mab-7 encodes a novel transmembrane protein that orchestrates sensory ray morphogenesis in C. elegans

The tapered sensory rays of the male Caenorhabditis elegans are important for successful male/hermaphrodite copulation. A group of ram (ray morphology abnormal) genes encoding modifying enzymes and transmembrane protein have been reported as key regulators controlling ray morphogenesis. Here we report the characterization of another component essential for this morphogenetic process encoded by mab-7. This gene is active in the hypodermis, structural cells, the body seam and several head neurons. It encodes a novel protein with a hydrophobic region at the N-terminus, an EGF-like motif, an ShKT motif and a long C-terminal tail. All these domains are shown to be critical to MAB-7 activity except the EGF-like domain, which appears to be regulatory and dispensable. MAB-7 is shown to be a type II membrane protein, tethered on the cell surface by the N-terminal transmembrane domain with the remainder of the protein exposed to the extracellular matrix. Since ectopic mab-7 expression in any ray cell or even in touch neurons of non-ray lineage can rescue the mutant phenotype, mab-7 is probably acting non-autonomously. It may facilitate intercellular communication among ray cells to augment normal ray morphogenesis.     

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.     

A novel thioredoxin-like protein encoded by the C. elegans dpy-11 gene is required for body and sensory organ morphogenesis

Sensory ray morphogenesis in C. elegans requires active cellular interaction regulated by multiple genetic activities. We report here the cloning of one of these genes, dpy-11, which encodes a membrane-associated thioredoxin-like protein. The DPY-11 protein is made exclusively in the hypodermis and resides in the cytoplasmic compartment. Whereas the TRX domain of DPY-11 displays a catalytic activity in vitro, mapping of lesions in different mutant alleles and functional analysis of deletion transgenes reveal that both this enzymatic activity and transmembrane topology are essential for determining body shape and ray morphology. Based on the abnormal features in both the expressing and non-expressing ray cells, we propose that the DPY-11 is required in the hypodermis for modification of its substrates. In turn, ray cell interaction and the whole morphogenetic process can be modulated by these substrate molecules.     

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.     

mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans

The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. We made and characterized a putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a). This mutant failed to complement mutants of mab-20 (Baird, S. E., Fitch, D. H., Kassem, I. A. A. and Emmons, S. W. (1991) Development 113, 515-526). In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. Errant epidermal cell migrations affect epidermal enclosure of the embryo, body shape and sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells of similar type and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells.     

Selective Lineage Specification by Mab-19 during Caenorhabditis Elegans Male Peripheral Sense Organ Development

The action of the gene mab-19 is required for specification of a subset of Caenorhabditis elegans male peripheral sense organ (ray) lineages. Two mab-19 alleles, isolated in screens for ray developmental mutations, resulted in males that lacked the three most posterior rays. Cell lineage alterations of male-specific divisions of the most posterior lateral hypodermal (seam) blast cell, T, resulted in the ray loss phenotype in mab-19 mutant animals. Postembryonic seam lineage defects were limited to male-specific T descendent cell divisions. Embryonic lethality resulted when either mab-19 mutation was placed over a chromosomal deficiency encompassing the mab-19 locus. The earliest detectable defect was aberrant hypodermal cell movements during morphogenesis. From these data, it is inferred that both mab-19 alleles described are hypomorphs, and further reduction of mab-19 function results in embryos that are unable to complete morphogenesis. Thus, mab-19 may play a larger role in developmental regulation of hypodermal cell fate, including sensory ray development in males. Body morphology mutations, passage through the dauer stage, and heat or CdCl(2) treatment suppressed mab-19 male phenotypes. A model is presented in which all three types of suppression result in a physiological stress response, which in turn leads to correction of the mab-19 defect.     

Ray-interray interactions during fin regeneration of Danio rerio

Teleost fin ray bifurcations are characteristic of each ray in each fin of the fishes. Control of the positioning of such morphological markers is not well understood. We present evidence suggesting that the interray blastema is necessary for a proper bifurcation of each ray during regeneration in Danio rerio (Hamilton-Buchanan) (Cyprinidae, Teleostei). We performed single ray ablations, heterotopical graftings of ray fragments and small holes in lateral rays which do not normally bifurcate, to generate recombinants in which the lateral rays are surrounded with ectopic interrays originating from different positions within the tail fin. These ray-interray recombinants do now bifurcate. Furthermore, we show that the interray tissue and surrounding epidermis can modulate the length of the ray. These results stress the role of the interray in inducing bifurcations of the ray blastema as well as modulating ray morphogenesis in general. In addition, gene expression analysis under these experimental conditions suggests that msxA and msxD expression in the ray and interray epidermis is controlled by the ray blastema and that bmp4 could be a candidate signal involved in these inductions.     

The roles of an ephrin and a semaphorin in patterning cell-cell contacts in C. elegans sensory organ development

Ephrins and semaphorins regulate a wide variety of developmental processes, including axon guidance and cell migration. We have studied the roles of the ephrin EFN-4 and the semaphorin MAB-20 in patterning cell-cell contacts among the cells that give rise to the ray sensory organs of Caenorhabditis elegans. In wild-type, contacts at adherens junctions form only between cells belonging to the same ray. In efn-4 and mab-20 mutants, ectopic contacts form between cells belonging to different rays. Ectopic contacts also occur in mutants in regulatory genes that specify ray morphological identity. We used efn-4 and mab-20 reporters to investigate whether these ray identity genes function through activating expression of efn-4 or mab-20 in ray cells. mab-20 reporter expression in ray cells was unaffected by mutants in the Pax6 homolog mab-18 and the Hox genes egl-5 and mab-5, suggesting that these genes do not regulate mab-20 expression. We find that mab-18 is necessary for activating efn-4 reporter expression, but this activity alone is not sufficient to account for mab-18 function in controlling cell-cell contact formation. In egl-5 mutants, efn-4 reporter expression in certain ray cells was increased, inconsistent with a simple repulsion model for efn-4 action. The evidence indicates that ray identity genes primarily regulate ray morphogenesis by pathways other than through regulation of expression of semaphorin and ephrin.     

Integration of semaphorin-2A/MAB-20, ephrin-4, and UNC-129 TGF-beta signaling pathways regulates sorting of distinct sensory rays in C. elegans

Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes an axon guiding TGF-beta, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. We present a model for the integrated control of TGF-beta, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail.     

Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene

We have investigated the mechanism that patterns dopamine expression among Caenorhabditis elegans male ray sensory neurons. Dopamine is expressed by the A-type sensory neurons in three out of the nine pairs of rays. We used expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway, the DBL-1 pathway, dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability.     

dmd-3, a doublesex-related gene regulated by tra-1, governs sex-specific morphogenesis in C. elegans

Although sexual dimorphism is ubiquitous in animals, the means by which sex determination mechanisms trigger specific modifications to shared structures is not well understood. In C. elegans, tail tip morphology is highly dimorphic: whereas hermaphrodites have a whip-like, tapered tail tip, the male tail is blunt-ended and round. Here we show that the male-specific cell fusion and retraction that generate the adult tail are controlled by the previously undescribed doublesex-related DM gene dmd-3, with a secondary contribution from the paralogous gene mab-3. In dmd-3 mutants, cell fusion and retraction in the male tail tip are severely defective, while in mab-3; dmd-3 double mutants, these processes are completely absent. Conversely, expression of dmd-3 in the hermaphrodite tail tip is sufficient to trigger fusion and retraction. The master sexual regulator tra-1 normally represses dmd-3 expression in the hermaphrodite tail tip, accounting for the sexual specificity of tail tip morphogenesis. Temporal cues control the timing of tail remodeling in males by regulating dmd-3 expression, and Wnt signaling promotes this process by maintaining and enhancing dmd-3 expression in the tail tip. Downstream, dmd-3 and mab-3 regulate effectors of morphogenesis including the cell fusion gene eff-1. Together, our results reveal a regulatory network for male tail morphogenesis in which dmd-3 and mab-3 together occupy the central node. These findings indicate that an important conserved function of DM genes is to link the general sex determination hierarchy to specific effectors of differentiation and morphogenesis.     

The Caenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing the selector Hox gene egl-5 and the effector gene mec-3

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which we find is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; we show that a subset also express egl-5. We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.     

Morphogenesis of the Caenorhabditis elegans male tail tip

Using electron microscopy and immunofluorescent labeling of adherens junctions, we have reconstructed the changes in cell architecture and intercellular associations that occur during morphogenesis of the nematode male tail tip. During late postembryonic development, the Caenorhabditis elegans male tail is reshaped to form a copulatory structure. The most posterior hypodermal cells in the tail define a specialized, sexually dimorphic compartment in which cells fuse and retract in the male, changing their shape from a tapered cone to a blunt dome. Developmental profiles using electron microscopy and immunofluorescent staining suggest that cell fusions are initiated at or adjacent to adherens junctions. Anterior portions of the tail tip cells show the first evidence of retractions and fusions, consistent with our hypothesis that an anterior event triggers these morphogenetic events. Available mutations that interfere with morphogenesis implicate particular regulatory pathways and suggest loci at which evolutionary changes could have produced morphological diversity.     

The Caenorhabditis elegans innexin INX-3 is localized to gap junctions and is essential for embryonic development

Innexins are the proposed structural components of gap junctions in invertebrates. Antibodies that specifically recognize the Caenorhabditis elegans innexin protein INX-3 were generated and used to examine the patterns of inx-3 gene expression and the subcellular sites of INX-3 localization. INX-3 is first detected in two-cell embryos, concentrated at the intercellular interface, and is expressed ubiquitously throughout the cellular proliferation phase of embryogenesis. During embryonic morphogenesis, INX-3 expression becomes more restricted. Postembryonically, INX-3 is expressed transiently in several cell types, while expression in the posterior pharynx persists throughout development. Through immuno-EM techniques, INX-3 was observed at gap junctions in the adult pharynx, providing supporting evidence that innexins are components of gap junctions. An inx-3 mutant was isolated through a combined genetic and immunocytochemical screen. Homozygous inx-3 mutants exhibit defects during embryonic morphogenesis. At the comma stage of early morphogenesis, variable numbers of cells are lost from the anterior of inx-3(lw68) mutants. A range of terminal defects is seen later in embryogenesis, including localized rupture of the hypodermis, failure of the midbody to elongate properly, abnormal contacts between hypodermal cells, and failure of the pharynx to attach to the anterior of the animal.