Mir, A.B. et al. (2017) International Worm Meeting "Regulation of APL-1, the C. elegans ortholog of the Alzheimer's Disease associated APP protein."

Mir, A.B., Li, C., Chait, A.

[International Worm Meeting, 2017]

The neurodegenerative disorder Alzheimer's disease (AD) can be characterized by the presence of amyloid plaques in the brain. These plaques consist chiefly of the beta -amyloid peptide, a cleavage product of the poorly understood amyloid precursor protein (APP). The study of APP in mammalian systems is complicated by the presence of three separate genes encoding functionally overlapping proteins: APP, APLP1 and APLP2. We are using C. elegans, which has only a single APP-related gene apl-1, to examine the role of APP-related proteins. APL-1 is a single pass transmembrane protein; null alleles of apl-1 are lethal. This lethality can be rescued by expression of the APL-1 extracellular domain. Similarly, mutants in which the intracellular and transmembrane domains of APL-1 are deleted (apl-1(yn5)) are viable, demonstrating that the extracellular domain of APL-1 is necessary and sufficient for viability. In mammalian models, the extracellular domain of APP is released by the cleavage activity of the a-secretase ADAM complex. We investigated the cleavage of APL-1 in C. elegans by examining the ADAM mutants adm-4 and sup-17. apl-1(yn5) mutants show a general developmental delay as well as a limited larval lethality at 25 deg C. This temperature sensitive lethality is greatly enhanced by knockdown of the IGF/insulin receptor ortholog daf-2; the double mutants show L1 arrest. In a forward screen to isolate suppressors of this L1 arrest, we isolated six suppressor mutants. To further understand how apl-1 is regulated, we are comparing the promoter regions of Caenorhabditis strains to identify conserved regions. We have generated constructs that delete different regions and will assay whether they are able to rescue the apl-1 null phenotype.

Mir, Alexander et al. (2015) International Worm Meeting "Identifying the proteolytic and functional pathways of APL-1, the C. elegans homolog to human APP."

Li, Chris, Chait, Alexander, Mir, Alexander

[International Worm Meeting, 2015]

The progressive, neurodegenerative disorder Alzheimer's disease (AD) is characterized by deposition of amyloid plaques in the brain. These plaques are composed mainly of aggregated b-amyloid peptide, a cleavage product of the poorly understood amyloid precursor protein (APP). The study of APP in mammalian systems is complicated by the presence of three genes encoding functionally redundant proteins: APP, APLP1 and APLP2. By contrast, C. elegans has only a single APP-related gene apl-1.Like APP, APL-1 is a single pass transmembrane protein. apl-1(yn10) is a null allele that results in lethality. This lethality can be rescued by microinjection of the APL-1 extracellular domain, indicating that that the extracellular domain is sufficient for viability. The extracellular domain of mammalian APP is released during cleavage by ADAM10 as part of the a-secretase complex. To investigate the cleavage of APL-1, we examined the two ADAM10 homologues present in the C. elegans genome: ADM-4 and SUP-17. Knockdown of adm-4 decreased cleavage of APL-1, whereas knockdown of sup-17 did not. We conclude that ADM-4 is the major protease cleaving the extracellular domain of APL-1.The apl-1(yn5) allele is a deletion mutation which removes the coding region for the cytoplasmic and transmembrane segments of the APL-1 protein as well as much of the 3' UTR. apl-1(yn5) mutants are viable, confirming the apl-1(yn10) rescue experiments that the extracellular domain of APL-1 is sufficient for survival. These mutants show a delayed development that is greatly enhanced by the knockdown of the insulin/IGF-1 receptor ortholog daf-2. To investigate this interaction, we generated a daf-2(e1368);apl-1(yn5) double mutant that experiences a highly penetrant L1 arrest at 25degC. We are performing a forward mutagenic screen to identify mutations rescuing the apl-1(yn5);daf-2(e1368) phenotype to identify novel agents in the APL-1 pathway. In a preliminary screen of 3,000 haploid genomes, we have isolated three suppressor mutants that revert the L1 arrest. We are currently mapping these suppressors and will identify their molecular lesions.

Gabaldon, Carolaing et al. (2017) International Worm Meeting "mir-243, mir-51 y mir-52 are required for diapause formation in C. elegans as a transgenerational defense mechanism against bacterial pathogens."

Verdugo, Lidia, Calixto, Andrea, Martin, Alberto.J.M, Caris, Carlos, Gabaldon, Carolaing

[International Worm Meeting, 2017]

C. elegans feeds on a variety of bacteria, both pathogenic and non-pathogenic, and under starvation, high population density, and other stresses enters diapause by forming the dauer larvae. Our previous work showed that C. elegans, also forms dauers as a mechanism of transgenerational defense against the pathogens P. aeruginosa PAO1 and S. Typhimurium MST1. We proposed that in the bacteria-worm communication, bacterial dsRNAs trigger an RNAi-dependent process, generating endo-siRNA accumulation in the germ line subsequently transmitted to the progeny to promote diapause formation in C. elegans. To understand which genes (small RNAs and mRNA) accumulate in response to pathogens, we performed a comparative differential expression analysis of the worm transcriptome under pathogenesis and on non-pathogenic bacteria. We found that mir-243, mir-51 and mir-52 are overexpressed by 6.46, 2.27 and 2.26 fold respectively, in response to P. aeruginosa PAO1. In vivo gfp expression confirms the overexpression of the three miRNAs in worms fed with pathogens compared to non-pathogenic bacteria. Importantly, animals with mutations in mir-243, mir-51 and mir-52 do not form dauers in response to P. aeruginosa PAO1, strongly suggesting a role for these small RNAs in the behavioral response to pathogenesis. To construct the gene networks underlying dauer formation under pathogenesis, we used the miRNA-mRNA interactions predicted by five softwares simultaneously (INTA, RNA duplex, RNAplex, RNAup and PITA), and overlapped them with an additional layer formed by the transcription factors, known to regulate the miRNAs that appeared differentially expressed in our experiments.

Burke, Samantha et al. (2013) International Worm Meeting "Mir-34 and mir-83 protect C. elegans gonad morphogenesis against temperature fluctuations."

Hammell, Molly, Ambros, Victor, Burke, Samantha

[International Worm Meeting, 2013]

Mir-34 is a well conserved microRNA that has been implicated in senescence and apoptosis in mammals. We examined C. elegans mir-34 mutants for developmental phenotypes and determined that the mutants display incompletely penetrant gonad migration defects, specifically wandering gonad arms. The incomplete penetrance of this phenotype suggested co-regulation by additional microRNAs, therefore we tested for genetic enhancement of the mir-34 phenotype by the loss of other microRNAs sharing predicted targets. Deletion of one particular microRNA, mir-83, the ortholog of mammalian mir-29, causes a similar migration defect. Mir-34; mir-83 double mutants exhibit the migration defect with increased penetrance, suggesting that mir-34 and mir-83 function redundantly in the regulation of targets involved in gonad migration during larval development. The phenotype is further enhanced when worms are subjected to temperature fluctuations - for example, four cycles between 15 deg C and 25 deg C over the course of two hours. The sensitive period for temperature fluctuations occurs in the L1 stage concurrent with the birth of the distal tip cells, before the cells begin their migration. This enhancement is independent of both the magnitude and initial direction of temperature change. Epistatic analysis suggests that mir-34 and mir-83 help regulate the activity of the integrin and GTPase signaling network responsible for distal tip cell migration by targeting both pat-3 and cdc-42. Knockdown of either pat-3 or cdc-42 suppresses the migration defect seen in mir-34; mir-83 worms suggesting that the error-prone gonadal migration in mir-34; mir-83 double-mutant animals results at least in part from elevated integrin and GTPase signaling.

Lorenzo F Sempere et al. (2003) International Worm Meeting "Temporal regulation of related microRNAs mir-48, mir-84, and let-7 by upstream components of the heterochronic pathway"

Victor R Ambros, Lorenzo F Sempere

[International Worm Meeting, 2003]

lin-4 and let-7 are founding members of an extensive class of genes with small noncoding RNA transcripts, termed microRNAs. microRNAs are 18-25 nucleotides long, and are generated by the processing of a 70-nucleotide hairpin precursor. Mutations in lin-4 and let-7 disrupt the timing of developmental programs, causing heterochronic defects. Mutation of the let-7 locus affects the developmental program that specifies the cell fates of the lateral hypodermis. let-7 retarded mutants undergo an extra molt, and terminal differentiation of the hypodermis is delayed. Upregulation of let-7 RNA at the end L3 stage influences the time of terminal differentiation of the hypodermis by translationally repressing target genes, which include lin-41. mir-48 (Lau et al., 2001) and mir-84 (Lee and Ambros, 2001) encode microRNAs similar to let-7 in sequence and expression profile. Thus, mir-48 and mir-84 may collaborate with let-7 to effect the time of hypodermal differentiation. To test if upstream heterochronic genes control the timing of let-7, mir-48, and mir-84 upregulation during post-embryonic development, RNA levels of these three microRNAs were analyzed using northern blots of total RNA from staged populations of N2 and heterochronic mutants. In lin-4(e912) animals, which fail to execute terminal differentiation of the hypodermis, RNA levels of all three microRNAs are decreased compared to the wild type (N2). However, in precocious lin-28(n719) or suppressed lin-28(n719);lin-46(ma164) mutants, each of these three microRNAs exhibits a different response. mir-84 RNA remains at levels comparable to N2 in lin-28(n719) and lin-28(n719);lin-46(ma164). Conversely, let-7 and mir-48 RNAs are detected at earlier stages in lin-28(n719) than in N2. Interestingly, the expression of let-7 and mir-48 in lin-28(n719) mutants differs in their dependence on lin-46. let-7 expression still occurs early in lin-28(n719);lin-46(ma164), even though these animals do not present heterochronic defects on the hypodermis. By contrast, the precocious expression of mir-48 is abolished in lin-28(n719);lin-46(ma164) suppressed animals. We interpret these results as follows: i) lin-4 acts to promote the expression of mir-48, mir-84 and let-7, probably via its role as a regulator of lin-28 (in the case of let-7 and mir-48), and via a separate pathway (in the case of mir-84). ii) lin-28 functions to repress mir-48 and let-7 expression at early stages of development (L1-L2), and perhaps also later to limit the extent of their upregulation. iii) lin-28 affects mir-48 expression via the action of lin-46, but affects let-7 independently of lin-46.

Jennifer B Phillips et al. (2001) International C. elegans Meeting "mir-1 and mir-2 exhibit defects in pronuclear migration and spindle orientation in the P0 stage C. elegans embryo"

Jennifer B Phillips, Rebecca Lyczak, Bruce Bowerman

[International C. elegans Meeting, 2001]

The position of the first mitotic spindle in the C. elegans embryo is crucial for establishing the cellular asymmetry necessary for proper patterning . Events such as pronuclear migration, centration, and rotation of the centrosome/nuclear complex are important precursors to proper spindle orientation, but remarkably little is known about the machinery which enacts these processes. To investigate the genes responsible for these and other early events, we screened for temperature sensitive embryonic lethal mutations with defects in P 0 spindle orientation. We isolated alleles of mel-26, zyg-9, rot-2 (see abstract by Greg Ellis) , and dnc-1 (see abstract by John Willis), in addition to two new loci we have named mir-1 and mir-2 , for pronuclear mi gration and P 0 r otation defective. In mir-1 and 2 mutant embryos, the maternal pronucleus often fails to migrate to the posterior of the embryo before the first mitotic spindle sets up. In these embryos, the maternal DNA may not be integrated into the mitotic apparatus, and instead seems to be randomly segregated to one of the daughter cells, appearing as an extra nucleus. The pronuclei do not centrate in these mutants, and the centrosome/nuclear complex does not rotate, resulting in a posteriorly positioned, transversely oriented spindle and a cleavage plane through the long axis of the embryo. Initial polarity appears normal, but cytoplasmic determinants are subsequently mislocalized due to the aberrant cleavage plane. I am working to characterize these migration and rotation defects in detail, using time lapse video microscopy, immunocytochemistry, and GFP fusion lines in which we can visualize tubulin and histone in living embryos. mir-1 and 2 map to chromosomes I and II, respectively. I am continuing to map each mutant to small intervals on these linkage groups, and will be employing RNAi and transgenic rescue to clone mir-1 and mir-2 .

Burke, Samantha et al. (2011) International Worm Meeting "A function for mir-34 in gonad morphogenesis."

Hammell, Molly, Burke, Samantha, Ambros, Victor

[International Worm Meeting, 2011]

Mir-34 is a highly conserved microRNA whose function is not yet fully understood. Human cell culture and mouse model work has shown that mir-34 plays a role in cell cycle progression, senescence, and apoptosis [1,2], while work in C. elegans has implicated mir-34 in radiation-induced DNA damage response [3]. In transgenic C. elegans expressing GFP under the control of the mir-34 promoter, expression is seen broadly throughout neural and muscle cells and in the hypodermis. Levels of both mature mir-34 and the GFP reporter increase during adulthood. In starved L1 and dauer larvae mature mir-34 levels are elevated compared to the developing L1 and L2 stage larvae, respectively [4]. We examined mir-34 mutants for developmental phenotypes and determined that the mutants display incompletely penetrant gonad migration defect, including overextension of the gonad arms, improper contact between the arms and hypodermis, and extra turns. The incomplete penetrance of this phenotype suggested the presence of co-regulators. Therefore, we tested for genetic enhancement of the mir-34 phenotype by loss of other microRNAS that share predicted targets with mir-34. Mutation of one such microRNA, mir-83, also causes a slight gonad migration defect. In mir-34;mir-83 double mutant animals, the migration defect is significantly more pronounced, suggesting that mir-34 and mir-83 function redundantly in the regulation of targets involved in guiding the migration of the gonad during larval development. We are in the process of identifying transcripts co-targeted by mir-34 and mir-83 in order to better understand how the loss of both microRNAs can lead to such defects. References: [1] C. He et al., Biochemical and Biophysical Research Communications 388 (2009) 35-40. [2] M. Yamakuchi and C.J. Lowenstein, Cell Cycle 8 (2009) 712-715. [3] M. Kato et al., Oncogene 28 (2009) 2419-2424. [4] X. Karp et al., RNA 17 (2011) 639-651.

Grimme, A. et al. (2019) International Worm Meeting "Elucidating the role of the miR-100 Family in C. elegans."

McJunkin, K., Grimme, A.

[International Worm Meeting, 2019]

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are important post-transcriptional regulators of gene expression. A miRNA contains a "seed" sequence, typically located in nucleotides 2-7, that has complementarity to a region in a mRNA 3'UTR which allows for target recognition while the miRNA is loaded to the RNA-induced silencing complex (RISC). MiRNA families are comprised of microRNAs that share the same seed sequence, and due to the identical seed sequences, these family members can act redundantly while regulating targets. One of the most highly conserved miRNA families is the miR-100 family, which contains three family members in humans: miR-99a, miR-99b, and miR-100. These miRNAs are implicated in several medical conditions including cancer, obesity, heart disease, and others. In the model organism C. elegans, the miR-100 family microRNAs are known as the mir-51 family members, which includes six microRNAs: mir-51 through mir-56. Current knowledge of the essential functions of this family is relatively limited; one of the most important functions in C. elegans is its role in embryonic development. Given the highly conserved nature of the miR-100 family, understanding the vital functions of the mir-51 family may serve to elucidate how this family is involved in human diseases. While removing all mir-51 family members results in embryonic lethality, this project utilizes a hypomorphic strain with a slow growth phenotype to study the pathways regulated by this family. A portion of this project is dedicated to performing forward genetic screens to isolate suppressors of the slow growth phenotype. Reverse genetic screens using RNAi are performed alongside the forward screens to determine the roles of predicted mir-51 family targets in the slow growth phenotype. Preliminary data from these screens suggest that the slow growth phenotype can be alleviated through isolated suppressors as well as through performing RNAi against certain mir-51 family targets. In the future these results may help to elucidate specific functions of the mir-51 family in C. elegans, which may represent conserved functions of the miR-100 family that can be studied further to determine the connection between this family and human diseases.

Axel Bethke et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "LIN-29/ZnF co-activates DAF-12/NHR to drive miR-84 expression, placing miR-84 towards the end of the heterochronic pathway"

Nicole Fielenbach, Axel Bethke, Adam Antebi, Linyan Meng

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

The nuclear hormone receptor DAF-12 acts as the switch between continuous development or arrest at the dauer diapause during the L2 stage. It also works in the heterochronic circuit, promoting L3-stage specific developmental programs. Another heterochronic factor, the zinc finger protein LIN-29 acts during L4 to promote terminal differentiation of various tissues like gonad or hypodermis, a function referred to as the larval-to-adult switch. Despite this terminal position, here we present evidence that LIN-29 is involved in transcriptional activation of the let-7 sister mir-84, which was thought to act at an earlier step. Previously, we had shown DAF-12 and ligand dependent activation of mir-84 promoter constructs in human cell culture and in worms. We wondered whether any of the heterochronic factors could serve as DAF-12 co-activators to augment transcription. Remarkably, we found that co-transfection of LIN-29 increased DAF-12 dependent transcription two-fold, but had no such activity alone, suggesting LIN-29 may be a DAF-12 co-activator. Accordingly, point mutation of DAF-12 response elements strongly decreased DAF-12 and ligand dependent activation as well as the co-activator effect, The DNA-motive that LIN-29 binds to is unknown, but EMSA with an oligo containing two functional DAF-12 sites show no LIN-29 binding, making it unlikely that LIN-29 can bind directly to DAF-12 response elements. Taken together, the experiments suggest that LIN-29 works through DAF-12 to exert these transcriptional effects. The mir-84 promoter also shows DAF-12 and LIN-29 dependence in vivo. In particular, the lin-29 null mutants exhibited decreased pharyngeal mir-84p::GFP expression, even below the level observed in a daf-12 null. Moreover, a lin-29, daf-12 double null nearly lost all GFP expression. These in vivo data are consistent with the notion that LIN-29 acts through DAF-12 and its response elements, but also hint at a DAF-12 independent function of LIN-29 on the mir-84 promoter. The placement of LIN-29 upstream of mir-84 inverts its conventional placement as the terminal factor in the heterochronic pathway. Interestingly, Hayes et al showed that mir-84 over expression can suppress lin-29 for supernumerary molting heterochronic phenotypes. This, together with the data presented here, suggests that LIN-29 could drive terminal differentiation in the hypodermis through activating mir-84 expression, placing miR-84 downstream of LIN-29 to the very end of the heterochronic signaling cascade. We are currently testing this hypothesis.

Alejandra Clark et al. (2008) C.elegans Neuronal Development Meeting "Investigating the Role of miR-124 in C. elegans"

Eric Miska, Funda Sar, Alejandra Clark

[C.elegans Neuronal Development Meeting, 2008]

miRNAs constitute a class of small non-coding RNAs shown to play critical roles in cell fate, developmental timing and apoptosis. Unlike most other miRNAs in C. elegans that share only partial sequence conservation across species, miR-124 is 100% conserved from worms to humans. Moreover its expression pattern is also highly conserved in all species so far investigated. In mice, miR-124 is estimated to be the most abundant miRNA in the brain, expressed throughout the embryonic and adult central nervous system. This high degree of conservation across species suggests a critical role in neural differentiation and or function. However, at present no loss of function mutants have been described in any species to support this hypothesis. To address this question, we are investigating a C. elegans mir-124 deletion mutant (Miska et al. 2007). mir-124 mutants are viable and superficially wild-type. A mir-124::GFP reporter transgene suggests that miR-124 expression is restricted to the nervous system. However, it is not pan-neuronal, but expressed strongly in sensory neurons. We are currently investigating the morphology and differentiation state of miR-124 expressing neurons in mir-124 mutants. In addition we are carrying out a number of behavioural assays to address the function of sensory neurons in this mutant. In parallel, to test whether other genes might act redundantly with mir-124, we are carrying out an RNAi genetic interaction screen.