Chow, Elly S et al. (2011) International Worm Meeting "Cis-regulating network in the ALA neuron."

Van Buskirk, Cheryl, Chow, Elly S, Sternberg, Paul

[International Worm Meeting, 2011]

The ALA neuron is so far the only neuron known to be involved in regulating the EGF-induced sleep-like state in C. elegans. We have previously discovered a network of three homeobox-containing transcriptional regulatory proteins that regulate expression of let-23/EGFR and other ALA-expressed genes. By comparing multiple nematode genomic sequences, we have identified highly conserved regions in the vicinity of coding sequence of two transcription factors (ceh-14 and ceh-17) and three of the functional genes (plc-3, ida-1, ver-3). These elements contain a MEME motif and a cluster of putative homeobox transcription factor binding sites. We analyzed 150 bp genomic elements for their ability to confer cell-type specific gene expression on the pes-10 basal promoter using a reporter gene assay. In each case, the element directed expression in the ALA neuron specifically, except in the case of the plc-3 element, expression is also found in two vulva cells. These elements thus contain ALA-specific regulatory sequences. We believe these elements may serve as probes for finding ALA -expressing genes in the genome. To verify our hypothesis, we continue to identify ALA-specific elements in all the known genes expressed in the ALA. To identify additional ALA-expressed genes, we also seek to profile enriched transcripts in the ALA neuron. We hope to identify crucial cis-regulatory elements and the logic of gene expression regulation in the ALA neuron.

Chow, Elly S. et al. (2013) International Worm Meeting "Deciphering a genetic regulatory network of the ALA neuron."

Van Buskirk, Cheryl, Schwarz, Erich M., Sternberg, Paul W., Chow, Elly S.

[International Worm Meeting, 2013]

The single, unpaired C. elegans ALA neuron responds to EGF-receptor activation and induces sleep-like behavior, including cessation of feeding and locomotion. We previously discovered a regulatory network comprising one LIM homeodomain transcription factor, ceh-14, and two paired-like homeodomain transcription factors, ceh-10 and ceh-17, that co-regulate EGF signaling pathway components, let-23/EGFR and phospholipase C-g, as well as other ALA-expressed genes. We sought to identify crucial cis-regulatory elements and the logic of gene regulation in the ALA neuron. We identified an 11-bp motif (the ALA-motif) from the regulatory regions of eight known ALA-expressing genes. The ALA-motif is necessary and sufficient to drive green fluorescent protein expression in the ALA neuron when attached to a basal promoter in a reporter construct. This motif harbors putative transcription binding sites for LIM homeodomain Lhx3 (an ortholog of ceh-14) and a paired-like homeodomain (like ceh-17 and ceh-10). Genetic analysis showed that the ALA-motif::GFP expression requires ceh-14 and ceh-17. A genome-wide search identified a set of 597 genes that have the ALA-motif in conserved neighboring non-coding DNA sequences, which may include regulatory regions. Of four neuropeptide genes in this set, we found a least one ALA-motif in the intron or in the 5' non-coding region that are sufficient to drive reporter expression in the ALA. Concurrently, we characterized the ALA transcriptomes from wild-type animals and ceh-14 mutants. We detected 8110 genes in the ALA and a set of these genes overlaps with motif-searched genes, which are also ceh-14-dependent. These results demonstrate that cell-specific cis-regulatory motif can identify co-expressed genes and single cell RNA-seq analysis in wild-type and mutant animals can sort out genes that are sharing the same regulatory inputs. Our combined approach may be used to identify the cis-regulatory network of a cell.

KL Chow (1999) International C. elegans Meeting "Dosage of ram-5 gene is critical for sensory ray morphogenesis in C. elegans"

KL Chow

[International C. elegans Meeting, 1999]

During normal tissue morphogenesis, cell adhesion and repellent molecules have to be expressed in the right level at the right place and right time. Deviation from the normal expression level may not be tolerated for tissue function nor animal vitality. In our study of ram-5 mutation, it was noticed that ram-5 mutant animals rescued by transformation constantly threw progeny with missing ray phenotype. A strong correlation of missing ray phenotype and the rescue activity was established. We speculate that there was a causative relationship between the ram-5 gene dosage and missing ray phenotype and thus employed both genetic and molecular approaches to test this notion. In the genetic analysis, two stable transgenic lines, KC61 and KC62 were used. These stable lines carried truncated and wild type ram-5 genes in a him-5 background. They showed 18% and 100% ray loss respectively. When heterozygous mutant transgenic worms were generated from them to lower the transgene dosage, the percentage of ray loss dropped to 0% and 53% respectively. A positive correlation between ray loss and transgene copy number was established in these lines by Southern analysis. This result argues that high level of ram-5 product might interfere the regular ray assembly process. When truncated ram-5 gene product was expressed in him-5 worms, both swollen ray tip and lumpy ray phenotype were observed. However, when the WT ram-5 gene was injected into the him-5 worm, it did not give these phenotypes. The results suggest that truncated RAM-5 protein can compete with the WT RAM-5 protein for its interacting components to form inactive complexes. By lowering of functional complex level, swollen ray tip or lumpy ray phenotype was resulted. Further over-expression of the truncated protein would lead to severe ray loss. We propose that ram gene products function as a multi-protein complex in a specific stoichiometry. Alteration of the ratio of these proteins will adversely affect the formation of functional complexes. As a result, ray cell assembly cannot take place and rays fail to attach to the cuticle to establish proper morphology.

Chow, Elly S. et al. (2013) International Worm Meeting "Single-cell transcriptomic analysis identifies quiescence-inducing neuropeptides."

Sternberg, Paul W., Schwarz, Erich M., Chow, Elly S.

[International Worm Meeting, 2013]

Neuropeptides are found throughout animal nervous systems and are important in regulating many behaviors and physiological functions. We used the nematode Caenorhabditis elegans to explore the interaction of neuropeptides and a conserved sleep regulator, Epidermal Growth Factor Receptor (EGFR) signaling, in a prolonged and reversible sleep-like state in young adults, when animals are normally active. RNA-seq reveals that 12 neuropeptide-encoding genes in C. elegans are enriched in the ALA neuron. Using conditional activation of neuropeptides and genetic screening, we identified a behavioral quiescence regulatory system consisting of two FMRFamide (Phe-Met-Arg-Phe-NH2)-like neuropeptide encoding genes, flp-13 and flp-24, and a neuropeptide-related receptor, npr-22, orthologous to human neuropeptide Y receptor (24.7% identity) and human opiate receptor-like 1 (19% identity). Overexpression of flp-13 and flp-24 reduces locomotion, feeding, and sensory responsiveness. In particular, hs:FLP-13 animals are inert to tactile and chemosensory stimuli while hs:FLP-24 animals exhibit delayed responses to them; and joint induction of hs:FLP-13 and hs:FLP-24 is stronger than either alone, suggesting functional redundancy. Loss of npr-22 function blocks the effects of neuropeptide-induced quiescence behavior. Our results indicate that nematodes and vertebrates share conserved neural signals that regulate sleep.

KL Chow (1999) International C. elegans Meeting "Cloning and characterization of C. elegans ram-5: strucutral organization and expression pattern of the gene"

KL Chow

[International C. elegans Meeting, 1999]

In C. elegans , nine pairs of bilateral sensory rays are formed during the late L4 larval stage. This morphogenesis process involves diverse cellular activities such as the formation of junctions with correct neighbors, cell migration, changes in cell-shape and cell-volume. A number of mutations were isolated to affect this morphogenetic process. The major group of these loci, the ram 's, showed a lumpy ray phenotype when they are mutated. In mutant ram animals, tight cylindrical morphology unique to individual ray is not maintained. Instead, rays are swollen with highly variable morphology. We attempted to characterize the morphogenesis process by cloning these ram genes. ram-5 mutation representing one of them was rescued by cosmid transformation. Three overlapping cosmids, H01A2, H02I07 and T24C2 could restore the wild type phenotype in ram-5 mutants. The result suggested that ram-5 gene was located in the overlapping region. Deletion analyses defined a minimal rescuing fragment of 9.5 Kb. More detailed deletion study suggested that the ram-5 gene was encoded in the minus strand, and the promoter was defined in a 2.5 Kb region on this genomic fragment. The identity of this locus will be further confirmed by genomic mapping of mutant alleles and RNA interference. It is interesting to note that a number of these deletion constructs caused a ray loss phenomenon when they carried high copy of the transgene (see the accompanying abstract by Yu and Chow). While no known cDNA was isolated in the library screened and the EST database, we used mutational strategy, inverse PCR, 5' and 3' RACE to define the transcript(s). Sequence analysis predicts that the encoded protein is a transmembrane glycoprotein with a short domain sharing similarity with cuticulin, a nematode cuticular protein. Promoter reporter constructs in transgenic animals revealed that ram-5 was expressed in ray structural cells in the male tail and a number of neuronal cells in the amphids, and pharyngeal region. These expression patterns suggest that this gene may have additional neuronal functions in worm.

KL Chow (1999) International C. elegans Meeting "Synthesis of glycoprotein is important for molting and male tail sensory ray morphogenesis in Caenorhabditis elegans"

KL Chow

[International C. elegans Meeting, 1999]

C. elegans male tail is a complex structure specialized for reproductive function. The morphogenetic events occurring at the late L4 larval stage represents a process of tissue differentiation. Active cellular movement occurs and new cell-cell association need to be established. It offers an excellent paradigm for the study of tissue remodelling. Recent characterization of mutants with ray morphological defects revealed that epitope recognized by FITC-WGA was detected at high level in mutants with lumpy ray (Ram) phenotype. We postulated that overexpression of this epitope might be a result of the mutations, blocking the synthesis of these glycoprotein epitope would revert the phenotype. This hypothesis was tested by treatment of mutant worm with glycosylation inhibitors. Failure of reverting the mutant phenotype in these experiments suggests that Ram phenotype is not caused by overexpression of the GlcNAc containing molecules. On the contrary, blocking of glyco-conjugate synthesis in wild-type animal generated Ram-like phenotype. We defined the window of the inhibitor effect on ray morphogenesis to be around late L4 stage, at the time of male tail retraction, where active cell migration and cell-cell association took place. In addition, glycosylation inhibitor blocked the molting of L4 cuticle implicating a role of glycoprotein in this molting process. Previous studies have suggested that WGA binding antigen is masked by components of the cuticle. srf mutants lose the cuticular components and give to high WGA staining signals on the body. We have further characterized these ram mutants by protein analysis to ascertain the role of glycosylation. Our result suggests that glyco-conjugates are constantly expressed beneath the surface cuticle. Similar to the features of the srf genes, ram genes may represent another group of cell surface glycoproteins, the presence of which ensure normal matrix-cell and cell-cell association for an intact body outer layer. When they are mutated, cuticular matrix and cell-cell interaction will be disrupted revealing the glyco-antigens beneath the surface.

Chow K-L et al. (1993) International C. elegans Meeting "mab-21 mutations cause abnormal cellular association in a fused ray."

Chow K-L, Hall DH, Emmons SW

[International C. elegans Meeting, 1993]

Yit-Lai Chow et al. (2010) East Asia Worm Meeting "Caenorhabditis elegans as a screening model for biological activities of plant natural chemicals"

Yit-Lai Chow, Fumihiko Sato

[East Asia Worm Meeting, 2010]

Higher plants produce divergent array of natural chemicals. Metabolic engineering of biosynthetic pathway further enhances chemical diversity of metabolites and would provide more biologically active chemicals for the development of novel pharmaceutical compounds. For the screening of biological active chemicals, the development of a rapid and broad-based system using whole body would be needed for the preliminary screening. In this study, Caenorhabditis elegans is used as a model to detect biological activities of isoquinoline alkaloids, which are one of the largest group of plant alkaloids. As a start, we have monitored the changes of lipid accumulation in C. elegans after the treatment with isoquinoline alkaloids both qualitatively and quantitatively. Microscopic observation such as Nile Red and Sudan Black staining of lipid droplets and chemical and biochemical determination of lipid content were performed to elucidate the effects of various alkaloids on lipid metabolism. Since some chemicals showed reduction of lipid accumulation, microarray analysis was performed to determine the cellular response to alkaloid treatment at the molecular level. qRT-PCR experiments further confirmed that several classes of detoxification genes were specifically induced, whereas several genes in the lipid synthesis pathway were down-regulated upon treatment with alkaloids. Currently the mechanism of lipid reduction induced by alkaloid treatment is being investigated.

Chow, King L. et al. (2013) International Worm Meeting "The role of the claudin-like gene nsy-4 in C. elegans sensory ray development."

Chow, King L., Fan, Kei C.

[International Worm Meeting, 2013]

During the ray development, homeobox protein CEH-43 and T-box transcription factor TBX-2 are key transcription factors regulating the cellular assembly of the organ structure. Yet, downstream structural genes that execute the process are largely unknown. Here we present our recent findings on a potential structural gene, nsy-4. Based on our genome-wide RNAi screen, nsy-4 is required for the ray differentiation. We showed with structural cell (Rnst)-specific marker that Rnsts of most missing ray in nsy-4RNAi males are born, indicating that nsy-4 is essential for the assembly but not the lineage of ray cells. Reporter gene analyses revealed that nsy-4 is expressed in both the hypodermis and Rnst. In Rnst, nsy-4 expression is positively regulated by ceh-43 and tbx-2. The nsy-4 reporter signal in both ceh-43(RNAi) and tbx-2 mutants was reduced significantly, suggesting that the expression of nsy-4 is dependent of CEH-43 and TBX-2. These results collectively imply nsy-4 to be a ray structural gene downstream of this transcription factor network. As for its function, though nsy-4 is a worm-specific protein with no obvious homolog outside nematodes, it structurally resembles claudin in the vertebrates. Studies done on claudin-like molecules in C. elegans had shown that they have similar functions as their mammalian counterparts in establishing distinct apical junctions. This type of cell junctions are found in rays and is important for the adhesion between ray cells. Using two translational reporters, NSY-4 was confirmed to be subcellularly localized at the apical junctions in all rays throughout the assembly process. Phenotypic analysis reveals morphological defects in the developing ray, including malformation of papillae and lack of Rnst in the extending rays, both of which are associated with improper apical junction establishment. We will continue to confirm the function of nsy-4 as a claudin in establishing cellular adhesion between ray cells during the ray development. Ultimately the study would shed light on detailed mechanism guiding the assembly of this simple sensory organ. (The study is supported by Research Grants Council, Hong Kong).

Chow K-L et al. (1996) East Coast Worm Meeting "ANALYSIS OF SENSORY ORGAN DIFFERENTIATION BY STRESS INDUCED PHENOCOPY IN CAENORHABDITIS ELEGANS"

Chan KW, Wong WL, Chow K-L

[East Coast Worm Meeting, 1996]

Differentiation of sensory organs in Caenorhabditis elegans is a complex process. In addition to the determination of neural cell fate, identity choice has been shown to involve multiple genetic functions and regulatory steps. We attempt to delineate the process of identity determination by an unconventional approach addressing the timing of these functional steps by stress induced phenocopy. Results from the heat shock study reveal that critical time for formation of specific ray identity varies from ray to ray. The determination of ray 6 identity is at late L3 stage while the differentiation between ray 3 and ray 4 appears slightly later in early L4 stage. The clear definition of ray 8 and ray 9 is defined in mid L4 stage. In all these cases, heat shock treatments results in ray fusion. Ray cells extension and stabilization of the ray morphology are the latest steps for the formation of the adult sensory ray. While the features of phenocopy observed resembles that of mutations of documented genetic loci, this finding lends support to the hypothesis that sensory ray formation requires neuronal identity determination, morphogenesis and stabilization of structures dictated by genetic functions acting at different time points. We have examined the phenocopy phenomenon using various stress inducing agents. While their effect is not as dramatic as that caused by heat shock, all tested agents can induce the same phenocopy at the same developmental time frame as heat shock does. It implies that phenocopy is caused by a general stress response of cell but not due to specific effect by heat. In addition, tolerance can be induced by prior treatment of stress before the testing treatment. Using mab-21 and mab-18 promoter-driven galactosidase gene reporter as a molecular probe in transgenic worm, we show that stress response may converge on one of these regulatory genes. The result indicates that the features of phenocopy is gene specific. The mutant phenotype observed after stress is due to shut down of the expression of a genetic locus at transcription, translational, or degradation level. We propose that uncoupling of gene activation and competence of development is the cause of the phenomenon. Detail analysis of the underlying mechanism is in progress.