Maduro MF et al. (1993) International C. elegans Meeting "Analysis of unc-119."

Pilgrim DB, Maduro MF

[International C. elegans Meeting, 1993]

Maduro MF et al. (1995) International C. elegans Meeting "UNC-119 IS EXPRESSED THROUGHOUT THE NERVOUS SYSTEM"

Maduro MF, Pilgrim DB

[International C. elegans Meeting, 1995]

Animals mutant in unc-119 exhibit a diversity of neuronal abnormalities. Aside from a primary movement defect, mutants lay eggs even in the absence of food, are unable to enter dauer, and show varying degrees of decreased pharyngeal pumping. Analysis of muscle arrangement and sarcomere ultrastructure shows no muscular defects, suggesting that the phenotype results from a disruption of neuronal function. We localized the gene on the physical map based on its failure to complement eDf2; eDp6, which is homozygous for a break that deletes most of unc-119. A 5.5 kbp rescuing fragment contains a single transcription unit of ~1.0 kbp, encoding a polypeptide of 216 a.a. with no significant similarity to other known proteins. This fragment rescues all aspects of the mutant phenotype and also rescues the eDf2; eDp6 strain. The original allele, e2498, contains an insertion of Tc1. Two EMS-induced alleles, ed3 and ed4, contain nonsense mutations. All of the known alleles of unc-119 are therefore putative nulls. We cloned the C. briggsae homologue and found it encodes a protein 90% identical to the C. elegans gene product. Fusions of unc-119 with lacZ and GFP show that the gene is expressed shortly before comma stage and continues for the remainder of the life cycle. In adults, many if not all neurons show expression, including cell bodies and processes in the nerve cord, pharynx, tail and male sensory rays, consistent with requirement of Unc-119 in neuronal function.

Maduro, M.F. et al. (2017) International Worm Meeting "Metabolic defects caused by hypomorphic gut specification are mediated by an NHR gene."

Maduro, M.F., Choi, Hailey, Broitman-Maduro, Gina, Doan, Kollan

[International Worm Meeting, 2017]

Perturbations that compromise specification generally result in developmental arrest, making it difficult to address questions of whether or not differentiation gene networks can correct for these in later development. We are using "hypomorphic gut specification" (HGS) strains in which activation of the gut specification factors end-1 and/or end-3 in the E (gut) lineage has been compromised. In HGS strains, development of gut becomes stochastic, manifested as a delay in activation of elt-2, a gene whose product maintains the intestinal fate, and changes in the E lineage. Surviving HGS adults have normal levels of ELT-2, but they variably have excess numbers of gut cells (hyperplasia) and store excess fat, as detected by the presence of abnormally large lipid storage granules revealed by Oil Red O staining and localization of ATGL-1::GFP. Other pleiotropic effects, including a shorter life span, variable sterility and morphological changes, suggest that animals are undernourished. We do not find hyperlipidemia in cdc-25.1(rr31) adults, which contain an even greater number of gut cells than HGS animals, suggesting that hyperplasia is not the cause of hyperlipidemia. We observe an increase in expression and nuclear localization of DAF-16, a primary regulator of lipid storage. However, a daf-16 null mutant background only partially reduces lipid accumulation in HGS animals, suggesting that other genes are the primary cause of the hyperlipidemia. To identify such genes, we performed tissue-specific RNA-Seq on isolated intestines from control and HGS adults. Only a small set of intestinal genes appears to be differentially expressed. One of these, a previously uncharacterized nuclear hormone receptor (NHR) gene, is strongly activated in the anterior gut in controls but downregulated in HGS animals. A null mutant in the gene induced by CRISPR/Cas9 results in adults that retain abnormally high amounts of lipids, suggesting that a major component of the fat accumulation in HGS strains is lack of expression of this NHR. Our results suggest two models by which HGS leads to hyperlipidemia and changed metabolism. First, compromised activation of specification through END-1,3 may prevent robust establishment of normal metabolism due to a failure to activate some early factors other than elt-2. In a second model, the anterior cells of the gut may undergo a transformation in fate to a more posterior type of gut cell, resulting in a failure to adequately digest bacteria arriving from the pharynx. This might cause a decrease in digestive capacity of the gut, resulting in starvation-like defects. Together, our results connect progenitor specification with function of the differentiated organ, showing that a failure of robust early gene expression can result in abnormalities that are not corrected by activation of a terminal organ identity gene.

Maduro MF et al. (2000) Worm Breeder's Gazette "INFLUENCE OF 3'UTR ON TIMING OF GFP REPORTER EXPRESSION ONSET"

Maduro MF, Rothman JH, Lin R, Robertson SM

[Worm Breeder's Gazette, 2000]

Reporter transgenes are frequently used to obtain a preliminary idea of the expression pattern of an endogenous gene and to assess the in vivo function of cis-acting control elements. The most widely used reporter plasmids, made available by the Fire lab, encode the S65C form of GFP coupled with the 3'UTR of the unc-54 gene. We have been studying the expression of med-1,2 and are now able to make some generalizations that might be useful for the study of genes with early zygotic expression. The med-1,2 genes are direct targets of maternal SKN-1, and are required for SKN-1-dependent specification of EMS cell fates. Global RT-PCR/Southern analysis on individual embryos has shown that med-1 message accumulates in 4-cell (EMS) stage embryos. Our first reporter transgenes, however, showed later expression onset than might be expected from these data. Reporters were constructed by cloning DNA upstream of the translation start sites of med-1 or med-2. The GFP transgenes showed onset of expression in E and MS, just as they are dividing to form MSa/p and Ea/p. Somewhat inexplicably, all med transgenes made to date with lacZ, either alone or fused to GFP, express very weakly or not at all. We next found that translational fusion 'tag' reporters containing the med-1 3'UTR show expression onset earlier than similar constructs with the unc-54 3'UTR. Such GFP-tagged med-1 transgenes show GFP fluorescence at the birth of E and MS. With a Chemicon anti-GFP antibody, transgene product first appears on the chromosomes of EMS at anaphase. We replaced the GFP coding region of the med-1::GFP::MED-1 reporter with the 35 carboxyl-terminal amino acids of c-MYC, which contains the MYC epitope EQKLISEEDL. Using Boehringer mAb 9E10, we have seen nuclear MYC::MED-1 in late prophase of EMS at the 6-cell stage. Similar results were obtained with the end-3 gene, which along with end-1 is required for E specification. Both end-3::GFP and end-3::lacZ fusions begin expressing in the E daughters, Ea and Ep, while expression of an end-3::END-3::MYC transgene containing the end-3 3'UTR can be seen in the nucleus and cytoplasm of E. We conclude that (1) expression of some reporters can occur earlier when their own 3'UTR, rather than that of unc-54, is used, and (2) an epitope tag, such as MYC, may allow even earlier detection of transgene product.

Maduro MF et al. (2000) West Coast Worm Meeting "Multiple regulatory elements activate end-1 expression in the E lineage"

Kasmir JJ, Maduro MF, Rothman JH

[West Coast Worm Meeting, 2000]

end-1 encodes a GATA factor that is sufficient to specify the identity of the sole endoderm precursor, the E blastomere. By dissecting the end-1 regulatory region, we have identified elements that control the levels and specificity of end-1 expression. end-1 is activated by at least one maternal factor, SKN-1. Heat-shock-driven expression of SKN-1 results in widespread end-1 expression. In addition, end-1 appears to be activated by zygotically expressed GATA factors: ectopic expression of either END-3, the genetically redundant partner of END-1, or MED-1, another zygotic GATA factor (see abstract by Maduro and Rothman), also result in widespread end-1 expression. We are testing whether conserved SKN-1 and GATA consensus binding sites are directly responsive to SKN-1, END-3 and MED-1 action. While embryos lacking SKN-1 do not express detectable MED-1, end-1 is still expressed, albeit at low levels, and gut is still made 30% of the time. This observation suggests the existence of yet another activator of end-1. Previously, we reported that POP-1, a Lef-1 like protein that represses end-1 expression in MS, appears to activate end-1 and gut differentiation in the E lineage in response to the MAPK/Wnt signal that induces endoderm. However, we have been unable to identify a single region in the end-1 promoter that is necessary for this POP-1-dependent activation of end-1. Indeed, our genetic and biochemical data indicate that multiple sites may be necessary for activation of end-1 in the E lineage by Wnt/MAPK-activated POP-1. Our further analysis has uncovered several important regulatory elements that are necessary to establish normal levels of end-1 expression. Some of these elements contain no consensus sites for known regulators, further indicating the existence of multiple positive inputs.

Maduro MF et al. (1996) West Coast Worm Meeting "THE LG III DEFICIENCY eDf2 AND ASSOCIATED FREE DUPLICATION eDp6 ARE NOT MUTUALLY COMPLEMENTARY"

Maduro MF, Pilgrim DB

[West Coast Worm Meeting, 1996]

The deficiency eDf2 and the free duplication eDp6 were obtained concomitantly following acetaldehyde mutagenesis (Hodgkin, 1980). These two aberrations have been useful in mapping many LG III loci, which have demonstrated that eDp6 contains the right third of LG III and eDf2 contains the left portion. We used the eDf2 and eDp6 breakpoints as physical markers to clone unc-119, and showed that the genomic region corresponding to the unc-119 cDNA is completely absent. The gene unc-119 is the only LG III locus to not map to either eDf2 or eDp6. Our interest in these aberrations was renewed when we ran across a report by Starich et al. on the isolation of dyf-2 which maps to the far end of LG III(R) and is complemented by both eDf2 and eDp6. Using a plasmid mini-library approach, we walked across the eDf2 breakpoint, and probed the YAC grid to find that eDf2 contains sequences from the far right end of III. What is strange is that these same sequences are also present on eDp6, suggesting that eDf2 and eDp6 do not result from a simple chromosomal break, and the use of these rearrangements for mapping genes on LG III(R) should be approached with caution. The mapping evidence will be summarized and a model for the creation of eDf2 and eDp6 will be given.

Maduro MF et al. (1998) West Coast Worm Meeting "A SECOND GATA TRANSCRIPTION FACTOR, END-3, ACTS IN PARALLEL WITH END-1 TO DETERMINE ENDODERM"

Priess JR, Newman-Smith ED, Maduro MF, Hill RJ, Rothman JH

[West Coast Worm Meeting, 1998]

An ~250 kbp genomic region on LG V (the Endoderm Determining Region or EDR) is required for specification of the C. elegans endoderm; deletions that remove this region cause the E blastomere to adopt a fate similar to that of its cousin, C. Gut development can be rescued by end-1, a gene in the EDR encoding a single-finger GATA transcription factor that is expressed specifically in the E lineage. Extensive screens for point mutations in zygotic genes that result in the penetrant absence of gut have identified only large deletions that remove the entire EDR, suggesting that end-1 may act redundantly with other genes in the EDR. Indeed, a second gene, end-2, encoding a steroid receptor-like protein, is also capable of rescuing EDR deficiencies (see abstract by Newman-Smith et al.) An apparent point mutation in the EDR, zu247, causes an impenetrant gutless phenotype and frequently leads to an E Æ MS transformation. Although end-1 can rescue this mutant, no sequence alterations were found in either the end-1 or end-2 genes from the zu247 mutant. From the C. elegans genomic sequence we identified a single-finger GATA factor that is the closest known relative to END-1. This protein is encoded approximately 30 kbp to the right of end-1, within the EDR. We found that this gene, end-3, rescues the endoderm specification defect of an EDR deficiency. Moreover, the sequence of one residue in the zinc finger domain of END-3 is altered in the zu247 mutant; experiments are underway to examine whether this alteration is responsible for the phenotype of the zu247 mutant. These observations suggest that two GATA factors, END-1 and END-3, may act in parallel to specify endoderm and that the zu247 mutation may be neomorphic. Expression analysis and the effect of ectopically expressing end-3 will be presented. Further studies to address whether the end genes perform unique or entirely overlapping functions are in progress.

Maduro MF et al. (1998) West Coast Worm Meeting "GOTTA LOTTA GATAs: THE DIVERSE ROLES OF GATA FACTORS IN C. ELEGANS EMBRYOS"

Rothman JH, Koh K, Newman-Smith ED, Maduro MF, Strohmaier K

[West Coast Worm Meeting, 1998]

Zinc-finger transcription factors of the GATA type (which bind the consensus sequence WGATAR) are known in fungi, worms, flies and vertebrates. In animals, GATA factors are implicated in regulating differentiation of a range of different cell types. Six GATA factors, each with two fingers, have been identified in vertebrates, three of which are involved in hematopoesis, and three others in formation of organs such as heart and endodermal organs. To evaluate the range of developmental events that GATA factors might direct in an animal, we are analyzing the expression and function of a number of C. elegans GATA factors. From the C. elegans genomic sequence, we can now deduce how many GATA factors are required to make a metazoan. Five were known from genetic and molecular studies: ELT-1 and ELT-3 are involved in development of the embryonic epidermis (Page et al. 1997; Gilleard and Shafi, wm97e186) and ELT-2, END-1 and END-3 regulate specification of the endoderm and differentiation of the gut (Fukushige et al. wm97e169; Zhu et al. 1997; Maduro et al. this meeting). Blast searches of the C. elegans sequence database led us to identify at least four more apparent GATA factors, tentatively called ELT-4 through 7. All appear to be single-finger GATA factors. elt-4 and elt-5, adjacent genes on the left end of IV, appear to correspond to a polycistronic unit; these genes are expressed in, and appear to be involved in the development of, seam cells among other cell types (see abstract by Koh et al.). Although GFP constructs with elt-6 X (T24D3.1) have been uninformative, and no phenotype was observed by RNAi, ectopic expression of ELT-6 shows that it can promote pharynx development at the expense of other cell types: hs-elt-6 embryos often arrest with large numbers of pharynx muscles and no intestine (i.e., the reciprocal effect of the endoderm-specific GATA factors). Analysis of elt-7 V (cosmid C18G1) is in progress. Our tentative conclusion is that most, if not all GATA factors in C. elegans, function in distinct embryonic specification and/or differentiation events. References: Page et al. (1997) Genes & Dev. 11: 1651-1661. Zhu et al. (1997) Genes & Dev. 11: 2883-2896.

Maduro MF et al. (1997) International C. elegans Meeting "UNC-119 ENCODES A NOVEL, CONSERVED NEURAL GENE WITH A SUSPECTED ROLE IN GROWTH CONE EXTENSION"

Maduro MF, Pilgrim DB

[International C. elegans Meeting, 1997]

Animals mutant for unc-119 display poor locomotion, constitutive pharyngeal pumping, a weak egg laying defect and a daf-7-suppressible inability to form dauer larvae[1]. By using an unc-119::GFP transgene to mark the nervous system, we have revealed gross structural abnormalities in unc-119 mutants: A defasciculated ventral nerve cord and highly branched commissures. Consistent with a structural neuronal defect, animals also exhibit poor FITC uptake into sensory neurons, and an inability to increase egg laying in response to exogenous imipramine. These defects suggest a role for UNC-119 in axonal outgrowth, perhaps in specifying the cytoskeletal rearrangements that accompany growth cone extension in response to a gradient. The predicted sequence of UNC-119 is similar to that of HRG4, a novel human gene isolated from a retina cDNA library using a subtractive hybridization approach[2]. All of the unc-119 defects can be fully complemented by a fusion of the nematode promoter with HRG4, consistent with the functional conservation of this gene. Using the similarities between UNC-119 and HRG4, we have also identified a putative homolog from the fruit fly Drosophila melanogaster. We shall describe the mutant phenotype of unc-119 and the rescue by the human transgene, as well as present preliminary data from a yeast two-hybrid screen. [1]Maduro, M and Pilgrim, D. (1995) Genetics 141:977-988. [2]Higashide, T. et al. (1996) J. Biol. Chem. 271:1797-1804.

Maduro MF et al. (1996) West Coast Worm Meeting "unc-119 ENCODES A NOVEL, FUNCTIONALLY CONSERVED NEURONAL PROTEIN WITH AN APPARENT ROLE IN AXONAL OUTGROWTH"

Pilgrim DB, Maduro MF

[West Coast Worm Meeting, 1996]

Mutations in unc-119 result in worms with defects in movement, egg laying and dauer formation. Using unc-119::GFP as a marker for the nervous system, we have observed deformed neuronal processes in unc-119 mutants, while the same expression constructs display no abnormalities in wild type animals. Previous searches of the databases with the unc-119 ORF showed no significant similarities to database sequences. However, a novel retina-specific gene (HRG4) was recently added, and shows strong conservation to UNC-119 suggesting that they may be homologues. To test this hypothesis, a portion of the HRG4 cDNA was amplified from total human retina RNA and subcloned into an unc-119 promoter construct. We found that this construct can rescue the movement and egg laying defects of unc-119 mutants, but not the dauer-forming defect. We will present the preliminary evidence for an outgrowth defect in unc-119 mutants, describe the transgene experiments, and hopefully suggest a role for UNC-119 in nervous system development consistent with the mutant phenotype.