XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans

Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans. The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, we show that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, we show that abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. We also demonstrate a specific function for xnp-1 together with lin-35 or hpl-2 in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development.     

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, as neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. Here we show that the C. elegansdsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. We suggest that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ.     

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.     

lin-35/Rb cooperates with the SWI/SNF complex to control Caenorhabditis elegans larval development

Null mutations in lin-35, the Caenorhabditis elegans ortholog of the mammalian Rb protein, cause no obvious morphological defects. Using a genetic approach to identify genes that may function redundantly with lin-35, we have isolated a mutation in the C. elegans psa-1 gene. lin-35; psa-1 double mutants display severe developmental defects leading to early larval arrest and adult sterility. The psa-1 gene has previously been shown to encode a C. elegans homolog of yeast SWI3, a critical component of the SWI/SNF complex, and has been shown to regulate asymmetric cell divisions during C. elegans development. We observed strong genetic interactions between psa-1 and lin-35 as well as a subset of the class B synMuv genes that include lin-37 and lin-9. Loss-of-function mutations in lin-35, lin-37, and lin-9 strongly enhanced the defects of asymmetric T cell division associated with a psa-1 mutation. Our results suggest that LIN-35/Rb and a certain class B synMuv proteins collaborate with the SWI/SNF protein complex to regulate the T cell division as well as other events essential for larval growth.     

lin-35 Rb and cki-1 Cip/Kip cooperate in developmental regulation of G1 progression in C. elegans.

We have investigated the regulation of cell-cycle entry in C. elegans, taking advantage of its largely invariant and completely described pattern of somatic cell divisions. In a genetic screen, we identified mutations in cyd-1 cyclin D and cdk-4 Cdk4/6. Recent results indicated that during Drosophila development, cyclin D-dependent kinases regulate cell growth rather than cell division. However, our data indicate that C. elegans cyd-1 primarily controls G1 progression. To investigate whether cyd-1 and cdk-4 solely act to overcome G1 inhibition by retinoblastoma family members, we constructed double mutants that completely eliminate the function of the retinoblastoma family and cyclin D-Cdk4/6 kinases. Inactivation of lin-35 Rb, the single Rb-related gene in C. elegans, substantially reduced the DNA replication and cell-division defects in cyd-1 and cdk-4 mutant animals. These results demonstrate that lin-35 Rb is an important negative regulator of G1/S progression and probably a downstream target for cyd-1 and cdk-4. However, as the suppression by lin-35 Rb is not complete, cyd-1 and cdk-4 probably have additional targets. An additional level of control over G1 progression is provided by Cip/Kip kinase inhibitors. We demonstrate that lin-35 Rb and cki-1 Cip/Kip contribute non-overlapping levels of G1/S inhibition in C. elegans. Surprisingly, loss of cki-1, but not lin-35, results in precocious entry into S phase. We suggest that a rate limiting role for cki-1 Cip/Kip rather than lin-35 Rb explains the lack of cell-cycle phenotype of lin-35 mutant animals.     

mir-35 is involved in intestine cell G1/S transition and germ cell proliferation in C. elegans

MicroRNA (miRNA) regulates gene expression in many cellular events, yet functions of only a few miRNAs are known in C. elegans. We analyzed the function of mir-35-41 unique to the worm, and show here that mir-35 regulates the G1/S transition of intestinal cells and germ cell proliferation. Loss of mir-35 leads to a decrease of nuclei numbers in intestine and distal mitotic gonad, while re-introduction of mir-35 rescues the mutant phenotypes. Genetic analysis indicates that mir-35 may act through Rb/E2F and SCF pathways. Further bioinformatic and functional analyses demonstrate that mir-35 targets evolutionally conserved lin-23 and gld-1. Together, our study reveals a novel function of mir-35 family in cell division regulation.     

Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8

Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression.     

APH-2/Nicastrin Functions in LIN-12/Notch Signaling in the Caenorhabditis elegans Somatic Gonad

Nicastrin is a recently identified member of high-molecular weight complexes containing presenilin. The Caenorhabditis elegans homolog of nicastrin, aph-2, was shown to be required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. We show that this latter defect is related to the SEL-12/presenilin egg-laying defect. We also show that aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. We illustrate a new role for aph-2 in facilitating lin-12 signaling in the somatic gonad, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Finally, we demonstrate that nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins.     

Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42

LIN-42, the Caenorhabditis elegans homolog of the Period (Per) family of circadian rhythm proteins, functions as a member of the heterochronic pathway, regulating temporal cell identities. We demonstrate that lin-42 acts broadly, timing developmental events in the gonad, vulva, and sex myoblasts, in addition to its well-established role in timing terminal differentiation of the hypodermis. In the vulva, sex myoblasts, and hypodermis, lin-42 activity prevents stage-specific cell division patterns from occurring too early. This general function of timing stage-appropriate cell division patterns is shared by the majority of heterochronic genes; their mutation temporally alters stage-specific division patterns. In contrast, lin-42 function in timing gonad morphogenesis is unique among the known heterochronic genes: inactivation of lin-42 causes the elongating gonad arms to reflex too early, a phenotype which implicates lin-42 in temporal regulation of cell migration. Three additional isoforms of lin-42 are identified that expand our view of the lin-42 locus and significantly extend the homology between LIN-42 and other PER family members. We show that, similar to PER proteins, LIN-42 has a dynamic expression pattern; its levels oscillate relative to the molts during postembryonic development. Transformation rescue studies indicate lin-42 is bipartite with respect to function. Intriguingly, the hallmark PAS domain is dispensable for LIN-42 function in transgenic animals.     

Role of phosphatidylinositol-4-phosphate 5' kinase (ppk-1) in ovulation of Caenorhabditis elegans

During Caenorhabditis elegans ovulation, the somatic gonad integrates signals from germ cells and propels a mature oocyte into the spermatheca for fertilization. Previous work suggests that phosphoinositide signaling plays important roles in C. elegans fertility. To fully understand inositol-1,4,5-trisphosphate (IP(3)) signaling in ovulation, we have examined the function of phosphatidylinositol-4-phosphate 5' kinase (PIP5K) in C. elegans. Our results show that the C. elegans PIP5K homolog, ppk-1, is essential for ovulation in C. elegans; ppk-1 is mainly expressed in somatic gonad, and depletion of ppk-1 expression causes defective ovulation, reduced gonad sheath contractility, and sterility. Increased IP(3) signaling compensates for ppk-1 (RNAi)-induced sterility, suggesting that ppk-1 is linked to IP(3) signaling. These results demonstrate that ppk-1 plays an essential role in IP(3) signaling and cytoskeleton organization in somatic gonad.     

tcl-2 encodes a novel protein that acts synergistically with Wnt signaling pathways in C. elegans

Mutations in tcl-2 cause defects in the specification of the fates of the descendants of the TL and TR blast cells, whose polarity is regulated by lin-44/Wnt and lin-17/frizzled, during Caenorhabditis elegans development. In wild-type animals, POP-1/TCF/LEF, is asymmetrically distributed to the T cell daughters, resulting in a higher level of POP-1 in the nucleus of the anterior daughter. The POP-1 asymmetric distribution is controlled by lin-44 and lin-17. However, in tcl-2 mutants, POP-1 is equally distributed to T cell daughters as is observed in lin-17 mutants, indicating that, like lin-17, tcl-2 functions upstream of pop-1. In addition, tcl-2 mutations cause defects in the development of the gonad and the specification of fate of the posterior daughter of the P12 cell, both of which are controlled by the Wnt pathway. Double mutant analyses indicate that tcl-2 can act synergistically with the Wnt pathway to control gonad development as well as P12 descendant cell fate specification. tcl-2 encodes a novel protein. A functional tcl-2::gfp construct was weakly expressed in the nuclei of the T cell and its descendants. Our results suggest that tcl-2 functions with Wnt pathways to control T cell fate specification, gonad development, and P12 cell fate specification.     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

ARI-1, an RBR family ubiquitin-ligase, functions with UBC-18 to regulate pharyngeal development in C. elegans

The LIN-35 retinoblastoma protein homolog and the ubiquitin-conjugating enzyme UBC-18 function redundantly to control an early step of pharyngeal morphogenesis in C. elegans. In order to identify ubiquitin-ligases acting downstream of UBC-18, we carried out a two-hybrid screen using UBC-18 as the bait molecule. Our screen identified three putative ubiquitin-ligases, one of which, ARI-1, showed genetic interactions leading to defective pharyngeal development that were identical to that previously observed for UBC-18. ARI-1 is a member of the RBR family of ubiquitin-ligases and contains a C-terminal motif that places it within the highly conserved Ariadne subfamily of RBR ligases. Our analyses indicate that ARI-1 is the principal Ariadne family member in C. elegans that is involved in the control of pharyngeal development with UBC-18. Using GFP reporters, we find that ARI-1 is expressed dynamically in a wide range of tissues including muscles and neurons during embryonic and postembryonic development. We also provide evidence that dsRNA species containing 14 or fewer base pairs of contiguous identity with closely related mRNAs are sufficient to mediate off-target silencing in C. elegans.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.