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.The large number of protease genes in animal genomes reflects the widespread importance of proteolysis to animal physiology and development. The rich functional diversity of proteases can be attributed to the variety of catalytic mechanisms and protein structures, some of which are of a modular design, offering different levels of structural and functional complexity (1). The MEROPS data base (merops.sanger.ac.uk; release 6.3) (2) catalogs 551, 553, 563, 366, and 367 proteases for human, mouse, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans, respectively. The majority of these enzymes have not been studied at the physiological and biochemical level, but it has been possible to classify most of them into families based on statistically significant similarities in primary protein structure (2).Surprisingly, a significant proportion (12-25%) of the total number of the predicted protease-like genes in the human, mouse, D. melanogaster, A. gambiae, and C. elegans genomes code for proteins that lack one or more catalytic residues and are therefore classified as "non-peptidase" family members. Some of these genes may be nonfunctional pseudogenes, but many have probably lost the catalytic activity of an ancestral protein while acquiring new functions. These non-peptidase proteins are structurally related to proteases distributed across all classes, but homologues of metallopeptidases are particularly well represented. One such protein is UNC-71, a C. elegans member of the ADAMs family, which lacks a zinc binding site and yet has acquired important roles in development (3). Our attention has recently been drawn to the fact that several invertebrate members of the angiotensin-converting enzyme (ACE) 1 (EC 3.4.15.1, peptidyl dipeptidase A) famil...
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.
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 CBFβ, 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.
SummaryCaenorhabditis 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 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. J. Cell. Biochem. 108: 14-21, 2009. ß 2009 Wiley-Liss, Inc. KEY WORDS: RUNX; STEM CELLS; HEMATOPOIESIS; HAIR FOLLICLE; C. ELEGANSA central and recurrent theme in the development of multicellular organisms is the establishment of disparate cell and tissue types from a single fertilized zygote. Somatic stem cells provide the means by which from a limited pool of undifferentiated cells, multipotent daughters can arise, destined for a variety of tissue types. Stem cells are able to divide asymmetrically (self-renewal maintenance) to maintain overall stem cell number and provide more specialized progenitors, which may then embark on one or more specific developmental programmes before becoming terminally differentiated. Alternatively, stem cells may undergo symmetric (self-renewal expansion) division, to repopulate the stem cell niche. In tissue homeostasis, stem cells are pivotal since they are essential for supplying replacements for short-lived cells, as in the case of hematopoiesis, or in tissue repair. Stem cell developmental programmes therefore need to be tightly regulated; if developmental programmes of progenitors are not properly controlled they may fail to mature, or follow inappropriate paths to differentiation; if the homeostasis of the stem cell itself is disrupted, too few progenitors may be available. It is therefore apparent that at the heart of proper stem cell function are the mechanisms that regulate the developmental decision between cell division and differentiation.The RUNX family of transcription factors have recently emerged as major players in the control of stem cell differentiation and proliferation in both cancer and developmental biology. With the advent of genome sequencing, RUNX genes have now been identified in a wide range of animal species, from worms to fish, sea urchins to mice. In mouse, Runx1 has recently been identified as ...
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