Science Spotlight

Two nuclei? You’re out!

From the Priess Lab, Basic Sciences Division

In organisms ranging from worms to mammals, generation of female egg cells (oocytes) is syncytial, meaning that cell boundaries are incomplete and many nuclei share a continuous cytoplasm. In the nematode Caenorhabditis elegans, over half of oocyte precursor cells die during development in a process known as physiological apoptosis. It is thought that these precursors can act as “nurse cells” by contributing nutrients to the communal cytoplasm, but how germ cells are selected for apoptosis is not known since those that die are not necessarily defective.

The Priess Laboratory in the Basic Sciences Division studies C. elegans development and recently performed a detailed study of oogenesis to better characterize the process of physiological apoptosis. Led by technician Stephan Raiders, now a graduate student in the Singhvi Lab, the researchers used a combination of live-cell imaging and transmission electron microscopy (TEM) of fixed cells to observe oogenesis in real time and track apoptotic cells. Their findings were published last month in PLOS Genetics.

Apoptotic cells are normally cleared very quickly via engulfment by neighboring sheath cells (Figure 1), so the authors first needed to generate a system to identify and track them. To increase the persistence of apoptotic cells, the researchers used C. elegans strains that had been rendered engulfment-defective by deletion of the transmembrane receptor gene ced-1. Since previous studies relied on markers that are engulfment-dependent, the Priess lab also needed a new apoptosis reporter. They found that the protein PGL-1 disappears from germ cells soon after they become committed to apoptosis and that these cells begin to shrink at the same time as PGL-1 loss.

Figure 1: Schematic of the C. elegans gonad. Germ cells move in the distal-to-proximal direction as they progress through various stages of meiosis, and slowly and intermittently contribute cytoplasm (grey arrows) to the core of the syncytium. Cells nearing the gonad loop undergo apoptosis (black cells) and engulfment by sheath cells (green), or rapidly intercalate and enlarge as they take up core cytoplasm (purple arrows). Image taken from the publication

Using live imaging, the researchers observed that oocyte precursors destined for apoptosis shrink in two phases: first, they rapidly expel their cytoplasm into the core of the syncytium but maintain a normal-sized nucleus, and second, the they close off from the syncytium and undergo nuclear and cytoplasmic compaction. Prior to shrinkage, the authors observed that apoptotic cells selectively eject their mitochondria by transporting them along microtubules in a process that requires the motor protein kinesin. The authors propose that active transport of the mitochondria may prevent coiling and entanglement during exit from cells committed to apoptosis.

By staining cells for F-actin, the authors observed that apoptotic cells remodel their cytoskeleton following shrinkage and closure from the syncytium. Actin levels were significantly higher in apoptotic cells compared to the surrounding cells, and TEM analysis revealed the existence of large bundles of actin microfilaments that co-localized with the actin disassembly factor cofilin. These cofilin-actin rods were found in about 70% of engulfment-defective apoptotic cells and 10% of wild-type apoptotic cells, indicating that normal clearance of apoptotic cells may preclude formation of these structures.

The researchers were surprised to notice that many of the cells containing the giant cofilin-actin rods had two—and sometimes three—nuclei. Further investigation revealed that the double nuclei arose due to fusion of post-mitotic germ cells prior to initiation of apoptosis. Because membranes tend to fold and unfold during morphogenesis and binucleate cells were enriched near folds that are in the process of resolving, “we propose that cells with more than one nucleus arise from folds that appear in the gonad of the worm as it grows larger with age,” says Mr. Raiders (Figure 2). In apoptosis-defective strains, binucleate germ cells were capable of becoming binucleate oocytes, but upon fertilization by wild-type sperm yielded triploid offspring only in very rare cases.

Figure 2: 3D rendered view of wild-type hermaphrodite gonads from the L4 stage to the 72-hr adult. The core of the L4 gonad shows little expansion before, in, or after the transition zone (TZ). By contrast, there is a large expansion of the core diameter in adults, and the complexity of core morphology increases markedly with age. The extracellular matrix protein HIM-4/hemicentin (yellow) is depicted in red in Figure 1. Image taken from the publication

The researchers were surprised to notice that many of the cells containing the giant cofilin-actin rods had two—and sometimes three—nuclei. Further investigation revealed that the double nuclei arose due to fusion of post-mitotic germ cells prior to initiation of apoptosis. Because membranes tend to fold and unfold during morphogenesis and binucleate cells were enriched near folds that are in the process of resolving, “we propose that cells with more than one nucleus arise from folds that appear in the gonad of the worm as it grows larger with age,” says Mr. Raiders (Figure 2). In apoptosis-defective strains, binucleate germ cells were capable of becoming binucleate oocytes, but upon fertilization by wild-type sperm yielded triploid offspring only in very rare cases.

Because the binucleate germ cells were always observed to be apoptotic, the Priess lab concluded that they are efficiently recognized and cleared. Thus, selection for physiological apoptosis is not completely random, meaning that this process functions not only as a means of transferring nutrients from nurse cells to developing oocytes; it is also a quality control mechanism. In future work, the Priess lab plans to investigate the possibility that formation and apoptosis of binucleate cells provides a mechanism for reducing cell numbers when folds become too large, thus contributing to gonad homeostasis.



Raiders SA, Eastwood MD, Bacher M and Priess JR. (2018) Binucleate germ cells in Caenorhabditis elegans are removed by physiological apoptosis. PLOS Genetics 14(7):e1007417.

This work was supported by the National Institutes of Health and the Human Frontiers Science Program.

Science Spotlight Editors
From the left: Science Spotlight editors Yiting Lim (Basic Sciences), Kyle Woodward (Clinical Research), Nicolas Chuvin (Human Biology), Maggie Burhans (Public Health Sciences) and Brianna Traxinger (Vaccine and Infectious Disease) Photo by Robert Hood / Fred Hutch

EDITORS

Yiting Lim
Basic Sciences Division

Nicolas Chuvin
Human Biology Division

Maggie Burhans, Ph.D.
Public Health Sciences Division

Brianna Traxinger
Vaccine and Infectious Disease Division

Kyle Woodward
Clinical Research Division

Julian Simon, Ph.D.
Faculty Mentor
Clinical Research Division
and Human Biology Division

Allysha Eyler
Publication Tracking
Arnold Digital Library

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