Elegans Development Research Articles

Poly(U) polymerase activity in Caenorhabditis elegans regulates abundance and tailing of sRNA and mRNA.

Terminal nucleotidyltransferases add nucleotides to the 3' end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, the expression of PUP-2, PUP-3, and especially PUP-4 limits uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that the addition of U-tails to nonadenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development.

Caenorhabditis elegans SEL-5/AAK1 regulates cell migration and cell outgrowth independently of its kinase activity.

During Caenorhabditis elegans development, multiple cells migrate long distances or extend processes to reach their final position and/or attain proper shape. The Wnt signalling pathway stands out as one of the major coordinators of cell migration or cell outgrowth along the anterior-posterior body axis. The outcome of Wnt signalling is fine-tuned by various mechanisms including endocytosis. In this study, we show that SEL-5, the C. elegans orthologue of mammalian AP2-associated kinase AAK1, acts together with the retromer complex as a positive regulator of EGL-20/Wnt signalling during the migration of QL neuroblast daughter cells. At the same time, SEL-5 in cooperation with the retromer complex is also required during excretory canal cell outgrowth. Importantly, SEL-5 kinase activity is not required for its role in neuronal migration or excretory cell outgrowth, and neither of these processes is dependent on DPY-23/AP2M1 phosphorylation. We further establish that the Wnt proteins CWN-1 and CWN-2, together with the Frizzled receptor CFZ-2, positively regulate excretory cell outgrowth, while LIN-44/Wnt and LIN-17/Frizzled together generate a stop signal inhibiting its extension.

Essential function of transmembrane transcription factor MYRF in promoting transcription of miRNA lin-4 during C. elegans development.

Precise developmental timing control is essential for organism formation and function, but its mechanisms are unclear. In C. elegans, the microRNA lin-4 critically regulates developmental timing by post-transcriptionally downregulating the larval-stage-fate controller LIN-14. However, the mechanisms triggering the activation of lin-4 expression toward the end of the first larval stage remain unknown. We demonstrate that the transmembrane transcription factor MYRF-1 is necessary for lin-4 activation. MYRF-1 is initially localized on the cell membrane, and its increased cleavage and nuclear accumulation coincide with lin-4 expression timing. MYRF-1 regulates lin-4 expression cell-autonomously and hyperactive MYRF-1 can prematurely drive lin-4 expression in embryos and young first-stage larvae. The tandem lin-4 promoter DNA recruits MYRF-1GFP to form visible loci in the nucleus, suggesting that MYRF-1 directly binds to the lin-4 promoter. Our findings identify a crucial link in understanding developmental timing regulation and establish MYRF-1 as a key regulator of lin-4 expression.

Apoptotic and Nonapoptotic Cell Death in Caenorhabditis elegans Development.

Programmed cell death (PCD) is an essential component of animal development, and aberrant cell death underlies many disorders. Understanding mechanisms that govern PCD during development can provide insight into cell death programs that are disrupted in disease. Key steps mediating apoptosis, a highly conserved cell death program employing caspase proteases, were first uncovered in the nematode Caenorhabditis elegans, a powerful model system for PCD research. Recent studies in C. elegans also unearthed conserved nonapoptotic caspase-independent cell death programs that function during development. Here, we discuss recent advances in understanding cell death during C. elegans development. We review insights expanding the molecular palette behind the execution of apoptotic and nonapoptotic cell death, as well as new discoveries revealing the mechanistic underpinnings of dying cell engulfment and clearance. A number of open questions are also discussed that will continue to propel the field over the coming years.

Orsay Virus Infection in Caenorhabditis elegans.

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: Synchronize C. elegans development by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

Neuronal IL-17 controls Caenorhabditis elegans developmental diapause through CEP-1/p53

During metazoan development, how cell division and metabolic programs are coordinated with nutrient availability remains unclear. Here, we show that nutrient availability signaled by the neuronal cytokine, ILC-17.1, switches Caenorhabditis elegans development between reproductive growth and dormancy by controlling the activity of the tumor suppressor p53 ortholog, CEP-1. Specifically, upon food availability, ILC-17.1 signaling by amphid neurons promotes glucose utilization and suppresses CEP-1/p53 to allow growth. In the absence of ILC-17.1, CEP-1/p53 is activated, up-regulates cell-cycle inhibitors, decreases phosphofructokinase and cytochrome C expression, and causes larvae to arrest as stress-resistant, quiescent dauers. We propose a model whereby ILC-17.1 signaling links nutrient availability and energy metabolism to cell cycle progression through CEP-1/p53. These studies describe ancestral functions of IL-17 s and the p53 family of proteins and are relevant to our understanding of neuroimmune mechanisms in cancer. They also reveal a DNA damage-independent function of CEP-1/p53 in invertebrate development and support the existence of a previously undescribed C. elegans dauer pathway.

Chemical basis of microbiome preference in the nematode C. elegans

Animals are exposed to many microbes in their environment, some of which have been shown to colonize various tissues including the intestine. The composition of the intestinal microbiota affects many aspects of the host’s physiology and health. Despite this, very little is known about whether host behavior contributes to the colonization. We approach this question in the nematode C. elegans, which feeds on bacteria and also harbors an intestinal microbiome. We examined the behavior of C. elegans towards CeMbio, a simplified microbiome consisting of twelve strains that represent the bacteria found in the animal’s natural environment. We observed that C. elegans raised on E. coli shows a strong preference for three members of CeMbio (Lelliottia amnigena JUb66, Enterobacter hormaechei CEent1, and Pantoea nemavictus BIGb0393) compared to E. coli. Previously, these three bacterial strains have been shown to support faster C. elegans development time than E. coli OP50 and are low colonizers compared to eight other members of CeMbio. We then used gas chromatography coupled to mass spectrometry to identify that these three bacteria release isoamyl alcohol, a previously described C. elegans chemoattractant. We suggest that C. elegans seeks bacteria that release isoamyl alcohol and support faster growth.

A genetic screen identifies C. elegans eif-3.H and hrpr-1 as pro-apoptotic genes and potential activators of egl-1 expression.

During C. elegans development, 1090 somatic cells are generated of which 131 reproducibly die, many through apoptosis. The C. elegans BH3-only gene egl-1 is the key activator of apoptosis in somatic tissues, and it is predominantly expressed in 'cell death' lineages i.e. lineages in which apoptotic cell death occurs. egl-1 expression is regulated at the transcriptional and post-transcriptional level. For example, we previously showed that the miR-35 and miR-58 families of miRNAs repress egl-1 expression in mothers of 'unwanted' cells by binding to the 3' UTR of egl-1 mRNA, thereby increasing egl-1 mRNA turnover. In a screen for RNA-binding proteins with a role in the post-transcriptional control of egl-1 expression, we identified EIF-3.H (ortholog of human eIF3H) and HRPR-1 (ortholog human hnRNP R/Q) as potential activators of egl-1 expression. In addition, we demonstrate that the knockdown of the eif-3.H or hrpr-1 gene by RNA-mediated interference (RNAi) results in the inappropriate survival of unwanted cells during C. elegans development. Our study provides novel insight into how egl-1 expression is controlled to cause the reproducible pattern of cell death observed during C. elegans development.

SKN-1 isoform-c is essential for C. elegans development.

The transcription factor SKN-1 in Caenorhabditis elegans is a critical regulator of various biological processes, impacting development, diet and immune responses, cellular detoxification, and lipid metabolism; thereby playing a pivotal role in regulating the health and lifespan of the organism. The primary isoforms of SKN-1 ( SKN-1 a, SKN-1 b, and SKN-1 c) exhibit distinct functions resembling mammalian Nrf transcription factors. This study investigates the specific role of the SKN-1 c isoform in development by generating mutants with targeted missense mutations in the skn-1 c and skn-1 a isoforms. The skn-1 c Met1Ala mutants, which replaces a start methionine with alanine, renders SKN-1 c non-functional while preserving other isoforms, produced inviable embryos, requiring a balancer chromosome for proper embryonic development. In contrast, skn-1 a Met1Ala mutants, which replaces the start methionine with alanine for this isoform, displayed normal embryonic development and hatching. Moreover, the data suggest that SKN-1 c plays a crucial role in embryonic development, as strains without maternally deposited SKN-1 c lead to embryos that are developmentally arrested. Together, these findings contribute to our understanding of SKN-1 c's specific role in influencing embryogenesis and development in C. elegans.

Early life changes in histone landscape protect against age-associated amyloid toxicities through HSF-1-dependent regulation of lipid metabolism.

Transient events during development can exert long-lasting effects on organismal lifespan. Here we demonstrate that exposure of Caenorhabditis elegans to reactive oxygen species during development protects against amyloid-induced proteotoxicity later in life. We show that this protection is initiated by the inactivation of the redox-sensitive H3K4me3-depositing COMPASS complex and conferred by a substantial increase in the heat-shock-independent activity of heat shock factor 1 (HSF-1), a longevity factor known to act predominantly during C. elegans development. We show that depletion of HSF-1 leads to marked rearrangements of the organismal lipid landscape and a significant decrease in mitochondrial β-oxidation and that both lipid and metabolic changes contribute to the protective effects of HSF-1 against amyloid toxicity. Together, these findings link developmental changes in the histone landscape, HSF-1 activity and lipid metabolism to protection against age-associated amyloid toxicities later in life.

Transmembrane protein 120A (TMEM-120A/TACAN) coordinates with PIEZO channel during Caenorhabditis elegans reproductive regulation.

Membrane protein TMEM120A (also known as TACAN) was presumed to be both a mechanically activated molecule and a lipid-modifying enzyme. TMEM120A has been identified as a negative regulator of the essential excitatory mechanosensitive protein PIEZO2. However, the extent to which TMEM120A mediates PIEZO2's activity during physiological processes remains largely unknown. In this study, we used the Caenorhabditis elegans reproductive tract to explore the functional contribution of tmem-120, the sole TMEM120A/B ortholog, and its genetic interaction with pezo-1 in vivo. tmem-120 was expressed throughout the C. elegans development, particularly in the germline, embryos, and spermatheca. A tmem-120 mutant with a full-length deletion (tmem-120Δ) displayed deformed germline, maternal sterility, and a reduced brood size. In vivo live imaging revealed that pinched zygotes were frequently observed in the uterus of tmem-120Δ mutant animals, suggesting damage during spermathecal contraction. We then employed the auxin-inducible degradation system to degrade TMEM-120 protein in all somatic tissues or the germline, both of which resulted in reduced brood sizes. These findings suggested that multiple inputs of tmem-120 from different tissues regulate reproduction. Lastly, the loss of tmem-120 alleviated the brood size reduction and defective sperm navigation behavior in the pezo-1Δ mutant. Overall, our findings reveal a role for tmem-120 in regulating reproductive physiology in C. elegans, and suggest an epistatic interaction between pezo-1 and tmem-120 when governing proper reproduction.

A Multisystemic Approach Revealed Aminated Polystyrene Nanoparticles-Induced Neurotoxicity.

Exposure to plastic nanoparticles has dramatically increased in the last 50 years, and there is evidence that plastic nanoparticles can be absorbed by organisms and cross the blood-brain-barrier (BBB). However, their toxic effects, especially on the nervous system, have not yet been extensively investigated, and most of the knowledge is based on studies using different conditions and systems, thus hard to compare. In this work, physicochemical properties of non-modified polystyrene (PS) and amine-functionalized PS (PS-NH2 ) nanoparticles are initially characterized. Advantage of a multisystemic approach is then taken to compare plastic nanoparticles effects in vitro, through cytotoxic readouts in mammalian cell culture, and in vivo, through behavioral readouts in the nematode Caenorhabditis elegans (C. elegans), a powerful 3R-complying model organism for toxicology studies. In vitro experiments in neuroblastoma cells indicate a specific cytotoxic effect of PS-NH2 particles, including a decreased neuronal differentiation and an increased Amyloid β (Aβ) secretion, a sensitive readout correlating with Alzheimer's disease pathology. In parallel, only in vivo treatments with PS-NH2 particles affect C. elegans development, decrease lifespan, and reveal higher sensitivity of animals expressing human Aβ compared to wild-type animals. In summary, the multisystemic approach discloses a neurotoxic effect induced by aminated polystyrene nanoparticles.

Identification of pararosaniline as a modifier of RNA splicing in Caenorhabditis elegans.

Posttranscriptional splicing of premessenger RNA (mRNA) is an evolutionarily conserved eukaryotic process for producing mature mRNA that is translated into proteins. Accurate splicing is necessary for normal growth and development, and aberrant splicing is increasingly evident in various human pathologies. To study environmental factors that influence RNA splicing, we employed a fluorescent Caenorhabditis elegans in vivo splicing reporter as a biomarker for splicing fidelity to screen against the US EPA ToxCast chemical library. We identified pararosaniline hydrochloride as a strong modifier of RNA splicing. Through gene expression analysis, we found that pararosaniline activates the oxidative stress response and alters the expression of key RNA splicing regulator genes. Physiological assays show that pararosaniline is deleterious to C. elegans development, reproduction, and aging. Through a targeted RNAi screen, we found that inhibiting protein translation can reverse pararosaniline's effect on the splicing reporter and provide significant protection against long-term pararosaniline toxicity. Together, this study reveals a new chemical modifier of RNA splicing and describes translation inhibition as a genetic mechanism to provide resistance.

Modified citrus pectin confers a preventative effect on cancer-related pathways in CdCl2-treated C. elegans

Cadmium chloride (CdCl2) is a toxic compound found as a pollutant in the environment due to agricultural and industrial sources. Exposure to Cd2+ is known to promote malignant tumors such as lung cancer and leukemia. While the current medications for cadmium toxicity focus on treatments to promote the excretion from the body, treatments to improve health after cadmium exposure are less well studied. Modified citrus pectin (MCP) is a polysaccharide derived from citrus peels that has been shown to induce natural killer cell activity in myeloid leukemia cells and also act as a natural chelation agent to help excrete toxic metals from healthy human subjects. We hypothesized that MCP might have a counteracting effect against CdCl2 toxicity through cancer-related pathways. This study investigates the effects of MCP on CdCl2 toxicity in C. elegans, which shares a number of cancer-related pathways with mammals. The results indicated that MCP was able to significantly counter the toxic effects of CdCl2 on C. elegans lifespan and development. Our studies suggest that the beneficial effects of MCP may result from its ability to mitigate the effects of CdCl2 on gene expression, particularly in conserved pathways associated with apoptosis, tumor induction and suppression and inflammation-related pathways.

The C. elegans PUM1, 2-like RNA binding protein PUF-8 is required for robustness of the cell death fate.

During C. elegans development, 1090 somatic cells are generated of which 959 survive and 131 die, many through apoptosis. We present evidence that PUF-8, a C. elegans ortholog of the mammalian RNA binding proteins PUM1 and PUM2, is required for the robustness of this 'survival and death' pattern. We found that PUF-8 prevents the inappropriate death of cells that normally survive, and we present evidence that this anti-apoptotic activity of PUF-8 is dependent on PUF-8's ability to interact with ced-3caspase mRNA thereby repressing the activity of the pro-apoptotic ced-3caspase gene. PUF-8 also promotes the death of cells that are programmed to die, and we propose that this pro-apoptotic activity of PUF-8 may depend on PUF-8's ability to repress the expression of the anti-apoptotic ced-9Bcl-2 gene. Our results suggest that stochastic differences in the expression of genes within the apoptosis pathway can disrupt the highly reproducible and robust survival and death pattern during C. elegans development, and that PUF-8PUM1, 2 acts at the post-transcriptional level to level out these differences, thereby ensuring proper cell number homeostasis.

A circadian-like gene network programs the timing and dosage of heterochronic miRNA transcription during C. elegans development

Development relies on the exquisite control of both the timing and the levels of gene expression to achieve robust developmental transitions. How cis- and trans-acting factors control both aspects simultaneously is unclear. We show that transcriptional pulses of the temporal patterning microRNA (miRNA) lin-4 are generated by two nuclear hormone receptors (NHRs) in C.elegans, NHR-85 and NHR-23, whose mammalian orthologs, Rev-Erb and ROR, function in the circadian clock. Although Rev-Erb and ROR antagonize each other to control once-daily transcription in mammals, NHR-85/NHR-23 heterodimers bind cooperatively to lin-4 regulatory elements to induce a single pulse of expression during each larval stage. Each pulse's timing, amplitude, and duration are dictated by the phased expression of these NHRs and the C.elegans Period ortholog, LIN-42, that binds to and represses NHR-85. Therefore, during nematode temporal patterning, an evolutionary rewiring of circadian clock components couples the timing of gene expression to the control of transcriptional dosage.

The Highs and Lows of FBXW7: New Insights into Substrate Affinity in Disease and Development.

FBXW7 is a critical regulator of cell cycle, cell signaling, and development. A highly conserved F-box protein and component of the SKP1-Cullin-F-box (SCF) complex, FBXW7 functions as a recognition subunit within a Cullin-RING E3 ubiquitin ligase responsible for ubiquitinating substrate proteins and targeting them for proteasome-mediated degradation. In human cells, FBXW7 promotes degradation of a large number of substrate proteins, including many that impact disease, such as NOTCH1, Cyclin E, MYC, and BRAF. A central focus for investigation has been to understand the molecular mechanisms that allow the exquisite substrate specificity exhibited by FBXW7. Recent work has produced a clearer understanding of how FBXW7 physically interacts with both high-affinity and low-affinity substrates. We review new findings that provide insights into the consequences of "hotspot" missense mutations of FBXW7 that are found in human cancers. Finally, we discuss how the FBXW7-substrate interaction, and the kinases responsible for substrate phosphorylation, contribute to patterned protein degradation in C. elegans development.

Praziquantel inhibits Caenorhabditis elegans development and species-wide differences might be cct-8-dependent.

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals. Caenorhabditis elegans is an established model to investigate anthelmintics used to treat roundworms. In this study, we use C. elegans to examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective against C. elegans, but the right-handed PZQ (R-PZQ) is most effective against schistosome infections. We conducted a genome-wide association mapping with 74 wild C. elegans strains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region: cct-8, znf-782, Y104H12D.4, Y104H12D.2, and cox-18, might underlie this variation. The gene cct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of both cct-8 and hsp-70. Acute exposure to PZQ caused increased expression of hsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows that C. elegans can be used to study PZQ mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses in C. elegans.

EAT-2 attenuates C. elegans development via metabolic remodeling in a chemically defined food environment.

Dietary intake and nutrient composition regulate animal growth and development; however, the underlying mechanisms remain elusive. Our previous study has shown that either the mammalian deafness homolog gene tmc-1 or its downstream acetylcholine receptor gene eat-2 attenuates Caenorhabditis elegans development in a chemically defined food CeMM (C. elegans maintenance medium) environment, but the underpinning mechanisms are not well-understood. Here, we found that, in CeMM food environment, for both eat-2 and tmc-1 fast-growing mutants, several fatty acid synthesis and elongation genes were highly expressed, while many fatty acid β-oxidation genes were repressed. Accordingly, dietary supplementation of individual fatty acids, such as monomethyl branch chain fatty acid C17ISO, palmitic acid and stearic acid significantly promoted wild-type animal development on CeMM, and mutations in either C17ISO synthesis gene elo-5 or elo-6 slowed the rapid growth of eat-2 mutant. Tissue-specific rescue experiments showed that elo-6 promoted animal development mainly in the intestine. Furthermore, transcriptome and metabolome analyses revealed that elo-6/C17ISO regulation of C. elegans development may be correlated with up-regulating expression of cuticle synthetic and hedgehog signaling genes, as well as promoting biosynthesis of amino acids, amino acid derivatives and vitamins. Correspondingly, we found that amino acid derivative S-adenosylmethionine and its upstream metabolite methionine sulfoxide significantly promoted C. elegans development on CeMM. This study demonstrated that C17ISO, palmitic acid, stearic acid, S-adenosylmethionine and methionine sulfoxide inhibited or bypassed the TMC-1 and EAT-2-mediated attenuation of development via metabolic remodeling, and allowed the animals to adapt to the new nutritional niche.

Stability of asymmetric cell division: A deformable cell model of cytokinesis applied to C. elegans

Cell division during early embryogenesis is linked to key morphogenic events such as embryo symmetry breaking and tissue patterning. It is thought that the physical surrounding of cells together with cell intrinsic cues act as a mechanical “mold,” guiding cell division to ensure these events are robust. To quantify how cell division is affected by the mechanical and geometrical environment, we present a novel computational mechanical model of cytokinesis, the final phase of cell division. Simulations with the model reproduced experimentally observed furrow dynamics and describe the volume ratio of daughter cells in asymmetric cell divisions, based on the position and orientation of the mitotic spindle. For dividing cells in geometrically confined environments, we show how the orientation of confinement relative to the division axis modulates the volume ratio in asymmetric cell division. Further, we quantified how cortex viscosity and surface tension determine the shape of a dividing cell and govern bubble-instabilities in asymmetric cell division. Finally, we simulated the formation of the three body axes via sequential (a)symmetric divisions up until the six-cell stage of early C. elegans development, which proceeds within the confines of an eggshell. We demonstrate how model input parameters spindle position and orientation provide sufficient information to reliably predict the volume ratio of daughter cells during the cleavage phase of development. However, for egg geometries perturbed by compression, the model predicts that a change in confinement alone is insufficient to explain experimentally observed differences in cell volume. This points to an effect of the compression on the spindle positioning mechanism. Additionally, the model predicts that confinement stabilizes asymmetric cell divisions against bubble-instabilities.