Mason, D Adam et al. (2009) International Worm Meeting "The DM-domain gene dmd-3 functions in multiple male-specific processes."

Murphy, Mark W, Portman, Douglas S, Nelson, Matthew D, Zarkower, David, Fitch, David HA, Mason, D Adam

[International Worm Meeting, 2009]

The majority of animal species generate two morphologically distinct sexes. In contrast to most developmental pathways, genetic pathways controlling sex differences are highly divergent between different taxa. The exception to this is the conserved use of DM domain transcription factors in determining sexual phenotypes in a variety of animals. Despite this conservation, the molecular mechanisms underlying DM domain gene function are still being elucidated. Therefore, characterizing the function of DM domain genes in C. elegans may inform our understanding of sex-specific development in all animals. C. elegans males and hermaphrodites are dimorphic for a number of traits including body size, tail morphology and gonad development. The C. elegans DM domain gene dmd-3 is expressed in a number of tissues that undergo male-specific development. For example dmd-3 is expressed strongly in the male, but not hermaphrodite L4 tail tip. The male tail tip undergoes a remodeling process that results in the male tail being blunt ended. Previous work has demonstrated that DMD-3 plays a critical role in regulating sex-specific male tail tip remodeling. Specifically, DMD-3 activity is both necessary and sufficient to induce the changes in cell shape, migration and fusion that drive retraction of the tail tip. Preliminary work suggests that DMD-3 activates a battery of genes that direct this morphogenetic process. Using a combination of biochemistry, genetics and bioinformatics we have identified a list of candidate genes that are potentially directly activated by DMD-3. These genes include the chondroitin sulfate synthase sqv-5 and the rab GTPase rab-6.2. We are further characterizing these genes to determine if they function as downstream effectors of DMD-3 activity in the male tail tip. In addition, preliminary evidence suggests that dmd-3 and mab-3, a similar DM domain gene, function together to control aspects of male gonad development. In male larvae, the linker cell guides the developing gonad to the hindgut. The linker cell is then engulfed by the hindgut cells and undergoes programmed cell death. Both dmd-3 and mab-3 are expressed in the migrating linker cell and hindgut. Furthermore, aspects of linker cell migration and programmed cell death are defective in mab-3; dmd-3 double mutant males. These findings suggest that DMD-3 mediates multiple male-specific developmental processes.

D. Adam Mason et al. (2008) Development & Evolution Meeting "The DM domain protein DMD-3 Controls Sex-Specific Morphogenesis"

Douglas Portman, D. Adam Mason

[Development & Evolution Meeting, 2008]

Morphogenesis involves complex changes in cell shape and movement that are essential for the development of the adult body form. During animal development these cellular changes are tightly regulated by upstream developmental signaling pathways to ensure that morphogenetic changes occur only in the right time and place. The mechanisms linking upstream developmental signaling pathways to downstream effector genes that direct cellular changes have not been well described. To address these issues we are studying the morphogenetic processes that shape the adult male tail. During the last larval stage the pointed tail tip of L4 males is remodeled into the blunt ended adult male tail by a complex process known as male tail morphogenesis. We have discovered that the doublesex related DM domain gene dmd-3 occupies the central node in the regulatory network controlling this remodeling. DMD-3 activity is both necessary and sufficient to induce two critical steps in male tail morphogenesis: the fusion and anteriorward retraction of the four tail tip hypodermal cells (hyp8-11). Furthermore, we have found that multiple upstream regulatory pathways control male tail morphogenesis by regulating dmd-3 expression in hyp8-11. A lin-41 mediated heterochronic pathway controls the timing of tail tip retraction by repressing dmd-3 expression until the mid-L4 larval stage. Likewise, the male specificity of morphogenesis results from the inhibition of dmd-3 expression in the hermaphrodite tail tip by the master sex determination protein TRA-1A. Finally, a pathway mediated by the Wnt target gene tlp-1 is necessary for the proper amplification/maintenance of dmd-3 expression in the tail tip. While these results yield important insights into the regulation of tail tip retraction, our data indicate that other regulatory pathways must function to control dmd-3 expression. To identify additional genes involved in the regulation of dmd-3 we are performing a genetic screen to isolate suppressors of a dmd-3 induced hermaphrodite tail tip retraction phenotype. Downstream of dmd-3 lie cellular effectors of morphogenesis. DMD-3 causes the sex-specific fusion of hyp8-11 by regulating the expression of the cell fusogen EFF-1. However, tail tip retraction proceeds in the absence of cell fusion, indicating that distinct effector genes mediate the cellular processes that cause tail tip retraction. We are taking a candidate gene approach to identify DMD-3 targets that mediate retraction. Together these results indicate that DMD-3 functions in the male tail tip to convert upstream regulatory information into downstream changes in cell shape, motility, fusion and adhesion that drive morphogenesis.

D. Adam Mason et al. (2007) International Worm Meeting "The DM domain protein DMD-3 is a direct TRA-1A target that coordinates C. elegans male tail morphogenesis."

D. Adam Mason, Douglas S. Portman

[International Worm Meeting, 2007]

The mechanism by which sexual identity is determined has been extensively studied in C. elegans and is relatively well understood. In contrast, much less is known about how this identity is communicated to downstream tissue-specific pathways to generate sexually dimorphic traits. DM domain factors, as the only known conserved family of sexual regulators, are excellent candidates for genes that function at the interface between the sex determination pathway and the development of sex-specific characteristics. Coordinated sex-specific remodeling of the larval male tail generates the sexual dimorphism of the adult tail. We have found that two DM domain proteins, MAB-3 and DMD-3, act in concert to direct this morphogenesis. mab-3; dmd-3 double mutants display a striking and complete failure in male tail tip cell fusion and retraction, resulting in a transformation of the blunt-ended male tail to that of a long, tapered hermaphrodite-like tail. dmd-3 single mutants display completely penetrant but less severe defects in male tail morphogenesis, while mab-3 mutants have only mild, low-penetrance defects. mab-3 and dmd-3 control one aspect of tail tip morphogenesis, the sex-specific fusion of tail tip hypodermal cells, by regulating the expression of the fusogen EFF-1. Thus, these two DM domain factors together are necessary to guide the tissue-specific sexual differentiation pathway that sculpts the male tail. Several findings indicate that dmd-3 is also sufficient to bring about tail tip retraction. dmd-3 expression closely coincides with tail tip morphogenesis: it is activated male-specifically in tail tip hypodermal cells immediately preceding the onset of cell fusion and retraction. We find that the timing of dmd-3 expression in these cells is controlled by the heterochronic pathway via lin-41. The spatial control of dmd-3 expression in the tail tip is regulated by lin-44/Wnt signaling through its effector tlp-1. Interestingly, the sex-specificity of dmd-3 expression is directly controlled by the master regulator of C. elegans sex determination, tra-1. Disruption of a putative TRA-1A binding site in the dmd-3 promoter results in expression of DMD-3 in the hermaphrodite tail tip hypodermis. Moreover, expression of functional DMD-3 under the control of this promoter is able to trigger some male-like tail tip retraction in hermaphrodites, demonstrating that DMD-3 expression in the tail tip is sufficient to promote male-specific morphogenesis. Together, these results indicate that dmd-3 and, to a lesser extent, mab-3, are master regulators of the tail tip morphogenetic program, linking the sex determination pathway to multiple downstream effectors of tail tip cell fusion and retraction.

D. Adam Mason et al. (2006) Development & Evolution Meeting "DMD-3 is a novel C. elegans DM domain protein that coordinates multiple aspects of male tail morphogenesis"

Douglas Portman, D. Adam Mason, Jeremy Rabinowitz

[Development & Evolution Meeting, 2006]

The large-scale remodeling of the C. elegans larval male tail provides an ideal opportunity to study the integration of the sex determination pathway with cellular differentiation and morphogenesis programs. Despite the apparent dissimilarity between sex determination pathways across phyla, proteins containing a divergent Zn-finger motif called the DM domain have been found to play key roles as sex determination effectors in animals as diverse as worms, flies and fish. Thus DM-domain factors may be thought of as sexual analogs of Hox genes, specifying sexual information in the way that Hox genes specify positional information. We have recently identified a novel C. elegans DM-domain gene, dmd-3, that is expressed male-specifically in the tail tip hypodermal cells hyp8-hyp11 during morphogenesis. dmd-3 mutant males exhibit a striking defect in male tail morphogenesis such that the blunt-ended male tail is partially transformed to a tapered hermaphrodite-like tail. In addition to this morphological defect, the fusion of the hyp8-hyp11 cells that characterizes normal tail remodeling fails to occur in dmd-3 males. Consistent with this, we have found that dmd-3 is required for the normal male-specific tail-tip expression of EFF-1, a critical regulator of developmental cell fusion. The expression pattern of dmd-3 reporters suggests that sexual, temporal and spatial inputs converge at the level of dmd-3 transcription. Spatial regulation of dmd-3 in the tail tip requires TLP-1, a transcription factor necessary for tail tip morphogenesis. We are examining the roles of the heterochronic and sex-determination pathways in regulating the temporal and sexual specificity of dmd-3. The phenotype of dmd-3 males partially overlaps with, yet is distinct from, the mutant phenotypes of mab-3 and mab-23, two other DM domain genes. We have found that mab-3 strongly enhances the tail retraction defect of dmd-3 males, indicating that these DM domain proteins act in concert to coordinate male tail morphogenesis. Our findings suggest that dmd-3 is a critical regulator of male tail morphogenesis, linking multiple upstream inputs to downstream effectors that control tail tip cell fusion and retraction.

Primakoff P et al. (2000) Trends Genet "The ADAM gene family: surface proteins with adhesion and protease activity."

Primakoff P, Myles DG

[Trends Genet, 2000]

An ADAM is a transmembrane protein that contains a disintegrin and metalloprotease domain and, therefore, it potentially has both cell adhesion and protease activities. Currently, the ADAM gene family has 29 members, although the function of most ADAM gene products is unknown. We discuss the ADAM gene products with known functions that act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic TGF-alpha release and the inflammatory response.

Monoson HL et al. (1973) Mycopathologia et Mycologia Applicata "Endoparasitic nematode-trapping fungi of Mason State Forest."

Monoson HL, Williams SA

[Mycopathologia et Mycologia Applicata, 1973]

An initial survey involving the endoparasitic nematode-trapping fungi of Mason State Forest in Illinois was conducted from 15 May to 26 July 1971. Three endoparasitic forms were isolated from the soil samples collected; one non-endoparasitic form was also isolated. Soil pH's and soil nutrient levels were determined for the investigation period.

Vuong, Edward et al. (2009) International Worm Meeting "fs8 is a new mutant that disrupts the timing of male tail morphogenesis."

Mason, Adam, Vuong, Edward, Portman, Douglas

[International Worm Meeting, 2009]

The morphogenesis of the male tail in C. elegans provides an excellent system to study the integration of sexual, spatial and temporal cues in the regulation of a developmental process. During the L4 stage, the male tail undergoes a dramatic transformation in which the the tail tip and anterior tail retract, resulting in the blunt-ended tail of the adult. As a master regulator of tail tip retraction, the DM-domain protein DMD-3 is both necessary and sufficient to specify this process. In WT males, dmd-3 is expressed in hyp8-11 starting in early L4 and its expression is quickly extinguished as retraction completes during late L4. Temporal, sexual and spatial signals act to control male tail morphogenesis by regulating dmd-3 expression. In order to better understand this process, we are studying fs8, a mutant in which males display a highly penetrant, severely unretracted Lep (Leptoderan) tail tip phenotype. In fs8 males, we found a loss of dmd-3 expression specifically in hyp10 during L4 and ectopic expression of dmd-3 in the unretracted tail tip of adults. This suggests that fs8 acts upstream of dmd-3 to affect the timing of its expression. Other evidence that indicates fs8 might be a developmental timing gene is that the Lep phenotype of fs8 mutants is suppressed by post dauer development. To place fs8 into the lin-41 mediated heterochronic pathway, we constructed lin-41(lf); fs8 double mutant males. These animals did not have Lep tails, suggesting that fs8 functions upstream/in parallel to lin-41. We have found that fs8 is on the X chromosome between +2 and +11. Further mapping is in progress. Characterization of this new mutant will provide greater insight into the complex process of male tail morphogenesis as well as the developmental timing pathways regulating it.

Yang, Ji-Sup et al. (2015) International Worm Meeting "Identification of DMD-3 targets in the C. elegans male tail tip."

Williams, Emily, Herrera, R Antonio, Ercan, Sevinc, Kiontke, Karin, Yang, Ji-Sup, Fitch, David, Mason, Adam, Littleford, Hana

[International Worm Meeting, 2015]

Cellular morphogenesis is a fundamental developmental mechanism that governs how cells migrate, change identity, change shape, and fuse. The C. elegans male tail tip undergoes a morphogenetic event in just four cells during late L4, making this a powerful model for elucidating linkage between regulators of timing, position, and epidermal fate and the cell-biological mechanisms required for morphogenesis. Male tail tip morphogenesis (TTM) is governed by DMD-3, a conserved DM-domain transcription factor that is homologous to DMRT1, which regulates male fate in humans. Expression of dmd-3 is necessary and sufficient for TTM to occur (Mason et al. 2008). Previous work done in the lab has shown that DMD-3 is at the center of a genetic network with a bow-tie structure, regulated by pathways including Wnt signaling and heterochronic regulation, and in turn regulating genes involved in cellular processes such as vesicle trafficking and cytoskeletal rearrangement (Nelson et al 2011). However, while some of the regulatory pathways involved in specifying DMD-3 expression are known, the structure of the network downstream of DMD-3 is uncharacterized. We are using two methods to investigate downstream targets of DMD-3: To identify tail tip-specific targets, we are performing transcriptome profiling of isolated tail tip tissue from worms that undergo tail tip morphogenesis (wild-type males and hermaphrodites ectopically expressing dmd-3), and worms that do not (wild-type hermaphrodites and dmd-3 mutant males). To identify direct DMD-3 transcriptional targets, we are employing chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq), utilizing a CRISPR-generated dmd-3 allele tagged with GFP and 3xFLAG. Preliminary microarray transcriptome analysis on tail tip-specific RNA identified 134 significantly differentially expressed genes in the dmd-3 mutant relative to wild-type. Combining the results of both approaches will yield direct targets of DMD-3 that act in the male tail tip.References: Mason et al. 2008. Development 135:2373-2382. Nelson et al. 2011. PLoS Genetics 7(3):e1002010.

Coates D et al. (2000) European Worm Meeting "The ADAMs family of C. elegans: what will the new members do?"

Coates D, Daulby J, Hooper NM, Hope IA

[European Worm Meeting, 2000]

ADAMs are a family of integral membrane glycoproteins containing a disintegrin and a metalloprotease domain. The physiological roles of the majority of ADAMs have yet to be elucidated, however some mammalian ADAMs are known to be involved in many diverse processes including sperm migration, sperm-egg binding and fusion, myoblast fusion, the processing of adhesion molecules, cytokines, cytokine receptors and extracellular protein domains, and in neural development. In C. elegans ADAMs are thought to be involved in cell fusion events in sperm and in epithelial cells (ADM-1), in early embryonic development (ADM-2) and in vulval development (SUP-17). In addition to these membrane anchored proteins C. elegans also has soluble ADAM-like proteases, for example GON-1, which plays an essential role in gonadal morphogenesis. We have identified four novel ADAM-like sequences in C. eleganswhich encode a TNFa converting enzyme (TACE) homologue and three soluble ADAM-like proteinases, and our aim is to determine the precise functions of these proteins. Initially we will examine their cellular location with reporter gene constructs and assess the importance of these proteinases by generating gene knockout mutants. We are also interested in finding out what the substrates of these ADAMs are and will biochemically characterise the recombinant proteins. The data we will attain will be related to the ADAM homologues found in humans.

Joanna Daulby et al. (2001) International C. elegans Meeting "Expression of C. elegans ADAM proteins in a mammalian system."

Nigel M Hooper, Joanna Daulby, David Coates, Ian A Hope

[International C. elegans Meeting, 2001]

ADAMs are integral membrane proteins which contain a d isintegrin a nd m etalloprotease domain, and have the potential to participate in proteolysis, adhesion, fusion and signaling events. So far thirty ADAM genes have been described from across the animal kingdom, but their precise functions and mechanisms remain largely unknown. A small number of mammalian ADAMs however, have been shown to be important in development, signal transduction, sperm and egg binding and muscle cell fusion. In addition, some C. elegans ADAMs have been investigated recently and these are thought to be involved in sperm and epithelial cell fusion events ( adm-1 ), in early embryonic development ( adm-2 ), and in vulval development ( sup-17 ). C. elegans also possesses homologues of ADAM-like genes known as ADAM-TS. Proteins encoded by this gene family are not membrane bound and have an additional thrombospondin-like motif, for example GON-1 and MIG-17. These proteins play essential roles in the morphogenesis of the C. elegans gonad. We are studying three other ADAMs genes in C. elegans : adm-4 (a tumour necrosis factor alpha converting enzyme or TACE homologue); and two soluble ADAM-TSs, ( C02B4.1 and T19D2.1 ). GFP reporter data indicates that adm-4 is highly expressed in most tissues in all post-embryonic stages. The expression pattern for C02B4.1 :: GFP however is more confined and is observed in body wall and pharyngeal muscle. We aim to complement the reporter data with RNAi/knockout mutagenesis work, but our main goal is to biochemically analyse the recombinant proteins in a mammalian expression system. Using this approach we hope to identify which proteins are cleaved by these ADAMs proteins, as well as providing information about their targeting and processing. Ultimately we hope to elucidate their physiological function and relate this information to mammalian ADAM proteins.