Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number.

Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens.

The Caenorhabditis elegans GATA factor ELT-1 works through the cell proliferation regulator BRO-1 and the Fusogen EFF-1 to maintain the seam stem-like fate.

Seam cells in Caenorhabditis elegans provide a paradigm for the stem cell mode of division, with the ability to both self-renew and produce daughters that differentiate. The transcription factor RNT-1 and its DNA binding partner BRO-1 (homologues of the mammalian cancer-associated stem cell regulators RUNX and CBFß, respectively) are known rate-limiting regulators of seam cell proliferation. Here, we show, using a combination of comparative genomics and DNA binding assays, that bro-1 expression is directly regulated by the GATA factor ELT-1. elt-1(RNAi) animals display similar seam cell lineage defects to bro-1 mutants, but have an additional phenotype in which seam cells lose their stem cell-like properties and differentiate inappropriately by fusing with the hyp7 epidermal syncytium. This phenotype is dependent on the fusogen EFF-1, which we show is repressed by ELT-1 in seam cells. Overall, our data suggest that ELT-1 has dual roles in the stem-like seam cells, acting both to promote proliferation and prevent differentiation.

Identification of cis-regulatory elements from the C. elegans T-box gene mab-9 reveals a novel role for mab-9 in hypodermal function.

We have identified Conserved Non-coding Elements (CNEs) in the regulatory region of Caenorhabditis elegans and Caenorhabditis briggsae mab-9, a T-box gene known to be important for cell fate specification in the developing C. elegans hindgut. Two adjacent CNEs (a region 78 bp in length) are both necessary and sufficient to drive reporter gene expression in posterior hypodermal cells. The failure of a genomic mab-9::gfp construct lacking this region to express in posterior hypodermis correlates with the inability of this construct to completely rescue the mab-9 mutant phenotype. Transgenic males carrying this construct in a mab-9 mutant background exhibit tail abnormalities including morphogenetic defects, altered tail autofluorescence and abnormal lectin-binding properties. Hermaphrodites display reduced susceptibility to the C. elegans pathogen Microbacterium nematophilum. This comparative genomics approach has therefore revealed a previously unknown role for mab-9 in hypodermal function and we suggest that MAB-9 is required for the secretion and/or modification of posterior cuticle.

RUNX genes find a niche in stem cell biology.

The RUNX family of transcriptional regulators are well conserved throughout the animal kingdom, from the simple nematode worm Caenorhabditis elegans to vertebrates. Interest in the RUNX genes emerged principally as a result of the finding that chromosomal translocations disrupting RUNX protein function are observed in a large number of patients suffering with acute myeloid leukemia (AML). In the 20 years that RUNX genes have been under investigation, they have emerged as central players in the control of developmental decisions between proliferation and differentiation in a wide variety of biological situations. This review focuses on recent data highlighting the roles of RUNX genes in stem cells and illustrates the diversity of processes in which the RUNX proteins play a critical role. In particular, we focus on the role of RUNX1 in hematopoietic stem cells (HSCs) and hair follicle stem cells (HFSCs) and the importance of the solo C. elegans RUNX factor rnt-1 in stem cell proliferation in the worm. Observations in a variety of stem cell systems have developed to the point where useful comparisons can be made, from which guiding principles may emerge.

CEH-20/Pbx and UNC-62/Meis function upstream of rnt-1/Runx to regulate asymmetric divisions of the C. elegans stem-like seam cells.

Caenorhabditis elegans seam cells divide in the stem-like mode throughout larval development, with the ability to both self-renew and produce daughters that differentiate. Seam cells typically divide asymmetrically, giving rise to an anterior daughter that fuses with the hypodermis and a posterior daughter that proliferates further. Previously we have identified rnt-1 (a homologue of the mammalian cancer-associated stem cell regulator Runx) as being an important regulator of seam development, acting to promote proliferation; rnt-1 mutants have fewer seam cells whereas overexpressing rnt-1 causes seam cell hyperplasia. We isolated the interacting CEH-20/Pbx and UNC-62/Meis TALE-class transcription factors during a genome-wide RNAi screen for novel regulators of seam cell number. Animals lacking wild type CEH-20 or UNC-62 display seam cell hyperplasia, largely restricted to the anterior of the worm, whereas double mutants have many additional seam cells along the length of the animal. The cellular basis of the hyperplasia involves the symmetrisation of normally asymmetric seam cell divisions towards the proliferative stem-like fate. The hyperplasia is completely suppressed in rnt-1 mutants, and rnt-1 is upregulated in ceh-20 and unc-62 mutants, suggesting that CEH-20 and UNC-62 function upstream of rnt-1 to limit proliferative potential to the appropriate daughter cell. In further support of this we find that CEH-20 is asymmetrically localised in seam daughters following an asymmetric division, being predominantly restricted to anterior nuclei whose fate is to differentiate. Thus, ceh-20 and unc-62 encode crucial regulators of seam cell division asymmetry, acting via rnt-1 to regulate the balance between proliferation and differentiation.

The SFT-1 and OXA-1 respiratory chain complex assembly factors influence lifespan by distinct mechanisms in C. elegans.

BACKGROUND: C. elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain function, yet, surprisingly, they are often long-lived, a property that has offered unique insights into the molecular mechanisms of aging. In this study, we examine the phenotypic consequences of reducing the expression of the respiratory chain complex assembly factors sft-1 (homologous to human SURF1) and oxa-1 (homologous to human OXA1) by RNA interference (RNAi). Mutations in human SURF1 are associated with Leigh syndrome, a neurodegenerative condition of the brain caused by cytochrome oxidase (COX) deficiency. Both SURF1 and OXA1 are integral proteins of the inner mitochondrial membrane, functioning in the COX assembly pathway. RESULTS: RNAi of both of these genes in C. elegans is associated with increased longevity, but the mechanism by which lifespan is extended is different in each case. sft-1(RNAi) animals display lifespan extension that is dependent on the daf-16 insulin-like signaling pathway, and associated with sensitivity to oxidative stress. oxa-1(RNAi) animals, in contrast, exhibit increased longevity that is at least partially independent of daf-16, and associated with a reduced developmental rate and increased resistance to oxidative stress. CONCLUSIONS: This study further delineates the consequences of mitochondrial dysfunction within a whole organism that will ultimately help provide new models for human mitochondrial-associated diseases. The difference in phenotype observed upon down-regulation of these two COX assembly factors, as well as phenotypic differences between these factors and other respiratory chain components analyzed thus far, illustrates the complex inter-relationships that exist among energy metabolism, reproduction and aging even in this simplest of metazoan model organisms.

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans.

The T-box transcription factor mab-9 has been shown to be required for the correct fate of the male-specific blast cells B and F, normal posterior hypodermal morphogenesis, and for the correct axon migration of motor neurons that project circumferential commissures to dorsal muscles. In this study, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat-shock promoter has the opposite effect, causing repression of mab-9 in various cells. We find that mab-9 expression in unc-37 mutants is also elevated in DA motor neurons, consistent with known roles for UNC-37 as a co-repressor with UNC-4. These results identify mab-9 as a novel target of the UNC-4/UNC-37 repressor complex in motor neurons, and suggest that mis-expression of mab-9 may contribute to the neuronal wiring defects in unc-4 and unc-37 mutants.

An essential role in molting and morphogenesis of Caenorhabditis elegans for ACN-1, a novel member of the angiotensin-converting enzyme family that lacks a metallopeptidase active site.

Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes.

Functional redundancy of two nucleoside transporters of the ENT family (CeENT1, CeENT2) required for development of Caenorhabditis elegans.

The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.