Hornsten AM et al. (2000) East Coast Worm Meeting "The extracellular domain of the C. elegans amyloid precursor-related protein is essential for viability"

Li C, Hornsten AM

[East Coast Worm Meeting, 2000]

Alzheimers disease is a neurodegenerative disorder characterized by the accumulation of dense extracellular plaques in the brain. The major component of these plaques is the b-amyloid peptide, which is a cleavage product of the Amyloid Protein Precursor (APP). The early-onset form of Alzheimers Disease has been linked to mutations in several genes, including APP. The function of APP remains unclear. There is a large family of APP-related proteins, suggesting that their function is conserved throughout evolution. These proteins are putative single pass transmembrane proteins that contain two highly conserved regions, E1 and E2, in the extracellular domain. We are studying the function(s) of the APP-related gene, apl-1, in the nematode Caenorhabditis elegans. APL-1 shares many similarities with the APP family of proteins, although it does not contain the b-amyloid peptide. apl-1 is expressed in over 50 cells throughout the animal. To analyze the function of APL-1, we have isolated three apl-1 alleles, yn10, yn23, and yn5. Western blot analysis indicates that yn10 and yn23 animals do not produce an APL-1 protein, suggesting that these alleles represent complete loss-of-function alleles. Homozygous yn10 and yn23 animals have a larval lethal phenotype. The lethality is rescued by expression of APL-1 in neurons. The extracellular domain of APL-1 appears to be essential for viability since expression of only the extracellular domain of APL-1 is sufficient to rescue the lethality. We are analyzing which regions of the extracellular domain are essential for APL-1 function. The E2 region of the extracellular domain does not appear to be important since microinjection of a construct encoding the APL-1 protein with a deletion of the E2 region rescues the lethality. By western blot analysis, yn5 animals produce a truncated APL-1 protein that presumably corresponds to only the extracellular domain of APL-1. yn5 animals show several phenotypes that appear to be due to an increased production of the extracellular domain of APL-1. These results suggest that the extracellular domain of APL-1 is involved in multiple behaviors.

Hornsten AM et al. (1997) International C. elegans Meeting "FUNCTION OF C. ELEGANS APL-1, A GENE ENCODING A PROTEIN RELATED TO THE HUMAN AMYLOID PROTEIN PRECURSOR"

Hornsten AM, O'Connor G, Li C

[International C. elegans Meeting, 1997]

Alzheimer's disease is characterized pathologically by the accumulation of dense extracellular plaques in the brain. The major component of these plaques is the beta-amyloid peptide, which is a cleavage product of the Amyloid Protein Precursor (APP). The C. elegans apl-l gene encodes a protein with similarities to APP. Unlike APP, however, APL-1 does not contain the beta-amyloid peptide. APP-related genes that do not contain the beta-amyloid peptide have also been found in humans, mice, and Drosophila. To determine the function of apl-l, a strain containing a Tc1 in exon 12 of apl-1 was obtained from Ron Plasterk. Several mutant forms of the apl-1 transcript are produced due to a variable "RNA footprint" from Tc1 splicing. The majority of the mutant transcripts maintain the reading frame, but produce larger proteins. apl-1::Tc1 animals have slightly less numbers of progeny when compared to wild-type animals. To obtain a deletion in apl-1, populations of apl-1::Tc1 animals were screened for imprecise Tc1 excision. One deletion strain was isolated. Southern blots revealed the presence of two copies of apl-1, both of which contained a deletion. The 1.1 kbp in-frame deletion removes most of the extracellular domain, but retains the transmembrane and cytoplasmic domains, while tbe 800 bp frame-shift deletion only produces the extracellular domain. Deletion animals show the same behavioral defect seen in Tcl insertion animals, but to a greater extreme. We are planning to rescue this phenotype with apl-1, and the human and mouse APP and APP-related genes. To determine the cell-specific expression of apl-1, lacZ and GFP reporter constructs under the transcriptional regulation of 4.8 kbp of the apl-1 upstream region have been made. Beta-galactosidase activity and GFP staining are seen mainly in neuronal cells and muscles. Exact identification of these cells is underway. To determine the effects of apl-1 overexpression, we have created stable transgenic lines carrying heat shock or neural-specific promoters controlling APL-1 expression. More extensive characterization of these strains is in progress.

Hornsten AM et al. (1998) East Coast Worm Meeting "DISRUPTION OF C. elegans apl-1, AN AMYLOID PROTEIN PRECURSOR-RELATED GENE, IS CORRELATED WITH MULTIPLE DEFECTS"

Li C, Hornsten AM

[East Coast Worm Meeting, 1998]

The early-onset form of Alzheimer's Disease has been linked to mutations in several genes, including the Amyloid Protein Precursor gene (APP). APP-related genes have been found in humans, mice, and Drosophila. The functions of APP and APP-related proteins, however, have not been clearly elucidated. We are studying the function of an APP-related gene, apl-1, in C. elegans. apl-1, which is located on the X chromosome, encodes a putative single pass transmembrane protein which shares many similarities with the APP family of proteins. To determine where apl-1 is expressed, animals carrying apl-1::lacZ and apl-1::GFP transcriptional fusion constructs were generated. B-galactosidase activity and GFP staining are seen mainly in neuronal cells and muscles. To generate a mutation in apl-1, we obtained a Tc1 transposon insertion strain from Ron Plasterk. The Tc1 was inserted into the last exon, exon 12, of apl-1. Using RT-PCR, we found that these animals produce several mutant forms of the apl-1 transcript due to variable splicing within the Tc1 transposon. The majority of the isolated transcripts would maintain the APL-1 reading frame. To obtain a deletion in apl-1, Tc1 insertion animals were screened for improper repair of a Tc1 excision. The yn3 and yn6 strains contain a duplication of the apl-1 locus with each apl-1 locus containing a different deletion. Because two independent strains containing a duplication of the apl-1 locus were isolated, we assume that the original Tc1 insertion strain has two apl-1 loci, each with a Tc1 insertion. By RT-PCR, we verified that two mutant apl-1 transcripts are made in each strain. We are currently screening the yn6 strain for a recombination event that separates the two deleted apl-1 loci. yn5 was isolated as a recombinant line of yn3, and contains only a single deleted apl-1 locus. The deletion in yn5 removes sequences encoding the cytoplasmic and transmembrane regions of the APL-1 protein. yn5 animals are defective in egg-laying, are resistant to serotonin-induced egg-laying, and show developmental defects. yn5 animals also have an increased rate of pharyngeal pumping and a decreased rate of defecation. To verify that the yn5 phenotypes are due to the loss of APL-1, a cosmid containing the apl-1 locus has been microinjected into yn5 animals; transgenic animals will be assayed for rescue of the deletion phenotypes. We will also use the apl-1 genomic region and the human and mouse APP and APP-related genes to determine whether the encoded proteins can rescue the deletion phenotypes. To determine whether yn5 represents a genetic null, we are crossing the yn5 animals with a strain containing a deficiency covering apl-1. Western blots will also be done on extracts from yn5 animals to determine if an APL-1 protein is being made.

AM Hornsten (1999) International C. elegans Meeting "The C. elegans Amyloid Protein Precursor-Related Gene Has An Essential Function"

AM Hornsten

[International C. elegans Meeting, 1999]

The early-onset form of Alzheimer's Disease has been linked to mutations in several genes, including the Amyloid Protein Precursor gene ( APP ). APP -related genes have been found in humans, mice, and Drosophila . The function(s) of APP and APP-related proteins, however, have not been clearly elucidated. We are studying the function(s) of the APP -related gene, apl-1 , in C. elegans . apl-1 encodes a putative single pass transmembrane protein which shares many similarities with the APP family of proteins. Since other members of this family are cleaved to release the extracellular domain from the membrane, it is likely that APL-1 will be processed similarly. apl-1 is expressed in over 50 cells, including neurons, muscles, and supporting cells, in the head region, ventral cord, and tail region. To generate a mutation in the apl-1 gene, we obtained a Tc1 transposon insertion strain ( apl-1 ::Tc1) from Ron Plasterk. Tc1 insertion animals were screened for improper repair of a Tc1 excision. Two deletion alleles, yn10 and yn5 , were isolated. yn10 animals contain a deletion that removes most of the apl-1 coding region. Homozygous yn10 animals show a larval lethal phenotype. These data suggest that yn10 represents a complete loss-of-function allele. The yn10 larval lethality is rescued by microinjection of the apl-1 genomic region and by expression of APL-1 in neurons. We will be attempting to rescue the yn10 larval lethality with the human APP gene. We are currently doing an EMS screen to generate other mutations in apl-1 that cause larval lethality. We are also performing genetic mosaic analysis on yn10 animals to determine the specific cells in which apl-1 gene function is necessary. yn5 animals contain a deletion which removes sequences encoding the cytoplasmic and transmembrane regions of the APL-1 protein. yn5 animals show developmental, egg-laying, and motor-related defects. apl-1 ::Tc1 insertion animals show similar phenotypes, but to a lesser degree. By analyzing the yn5 phenotypes, we have determined that resistance to serotonin-induced egg-laying and a decreased defecation rate are due to a partial loss-of-function and a gain-of-function mutation, respectively. These data suggest that APL-1 acts both as a membrane-bound protein and as a secreted protein.

Saenz-Narciso, Beatriz et al. (2011) International Worm Meeting "Characterizing the adrenomedullin homologue in C. elegans."

Martinez, Alfredo, Cabello, Juan, Gomez-Orte, Eva, Saenz-Narciso, Beatriz

[International Worm Meeting, 2011]

Adrenomedullin (AM) is a peptide hormone that shows multiple physiological functions in vertebrates, such as bronchodilation, neurotransmission, hormone regulation, antimicrobial activity or growth regulation. Deregulation of AM has been shown in different human pathologies such as diabetes, cancer, hypertension, heart failure, and sepsis. Research on molecules capable of modulating AM and its effects may lead to the discovery of new pharmacological agents to treat these important human diseases. Highly interesting is the role of AM in cancer. It has been proven that AM acts as an autocrine/paracrine tumor cell survival factor and its elevated expression in cancer cells can promote angiogenesis leading to tumor progression. While AM has been well characterized in other research models, little is known about this molecule in C. elegans. We have found and characterized a mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy analysis of wam-1 mutants shows defects in the migration of the hermaphrodite gonad. There are also several defects in embryonic development. Some embryos have cells that are excluded out of the worm during development while fate specification, as well as proliferation is normal. This suggests a function of wam-1 in cell adhesion. To localize the expression of the worm AM homologue protein, we performed inmunostaining using mouse anti-AM antibodies. The AM protein was localized in the hypodermis. In contrast, we also generated transgenic lines expressing GFP under the control of the wam-1 promoter. GFP expression was detected in the mouth of the worm, although due to its homology with secreted hormones, the protein could play a role far away from the secretory cells. At the moment, we are performing a detailed molecular and genetic characterization, in order to understand the function of this molecule.

Cristina Nieto et al. (2008) European Worm Meeting "Analysis of the adrenomedullin homologue in C.elegans."

Cristina Nieto, Alfredo Martinez, Sergio Moreno, Juan Cabello

[European Worm Meeting, 2008]

In vertebrates, adrenomedullin (AM) is a peptide hormone that shows. multiple physiological functions, such as bronchodilation,. neurotransmission, hormone regulation, antimicrobial activity or growth. regulation. Deregulation of AM has been shown in different human. pathologies such as diabetes, cancer, hypertension, heart failure, and. sepsis.. Research on molecules capable of modulating AM and its effects may lead to. the discovery of new pharmacological agents to treat these important human. diseases. Thus, the injection of a monoclonal antibody against AM produces. an antidiabetic effect, by reducting the glycemic level in diabetic. experimental animals, since AM has been shown to reduce insulin secretion.. Highly interesting is the role of AM in cancer. It has been proven that AM. acts as an autocrine/paracrine tumor cell survival factor and its elevated. expression in cancer cells can promote angiogenesis leading to tumor. progression.. While AM has been well characterized in other research models, little is. known about this molecule in C. elegans. We have found and characterized a. mutant in the AM homologue of C.elegans, here called wam-1. 4D microscopy. analysis of wam-1 mutants shows defects in the migration of the. hermaphrodite gonad. There are also several defects in embryonic. development. Some embryos have cells that are excluded out of the worm. during development while fate specification, as well as proliferation are. normal. This suggests a function of wam-1 in cell adhesion.. To localize the expression of the worm AM homologue protein, we generated. transgenic lines expressing GFP under the control of the wam-1 promoter.. GFP expression is detected in the mouth of the worm although due to its. homology with secreted hormones, the protein could play a role far away. from the secretory cells.. At present, we are performing a detailed molecular and genetic. characterization, in order to understand the function of this molecule.

Waring DA (1996) West Coast Worm Meeting "A C. ELEGANS HOMOLOG OF NEUROD"

Waring DA

[West Coast Worm Meeting, 1996]

The neuroD gene was identified in our lab as a bHLH transcription factor controlling neuronal differentiation in mouse and Xenopus. Extopic expression in Xenopus embryos leads to production of ectopic neurons within the epidermis. It is proposed that neuroD acts downstream of the proneural genes and negative regulators of the Notch/Delta pathway. I am characterizing a C. elegans homolog of neuroD cnd-1. In order understand the function of the gene I am in the process of knocking the gene out. I have identified an insertion of a transposable element within an intron of the gene. This has no phenotype and is apparently spliced out without effecting the gene. I am presently screening for imprecise excision of the transposable element that will knock out cnd-1. In addition I am characterizing larger deficiencies and lethal mutation in the region. I have generated antibodies to cnd-1 and I am beginning to characterize the expression pattern. Neurogenesis in C. elegans is still poorly characterized. Several bHLH proteins related to the Achaete/Scute family, have been found in C. elegans, but as yet there is functional data for only one of these, lin-32 which was identified by mutations lacking a subset of neuroblasts, suggesting that as in other species bHLH proteins may regulate neurogenesis. If cnd-1 functions in C. elegans as its homologs do in vertebrates, the single cell resolution that C. elegans affords should be very useful in understanding its role in neurogenesis, and its relationship to the proneural genes.

Negishi, Takefumi et al. (2019) MicroPubl Biol

Sawa, Hitoshi, Kanemaki, Masato T, Negishi, Takefumi, Asakawa, Masayo

[MicroPubl Biol, 2019]

The targeted protein degradation systems in which a protein accompanying with specific tags can be degraded are developed as an approach of conditional loss of function analyses (Natsume and Kanemaki 2017). The insertion of tags into the gene loci by the CRISPR/Cas9 system allows us to deplete endogenous proteins in stage and cell specific manners. So far, four systems can be used to deplete the tagged proteins in C . elegans (Armenti et al. 2014; Zhang et al. 2015; Wang et al. 2017; Wu et al. 2017). The auxin-inducible degradation (AID) system can degrade the tagged protein by administration of the phytohormone auxin. In the AID system, a plant-derived TIR1 F-box protein can form an E3 ubiquitin ligase complex with the endogenous Skp and Cullin proteins, and in the presence of auxin, the complex interacts with a degron tag derived from the IAA17 transcriptional repressor (Nishimura et al. 2009; Yesbolatova et al. 2019). Consequently, the degron-tagged proteins are polyubiquitinated for degradation by the proteasome. With the controlled expression of TIR1 and administration of auxin, the AID system can allow us to perform spaciotemporal protein depletion. Although this system can be a powerful tool for conditional loss of function analyses, it appears to be difficult to degrade proteins in embryos, especially at late embryonic stages, since efficiencies of degradation in laid embryos surrounded by the eggshell is low compared to those in hatched larvae or adults (Zhang et al. 2015). Here, we report that acetoxymethyl indole-3-acetic acid (IAA-AM), a cell permeable analog of natural auxin IAA (Figure1A), is more effective in triggering the degradation of tagged proteins in embryos. To test the efficiency of protein degradation, we used the previously reported strain ubiquitously expressing both a 44-amino acid (-aa) degron derived from the Arabidopsis IAA17 protein (known as AID*) fused with GFP and Arabidopsis TIR1 (AtTIR1) fused with mRuby (ieSi58 [Peft-3::degron (AID*)::GFP] and ieSi57 [Peft–3::AtTIR1::mRuby])(Morawska and Ulrich 2013; Zhang et al. 2015). When we put laid embryos into the drop of M9 buffer containing 1 mM IAA-AM (AM form of IAA), we observed rapid decrease of green fluorescence (GF) intensity (Figure1B, C and D). In contrast to IAA-AM, the embryos treated with IAA showed increased GF intensity due to elevated expression of degron-tagged GFP (Figure1B, C and D). We also confirmed that IAA-AM did not cause the decreased GF intensity without the AtTIR1 transgene (Figure1C). Next, we tried to deplete the endogenous protein in laid embryos. WRM-1 is the C. elegans homolog of -catenin and is essential for asymmetry of most cell divisions (Mizumoto and Sawa 2007). Since WRM-1 is required for asymmetric division of EMS in the 4-cell stage embryo (Rocheleau et al. 1997), conditional knockdown approaches are required for the investigation of its function in later stages. To test whether the AID system with IAA-AM allows us to perform conditional knockdown of wrm-1 in embryos, we inserted a fusion tag composed of the mini-auxin inducible degron (mAID), a 68-aa degron derived from the Arabidopsis IAA17 protein that showed similar degradation efficiency with that of AID* (Li et al. 2019), and mClover at the 3 end of wrm-1 endogenous locus by the CRISPR/Cas9 system (Natsume et al 2016) (Dickinson et al. 2015). We put laid embryos of the strain carrying wrm-1::mAID::mClover (os169) and Peft-3::AtTIR1::mRuby (ieSi57) into drops of 1 mM IAA-AM or IAA (both with 0.2% DMSO in M9 buffer) for two hours. After washing with M9 buffer two times, we placed embryos on NGM plates without the inducing ligand. At the young adult stage, we scored elongation of gonadal arms guided by the distal tip cells (DTCs) whose production requires wrm-1-regulated asymmetric divisions (Siegfried et al. 2004). When we treated os169 ieSi57 embryos with 1 mM IAA-AM, all animals (13/13) shows no gonadal extension. In contrast, treating with 1 mM IAA caused this phenotype only in 2/17 animals (15/17 showed normal gonad) (Figure 1E). In addition, all os169 ieSi57 embryos treated with 0.2% DMSO (16/16) or ieSi58 ieSi57 embryos treated with 1mM IAA-AM (19/19) produced animals with normal gonads. These results show that the AM modification of IAA induces efficient protein degradation in laid embryos with the AID system. Since the AM modification is widely used to improve cell membrane permeability (Schultz 2003), IAA-AM is likely to penetrate into the eggshell more easily than IAA. After penetration into cells, endogenous cytoplasmic esterases are known to cleave the AM bond, making the compound again impermeant to cell membrane (Schultz 2003). We note that the chemical cleavage reaction of the AM bond produces formaldehyde (Schultz 2003), which may be toxic to cells at high concentrations. In this study, however, we found the application of IAA-AM at a concentration of 1 mM for two hours allowed normal development including gonad elongation. Our data presented here show IAA-AM will broaden the possible applications of the AID system for conditional loss of function analyses in C. elegans.

Meneely PM et al. (1982) Worm Breeder's Gazette "embryonic lethal amber mutations on linkage group II."

Meneely PM, Wood WB, Pesavento P

[Worm Breeder's Gazette, 1982]

In the last newsletter, we described a selection of lethal amber mutations linked to dpy-10, using sup-7. We have revised this procedure slightly to take advantage of the balancer chromosome C1. Our current procedure is to mutagenize unc-4(e120); ) hermaphrodites and mate them with sup-7 males. Neither marker is suppressed by sup-7. From the F1 progeny, we pick wild-type animals and score their F2 progeny. Broods that have fertile Uncs and Dpys, but no fertile Unc Dpys are candidates for an amber lethal linked to unc-4. We have been outcrossing such candidates with C1/unc-4 males to establish stocks of genotype C/unc-4 am, which segregate am am mutant homozygotes. We also have been establishing stocks of genotype C1/unc-4 am; sup-7/sup-7. This is readily distinguished from the former stock not only by the suppressible lethal, but also by the suppression of unc-52(e669), one of the markers on the C1 balancer. Thus far, we have isolated more than thirty candidates, and have outcrossed about half. From these, two separate amber mutants have been maintained in both suppressed and nonsuppressed stocks. The balancer makes stock maintenance and mutant complementation much easier. We hope to identify amber mutations of genes known only by temperature-sensitive alleles, as well as new essential genes.

Riberu WA et al. (1990) Am J Trop Med Hyg "Cultivation of sexually mature Brugia malayi in vitro."

Baird JK, Bangs MJ, Atmosoedjono S, Tirtokusumo S, Riberu WA

[Am J Trop Med Hyg, 1990]

Sexually mature male and female Brugia malayi were developed from third stage larvae after 60 days in the in vitro culture system described by Franke and others in 1987 (Am J Trop Med Hyg 37: 370-375). Between 75 and 100 days in culture, many worms produced living microfilariae. Each gravid female produced 200-1,500 microfilariae/day.