Elegans Male Tail Research Articles

Quantifying the Over-retraction (Ore) phenotype in C. elegans male tail tip morphogenesis.

Morphometrics, the quantitative analysis of biological structures, reduces subjectivity and increases reproducibility in characterizing morphological phenotypes. In C. elegans males, the rounded adult tail tip emerges from a stage-specific retraction of epidermal cells regulated by the heterochronic pathway via LIN-41/TRIM71. Precocious tail tip morphogenesis in lin-41 reduction-of-function conditions results in a blunted tail (Ore) phenotype, previously described qualitatively (Del Rio-Albrechtsen et al., 2006). We present a quantitative method to assess the Ore phenotype by measuring the tail tip position relative to the cloacal opening. This method can be used to study variation in Ore phenotypes and to validate lin-41 loss-of-function reagents.

The novel gene, mtre-1 , is expressed downstream of MAB-3 and DMD-3 in the male tail tip at the termination of male tail tip retraction.

The development of the adult C. elegans male tail involves an extensive remodeling during the last larval stage where the pointed tail of the L4 male is converted into the blunt-ended adult tail with its collection of mechano-sensitive rays. The first step in this remodeling is the retraction of the four hypodermal cells of the tail tip to generate the blunt-ended tail. Male tail tip retraction is an excellent model for characterizing how upstream regulatory networks interact with the downstream cell biological effectors that drive morphogenetic changes in all animals. Previously, we've shown that two DM-domain transcription factors, MAB-3 and DMD-3 , are central regulators of male tail tip retraction. Using a microarray-based approach we have identified ~400 genes that are more highly expressed in the L4 male tail tip relative to the hermaphrodite L4 tail tip. The uncharacterized gene T05H10.3 , which we've named mtre-1 , was highly over-represented in the male tail tip vs. the hermaphrodite tail tip and was under-represented in mab-3 ; dmd-3 mutant male tail tips vs. wild-type male tail tips. A transcriptional reporter for mtre-1 shows clear expression in the male tail tip cells for a short period (~3 hours) at the end of retraction. This expression is dependent on the activity of MAB-3 and DMD-3 , since expression is reduced in dmd-3 single mutant males and absent in mab-3 ; dmd-3 mutant males. Finally, males homozygous for a putative null allele of mtre-1 display a phenotypically wild-type adult male tail, indicating that mtre-1 is not essential for male tail morphogenesis.

Function of cell adhesion molecules in differentiation of ray sensory neurons in C. elegans.

For proper functioning of the nervous system, it is crucial that neurons find their appropriate partners and build the correct neural connection patterns. Although cell adhesion molecules (CAMs) have been studied for many years as essential players in neural connections, we have yet to unravel the code by which CAMs encode synaptic specificity. We analyzed the effects of mutations in CAM genes on the morphology and synapses of a set of sensory neurons in the Caenorhabditis elegans male tail. B-type ray sensory neurons express 10 genes encoding CAMs. We examined the effect on axon trajectory and localization of pre-synaptic components in viable mutants of nine of these. We found axon trajectory defects in mutants of UNC-40/DCC, SAX-3/ROBO, and FMI-1/Flamingo/Celsr1. None of the mutations caused loss of pre-synaptic components in axons, and in several the level even appeared to increase, suggesting possible accumulation of pre-synapses. B-type sensory neurons fasciculate with a second type of ray sensory neuron, the A-type, in axon commissures. We found a CAM expressed in A-type functions additively with a CAM expressed in B-type in axon guidance, and lack of a CAM expressed in B-type affected A-type axon guidance. Overall, single and multiple mutants of CAM genes had limited effects on ray neuron trajectories and accumulation of synaptic components.

C. elegansPVF-1 inhibits permissive UNC-40 signalling through CED-10 GTPase to position the male ray 1 sensillum

Graded distributions of netrin and semaphorin guidance cues convey instructive polarity information to migrating cells and growth cones, but also have permissive (i.e. non-polarity determining) functions in mammalian development and repair. The permissive functions of these cues are largely uncharacterised at a molecular level. We found previously that UNC-6 (netrin) signals permissively through UNC-40 (DCC) and UNC-5 receptors to prevent anterior displacement of the ray 1 sensillum in the C. elegans male tail. UNC-6/UNC-40 signalling functions in parallel with SMP-1 (semaporin 1)/PLX-1 (plexin) signalling to prevent this defect. Here, we report that a deletion allele of pvf-1, which encodes a VEGF-related protein, causes no ray 1 defects, but enhances ray 1 defects of a plx-1 mutant, and unexpectedly also suppresses unc-6(ev400)-null mutant ray 1 defects. These mutant ray 1 inductive and suppressive effects are mimicked by the ability of unc-40(+) and ced-10(gain-of-function) multi-copy transgene arrays to induce ray 1 defects or suppress unc-6 mutant ray 1 defects, depending on their dosage, suggesting the pvf-1 mutation causes UNC-40 overactivity that interferes with signalling but is partially sensitive to UNC-6. Additional data suggest PVF-1 functions through four VEGF receptor-related proteins and inhibits only CED-10 (a GTPase), but not MIG-2-dependent UNC-40 activity, even though UNC-40 functions through both GTPases to position ray 1. pvf-1 and receptor mutant ray 1 defects are rescued by transgenes expressing mouse VEGF164 and human VEGF receptors, respectively. These data report the first case of VEGF-induced inhibition of the netrin signalling and a molecular conservation of VEGF function from worms to humans.

Semaphorin-1 and Netrin Signal in Parallel and Permissively to Position the Male Ray 1 Sensillum in Caenorhabditis elegans

Netrin and semaphorin axon guidance cues have been found to function in the genesis of several mammalian organs; however, little is known about the underlying molecular mechanisms involved. A genetic approach could help to reveal the underpinnings of these mechanisms. The most anterior ray sensillum (ray 1) in the Caenorhabditis elegans male tail is frequently displaced anterior to its normal position in smp-1/semaphorin-1a and plexin-1/plx-1 mutants. Here we report that UNC-6/netrin and its UNC-40/DCC receptor signal in parallel to SMP-1/semaphorin-1a and its PLX-1/plexin-1 receptor to prevent the anterior displacement of ray 1 and that UNC-6 plus SMP-1 signaling can account entirely for this function. We also report that mab-20/semaphorin-2a mutations, which prevent the separation of neighboring rays and cause ray fusions, suppress the anterior displacements of ray 1 caused by deficiencies in SMP-1 and UNC-6 signaling and this is independent of the ray fusion phenotype, whereas overexpression of UNC-40 and PLX-1 cause ray fusions. This suggests that for ray 1 positioning, a balance is struck between a tendency of SMP-1 and UNC-6 signaling to prevent ray 1 from moving away from ray 2 and a tendency of MAB-20/semaphorin-2a signaling to separate all rays from each other. Additional evidence suggests this balance involves the relative adhesion of the ray 1 structural cell to neighboring SET and hyp 7 hypodermal cells. This finding raises the possibility that changes in ray 1 positioning depend on passive movements caused by attachment to the elongating SET cell in opposition to the morphologically more stable hyp 7 cell. Several lines of evidence indicate that SMP-1 and UNC-6 function permissively in the context of ray 1 positioning.

A Bow-Tie Genetic Architecture for Morphogenesis Suggested by a Genome-Wide RNAi Screen in Caenorhabditis elegans

During animal development, cellular morphogenesis plays a fundamental role in determining the shape and function of tissues and organs. Identifying the components that regulate and drive morphogenesis is thus a major goal of developmental biology. The four-celled tip of the Caenorhabditis elegans male tail is a simple but powerful model for studying the mechanism of morphogenesis and its spatiotemporal regulation. Here, through a genome-wide post-embryonic RNAi-feeding screen, we identified 212 components that regulate or participate in male tail tip morphogenesis. We constructed a working hypothesis for a gene regulatory network of tail tip morphogenesis. We found regulatory roles for the posterior Hox genes nob-1 and php-3, the TGF-β pathway, nuclear hormone receptors (e.g. nhr-25), the heterochronic gene blmp-1, and the GATA transcription factors egl-18 and elt-6. The majority of the pathways converge at dmd-3 and mab-3. In addition, nhr-25 and dmd-3/mab-3 regulate each others' expression, thus placing these three genes at the center of a complex regulatory network. We also show that dmd-3 and mab-3 negatively regulate other signaling pathways and affect downstream cellular processes such as vesicular trafficking (e.g. arl-1, rme-8) and rearrangement of the cytoskeleton (e.g. cdc-42, nmy-1, and nmy-2). Based on these data, we suggest that male tail tip morphogenesis is governed by a gene regulatory network with a bow-tie architecture.

Semaphorin signaling in morphogenesis: found in translation: Figure 1.

Semaphorins play diverse roles in axon guidance and epithelial morphogenetic cell movements. In this issue of Genes & Development, Nukazuka and colleagues (1025-1036) show that semaphorins regulate Caenorhabditis elegans male tail morphogenesis by stimulating the translation of specific messages, including the actin-depolymerizing enzyme cofilin.

Novel gain-of-function alleles demonstrate a role for the heterochronic gene lin-41 in C. elegans male tail tip morphogenesis

To gain an understanding of the genes and mechanisms that govern morphogenesis and its evolution, we have analyzed mutations that disrupt this process in a simple model structure, the male tail tip of the rhabditid nematode C. elegans. During the evolution of rhabditid male tails, there have been several independent changes from tails with rounded tips (“peloderan”, as in C. elegans) to those with pointed tips (“leptoderan”). Mutations which produce leptoderan (Lep) tails in C. elegans thus identify candidate genes and pathways in which evolutionary changes could have produced leptoderan tails from peloderan ancestors. Here we report that two novel, gain-of-function (gf) alleles of lin-41 have lesions predicted to affect the N-terminus of the RBCC-domain LIN-41 protein. Both gf alleles cause the tail tip of adult males to retain the pointed shape of the juvenile tails, producing a Lep phenotype that looks like the tails of leptoderan species. Consistent with its role in the heterochronic pathway, we find that lin-41 governs the timing and extent of male tail tip morphogenesis in a dose-dependent manner. Specifically, the Lep phenotype results from a heterochronic delay in the retraction and fusion of the tail tip cells during L4 morphogenesis, such that retraction is not completed before the adult molt. Conversely, we find that tail tip morphogenesis and cell fusions begin precociously at the L3 stage in the reduced-function lin-41 mutant, ma104, resulting in over-retracted male tails in the adult. Because modulated anti-LIN-41 RNAi knockdowns in the gf mutants restore wild-type phenotype, we suggest that the leptoderan phenotype of the gf alleles is due to a higher activity of otherwise normal LIN-41. Additionally, the gf allele is suppressed by the wild-type allele, suggesting that LIN-41 normally regulates itself, possibly by autoubiquitination. We speculate that small changes affecting LIN-41 could have been significant for male tail evolution.

Axial patterning of C. elegans male sensilla identities by selector genes

The fan and rays of the C. elegans male tail constitute a compound sensory organ essential for mating. Within this organ, the individual sensilla, known as rays, have unique identities. We show that ray identities are patterned by a selector gene mechanism in a manner similar to other serially homologous axial structures. One selector gene that promotes the identities of a subset of the rays is the Hox gene egl-5. Within EGL-5-expressing rays, further patterning is provided by a Pax-6 homolog and a signal of the TGFβ family. These genes and pathway coordinately specify multiple ray properties affecting all three terminal ray cell types. These properties include complex patterns of FMRFamide-like (FaRP) neuropeptides, serotonin (5HT) and dopamine expression, and ray morphology. Differences in these differentiated characteristics give each sensillum a unique identity and potentially endow the compound ray organ with a higher-order information gathering capacity.

The Caenorhabditis elegans ADAMTS Family Gene adt-1 Is Necessary for Morphogenesis of the Male Copulatory Organs

Remodeling of the extracellular matrix (ECM) is pivotal for various biological processes, including organ morphology and development. The Caenorhabditis elegans male tail has male-specific copulatory organs, the rays and the fan. Ray morphogenesis, which involves a rapid remodeling of the ECM, is an important model of morphogenesis, although its mechanism is poorly understood. ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin type I motifs) is a novel metalloproteinase family that is thought to be an important regulator for ECM remodeling during development and pathological states. We report here that a new C. elegans ADAMTS family gene, adt-1, plays an important regulatory role in ray morphogenesis. Inactivation of the adt-1 gene resulted in morphological changes in the rays as well as the appearance of abnormal protuberances around the rays. In addition, mating ability was remarkably impaired in adt-1 deletion mutant males. Furthermore, we found that the green fluorescent protein reporter driven by the adt-1 promoter was specifically expressed throughout the rays in the male tail. We hypothesize that ADT-1 controls the ray extension process via remodeling of the ECM in the cuticle.

The basic helix-loop-helix transcription factors LIN-32 and HLH-2 function together in multiple steps of a C. elegans neuronal sublineage.

bHLH transcription factors function in neuronal development in organisms as diverse as worms and vertebrates. In the C. elegans male tail, a neuronal sublineage clonally gives rise to the three cell types (two neurons and a structural cell) of each sensory ray. We show here that the bHLH genes lin-32 and hlh-2 are necessary for the specification of multiple cell fates within this sublineage, and for the proper elaboration of differentiated cell characteristics. Mutations in lin-32, a member of the atonal family, can cause failures at each of these steps, resulting in the formation of rays that lack fully-differentiated neurons, neurons that lack cognate rays, and ray cells defective in the number and morphology of their processes. Mutations in hlh-2, the gene encoding the C. elegans E/daughterless ortholog, enhance the ray defects caused by lin-32 mutations. In vitro, LIN-32 can heterodimerize with HLH-2 and bind to an E-box-containing probe. Mutations in these genes interfere with this activity in a manner consistent with the degree of ray defects observed in vivo. We propose that LIN-32 and HLH-2 function as a heterodimer to activate different sets of targets, at multiple steps in the ray sublineage. During ray development, lin-32 performs roles of proneural, neuronal precursor, and differentiation genes of other systems.

Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail.

Tissue morphogenesis requires complex cellular interaction and communication. The sensory ray in the Caenorhabditis elegans male tail has a simple cellular make-up and a non-essential function, thus providing an ideal model for studying the mechanisms guiding morphogenesis. We present here the analysis of a novel gene, ram-5, mutations of which are characterized by abnormal lumpy rays in the male tail. Microscopic analysis and behavioral studies revealed that lumpy rays contain operational sensory neurons. However, abnormalities were observed in the hypodermis and structural cells as well as in appositions between these two cell types. Molecular cloning and expression studies revealed that the ram-5 gene encodes a transmembrane protein localized in sensory ray support cells, the structural cells. Expression of ram-5 in these cells is required for normal ray morphogenesis. ram-5-dependent cell-cell communication is implicated in organizing the structural cell and the hypodermis, potentially through adhesion at the structural cell-hypodermal cell border.

Mutations with sensory ray defect unmask cuticular glycoprotein antigens in Caenorhabditis elegans male tail.

Caenorhabditis elegans male tail has nine pairs of bilaterally symmetric ray processes extended into a cuticular fan. The formation of these structures involves both cell lineage differentiation and cellular morphogenesis. Nine mutations were examined, all of which presented an amorphous ray phenotype. Glycoconjugates carrying an N-acetylglucosamine (GlcNAc) epitope were detected at a high level in their male bursa. It was shown that these antigens are not responsible for the morphological defects. It was further demonstrated that these ram and mab gene products represent critical components for male tail cuticle organization. Mutations of them abolish the integrity of the male bursal cuticle and unmask the underlying GlcNAc epitope.

The mab-21 gene of Caenorhabditis elegans encodes a novel protein required for choice of alternate cell fates.

The gene mab-21, which encodes a novel protein of 386 amino acids, is required for the choice of alternate cell fates by several cells in the C. elegans male tail. Three cells descended from the ray 6 precursor cell adopt fates of anterior homologs, and a fourth, lineally unrelated hypodermal cell is transformed into a neuroblast. The affected cells lie together in the lateral tail epidermis, suggesting that mab-21 acts as part of a short-range pattern-formation mechanism. Each of the changes in cell fate brought about by mab-21 mutants can be interpreted as a posterior-to-anterior homeotic transformation. mab-21 mutant males and hermaphrodites have additional pleiotropic phenotypes affecting movement, body shape and fecundity, indicating that mab-21 has functions outside the tail region of males. We show that the three known alleles of mab-21 are hypomorphs of a new gene. Mosaic analysis revealed that mab-21 acts cell autonomously to specify the properties of the sensory ray, but non-autonomously in the hypodermal versus neuroblast cell fate choice. Presence of cell signalling in the choice of the neuroblast fate was confirmed by cell ablation experiments. Mutations in mab-21 were shown previously to be genetic modifiers of the effects of HOM-C/Hox gene mutations on ray identity specification. The results presented here support the conclusion that mab-21 acts as part of a mechanism required for correct cell fate choice, possibly involving the function of HOM-C/Hox genes in several body regions.

HOM-C/Hox genes and four interacting loci determine the morphogenetic properties of single cells in the nematode male tail.

The copulatory structure of the C. elegans male tail includes a set of nine bilaterally symmetrical pairs of sense organs known as rays. Each ray comprises three cells, which are generated by a stereotyped cell sublineage expressed by 18 epidermal ray precursor cells. A pattern formation mechanism in the epidermis guides the specification of morphogenetic differences between the rays necessary for correct organelle assembly at specific positions within the epidermis. Expression of these ray differences was altered in mutations we described previously, resulting in displaced and fused rays. Here we show that two genes of the C. elegans HOM-C/Hox gene complex play a role in the pattern formation mechanism. Increasing or decreasing the gene dosage of mab-5, an Antennapedia homolog, and egl-5, an Abdominal B homolog, results in displacement and fusion of specific rays. These changes are interpreted as anterior or posterior transformations in ray identities. Mutations in the genes previously described are dominant modifiers of these effects. This suggests that these genes act in the same morphogenetic pathway as mab-5 and egl-5. Several lines of evidence, including cell ablation experiments, argue that the identity of each ray is specified cell-autonomously in the terminal cells of the ray lineages. mab-5 and egl-5, therefore, specify the morphogenetic properties of differentiating cells, without change in cell lineage or apparent cell type. Modifier genes may act upstream of mab-5 and egl-5 to regulate their expression. Alternatively, they may act at the same step in the pathway, as cofactors, or they may be target genes. Target genes could include genes specifying cell recognition and adhesion molecules governing ray organelle assembly.

From cell fates to morphology: developmental genetics of the Caenorhabditis elegans male tail.

The C. elegans male tail is being studied as a model to understand how genes specify the form of multicellular animals. Morphogenesis of the specialized male copulatory organ takes place in the last larval stages during male development. Genetic analysis is facilitated because the structure is not necessary for male viability or for strain propagation. Analysis of developmental mutants, isolated in several functional and morphological screens, has begun to reveal how fates of cells are determined in the cell lineages, and how the specification of cell fates affects the morphology of the structure. Cytological studies in wild type and in mutants have been used to study the mechanism of pattern formation in the tail peripheral nervous system. The ultimate goal is to define the entire pathway leading to the male copulatory organ.

Pattern formation in the nematode epidermis: determination of the arrangement of peripheral sense organs in the C. elegans male tail

The developmental process that determines the arrangement of ray sensilla in the Caenorhabditis elegans male tail has been studied. It is shown that the adult arrangement of rays is determined by the placement of ray cells at specific sites in the epidermis of the last larval (L4) stage. Placement of ray cells at specific epidermal sites results from the generation of neurons and support cells in the epidermis near to their final positions, and the subsequent refinement of these positions by an active mechanism involving specific cellular associations. Positions of ray cells and adjacent epidermal cells have been studied during ray development by means of indirect immunofluorescence staining with an antibody to a cell junctional antigen. Mutations are described in six genes that alter the adult arrangement of the rays, frequently resulting in fusion of rays. Changes in the adult pattern of rays in mutants appear to result from prior changes in the epidermal positions of ray cells, and for two mutants it is suggested that this may be due to the inappropriate clustering of processes from neurons and support cells of adjacent rays. Development of the wild-type arrangement of rays appears to require the specification of molecular differences between the rays that affect the specificity of their cellular associations.