C Clucas et al. (1999) International C. elegans Meeting "GFP transgene expression pattern based mutant screens to identify genes involved in endoderm development in Caenorhabditis elegans."

I Johnstone, C Clucas

[International C. elegans Meeting, 1999]

The C. elegans gut is exclusively derived from the E lineage. At hatch, the gut consists of 20 mononucleate cells. Although the gut cells do not divide post embryonically, DNA replication and nuclear division occurs in most gut cells during L1 producing bi-nucleate cells, and endoreduplication of all gut nuclei occurs during all larval stages. We have recently performed forward genetic screens on worms containing an integrated gut specific GFPlacZ reporter gene to identify genes involved in various aspects of gut development. The pattern of GFP fluorescence was used to detect mutants and for preliminary phenotype characterisation. Various interesting classes of phenotypes were obtained including mutants with extra gut cells, mutants with multi-nucleate cells (possibly tetra-nucleate), mutants with fewer gut nuclei (but correct number of cells) and mutants with an altered GFP/gut nuclei integrity. We have so far characterised one mutation belonging to the class with extra gut cells (as indicated by MH27 cell boundary staining). lin-60 ( ij048 ) maps 0.5MU to the right of dpy-5 on chromosome I. Further experiments are underway to investigate the nature of alterations to the E lineage in this mutant. lin-60 ( ij048 ) displays some interesting genetics. Current data would suggest both maternal and zygotic components to function. ij048 is at least partly dominant.

Wiegner O et al. (1997) International C. elegans Meeting "CELL SPECIFICATION IN NEMATODES: CONSIDERABLE DIFFERENCES BETWEEN C. ELEGANS AND CEPHALOBUS"

Schierenberg E, Dinger C, Wiegner O

[International C. elegans Meeting, 1997]

Experimental analysis of C. elegans development has revealed that specification of cell fate requires a number of cell-cell interactions between adjacent early blastomeres. We compared the pattern of gut specification in C. elegans with that in Cephalobus spec., another representative of free-living rhabditid nematodes. Surprisingly, we found considerable differences. a) In C. elegans gut specification requires an inductive interaction in the 4-cell embryo between the germline cell P2 and its somatic sister EMS (gut precursor). In contrast, in Cephalobus gut specification does not require induction: The EMS blastomere produces gut cells autonomously. b) Cell isolation experiments show that in C. elegans EMS is the only cell which carries the potential to form gut. In Cephalobus, however, EMS and its somatic neighbour AB, both, are able to generate gut cells, if cultured in isolation. c) Since in normal Cephalobus development, as in C. elegans, gut derives exclusively from descendants of the EMS blastomere, AB and EMS seem to compete for gut fate. Thus, gut specification in Cephalobus does not depend on an inductional event as in C. elegans but instead requires inhibitory cell-cell interaction.

Yanjun, Shi et al. (2021) International Worm Meeting "A gut neuroendocrine signal regulates synaptic assembly in the brain"

Yanjun, Shi, Zhiyong , Shao, Lu, Qin

[International Worm Meeting, 2021]

The gut-brain axis plays an essential role in regulating neural development in response to internal environmental stimuli, such as microbes or nutrients. Defects in gut-brain communication can lead to various neurological disorders. However, it remains unknown whether gut plays an intrinsic role in regulating neuronal development. Through a genetic screen in C. elegans, we uncovered that an intrinsic Wnt-endocrine pathway in gut regulates synaptic development and neuronal activity in brain. Specifically, the Wnt signaling upregulates the expression of the neuropeptide NLP-40 in the gut and facilitates presynaptic assembly through the neuronal expressed GPCR AEX-2 receptor during development. The NLP-40 acts most likely through modulating neuronal activity and promoting synaptic protein trafficking. Our study reveals a novel role of gut in synaptic development in the brain and provides additional molecular basis for gut-brain interactions. Key words: Gut-brain axis; Synaptic development; Wnts; Endocrine; Neuropeptide/NLP-40; GPCR/AEX-2; Neuronal activity

JD Kormish et al. (1999) International C. elegans Meeting "A genetic screen for genes involved in gut development and differentiation"

JD McGhee, JD Kormish

[International C. elegans Meeting, 1999]

To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific elt-2 GATA factor appears necessary for correct gut cell development. elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302

Morris, C. et al. (2017) International Worm Meeting "Screening the Million Mutation collection for defects in gut granule biogenesis."

Hermann, G., Dewey, M., Morris, C., Voss, L., Delahaye, J.

[International Worm Meeting, 2017]

C. elegans intestinal cells are characterized by the presence of gut granules, lysosome-related organelles (LROs) that contain autofluorescent and birefringent material. Gut granule biogenesis requires the activity of evolutionarily conserved genes that function in directing and transporting proteins to LROs. There are likely many undiscovered genes necessary for the generation of LROs. To identify these genes, we have screened 1336 Million Mutation Project (MMP) strains and found 137 strains with altered gut granule formation, size, and/or positioning. We identified 78 MMP strains with altered gut granule biogenesis. Seven strains have nonsense mutations likely to disrupt the function of 5 genes known to function in gut granule formation. 23 strains have missense mutations in 10 genes that act in gut granule formation. The 48 remaining strains lack mutations in known gut granule biogenesis genes. In 21 of these strains, we have characterized multiple alleles and used rescue experiments to identify mutations in glo-9, glo-10, lyst-1, and wht-2 as causing the defects in gut granule formation. These four genes represent new factors promoting gut granule biogenesis. We are continuing experiments to identify the genes causing altered gut granule formation in the 27 remaining strains. 90 of the MMP strains have increased or decreased gut granule size. Four of these strains have nonsense mutations likely to disrupt function of 2 genes known to play a role in gut granule morphology. 13 strains have missense mutations in 9 genes known to play a role in both gut granule morphology and biogenesis. In 11 strains we have used multiple alleles to identify mutations in glo-9 and glo-10 as causing alterations in gut granule morphology. We are continuing our analyses of the remaining 62 strains. We identified two strains with altered gut granule positioning and have used rescue experiments to show that they result from mutations in wht-2 and wht-7. Here we present our analyses of glo-9, which encodes a regulatory protein that likely impacts Arf GTPase activity. glo-9 mutants display enlarged gut granules that mislocalize the gut granule proteins LMP-1 and CDF-2::GFP. Introduction of N- or C-terminally GFP tagged forms of GLO-9 rescue these phenotypes. Introduction of GLO-9 containing a point mutation predicted to alter its effects on Arf activity no longer rescues, consistent with GLO-9 acting as an Arf regulator in gut granule formation. The GFP tagged forms of GLO-9 localize to the Golgi, which is often closely associated with gut granules. Together this work implicates Arf mediated trafficking between the Golgi and gut granules in LRO biogenesis.

Greg J Hermann et al. (2001) International C. elegans Meeting "Analysis of glo-1 which lacks autofluorescent and birefringent intestinal granules"

James R Priess, Greg J Hermann, Russell J Hill

[International C. elegans Meeting, 2001]

GFP reporters are very useful in determining the expression pattern, localization, and function of genes in C. elegans . However, the analysis of GFP expression can be hindered by the strong fluorescence emitted from the birefringent and autofluorescent granules (called gut granules) present within the cytoplasm of intestinal cells. Here we report the identification and characterization of glo-1 , a mutant that lacks gut granules. The loss of gut granules in the glo-1 strain make it ideal for the analysis of GFP expression patterns. Gut granules first appear in the intestine of bean stage embryos at a time when the polarizing intestinal cells associate into an epithelium. During epithelial polarization, gut granules become asymmetrically positioned near the future basal membrane. In screens for intestinal polarity mutants, we identified a mutant called glo-1 (for Gut granule LOss) that causes gut granules to be targeted apically and to be secreted into the intestinal lumen during embryogenesis. glo-1 mutants also lack autofluorescent and birefringent gut granules in larval and adult stages. Homozygous glo-1 animals are reasonably healthy and fertile indicating that gut granules are not essential for viability. We are currently cloning the glo-1 gene. Gut granules may represent a specialized endocytic compartment involved in storage or degradation. Therefore, defects in polarized vesicular transport or targeting might result in the secretion of gut granules seen in glo-1 mutants. In the future, we will analyze the role of glo-1 in these processes.

A Davies et al. (1999) International C. elegans Meeting "gut-2 and gut-4 Encode Proteins with Similarity to Sm-like Proteins"

A Davies, J Shaw, M Moseley

[International C. elegans Meeting, 1999]

Maternal effect lethal mutations in gut-2 and gut-4 lead to very similar embryonic defects including a very penetrant failure to produce differentiated gut cells and pharyngeal muscle cells, a failure of E cells to elongate their cell-cycle at the 26-cell stage of embryogenesis and a failure to gastrulate. Embryos arrest development with an approximately wild-type number of cells and differentiated hypodermal, body-wall muscle and neuronal tissues. Laser ablation of early blastomeres in embryos from mutant mothers indicate that E and MS produce hypodermal, body-wall muscle and neuronal cells. These tissues are normally produced by the C blastomere; however, the number of neuronal cells derived from the mutant E and MS blastomeres suggests that they do not take on precisely a C-like fate. Deletion mutations of gut-2 have a slow-growing, sterile phenotype and are thus stronger than the maternal effect lethal mutations. Cloning of gut-2 and of gut-4 has revealed that both of these genes encode proteins with similarity to Sm-like proteins, which are related to core spliceosomal snRNP proteins. A functional GUT-2::HA protein is localized to nuclei and is expressed in most cells, including the germline. The lesions in two gut-2 alleles and four gut-4 alleles have been analyzed; all six mutations affect the same conserved glycine residue in the GUT-2 and GUT-4 proteins. Thus the maternal effect lethal mutations are special alleles of these genes. It is possible that these special alleles affect a particular function of gut-2 and gut-4 leading to the observed defects in E- and MS-derived cell fate. Alternatively, these special alleles may reduce the efficiency of general splicing so that the production of mature mRNA is reduced for a few transcripts whose levels are critical for specifying the fates of these cells.

Marcela Kokes et al. (2008) Development & Evolution Meeting "GLO-3, a Novel Gut Granule Associated Protein, Regulates Gut Granule Formation in C. elegans"

Greg Hermann, Beverley Rabbitts, Steven Levitte, Marcela Kokes

[Development & Evolution Meeting, 2008]

Caenorhabditis elegans intestinal cells are characterized by the presence of gut granules, lysosome-related organelles that contain autofluorescent and birefringent material. Gut granule formation requires the activity of AP-3 subunits, HOPS, and GLO-1/Rab38, genes whose homologues function in trafficking to lysosomes and lysosome-related organelles. To define the molecular and cellular pathways that function in gut granule biogenesis, we have isolated a collection of mutants that lack and/or mislocalize birefringent material into the embryonic intestinal lumen. The glo (gut granule loss) mutants define at least eight different genes. Here we present our phenotypic and molecular analysis of glo-3. We have cloned glo-3 and find that it encodes a novel predicted membrane protein that is associated with gut granules. Interestingly, glo-3(-) alleles fall into three different phenotypic classes based upon the number of birefringent gut granules present within embryonic intestinal cells. "Strong" glo-3(-) alleles completely lack the compartment while "weak" glo-3(-) alleles contain a reduced number of enlarged gut granules. All glo-3(-) alleles contain a reduced number of gut granules at the adult stage. We are using this characteristic of glo-3(-) alleles to identify other pathways that function in gut granule formation.

Greg J Hermann et al. (2002) West Coast Worm Meeting "glo-1 is necessary for lysosome-related organelle biogenesis in C. elegans"

Greg J Hermann, James R Priess, Caroline A Hieb

[West Coast Worm Meeting, 2002]

Lysosome-related organelles represent a diverse group of specialized, cell type specific organelles that share a significant number of characteristics with conventional lysosomes. The C. elegans intestinal-specific gut granule is a member of this group of organelles. Gut granules are acidified, contain lysosomal proteins, and are the terminal endocytic compartment. However, unlike other lysosomes in C. elegans, gut granule contents are birefringent and autofluorescent, and they are likely to have a specialized role in the digestive physiology of the animal. To identify the pathways involved in the biogenesis of lysosome-related organelles, we screened for mutants with defects in gut granule assembly. From our screens we identified three glo (for Gut granule LOss) genes that are necessary for gut granule biogenesis. Here we present our analysis of glo-1. glo-1 mutants mislocalize gut granule contents to the intestinal lumen during embryogenesis. In addition, glo-1 larvae and adults lack birefringent and autofluorescent gut granules and do not contain acidified and terminal endocytic compartments in the intestine. Surprisingly, despite the loss of functional lysosomes in the intestine, glo-1 animals are viable and fertile.

Maxfield, A. et al. (2019) International Worm Meeting "The regulation of Rab activity in trafficking to gut granules."

Morris, C., Voss, L., Lavoy, S., Maxfield, A., Foster, O., Handa, S., Hermann, G.

[International Worm Meeting, 2019]

C. elegans gut granules are intestinally restricted lysosome-related organelles (LROs) that are distinct from, and coexist with, conventional lysosomes. The formation of gut granules requires the Rab32/38 family member GLO-1. Our genetic and cellular data suggest that GLO-3 and CCZ-1 function as a guanine nucleotide exchange factor (GEF) for GLO-1 recruiting it to the gut granule membrane. We present yeast 2- and 3-hybrid results that point to direct physical interactions between these three proteins. Using rescuing forms of glo-1 and glo-3, where gfp was inserted into its genomic locus, we have identified the subcellular sites where GLO-1 is likely activated and localized. By screening our collection of gut granule biogenesis mutants we found that wht-2(-) mutants lead to the loss of GLO-1 from gut granules similar to glo-3(-) mutants. WHT-2 is an ABCG family membrane transporter, which localizes to the gut granule membrane. wht-2(-) mutants contain partially formed gut granules and in combination with other LRO biogenesis mutants, leads to the disruption of gut granule protein trafficking. Unlike glo-3(-) and ccz-1(-) mutants, a fast nucleotide exchange GLO-1 mutant does not restore the gut granule association of GLO-1 or gut granule formation in wht-2(-) mutants. Instead, WHT-2 is required for the ability of GLO-1 fast nucleotide exchange mutants to bypass the requirement of CCZ-1 and GLO-3 in gut granule biogenesis. Together, these data point to a GEF independent role of WHT-2 in GLO-1 organelle recruitment and activation. This likely requires the membrane transport function of WHT-2, as point mutations that disrupt its ATPase activity cause the loss of GLO-1 from gut granules.