MicroRNA-181a regulates local immune balance by inhibiting proliferation and immunosuppressive properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) exhibit extensive self-renewal potential and can modulate immunocyte activation. Our previous study reported that miR-181a expression was significantly increased in placenta from women with severe preeclampsia (PE), but the mechanisms by which miR-181a regulates MSCs are unknown. In this study, we asked if and how miR-181a regulates MSCs' proliferation and immunosuppressive properties. We found that the expression of miR-181a in the MSCs derived from the umbilical cord and decidua of PE patients increased relative to MSCs derived from normal patients. Transfection with miR-181a oligos prevented MSCs proliferation but did not affect MSCs apoptosis. Overexpression of miR-181a blocked activation of the TGF-ß signaling pathway and caused downregulation of target gene (TGFBR1 and TGFBRAP1) mRNA and protein expression. Reporter genes with putative miR-181a binding sites from the TGFBR1 and TGFBRAP1 3'-untranslated regions (3'-UTRs) were downregulated in the presence of miR-181a, suggesting that miR-181a binds to TGFBR1 and TGFBRAP1 3'-UTRs. In contrast, transfection of MSCs with miR-181a oligo enhanced expression of IL-6 and indoleamine 2,3-dioxygenase by activating p38 and JNK signaling pathways, respectively. MSCs transfected with miR-181a also enhanced the proliferation of T cells in a short-term culture. Additionally, treatment with control MSCs, but not miR-181a transfected MSCs, improved dextran sulfate sodium-induced experimental colitis, suggesting that miR-181a attenuates the immunosuppressive properties of MSCs in vivo. Together, our data demonstrate that miR-181a is an important endogenous regulator in the proliferation and immunosuppressive properties of MSCs.

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish.

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

The N- or C-terminal domains of DSH-2 can activate the C. elegans Wnt/beta-catenin asymmetry pathway.

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a beta-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/beta-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/beta-catenin, WRM-1/beta-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/beta-catenin asymmetry pathway and regulating anterior-posterior fate decisions required for proper morphogenesis.

gon-14 functions with class B and class C synthetic multivulva genes to control larval growth in Caenorhabditis elegans.

Previous work showed that C. elegans gon-14 is required for gonadogenesis. Here we report that gon-14 encodes a protein with similarity to LIN-15B, a class B synMuv protein. An extensive region of GON-14 contains blocks of sequence similarity to transposases of the hAT superfamily, but key residues are not conserved, suggesting a distant relationship. GON-14 also contains a putative THAP DNA-binding domain. A rescuing gon-14::GON-14::VENUS reporter is broadly expressed during development and localizes to the nucleus. Strong loss-of-function and predicted null gon-14 alleles have pleiotropic defects, including multivulval (Muv) defects and temperature-sensitive larval arrest. Although the gon-14 Muv defect is not enhanced by synMuv mutations, gon-14 interacts genetically with class B and class C synMuv genes, including lin-35/Rb, let-418/Mi-2beta, and trr-1/TRRAP. The gon-14; synMuv double mutants arrest as larvae when grown under conditions supporting development to adulthood for the respective single mutants. The gon-14 larval arrest is suppressed by loss of mes-2/E(Z), mes-6/ESC, or mes-4, which encodes a SET domain protein. Additionally, gon-14 affects expression of pgl-1 and lag-2, two genes regulated by the synMuv genes. We suggest that gon-14 functions with class B and class C synMuv genes to promote larval growth, in part by antagonizing MES-2,3,6/ESC-E(z) and MES-4.

The sys-1 and sys-3 genes cooperate with Wnt signaling to establish the proximal-distal axis of the Caenorhabditis elegans gonad.

To form the proximal-distal axis of the C. elegans gonad, two somatic gonadal precursor cells, Z1 and Z4, divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. Genes governing this process include the lin-17 frizzled receptor, wrm-1/beta-catenin, the pop-1/TCF transcription factor, lit-1/nemo-like kinase, and the sys-1 gene. Normally, all of these regulators promote the distal fate. Here we show that nuclear levels of a pop-1 GFP fusion protein are less abundant in the distal than in the proximal Z1/Z4 daughters. This POP-1 asymmetry is lost in mutants disrupting Wnt/MAPK regulation, but retained in sys-1 mutants. We find that sys-1 is haplo-insufficient for gonadogenesis defects and that sys-1 and pop-1 mutants display a strong genetic interaction in double heterozygotes. Therefore, sys-1 is a dose-sensitive locus and may function together with pop-1 to control Z1/Z4 asymmetry. To identify other regulatory genes in this process, we screened for mutants resembling sys-1. Four such genes were identified (gon-14, -15, -16, and sys-3) and shown to interact genetically with sys-1. However, only sys-3 promotes the distal fate at the expense of the proximal fate. We suggest that sys-3 is a new key gene in this pathway and that gon-14, gon-15, and gon-16 may cooperate with POP-1 and SYS-1 at multiple stages of gonad development.

amontillado, the Drosophila homolog of the prohormone processing protease PC2, is required during embryogenesis and early larval development.

Biosynthesis of most peptide hormones and neuropeptides requires proteolytic excision of the active peptide from inactive proprotein precursors, an activity carried out by subtilisin-like proprotein convertases (SPCs) in constitutive or regulated secretory pathways. The Drosophila amontillado (amon) gene encodes a homolog of the mammalian PC2 protein, an SPC that functions in the regulated secretory pathway in neuroendocrine tissues. We have identified amon mutants by isolating ethylmethanesulfonate (EMS)-induced lethal and visible mutations that define two complementation groups in the amon interval at 97D1 of the third chromosome. DNA sequencing identified the amon complementation group and the DNA sequence change for each of the nine amon alleles isolated. amon mutants display partial embryonic lethality, are defective in larval growth, and arrest during the first to second instar larval molt. Mutant larvae can be rescued by heat-shock-induced expression of the amon protein. Rescued larvae arrest at the subsequent larval molt, suggesting that amon is also required for the second to third instar larval molt. Our data indicate that the amon proprotein convertase is required during embryogenesis and larval development in Drosophila and support the hypothesis that AMON acts to proteolytically process peptide hormones that regulate hatching, larval growth, and larval ecdysis.

The C. elegans Chp/Wrch Ortholog CHW-1 Contributes to LIN-18/Ryk and LIN-17/Frizzled Signaling in Cell Polarity.

Wnt signaling controls various aspects of developmental and cell biology, as well as contributing to certain cancers. Expression of the human Rho family small GTPase Wrch/RhoU is regulated by Wnt signaling, and Wrch and its paralog Chp/RhoV are both implicated in oncogenic transformation and regulation of cytoskeletal dynamics. We performed developmental genetic analysis of the single Caenorhabditis elegans ortholog of Chp and Wrch, CHW-1. Using a transgenic assay of the distal tip cell migration, we found that wild-type CHW-1 is likely to be partially constitutively active and that we can alter ectopic CHW-1-dependent migration phenotypes with mutations predicted to increase or decrease intrinsic GTP hydrolysis rate. The vulval P7.p polarity decision balances multiple antagonistic Wnt signals, and also uses different types of Wnt signaling. Previously described cooperative Wnt receptors LIN-17/Frizzled and LIN-18/Ryk orient P7.p posteriorly, with LIN-17/Fz contributing approximately two-thirds of polarizing activity. CHW-1 deletion appears to equalize the contributions of these two receptors. We hypothesize that CHW-1 increases LIN-17/Fz activity at the expense of LIN-18/Ryk, thus making the contribution of these signals unequal. For P7.p to polarize correctly and form a proper vulva, LIN-17/Fz and LIN-18/Ryk antagonize other Wnt transmembrane systems VANG-1/VanGogh and CAM-1/Ror. Our genetic data suggest that LIN-17/Fz represses both VANG-1/VanGogh and CAM-1/Ror, while LIN-18/Ryk represses only VANG-1. These data expand our knowledge of a sophisticated signaling network to control P7.p polarity, and suggests that CHW-1 can alter ligand gradients or receptor priorities in the system.

Ras-related small GTPases RalA and RalB regulate cellular survival after ionizing radiation.

PURPOSE: Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases RalA and RalB are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole-body irradiation, but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR. METHODS AND MATERIALS: RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expression of short hairpin RNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, gammaH2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (PARP) cleavage for apoptosis. RESULTS: Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR, and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR. CONCLUSIONS: Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.

Fgfs control homeostatic regeneration in adult zebrafish fins.

Adult teleost fish and urodele amphibians possess a spectacular ability to regenerate amputated appendages, based on formation and maintenance of progenitor tissue called a blastema. Although injury-induced, or facultative, appendage regeneration has been studied extensively, the extent to which homeostatic regeneration maintains these structures has not been examined. Here, we found that transgenic inhibition of Fgf receptors in uninjured zebrafish caused severe atrophy of all fin types within 2 months, revealing a requirement for Fgfs to preserve dermal bone, joint structures and supporting tissues. Appendage maintenance involved low-level expression of markers of blastema-based regeneration, focused in distal structures displaying recurrent cell death and proliferation. Conditional mutations in the ligand Fgf20a and the kinase Mps1, factors crucial for regeneration of amputated fins, also caused rapid, progressive loss of fin structures in otherwise uninjured animals. Our experiments reveal that the facultative machinery that regenerates amputated teleost fins also has a surprisingly vigorous role in homeostatic regeneration.

Reciprocal asymmetry of SYS-1/beta-catenin and POP-1/TCF controls asymmetric divisions in Caenorhabditis elegans.

beta-Catenins are conserved regulators of metazoan development that function with TCF DNA-binding proteins to activate transcription. In Caenorhabditis elegans, SYS-1/beta-catenin and POP-1/TCF regulate several asymmetric divisions, including that of the somatic gonadal precursor cell (SGP). In the distal but not the proximal SGP daughter, SYS-1/beta-catenin and POP-1/TCF transcriptionally activate ceh-22 to specify the distal fate. Here, we investigate the distribution of SYS-1/beta-catenin and its regulation. Using a rescuing transgene, VNS::SYS-1, which fuses VENUS fluorescent protein to SYS-1, we find more VNS::SYS-1 in distal than proximal SGP daughters, a phenomenon we call "SYS-1 asymmetry." In addition, SYS-1 asymmetry is seen in many other tissues, consistent with the idea that SYS-1 regulates asymmetric divisions broadly during C. elegans development. In particular, SYS-1 is more abundant in E than MS, and SYS-1 is critical for the endodermal fate. In all cases, SYS-1 is reciprocal to POP-1 asymmetry: cells with higher SYS-1 have lower POP-1, and vice versa. SYS-1 asymmetry is controlled posttranslationally and relies on frizzled and dishevelled homologs, which also control POP-1 asymmetry. Therefore, upstream regulators modulate the SYS-1 to POP-1 ratio by increasing SYS-1 and decreasing POP-1 within the same cell. By contrast, SYS-1 asymmetry does not rely on WRM-1, which appears specialized for POP-1 asymmetry. We suggest a two-pronged pathway for control of SYS-1:POP-1, which can robustly accomplish differential gene expression in daughters of an asymmetric cell division.

A beta-catenin identified by functional rather than sequence criteria and its role in Wnt/MAPK signaling.

Wnt/MAPK signaling is a common variant of Wnt signaling in C. elegans and has been implicated in vertebrates. The sys-1 gene works with Wnt/MAPK signaling to control cell fates during C. elegans development. We report that the SYS-1 amino acid sequence is novel but that SYS-1 functions as beta-catenin: SYS-1 rescues a bar-1/beta-catenin null mutant, binds the POP-1/TCF beta-catenin binding domain, and coactivates POP-1-dependent transcription. Moreover, we provide genetic and molecular evidence that SYS-1 levels are crucial to POP-1 activity. Our results suggest that Wnt/MAPK signaling promotes POP-1 export from the nucleus to accommodate the limiting availability of its SYS-1/beta-catenin transcriptional coactivator. Discovery of SYS-1/beta-catenin extends our definition of beta-catenins and brings together aspects of the canonical mechanism for Wnt signaling with the noncanonical Wnt/MAPK mechanism. We discuss the idea that a similar pathway may be employed broadly in animal development.