Post-transcriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell and tissue-dependent.

The T-box proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. It is highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report post-transcriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla Luciferase reporter significantly reduced the activity of the Luciferase. Whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid mRNA between the two transgenes, with and without the 3'UTR, indicating the absence of post-transcriptional regulation of mid in MP2. Moreover, while elevated mid RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Over-expression of the two transgenes resulted in MP2-lineage defects about the same frequency. These results indicate a translational/post-translational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid RNA and the protein and had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the two transgenes.

Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity.

Proper muscle development and function depend on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/regulation via posttranslational modifications, protein-protein interactions, or some other unknown mechanism.

Restriction on self-renewing asymmetric division is coupled to terminal asymmetric division in the Drosophila CNS.

Neuronal precursor cells undergo self-renewing and non-self-renewing asymmetric divisions to generate a large number of neurons of distinct identities. In Drosophila, primary precursor neuroblasts undergo a varying number of self-renewing asymmetric divisions, with one known exception, the MP2 lineage, which undergoes just one terminal asymmetric division similar to the secondary precursor cells. The mechanism and the genes that regulate the transition from self-renewing to non-self-renewing asymmetric division or the number of times a precursor divides is unknown. Here, we show that the T-box transcription factor, Midline (Mid), couples these events. We find that in mid loss of function mutants, MP2 undergoes additional self-renewing asymmetric divisions, the identity of progeny neurons generated dependent upon Numb localization in the parent MP2. MP2 expresses Mid transiently and an over-expression of mid in MP2 can block its division. The mechanism which directs the self-renewing asymmetric division of MP2 in mid involves an upregulation of Cyclin E. Our results indicate that Mid inhibits cyclin E gene expression by binding to a variant Mid-binding site in the cyclin E promoter and represses its expression without entirely abolishing it. Consistent with this, over-expression of cyclin E in MP2 causes its multiple self-renewing asymmetric division. These results reveal a Mid-regulated pathway that restricts the self-renewing asymmetric division potential of cells via inhibiting cyclin E and facilitating their exit from cell cycle.

Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B.

UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. It has previously been demonstrated that the UCS domain is responsible for the chaperone-like properties of the UNC-45B. To better understand the chaperoning function of the UCS domain of the UNC-45B chaperone, we engineered mutations designed to 1) disrupt chaperone-client interactions by removing and altering the structure of a putative client-interacting loop and 2) disrupt chaperone-client interactions by changing highly conserved residues in a putative client-binding groove. We tested the effect of these mutations by using a, to our knowledge, novel combination of complementary biophysical assays (circular dichroism, chaperone activity, and small-angle x-ray scattering) and in vivo tools (Caenorhabditis elegans sarcomere structure). Removing the putative client-binding loop altered the secondary structure of the UCS domain (by decreasing the α-helix content), leading to a significant change in its solution conformation and a reduced chaperoning function. Additionally, we found that mutating several conserved residues in the putative client-binding groove did not alter the UCS domain secondary structure or structural stability but reduced its chaperoning activity. In vivo, these groove mutations were found to significantly alter the structure and organization of C. elegans sarcomeres. Furthermore, we tested the effect of R805W, a mutation distant from the putative client-binding region, which in humans, has been known to cause congenital and infantile cataracts. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic detrimental effect on the structure and organization of the worm sarcomeres, indicating a crucial role of R805 in UCS-client interactions. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin-chaperone interactions in mechanistic detail.

The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury.

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening.

The tyrosine kinase MET, a receptor for hepatocyte growth factor, is a key regulator for normal development and organ renewal via stem cell maintenance. Dysregulated MET signaling contributes to tumor progression and metastasis and is considered a potent therapeutic target for a growing number of malignancies. Toward that goal it is critical to develop high-throughput assays to identify candidate regulators for the termination of MET signaling. We describe here a rapid and efficient method for identifying cellular factors required for MET ubiquitination, which utilizes high-throughput RNA interference screening (HT-siRNA) with a receptor internalization assay and an In-Cell ELISA in a 96-well format. The assay is amenable to a large array of cell surface proteins as well as genome-wide siRNA libraries, with high signal-to-background ratio and low well-to-well variability.

The midline protein regulates axon guidance by blocking the reiteration of neuroblast rows within the Drosophila ventral nerve cord.

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.

Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer.

At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.

Neuralized mediates asymmetric division of neural precursors by two distinct and sequential events: promoting asymmetric localization of Numb and enhancing activation of Notch-signaling.

In the CNS, the evolutionarily conserved Notch pathway regulates asymmetric cell fate specification to daughters of ganglion mother cells (GMCs). The E3 Ubiquitin ligase protein Neuralized (Neur) is thought to activate Notch-signaling by the endocytosis of Delta and the Delta-bound extracellular domain of Notch. The intracellular Notch then initiates Notch-signaling. Numb blocks N-signaling in one of the two daughters of a GMC, allowing that cell to adopt a different identity. Numb is asymmetrically localized in a GMC and is segregated to only one of the two daughter cells. In the typical GMC-1→RP2/sib lineage, we found that loss of Neur activity causes symmetric division of GMC-1 into two RP2s. We further found that Neur asymmetrically localizes in a late GMC-1 to the Numb domain and Neur mediates asymmetric division via two distinct, sequential mechanisms: by promoting the asymmetric localization of Numb in a GMC-1 via down-regulation of the transcription factor Pdm1, followed by enhancing the Notch-signaling via trans-potentiation of Notch in a cell committed to become a sib. In neur mutants the GMC-1 identity is not altered but Numb is non-asymmetrically localized due to an up-regulation of Pdm1. Thus, both its daughters inherit Numb, which prevents Notch from specifying a sib identity. Neur also enhances Notch since in neur; numb double mutants, both sibling cells often adopt a mixed fate as opposed to an RP2 fate observed in Notch; numb double mutants. Furthermore, over-expression of Neur can induce both cells to adopt a sib fate similar to gain of function Notch. Our results tie Numb and Notch-signaling through a single player, Neur, thus giving us a more complete picture of the events surrounding asymmetric division of precursor cells. We also show that Neur and Numb are interdependent for their asymmetric-localizations.

Ancestry-independent fate specification and plasticity in the developmental timing of a typical Drosophila neuronal lineage.

In the Drosophila CNS, combinatorial, interdependent, sequential genetic programs in neuroectodermal (NE) cells, prior to the formation of neuroblasts (NBs), determine the initial identity of NBs. Temporal factors are then sequentially expressed to change the temporal identity. It is unclear at what levels this positional and temporal information integrates to determine progeny cell identity. One idea is that this is a top-down linear process: the identity of a NB determines the identity of its daughter, the ganglion mother cell (GMC), the asymmetric division of the GMC and the fate specification of daughter cells of the GMC. Our results with midline (mid), which encodes a T-box protein, in a typical lineage, NB4-2-->GMC-1-->RP2/sib, suggest that at least part of the process operates in GMCs. That is, a GMC or a neuronal identity need not be determined at the NB or NE level. This is demonstrated by showing that Mid is expressed in a row 5 GMC (M-GMC), but not in its parent NB or NE cell. In mid mutants, M-GMC changes into GMC-1 and generates an RP2 and a sib without affecting the expression of key genes at the NE/NB levels. Expression of Mid in the M-GMC in mid mutants rescues the fate change, indicating that Mid specifies neurons at the GMC level. Moreover, we found a significant plasticity in the temporal window in which a neuronal lineage can develop. Although the extra GMC-1 in mid mutants is born approximately 2 hours later than the bona fide GMC-1, it follows the same developmental pattern as the bona fide GMC-1. Thus, a GMC identity can be independent of parental identity and GMC formation and elaboration need not be strictly time-bound.

Regulation of axon guidance by slit and netrin signaling in the Drosophila ventral nerve cord.

Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.

Generating asymmetry: with and without self-renewal.

At some point during the history of organismal evolution, unicellular, unipotent and mitotically active cells acquired an ability to undergo a special type of cell division called asymmetric division. By this special type of cell division, these cells could divide to generate two different progeny or to self-renew and at the same time generate a progeny that is committed to become a cell different from the mother cell. This type of cell division, which forms the basis for the functioning of totipotent or multipotent stem cells, underlies the fundamental basis for the developmental evolution of organisms. It is not clear if the asymmetric division without self-renewal preceded the asymmetric division with self-renewal. It is reasonable to assume that the asymmetric division without self-renewal preceded the asymmetric division with self-renewal. In this review we explore the genetic regulation of these two types of asymmetric divisions using the Drosophila central nervous system (CNS) as a model system. The results from recent studies argue that for cells to undergo a self-renewing asymmetric division, certain "stem cell" proteins must be maintained or up-regulated, while genes encoding proteins responsible for differentiation must be repressed or down-regulated. As long as a balance between these two classes of proteins is maintained via asymmetric segregation and activation/repression, the progeny that receives stem cell proteins/maintains stem cell competence will have the potential to undergo self-renewing asymmetric division. The other progeny will commit to differentiate. In non-self-renewing asymmetric division, down-regulation of stem cell proteins/competence combined with asymmetric segregation of cell identity specifying factors (either cell-autonomous or a combination of cell autonomous and non-cell autonomous signals) cause the two progeny to assume different differentiated identities. Identification of mutations that confer a stem cell type of division to nonstem cell precursors, or mutations that eliminate asymmetric division, has led the way in elucidating the molecular basis for these divisions. Given that there is a considerable degree of conservation of genes and their function, these studies should provide clear insight into how the self-renewing asymmetric division of stem cells in neural and other lineages is regulated not only in Drosophila but also in vertebrates including humans.

Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila.

Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. We show that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. We conclude that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress.

Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis.

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless(1) (ecd(1)) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd(1) conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd(-) clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.

Mitochondrial and nuclear localization of topoisomerase II in the flagellate Bodo saltans (Kinetoplastida), a species with non-catenated kinetoplast DNA.

We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.