 65, 76.ent fish . βWnt/-catenin signaling pathway plays a variety of important roles in embryonic development, cell differentiation, and regulation of stem cells [βWnt/-catenin pathway [βWnt/-catenin signaling is activated during regeneration of the zebrafish caudal fin. It is required for the formation and subsequent proliferation of the progenitor cells in the blastema of the regenerating fin [The em cells , 78. It em cells , 80. Pat pathway . Wnt/β-cting fin .in vivo create “experimental windows” to assess effects of radiation and chemicals on specific melanocyte precursor populations. Alternations are manifested by abnormal pigment patterns or melanocyte proliferation (nevi and tumors).Several chemicals offer pharmacological methods to prevent development or activate MSC pathways. AG1478 prevents progression of a pre-MSC , ICI-118ERBB family of receptors [AG 178 is an epidermal growth factor receptor tyrosine kinase inhibitor of the eceptors . When theceptors . dct+), an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin, but never develop into fully differentiated melanocytes that express melanin [The regeneration specific drug, ICI-118,551, is a small molecule that blocks melanocyte regeneration in larvae by preventing the developmental progression of a MSC to the melanoblast stage. The melanoblasts express dopachrome tautomerase ( melanin .Neocuproine (NCP), when added to fish water, triggers apoptosis of the adult melanocyte population in zebrafish. It has no effect on either larval melanocytes or the resident MSC population. After drug washout, the pigment pattern is regenerated from resident MSCs within 4 weeks . Thus, tThis small pro-drug molecule, (2-morpholinobutyl)-4-thiophenol (MoTP), specifically ablates larval melanocytes or melanoblasts when administered before 6-7 dpf or at 15 dpf . Its cytWnt signaling [βWnt/-catenin signaling is important in the activation of stem cells in heart, muscle, bone and liver [βWnt/-catenin signaling regulates blastema formation and subsequent proliferation during regeneration of the caudal fin following amputation. IWR will temporarily stop regeneration of the caudal fin at any stage. It can be used to suspend the progression of MSC into mature melanocytes at any stage just by varying the time at which the chemical is added to the water. Fish maintained in water containing IWR-1 for 7 days are able to resume tissue regeneration to normal levels after IWR-1 washout, which suggests that transient inhibition of Wnt/B-catenin response does not permanently alter the ability of MSCs to self-renew and participate in the regeneration of melanocytes [Wnt signaling with IWR provides a powerful method to temporarily suspend developmental progression of a MSC at any period of time or stage, creating “experimental windows” to subject MSCs and melanocyte precursors to radiation. This small-molecule inhibitor acts as a negative regulator of canonical ignaling . Wnt/β-cnd liver , 84, 85.anocytes , 86. InhV600E mutation, the most common mutation in human nevi and melanoma, is sufficient to induce ectopic nevi in zebrafish and can collaborate with p53 mutations to promote melanoma [p53-dependent NER pathway to protect cells from UVB-induced DNA damage in the skin [The zebrafish model is becoming more widely used for melanoma research . Severalmelanoma , 89. It the skin . We are We use IWR-1 to “freeze” melanocyte precursors at specific times of development to discover stages in the MSC developmental pathway where a melanocyte precursor may be more vulnerable to the damaging rays of UVA or UVB radiation . Our preThe zebrafish model has significant potential for discovering how UV-induced damage to MSCs may play a role in the development of melanoma. It offers many experimental approaches for identifying the many important variables in the initiation, development, and progression of melanoma. Variables include fluence, UV spectra, and dosage. In a general way, the zebrafish model has great potential for assessing the effects of any chemical on the function, proliferation, and differentiation of adult stem cells. This may have major implications for understanding the overall role of stem cells in the genesis of tumors or as precursors of many degenerative diseases in humans.


Super-macromolecular complexes play many important roles in eukaryotic cells. Classical structural biological studies focus on their complicated molecular structures, physical interactions and biochemical modifications. Recent advances concerning intracellular electric fields generated by cell organelles and super-macromolecular complexes shed new light on the mechanisms that govern the dynamics of mitosis and meiosis. In this review we synthesize this knowledge to provide an integrated theoretical model of these cellular events. We suggest that the electric fields generated by synchronized oscillation of microtubules, centrosomes, and chromatin fibers facilitate several events during mitosis and meiosis, including centrosome trafficking, chromosome congression in mitosis and synapsis between homologous chromosomes in meiosis. These intracellular electric fields are generated under energy excitation through the synchronized electric oscillations of the dipolar structures of microtubules, centrosomes and chromosomes, three of the super-macromolecular complexes within an animal cell. The choreography of microtubules, centrosomes and chromosomes during mitosis and meiosis is beautifully designed by nature. Finely regulated and synchronized movements of these super-macromolecular complexes against the entropic forces within a dividing cell ensure the fidelity of the genetic material in both daughter cells. Currently, several models exist for the mechanisms of chromosome congression and spindle body assembly during M phase such as the search and capture model, kinetochore-mediated k-fibre formation, kinetochore motors contributing to congression, and the polar wind model. The mechanisms evoked by these models probably overlap, so there is redundancy among them, since mutations in the genes involved have only mild effects on chromosome congression during mitosis . Many opCellular electric fields have been studied in various cell types, and several studies have reported the existence of dielectrophoretic forces around cells -8; electCoherent oscillations in microtubules can be explained by Fröhlich’s theory, which describes a system of oscillators with energy supply, linear and nonlinear coupling with a heat bath. If a sufficient energy supply is provided to this system, condensation of energy occurs in the lowest mode leading to its coherent excitation ,24. ElecIn this article, we integrate research from several disciplines to provide an ‘electric’ view of the dynamics of these super-macromolecular complexes in mitosis, meiosis and other relevant cellular events. From our theoretical point of view, many of the unidentified forces regulating major cellular dynamic events during mitosis are probably electric forces generated by the synchronized oscillation of the electric dipoles within these super-macro organelle structures. Chronic exposure to extremely low frequency electric fields could affect several key steps of mitosis and neuronal cell physiology, resulting in an increased risk for cancer.The electric field of the microtubule is generated by the synchronized oscillation of α and β tubulins. These tubulins form electric dipoles during microtubule polymerization; under intracellular energy excitation, synchronized oscillation of α and β tubulin subunits generates a longitudinal electric field around the microtubule -15  . As in mMechanisms of centrosome separation and bipolar spindle body assembly have been discussed in a recent review . The proAndrews et al. have studied the effects of high frequency (range 2 to 50 MHz) electric fields on mammalian (human and Chinese hamster) chromosomes in vitro. They showed that such chromosomes can be oriented, aligned and translated by an oscillating electrical force. They also observed that above certain threshold field strengths the chromosomes orient themselves with their long axes along the field direction. The dependence of this threshold on frequency was measured and was found to be much larger at low than at high frequencies . Using eHowever, the physical mechanisms that regulate higher order packaging of M phase chromosomes are still not well characterized. Here we present a hypothesis of chromosome compaction. We apply a pulse-coupled oscillation clustering model to the dynamic events of chromosome packaging and inter-/intra-chromosomal organization. During chromosomal packaging, differentially compacted regions form partially synchronized electric oscillators interacting with an elastic electromagnetic field. According to the physical pulse-coupled oscillator model, unsynchronized pulse-coupled oscillators with proximal natural frequencies form synchronized oscillation clusters at a given coupling strength. As the coupling strength increases, these synchronized oscillation clusters merge with each other -40.During M phase chromosome compaction, the 30 nm chromatin fiber is initially formed by the electrostatic forces between neighboring nucleosomes. Under intracellular stochastic energy excitation, electric dipolar oscillation would be generated between neighboring nucleosomes. After oscillation synchronization and coupling, regulated electric oscillation is generated along the 30 nm chromatin fiber, and the oscillation coupling process further compacts that fiber -14,41. TTheoretically, the oscillation clustering model explains the closely juxtaposed configuration of duplicated chromosomes during M phase, which is counter-intuitive from the perspective of electrostatic repulsion between duplicated chromosome arms. As the homologous chromosomal regions develop synchronized oscillation clusters with identical natural frequencies, they tend to cluster together. The same scenario could apply to synapsis during meiosis; the electric oscillations of homologous chromosomes couple with each other, preventing synapsis between non-homologous chromosomes.The intracellular electric fields described in the foregoing sections could facilitate several cellular events during mitosis and meiosis. First, the metaphase bipolar spindle microtubules are formed through microtubule nucleation from the γ tubulin ring complexes (γ-TuRCs) at the centrosomes and retrograde delivery of peripheral microtubules by motor proteins ,44-46. TAccording to the physical organization of the duplicated chromosome arms, the condensed electric chromosomal fields around the centromeric regions could attract microtubule fragments to the sister kinetochores through electric interaction, which is consistent with observations of kinetochore movement along uncaptured microtubules, forming K-fibers (kinetochore associated microtubules) . Thus, eIn meiosis, the kinetochores are positioned at one side of the duplicated chromosome dimers, so the two sister chromosomes do not separate. The electric oscillation clustering between homologous chromosomal regions results in synapsis and recombination between homologous chromosomes; the electric fields generated by two duplicated homologous chromosomes can be viewed as two identical partially-entrained electric oscillation clusters, constituted by sub-chromosomal clusters throughout the chromosome arms. Such clusters in homologous chromosomes share identical electric frequencies, so the close juxtaposition between homologous chromosomes at synapsis is achieved through electric clustering and coupling among them. Synapsis does not occur during mitosis probably because the chromosome configuration caused by the opposing outward-pulling forces of the kinetochores at the opposite sides of duplicated centromere disfavors inter-chromosomal electric attraction. In addition, this event may be regulated by synaptonemal complex proteins .Magidson et al. reported that chromosomes adopt a toroidal/ring shape organization of after NE breakdown, which facilitates spindle assembly during M phase . Their oNumerous reports indicate that extremely low frequency electric fields can increase the risks of certain types of cancer . MicronuTo characterize these intracellular electric fields and study their cellular functions further, biophysicists should develop more detailed mathematical and physical models for chromosome electric fields and their role in M phase chromosome compaction, to allow these fields to be described and calculated more precisely and to predict the dynamics of related cellular events. The dynamic of changes of the electric fields in a living cell during mitosis and other cellular processes could be visualized using live cell imaging technologies such as nano-sized voltmeters . It woulThe authors declare they have no competing interests.Yue Zhao conceived the general concepts and wrote the article; Qimin Zhan shared insights and gave advice. Both authors read and approved the final version of the article.


Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies. While advanced cancers are diverse in phenotype, they often share essential functional properties ascribed to stem cells such as the capacity to become quiescent, acquire multi-lineage differentiation potential, survive and self-renew . Early iia (CML) –4. CML ria (CML) , 6; and ia (CML) .http://www.leukemia-lymphoma.org) is relatively low, it is expected to increase significantly over the next 20 years as a result of widespread use of BCR-ABL tyrosine kinase inhibitor (TKI) therapy. Previous reports showing preclinical and clinical data demonstrate that discontinuation of TKI therapy results in resurgence of disease [While the current CML prevalence of 24,000 affected patients in the United States  and p21(cip1). Under homeostatic conditions, the activities of these components are tightly regulated. In contrast, abnormal tyrosine kinase-directed phosphorylation of cell cycle regulatory proteins and mis-localization of cell cycle proteins have been implicated in de-regulation of the cell cycle in BCR-ABL expressing cells . AdditioIt is unclear why under certain conditions this may promote uncontrolled proliferation while in other contexts cell quiescence may be favored. However, it is possible that cues from the microenvironment might be involved. Several lines of evidence support the contention that LSC localized to the bone marrow niche are dormant and resistant to traditional cytotoxic therapies. Specific signals from the surrounding stromal cells might promote LSC cell cycle arrest and allow them to persist even during treatment with TKI therapies. Altered interactions between CML LSC and the bone marrow microenvironment can occur as a result of direct intercellular contact; secreted factors such as cytokines or chemokines; or through microenvironmental matrix proteins that regulate cellular adhesion and migration, and growth factor receptor and integrin signals , among oPrevious reports support the hypothesis that LSC fuel therapeutic resistance, relapse, and disease progression from CP to BC as a result of enhanced survival and self-renewal combined with a propensity to become dormant in supportive microenvironments . To dateThe role of the hematopoietic microenvironment in the development and progression of leukemic disorders has been a subject of intense investigation •, 17•. PDicer1 deleted mice also resulted in the development of myelodysplasia in the recipient animals. This supports a critical role for perturbations in the bone marrow microenvironment in the development of disorders of myeloid progenitors, however disease initiation and progression to BC driven by alterations intrinsic to malignant CML progenitors has also been well characterized.Seminal mouse transgenic studies reveal that niche-induced oncogenesis can drive the development of myelodysplasia •. IntereThe truth likely lies somewhere in between the two poles of niche-induced versus LSC-autonomous oncogenesis, wherein subtle changes in both HSC and the bone marrow microenvironmental stromal cells act in concert to drive generation of LSC and persistence of disease, and further exacerbate aberrant regulation of cellular pathways. Malignant CML progenitors harboring mutations and epigenetic alterations that drive enhanced survival and self-renewal might perpetuate pro-leukemic signals from the microenvironment via abnormal inter-cellular signaling with stromal cell populations. Moreover, recent reports indicate that imatinib treatment promotes migration of CML progenitors to the bone marrow via activation of inflammatory signaling receptors , which fosters the survival of quiescent LSC in the bone marrow niche .The precise molecular mechanisms driving malignant reprogramming of progenitors into LSC in BC CML have remained elusive. Myeloid LSC evolve from granulocyte-macrophage progenitors (GMP) that aberrantly activate self-renewal pathways and drive therapeutic resistance , 19, 20.Recent advances have been made toward targeting these signaling molecules with the aim of eradicating LSC or sensitizing them to TKI therapies. Candidate therapies under development include inhibitors of Wnt/β-catenin signaling. New evidence suggests that such therapeutic approaches may hold promise for targeting imatinib-resistant LSC populations, and may represent an important therapeutic strategy in CML and other leukemic disorders. However, current reports of therapeutic Wnt inhibition in models of CML are limited and will require further in vivo studies to elucidate the therapeutic efficacy of these approaches.Another potential therapeutic target that has received recent attention is cell survival gene pathways that are aberrantly activated in CML LSC. For example, deregulation of the anti-apoptotic B-cell lymphoma/leukemia-2 (BCL2) family of genes contributes to LSC apoptosis resistance in the bone marrow microenvironment. Overexpression of BCL2 family genes has been observed in human BC CML and may fuel LSC survival . Human stem cells express myriad pro-death and pro-survival BCL2 family members, each with alternative splice isoforms, rendering the study of human stem cells essential for predicting both the potential efficacy and toxicity of targeted BCL2 inhibition as a therapeutic strategy to eliminate apoptosis resistant LSC. Recent data demonstrate that human LSC express numerous anti-apoptotic BCL2 family splice isoforms. This family of proteins provides a promising candidate for LSC-targeted strategies aimed at selectively eliminating dormant CML LSC. Since multiple BCL2 family members appear to be involved, it is possible that a pan-BCL2 inhibitor will be required to abrogate LSC survival.BCL2 gene overexpression plays a key role in therapeutic resistance of quiescent BC CML LSC. Combination therapies targeting activated kinases in concert with anti-apoptotic factors such as the BCL2 family may represent a key strategy toward preventing therapeutic resistance and may be more broadly applicable to LSC in other malignancies.We have recently demonstrated that potent inhibitors of BCL2 pro-survival family proteins significantly inhibit LSC survival, at doses that spare normal hematopoietic progenitors in BC CML . Thus, anti-apoptotic Alternative important potential targets for therapeutic development in CML are pathways that regulate stem cell self-renewal. For example, one critical stem cell-associated self-renewal gene that has been implicated in the pathogenesis of myeloid and other malignancies is Shh . Shh actWhile in hematological malignancies, previous studies have linked Shh activation to cancer stem cell (CSC) generation and disease progression , others While thus far we have covered several examples of critical pathways that may drive LSC generation and maintenance, including cell cycle, survival, and self-renewal gene families, the reality is that a plethora of abnormally regulated pathways have been implicated in the pathogenesis of CML. This hints at the possibility that broader reprogramming events at the DNA and/or RNA level may be responsible for the downstream molecular events that drive disease progression. Although abnormal activation of signal transduction pathways promoting cell cycle de-regulation and malignant cues derived from LSC-supportive hematopoietic microenvironments may be responsible in part for the development and progression of CML, other post-transcriptional mechanisms may also contribute. Monumental efforts to date have resulted in the identification of DNA mutations and translocations  that constitute the initiating oncogenic events in the development of chronic leukemic disorders, however the persistence of LSC and the rise of therapeutic resistance among patients receiving long-term TKI therapies indicates that other mechanisms may fuel leukemic progression and relapse. This has provided the impetus for elucidating the role of genetic and epigenetic alterations fueling cancer progression.LSC dormancy, survival, and self-renewal, properties that drive both leukemic transformation and therapeutic resistance, occur as a consequence of cell type-specific and microenvironment-specific alterations in both DNA repair and RNA processing. Important clues to the molecular pathways involved in leukemia initiation and progression have been revealed previously by mutational analyses of tumors that described accumulated DNA mutations. In particular, alterations in TET2—a regulator of cytosine methylation—have been detected frequently in myelodysplastic syndromes (MDS) and more recently have been reported to occur as secondary cytogenetic abnormalities in acute and blast phase CML . While aAs discussed above, previous studies have implicated the generation of novel splice acceptor sites in critical signaling molecules, such as GSK3β, can result in the generation of novel splice isoforms that promote malignant reprogramming of progenitors into LSC that drive blastic transformation of CML , 36. HowGSK3β, Bcl-x, p53, and caspase-2) and drug resistance [Alternative splicing also plays a central role in the maintenance of fetal and adult HSC and may have important functions in LSC and disease progression. Cancer-associated alterations in splicing have been shown to affect genes controlling apoptosis  , in cellPrevious reports have proposed that general changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis ; howeverhttp://seer.cancer.gov/). Unfortunately, a growing proportion of patients are non-compliant because they become inured to the fact that they have leukemia or simply cannot afford full dose TKI therapy as a result of spiraling annual costs and thus, progress to advanced phase disease with a 5-year survival rate of less than 30 %. Thus, elimination of LSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance, and understanding the molecular mechanisms of CML LSC biology that drive the generation of LSC and their persistence is crucial.In the pathogenesis of CML, therapeutically recalcitrant LSC are thought to fuel disease progression and relapse. While targeted TKI therapies (BCR-ABL antagonists) have resulted in improved survival of patients with CP CML, the prevalence of CML doubled in the U.S. between 2001 and 2009 (Characteristic features of CML LSC, such as their enhanced capacity for survival, self-renewal, and persistence over time through dormancy, underlie their unique resilience to conventional therapies. Cell cycle alterations, abnormal activation of signaling pathways involved in cell survival and self-renewal, and microenvironmental cues from the malignant bone marrow microenvironment can facilitate the enhanced survival and malignant transformation of CML LSC. More recently, epigenetic alterations at the RNA level, including aberrant splicing and RNA processing, have been implicated in the pathogenesis of CML and MDS. Therapeutic strategies targeting regulators of cell survival and self-renewal, or RNA processing pathways, could represent vital novel components of a potentially curative strategy for advanced CML that may obviate the need for costly continuous TKI therapy by increasing sensitivity to therapy. Such therapeutic strategies could provide a valuable companion diagnostic with relevance to a broad group of patients suffering from leukemia and lymphoma disorders.


Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans.  Drosophila eye development relies on a network of retinal determination (RD) genes, which encode highly conserved transcription factors and cofactors eyes absent (eya), which is necessary for survival and normal eye formation, as well as sufficient for ectopic eye induction when overexpressed Eya homologues (Eya1-4) regulate the development of the kidneys, ears, craniofacial and skeletal structures, muscle, thymus, parathyroid, and lungs, as well as acting in the innate immune response, DNA damage repair, inhibition of apoptosis, angiogenesis, and photoperiodism Eya1-4 are implicated in several diseases in humans, such as the multi-organ developmental disorderbranchio-oto-renal (BOR) syndrome In vitro phosphatase assays show that Eya proteins derived from plant, mouse, and fly exhibit tyrosine phosphatase activity, although that of Drosophila Eya is very low and is difficult to detect EYA1Eya1, Eya2, or Eya3 results in increased proliferation, migration, invasion, and transformation of breast cancer cells. Interestingly, Eya1/2/3 phosphatase-dead mutations attenuate induction of migration, invasion, and transformation, suggesting that the tyrosine phosphatase activity promotes tumor cell invasiveness Eya is known to act as a transcriptional coactivator as well as a protein phosphatase. The highly conserved C-terminal region of Eya, referred to as the Eya domain (ED) Drosophila, cDNA-based eya mutant transgenes that disrupt the predicted tyrosine phosphatase active site (D493N and E728Q) have drastically decreased ability to induce ectopic eye formation and to rescue eye development in the 2eya eye-specific loss-of-function mutant eya transgenes in the developing eye suggests that Eya tyrosine phosphatase regulates photoreceptor axon targeting Drosophila Eya tyrosine phosphatase activity may play a role during normal development, this hypothesis has not been tested using a system that accurately reproduces endogenous levels, timing, and patterns of eya expression.In in vivo significance of two predicted MAPK target sites in the Eya protein. Previous experiments using the Gal4-UAS system had suggested that MAPK-mediated phosphorylation activates Eya during ectopic eye development. In contrast, our genomic rescue-based study found that the two MAPK target residues of Eya are not required for normal eye development or survival in DrosophilaDrosophila development. Surprisingly, we find that tyrosine phosphatase activity is not required for any known function of the Eya protein during normal Drosophila development.We recently reported a study that used a genomic DNA-based rescue system to evaluate the +GReya construct as described previously attP2 (68A) on the third chromosome attP2 was confirmed by genomic PCR with attP and attB primers eya transgenes were confirmed by genomic DNA PCR sequencing. Primer sequences are available on request.We used a two-step recombineering method to create the D493N (GAT->AAT) and E728Q (GAG->CAG) point mutations in the 1118w females or five males in each vial, respectively, and allowed to lay eggs for 48 hours before being discarded. Triplicate vials were set up for cli/CyO, Df/CyOeya and cli/Df; eya*GR/+eya. All resulting F1 progeny were counted during the first three days of eclosion.Flies were raised on standard media at 25°C. Fertility of rescued flies was tested as follows: five males or females of a given genotype were mated to 10 Tangential sections of three-day-old adult eyes were performed as described Larval imaginal discs . ERGs were performed as described previously Six three-day-old adults were assayed for each genotypeFor viability tests, goodness of fit was evaluated using the chi-square test. For fertility tests and scoring overshooting of axon bundles, data are presented as the mean ± standard deviation (s.d.). Statistical significance  of each data set was tested using ANOVA.eya genomic rescue constructs  fully rescues viability and eye formation in an eya mutant background, and is used as a positive control throughout our study. We generated three genomic rescue constructs encoding tyrosine phosphatase-dead Eya proteins. The residues Asp 493 and Glu 728 we chose to mutate have been reported previously to be required for Eya tyrosine phosphatase activity in vitroD493NGReya and E728QGReya, encode a protein with a single amino-acid substitution: D493N and E728Q, respectively. In the third construct, NQGReya, we engineered both D493N and E728Q mutations in +GReya. Hereafter D493NGReya, E728QGReya and NQGReya are collectively described as eya*GR.In this study, we utilized s eyaGR,  and siteDrosophila Eya leads to tyrosine phosphatase inactivation, a low frequency of ectopic eye induction, and incomplete genetic rescue using the Gal4-UAS system Drosophila E728Q, has severely decreased in vitro phosphatase activity D493NGReya and E728QGReya would either rescue eya mutants poorly or not at all. In order to investigate the effect of mutating both catalytic residues at once, we also generated the double-mutant genomic rescue construct NQGReya. Hereafter we only show data for NQGReya since all three eya*GR constructs behaved identically. We first tested the ability of eya*GR to rescue 2eya homozygous flies, which are viable and fertile but completely lack eyes due to a deletion of an enhancer required for eya expression during eye development +GReya or wild type flies  harbors a nonsense mutation and is embryonic lethal Df(2L)BSC354 (hereafter referred to as Df) deletes the entire eya gene and fails to complement clieyaeya*GR into the background of cli/Dfeya and found rescued adults are viable and fertile, both in males and females. Since Eya regulates muscle development cli/Df; eya*GR/+eya adults appear to move and fly normally, suggesting that they have no gross defects in muscle development (data not shown). To investigate the viability of rescued flies, we crossed cli/CyO; eya*GRw; eya males to w; Df/CyO virgin females. Progeny are present at Mendelian ratios , which marks the onset of differentiation in the eye disc. During the third instar larval stage, the MF progresses across the eye disc,with Eya expressed in a zone of undifferentiated cells anterior to the MF as well as in progressively differentiating cells posterior to the MF. In contrast, 2eya mutants have smaller eye discs due to massive apoptosis, loss of differentiation and reduced or no detectable Eya expression in vivo rescue results, we predicted the cli/Df; eya*GR/+eya larval eye discs would develop normally. As predicted, the level and pattern of Eya expression appear indistinguishable in the eye discs of positive control and rescued larvae , a physiological assay of retinal function. Three-day-old wild type, cordings , indicattauro-lacZ, which is driven by the R2–R5 specific rough enhancer and encodes a Tau-β-galactosidase fusion protein that marks R2–R5 photoreceptor axons eya*GR rescued animals have normal photoreceptor axon projections , in which the tyrosine phosphatase active site is located, is required for other functions during development. First, lethal missense and nonsense mutations within the ED of Drosophila Eya have been reported Drosophila ED binds to the transcription factor Sine oculis (So/Six), perhaps serving to recruit Eya to target genes, where it can act as a transcriptional co-activator within an Eya/So complex Drosophila, it appears that some other function of the ED has either been acquired or retained since the divergence of insects and vertebrates.As a dual-function protein, Eya has been best characterized as a transcriptional coactivator in vivoDrosophila survival as well as normal eye development . These results validate our genomic rescue system and indicate that, unlike the transactivation function, Eya tyrosine phosphatase activity is not necessary for Drosophila normal development.cDNA-based studies have also suggested that an internal proline-serine-threonine-rich (PST) domain in Eya is required for transcriptional activation in cell culture reporter assays and for efficient induction of ectopic eyes Gal4-UAS genetic rescue assays may be observed for several reasons. First, normal eye formation and reprogramming of non-retinal tissue into an ectopic eye involve overlapping but distinct genetic programs, as suggested by previous studies Gal4-UAS system does not recapitulate the wild-type pattern or timing of eya expression while our genomic rescue constructs are likely to contain the full complement of regulatory sequences needed, allowing the transgene to be expressed in the same spatiotemporal pattern as the endogenous gene of interest. Evidence supporting the genomic rescue strategy comes from our findings that, unlike ectopic eye formation in Drosophila, normal eye development and survival are unaffected by mutations in two previously studied MAPK target residues of Eya using genomic rescue constructs UAS-eya transgenes used in previous studies, we used site-specific integration, minimizing genomic position effects on different constructs. Previous studies have reported that the extent of rescue is indeed highly variable among four independent insertion lines of a D493NUAS-eya transgene D493Neya transgene fails to rescue 2eya using site-specific heat shock-mediated induction eya gene. This report highlights the importance of the genomic rescue system for evaluating the function of specific protein domains, motifs, and even single residues or base pairs in vivo.The differences between our findings based on a genomic rescue strategy and previous assays using ectopic eye induction and cDNA-based Drosophila development under standard laboratory conditions, it remains possible that Eya tyrosine phosphatase contributes to the animal’s response to environmental stressors. Previous studies reported that murine Eya1 and Eya3 can dephosphorylate H2AX, a histone variant associated with DNA damage, promoting efficient DNA repair Drosophila has an H2AX homologue, it will be interesting to determine if Drosophila Eya is also able to dephosphorylate H2AX. If this is the case, then our eya null mutants rescued with phosphatase-inactive eya*GR may be more sensitive to conditions that cause DNA damage.Although our studies suggest that Eya tyrosine phosphatase is dispensable for Drosophila. Whether the tyrosine phosphatase activity is required for mammalian Eya function in vivo remains to be tested. Our studies shed new light on a protein that plays a key role in a variety of developmental processes throughout the metazoans.To summarize, contrary to current models, the tyrosine phosphatase activity of Eya is dispensable for all known Eya functions in


Heliconius butterflies display an extraordinary diversity of wing patterns due in part to mimicry and sexual selection. Identifying the genetic drivers of this crucible of evolution is now within reach, as it was recently shown that cis-regulatory variation of the optix transcription factor explains red pattern differences in the adaptive radiations of the Heliconius melpomene and Heliconius erato species groups.While the ecological factors that drive phenotypic radiations are often well understood, less is known about the generative mechanisms that cause the emergence and subsequent diversification of novel features. Heliconius and Eueides genera. We also characterize novel Optix and Doublesex expression in the male-specific pheromone wing scales of the basal heliconiines Dryas and Agraulis, thus illustrating that within the Heliconinii lineage, Optix has been evolutionarily redeployed in multiple contexts in association with diverse wing features.Here, we compare the developmental expression of the Optix protein across a large phylogenetic sample of butterflies and infer that its color patterning role originated at the base of the neotropical passion-vine butterfly clade , shortly predating multiple Optix-driven wing pattern radiations in the speciose Our findings reveal that the repeated co-option of Optix into various aspects of wing scale specification was associated with multiple evolutionary novelties over a relatively short evolutionary time scale. In particular, the recruitment of Optix expression in colored scale cell precursors was a necessary condition to the explosive diversification of passion-vine butterfly wing patterns. The novel deployment of a gene followed by spatial modulation of its expression in a given cell type could be a common mode of developmental innovation for triggering phenotypic radiations. The corresponding DNA fragment was generated by gene synthesis  and cloned into the pQE-30 expression vector . A 6xHis-Optix fusion protein was expressed in bacterial cultures, extracted in 8 M Urea and purified using Ni2+ affinity column. An insoluble precipitate was recovered upon dialysis and approximately 1 mg was injected in a rat for immunization .The nucleotide sequence of the , those at an earlier stage of development) were immediately dissected. A portion of these then yielded pupal wing tissues at stages that were early enough for antibody permeability [see Additional fileButterfly pupae either originated from individuals collected in southern California, from laboratory colonies maintained at University of Texas - Austin, or from a butterfly farm in Costa Rica [see Additional filePupae were cold-anesthetized and dissected in PBS. Pupal forewings were incubated in fixative  for 30 min at room temperature while still attached to their cuticle. Similarly, and in order to maintain flat wing tissues, hindwings were kept attached to their pupal case and fixed for at least 5 min as such, detached by dissection, and then the remainder of the 30 min fixation was completed on free-floating wings. Fixed wings were washed five times with PBST  and stored at 4°C for up to two months. Wings were cleaned from remnants of peripodial membrane with fine forceps and incubated in blocking buffer for 2 hrs , incubated overnight at 4°C with anti-Optix polyclonal rat serum (dilution 1:3000), washed six times with PBST, incubated for 2 hrs at room temperature with an anti-rat IgG Alexa555 , and washed extensively in PBST. Nuclear counterstaining was performed with DAPI, and the wings were mounted in glycerol . F-actin counterstaining was performed using phalloidin conjugated to green fluorescent Oregon Green 488 dye . For immunostaining in eyes and optic lobes, whole heads were fixed in 4% paraformaldehyde, sucrose protected, and frozen for sectioning. Horizontal sections of 14-μm thickness were made using a cryostat and used for immunohistofluorescence using a previously described procedure. A rabbiStained tissues were imaged with a Zeiss Axioplan 2 epifluorescence microscope and with a Zeiss LSM 700 confocal microscope. Tiled images were assembled using either the Photomerge function of Adobe Photoshop CS3 (epifluorescence imaging), or were acquired using the Tiled Scan function in the Zeiss ZEN software . Images were submitted to a linear contrast adjustment to eliminate background illumination.Other methods are described in the Additional fileHeliconius Optix protein. The crude serum immunodetected a band of the expected size (30 kDa) in a western blot , suggesting that the polyclonal serum targets epitopes that are Lepidoptera-specific and is also unlikely to detect other lepidopteran SIX family transcription factors. Wing veins and scale sockets produced signals in several negative controls [see Additional fileA polyclonal antibody serum was generated in a rat immunized with the  Figure a, and im Figure b-c. No sH. erato petiverana differs on its proximal boundary by up to six scale rows between the ventral and dorsal sides. This difference is reflected by corresponding shifts of Optix immunolocalization  in what we term the ‘proximal complex’, consisting of the red costal forewing bars and of basal hindwing red spots, both situated on ventral wing surfaces  arguing that Optix-positive patterns are a relatively recent novelty that seemingly appeared by co-option of Optix expression in color scale cell precursors, and (b) disentangling different genetic modes of pattern diversification that followed the original co-option event.The evolutionary origin of red wing patterns in terflies. Here, wDryas iulia) or red (Heliconius spp.) pigmentation across Heliconiini. We propose that each of these three Optix-associated scale phenotypes forms a distinct evolutionary character due to the divergent scale morphologies they underlie. Importantly, the recruitment of Optix expression into colored scale cell precursors of the ‘proximal complex’ is a synapomorphy of neotropical passion-vine butterflies (Heliconiini) and was detected in the basal genus Dryas, thus marking an innovation event associated with the Heliconiini radiation  in Heliconius.1. cis-regulatory evolution of ra clade, two allmelpomene, and two species. It is p modules. Followi modules, and thaoptix locus tends to explain presence/absence polymorphism of a given pattern element , pattern shape variation can sometimes be explained by epistatic effects with unlinked loci. Several examples have been characterized at various levels. For instance, the early expression of WntA during larval development determines the shape of the red forewing band[N spatially regulates Optix pattern shapes in H. melpomene.2. While the ing band,64. We aoptix alleles have been shown to introgress across geographic barriers and to create taxonomic mosaics of pattern diversity, where a given pattern of single origin is scattered across the lineage phylogeny[virtually all departures from the common pattern themes within the cydno clade  can be explained by introgression of toolbox elements from H. melpomene. Optix appears to be a core component of this toolbox, and recent genetic data has since confirmed that cross-species introgressions of optix alleles drive multiple examples of mimetic convergence in the ‘melpomene-cydno-silvaniform’ clade[H. heurippa[Heliconius optix alleles emerge as a model system of choice to study this phenomenon[3. Adaptive hylogeny. More suhylogeny. Directlm’ clade,30,32,65 heurippa,65. Intrenomenon.optix and affect its localization, modifier loci acting downstream of optix may modulate its color output. A likely example occurs in H. melpomene plesseni, where an unidentified mechanism converts optix-positive scales into a highly-UV reflective scale field of white appearance[plesseni origin is visible in an artificial ‘Mixed white’ hybrid population, where an unbroken forewing patch of white and red scales expresses optix  introgressions of optix variants maximize the phylogenetic distribution of pre-existing patterns, sometimes resulting in novel, hybrid patterns due to epistatic effects; and 3) unidentified modifier genes can modulate the color output of Optix-positive scale cell precursors. Similar principles may apply to Optix-independent patterns.In summary, 1) cis-regulatory evolution of optix locus drives morphological radiations in the wings of H. melpomene and H. erato[Heliconius and Eueides. Multiple recruitments of Optix expression into the differentiation of derived wing scales - the red color scale cells, as well as the male-specific vein structures of basal heliconiines - provide compelling examples of developmental innovation, and the genetic co-option event associated to coloration was followed by a rapid radiation in wing patterns. By opening paths to unexplored valleys of the morphospace, gene co-option events may be a general mechanism explaining sudden bursts of phenotypic change.Multiple lines of evidence suggest that the regulatory evolution of the  H. erato,18,30,45 H. erato,36. In tThe authors declare that they have no competing interests.Heliconius pupae and carried out the spectrophotometer measurements. LEG performed the hybrid Heliconius crosses and contributed live Heliconius pupae. RDR supervised the project and helped to draft the manuscript. All authors read and approved the final manuscript.AM carried out wing development work, analyzed the data and drafted the manuscript. KJM carried out eye and brain immunostainings. NHP supervised antibody production. ADB provided live Martin_et_al-Supplementary.pdf. Supplementary Materials including Supplementary Methods, Supplementary Table, Supplementary Figures Click here for file


Our understanding of the structure and function of kinetochores has advanced dramatically over the past 10 years, yet how the plus end of spindle microtubules interacts with the kinetochore and establishes amphitelic attachment for proper sister chromatid segregation remains unresolved. However, several recent reports from different organisms have shed new light on this issue. A key player in microtubule-kinetochore interaction is the conserved Ndc80 outer kinetochore complex. In both yeast and human cells in particular, a ubiquitous internal ‘loop’ found in the Ndc80 molecule interrupting its C-terminal coiled-coil domain plays critical roles in protein-protein interaction, by recruiting microtubule-binding proteins to ensure proper kinetochore-microtubule attachment. In this commentary, we summarise the recent progress made and discuss the evolutionary significance of this loop’s role in microtubule dynamics at the kinetochore for accurate chromosome segregation. During mitosis, the kinetochore complex attaches to the mitotic spindle to ensure accurate chromosome segregation-3. The NStructural studies have revealed that the Ndc80 complex has a dumbbell-like architecture consisting of an internal rod-shaped coiled-coil structure and a globular domain on each end,5. In adndc80 mutants that contain mutations within the loop in Saccharomyces cerevisiae, the Tanaka lab showed that this region facilitates binding of the Dam1 kinetochore complex to the Ndc80 complex , the Dis1 protein delocalises from the kinetochore, resulting in an unstable mitotic spindle and early mitotic arrest with no stable kinetochore-microtubule attachment being observed in this mutant[ndc80 loop mutant with a different mutation site in the loop (F420S), that showed normal Dis1 protein localisation to the kinetochore and normal mitotic spindle structure. In this mutant however, the Alp7-Alp14 complex was specifically delocalised from the kinetochore, leading to chromosome missegregation during anaphase A[Our recent studies in s mutant. Interesaphase A. We specin vitro.Microtubules are intrinsically dynamic and bind to a cohort of MAPs. In particular, the mitotic spindle changes its polymerising/depolymerising activity throughout the different stages of mitosis. This leads us to suggest that the Ndc80 internal loop, by interacting with different proteins during different stages, helps to regulate microtubule dynamics. It would be interesting to test this idea by reconstituting each protein complex in the presence of microtubules To date, all the Ndc80 loop-interacting proteins identified have no reported microtubule depolymerising activity. Although the Dam1 and Ska complexes have been shown to track on the plus end of depolymerising microtubules and the TOG proteins are in fact microtubule polymerases, it remains enigmatic as to how microtubule depolymerisation per se is directionally driven during anaphase A,17. It sAlthough the proteins bound to the Ndc80 loop in different organisms show no sequence homology to one another, these studies showed the importance and capabilities of the Ndc80 internal loop region as a protein-protein interaction motif, thereby ensuring proper chromosome segregation, and that mitotic progression is conserved from yeasts to higher eukaryotes. Furthermore, these studies provided further insight into how the kinetochore, in particular the Ndc80 complex, regulates microtubule dynamics during mitosis.MAP: Microtubule-associated protein; TACC: Transforming acidic coiled coil; +TIP: Plus-end tracking protein; TOG: Tumor overexpressed geneThe authors declare that they have no competing interests.NHT and TT jointly wrote the paper. Both authors read and approved the final manuscript.