Genetic and molecular studies have provided considerable insight into how various tissue progenitors are specified in early embryogenesis, but much less is known about how those progenitors create three-dimensional tissues and organs. The C. elegans intestine provides a simple system for studying how a single progenitor, the E blastomere, builds an epithelial tube of 20 cells. As the E descendants divide, they form a primordium that transitions between different shapes over time. We used cell contours, traced from confocal optical z-stacks, to build a 3D graphic reconstruction of intestine development. The reconstruction revealed several new aspects of morphogenesis that extend and clarify previous observations. The first 8 E descendants form a plane of four right cells and four left cells; the plane arises through oriented cell divisions and VANG-1/Van Gogh-dependent repositioning of any non-planar cells. LIN-12/Notch signaling affects the left cells in the E8 primordium, and initiates later asymmetry in cell packing. The next few stages involve cell repositioning and intercalation events that shuttle cells to their final positions, like shifting blocks in a Rubik's cube. Repositioning involves breaking and replacing specific adhesive contacts, and some of these events involve EFN-4/Ephrin, MAB-20/semaphorin-2a, and SAX-3/Robo. Once cells in the primordium align along a common axis and in the correct order, cells at the anterior end rotate clockwise around the axis of the intestine. The anterior rotation appears to align segments of the developing lumen into a continuous structure, and requires the secreted ligand UNC-6/netrin, the receptor UNC-40/DCC, and an interacting protein called MADD-2. Previous studies showed that rotation requires a second round of LIN-12/Notch signaling in cells on the right side of the primordium, and we show that MADD-2-GFP appears to be downregulated in those cells.

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.

Many animal organs are composed largely or entirely of polarized epithelial tubes, and the formation of complex organ systems, such as the digestive or vascular systems, requires that separate tubes link with a common polarity. The Caenorhabditis elegans digestive tract consists primarily of three interconnected tubes-the pharynx, valve, and intestine-and provides a simple model for understanding the cellular and molecular mechanisms used to form and connect epithelial tubes. Here, we use live imaging and 3D reconstructions of developing cells to examine tube formation. The three tubes develop from a pharynx/valve primordium and a separate intestine primordium. Cells in the pharynx/valve primordium polarize and become wedge-shaped, transforming the primordium into a cylindrical cyst centered on the future lumenal axis. For continuity of the digestive tract, valve cells must have the same, radial axis of apicobasal polarity as adjacent intestinal cells. We show that intestinal cells contribute to valve cell polarity by restricting the distribution of a polarizing cue, laminin. After developing apicobasal polarity, many pharyngeal and valve cells appear to explore their neighborhoods through lateral, actin-rich lamellipodia. For a subset of cells, these lamellipodia precede more extensive intercalations that create the valve. Formation of the valve tube begins when two valve cells become embedded at the left-right boundary of the intestinal primordium. Other valve cells organize symmetrically around these two cells, and wrap partially or completely around the orthogonal, lumenal axis, thus extruding a small valve tube from the larger cyst. We show that the transcription factors DIE-1 and EGL-43/EVI1 regulate cell intercalations and cell fates during valve formation, and that the Notch pathway is required to establish the proper boundary between the pharyngeal and valve tubes.

Much of the patterning of early C. elegans embryos involves a series of Notch interactions that occur in rapid succession and have distinct outcomes; however, none of the targets for these interactions have been identified. We show that the REF-1 family of bHLH transcription factors is a major target of Notch signaling in all these interactions and that most examples of Notch-mediated transcriptional repression can be attributed to REF-1 activities. The REF-1 family is expressed and has similar functions in both Notch-dependent and Notch-independent pathways, and this dual mode of deployment is used repeatedly to pattern the embryo. REF-1 proteins are unusual in that they contain two different bHLH domains and lack the distinguishing characteristics of Hairy/Enhancer of Split (HES) bHLH proteins that are Notch targets in other systems. Our results show that the highly divergent REF-1 proteins are nonetheless HES-like bHLH effectors of Notch signaling.

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.

The centriole/basal body is a eukaryotic organelle that plays essential roles in cell division and signaling. Among five known core centriole proteins, SPD-2/Cep192 is the first recruited to the site of daughter centriole formation and regulates the centriolar localization of the other components in C. elegans and in humans. However, the molecular basis for SPD-2 centriolar localization remains unknown. Here, we describe a new centriole component, the coiled-coil protein SAS-7, as a regulator of centriole duplication, assembly and elongation. Intriguingly, our genetic data suggest that SAS-7 is required for daughter centrioles to become competent for duplication, and for mother centrioles to maintain this competence. We also show that SAS-7 binds SPD-2 and regulates SPD-2 centriolar recruitment, while SAS-7 centriolar localization is SPD-2-independent. Furthermore, pericentriolar material (PCM) formation is abnormal in sas-7 mutants, and the PCM-dependent induction of cell polarity that defines the anterior-posterior body axis frequently fails. We conclude that SAS-7 functions at the earliest step in centriole duplication yet identified and plays important roles in the orchestration of centriole and PCM assembly.

Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. Here we present a system to monitor histone turnover and chromatin remodeling during Caenorhabditis elegans development by following the developmental dynamics of H3.3. We generated worm strains expressing green fluorescent protein- or yellow fluorescent protein-fused histone H3.3 proteins, HIS-71 and HIS-72. We found that H3.3 is retained in mature sperm chromatin, raising the possibility that it transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. Our demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development.

During organogenesis of the C. elegans digestive system, epithelial cells within a cyst-like primordium develop diverse shapes through largely unknown mechanisms. We here analyze two adjacent, dorsal epithelial cells, called pm8 and vpi1, that remodel their shapes and apical junctions to become donut-shaped, or toroidal, single-cell tubes. pm8 and vpi1 delaminate from the dorsal cyst epithelium and migrate ventrally, across the midline of the cyst, on a transient tract of laminin. pm8 appears to encircle the midline by wrapping around finger-like projections from neighboring cells. Finally, pm8 and vpi1 self-fuse to become toroids by expressing AFF-1 and EFF-1, two fusogens that are each sufficient to promote crossfusion between other cell types. Notch signaling in pm8 induces AFF-1 expression, while simultaneously repressing EFF-1 expression; vpi1 expresses EFF-1 independent of Notch. Thus, the adjacent pm8 and vpi1 cells express different fusogens, allowing them to self-fuse into separate, single-cell tubes while avoiding crossfusion.

Retroviruses and closely related LTR retrotransposons export full-length, unspliced genomic RNA (gRNA) for packaging into virions and to serve as the mRNA encoding GAG and POL polyproteins. Because gRNA often includes splice acceptor and donor sequences used to splice viral mRNAs, retroelements must overcome host mechanisms that retain intron-containing RNAs in the nucleus. Here we examine gRNA expression in Cer1, an LTR retrotransposon in C. elegans which somehow avoids silencing and is highly expressed in germ cells. Newly exported Cer1 gRNA associates rapidly with the Cer1 GAG protein, which has structural similarity with retroviral GAG proteins. gRNA export requires CERV (C. elegans regulator of viral expression), a novel protein encoded by a spliced Cer1 mRNA. CERV phosphorylation at S214 is essential for gRNA export, and phosphorylated CERV colocalizes with nuclear gRNA at presumptive sites of transcription. By electron microscopy, tagged CERV proteins surround clusters of distinct, linear fibrils that likely represent gRNA molecules. Single fibrils, or groups of aligned fibrils, also localize near nuclear pores. During the C. elegans self-fertile period, when hermaphrodites fertilize oocytes with their own sperm, CERV concentrates in two nuclear foci that are coincident with gRNA. However, as hermaphrodites cease self-fertilization, and can only produce cross-progeny, CERV undergoes a remarkable transition to form giant nuclear rods or cylinders that can be up to 5 microns in length. We propose a novel mechanism of rod formation, in which stage-specific changes in the nucleolus induce CERV to localize to the nucleolar periphery in flattened streaks of protein and gRNA; these streaks then roll up into cylinders. The rods are a widespread feature of Cer1 in wild strains of C. elegans, but their function is not known and might be limited to cross-progeny. We speculate that the adaptive strategy Cer1 uses for the identical self-progeny of a host hermaphrodite might differ for heterozygous cross-progeny sired by males. For example, mating introduces male chromosomes which can have different, or no, Cer1 elements.

Cell death plays a major role during C. elegans oogenesis, where over half of the oogenic germ cells die in a process termed physiological apoptosis. How germ cells are selected for physiological apoptosis, or instead become oocytes, is not understood. Most oocytes produce viable embryos when apoptosis is blocked, suggesting that physiological apoptosis does not function to cull defective germ cells. Instead, cells targeted for apoptosis may function as nurse cells; the germline is syncytial, and all germ cells appear to contribute cytoplasm to developing oocytes. C. elegans has been a leading model for the genetics and molecular biology of apoptosis and phagocytosis, but comparatively few studies have examined the cell biology of apoptotic cells. We used live imaging to identify and examine pre-apoptotic germ cells in the adult gonad. After initiating apoptosis, germ cells selectively export their mitochondria into the shared pool of syncytial cytoplasm; this transport appears to use the microtubule motor kinesin. The apoptotic cells then shrink as they expel most of their remaining cytoplasm, and close off from the syncytium. Shortly thereafter the apoptotic cells restructure their microtubule and actin cytoskeletons, possibly to maintain cell integrity; the microtubules form a novel, cortical array of stabilized microtubules, and actin and cofilin organize into giant cofilin-actin rods. We discovered that some apoptotic germ cells are binucleate; the binucleate germ cells can develop into binucleate oocytes in apoptosis-defective strains, and appear capable of producing triploid offspring. Our results suggest that the nuclear layer of the germline syncytium becomes folded during mitosis and growth, and that binucleate cells arise as the layer unfolds or everts; all of the binucleate cells are subsequently removed by apoptosis. These results show that physiological apoptosis targets at least two distinct populations of germ cells, and that the apoptosis machinery efficiently recognizes cells with two nuclei.

Virus-like particles (VLPs) have not been observed in Caenorhabditis germ cells, although nematode genomes contain low numbers of retrotransposon and retroviral sequences. We used electron microscopy to search for VLPs in various wild strains of Caenorhabditis, and observed very rare candidate VLPs in some strains, including the standard laboratory strain of C. elegans, N2. We identified the N2 VLPs as capsids produced by Cer1, a retrotransposon in the Gypsy/Ty3 family of retroviruses/retrotransposons. Cer1 expression is age and temperature dependent, with abundant expression at 15°C and no detectable expression at 25°C, explaining how VLPs escaped detection in previous studies. Similar age and temperature-dependent expression of Cer1 retrotransposons was observed for several other wild strains, indicating that these properties are common, if not integral, features of this retroelement. Retrotransposons, in contrast to DNA transposons, have a cytoplasmic stage in replication, and those that infect non-dividing cells must pass their genomic material through nuclear pores. In most C. elegans germ cells, nuclear pores are largely covered by germline-specific organelles called P granules. Our results suggest that Cer1 capsids target meiotic germ cells exiting pachytene, when free nuclear pores are added to the nuclear envelope and existing P granules begin to be removed. In pachytene germ cells, Cer1 capsids concentrate away from nuclei on a subset of microtubules that are exceptionally resistant to microtubule inhibitors; the capsids can aggregate these stable microtubules in older adults, which exhibit a temperature-dependent decrease in egg viability. When germ cells exit pachytene, the stable microtubules disappear and capsids redistribute close to nuclei that have P granule-free nuclear pores. This redistribution is microtubule dependent, suggesting that capsids that are released from stable microtubules transfer onto new, dynamic microtubules to track toward nuclei. These studies introduce C. elegans as a model to study the interplay between retroelements and germ cell biology.

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. We show that the one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry.