RESUMEN

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the DM-domain transcription factor DMD-3. To find genes regulated by DMD-3, We performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males vs. dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the unfolded protein response pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are coregulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

RESUMEN

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

Asunto(s)

RESUMEN

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the transcription factor DMD-3. To find genes regulated by DMD-3, We performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males vs. dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the UPR (unfolded protein response) pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are coregulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

RESUMEN

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.

RESUMEN

The developmental processes that give rise to the animal body plan are exceedingly complex. Model systems such as Drosophila melanogaster have yielded profound insight into roles of conserved genes and genetic pathways in development. Drosophila development begins with the formation of sperm and eggs, and proceeds through several morphologically distinct stages including development of the early embryo, larval instars, formation of pupae, and differentiation of adult tissues. The nuclear transport of proteins and RNAs represents a critical step in the regulation of gene expression during every stage of development and tissue differentiation. Studies of the nuclear transport machinery in Drosophila refute the notion that nuclear transport is strictly a housekeeping process without specific regulatory roles in development. Rather, they support the idea that the basal nuclear transport machinery has adapted during the evolution of the metazoan body plan to play important regulatory roles in key developmental events.

Asunto(s)

RESUMEN

Importin alphas are import receptors for nuclear localization signal-containing proteins. Most animal importin alphas assort into alpha1, alpha2, and alpha3 groups. Studies in Drosophila melanogaster, Caenorhabditis elegans, and mouse suggest that the animal importin alpha gene family evolved from ancestral plant-like genes to serve paralog-specific roles in gametogenesis. To explore this hypothesis we extended the phylogenetic analysis of the importin alpha gene family to nonbilateral animals and investigated whether animal-like genes occur in premetazoan taxa. Maximum likelihood analysis suggests that animal-like importin alpha genes occur in the Choanoflaggelate Monosiga brevicollis and the amoebozoan Dictyostelium; however, both of these results are caused by long-branch attraction effects. The absence of animal-like alpha genes in premetazoan taxa is consistent with the hypothesis that they duplicated and then specialized to function in animal gametogenesis. The gene structures of the importin alphas provide insight into how the animal importin alpha gene family may have evolved from the most likely ancestral gene. Interestingly, animal alpha1s are more similar to plant and fungal alpha1-like sequences than they are to animal alpha2s or alpha3s. We show that animal alpha1 genes share most of their introns with plant alpha1-like genes, and alpha2s and alpha3s share many more intron positions with each other than with the alpha1s. Together, phylogenetics and gene structure analysis suggests a parsimonious path for the evolution of the mammalian importin alpha gene family from an ancestral alpha1-like progenitor. Finally, these results establish a rational basis for a unified nomenclature of the importin alpha gene family.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.

Asunto(s)

RESUMEN

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.

Asunto(s)

RESUMEN

The nuclear transport of classical nuclear localization signal (cNLS)-containing proteins is mediated by the cNLS receptor importin alpha. The conventional importin alpha gene family in metazoan animals is composed of three clades that are conserved between flies and mammals and are referred to here as alpha1, alpha2, and alpha3. In contrast, plants and fungi contain only alpha1 genes. In this study we report that Drosophila importin alpha3 is required for the development of both larval and adult tissues. Importin alpha3 mutant flies die around the transition from first to second instar larvae, and homozygous importin alpha3 mutant eyes are defective. The transition to second instar larvae was rescued with importin alpha1, alpha2, or alpha3 transgenes, indicating that Importin alpha3 is normally required at this stage for an activity shared by all three importin alpha's. In contrast, an alpha3-specific biochemical activity(s) of Importin alpha3 is probably required for development to adults and photoreceptor cell development, since only an importin alpha3 transgene rescued these processes. These results are consistent with the view that the importin alpha's have both overlapping and distinct functions and that their role in animal development involves the spatial and temporal control of their expression.

Asunto(s)

RESUMEN

Importin alpha's mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin alpha genes, most of which fall into three conventional phylogenetic clades, here designated alpha1, alpha2, and alpha3. Using degenerate PCR we cloned Drosophila melanogaster importin alpha1, alpha2, and alpha3 genes, demonstrating that the complete conventional importin alpha gene family arose prior to the split between invertebrates and vertebrates. We have begun to analyze the genetic interactions among conventional importin alpha genes by studying their capacity to rescue the male and female sterility of importin alpha2 null flies. The sterility of alpha2 null males was rescued to similar extents by importin alpha1, alpha2, and alpha3 transgenes, suggesting that all three conventional importin alpha's are capable of performing the important role of importin alpha2 during spermatogenesis. In contrast, sterility of alpha2 null females was rescued only by importin alpha2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin alpha2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin alpha2.

Asunto(s)