Richard W. Padgett

TGFβ-related pathways: roles in Caenorhabditis elegans development

Abstract Genetic and molecular analysis in Caenorhabditis elegans has produced new insights into how TGFβ-related pathways transduce signals and the developmental processes in which they function. These pathways are essential regulators of dauer formation, body-size determination, male copulatory structures and axonal guidance. Here, we review the insights that have come from standard molecular genetic experiments and discuss how the recently completed genome sequence has contributed to our understanding of these pathways.

Nomenclature: Vertebrate Mediators of TGFβ Family Signals

Anita B. Roberts,10 Jim Smith,11 Gerald H. Thomsen,14 Nomenclature: Vertebrate Bert Vogelstein,7,12 and Xiao-Fan Wang,13 Mediators of TGFb Family 1 Departments of Growth and Development, and Anatomy, Programs of Cell and Developmental Biology Signals University of California, San Francisco San Francisco, California 94143-0640 2 Department of Cellular Developmental Biology The Mad (mothers against decapentaplegic) gene in Harvard University Drosophila and the related Sma genes in Caenorhabditis Cambridge, Massachusetts 02138-2019 elegans have been implicated in signal transduction by 3 Department of Molecular and Cell Biology factors of the TGFb family (Sekelski et al., 1995; Savage Division of Biochemistry and Molecular Biology et al., 1996). Related genes have been identified recently University of California, Berkeley in vertebratesand shown to mediateTGFb family signals Berkeley, California 94720-3204 in these organisms as well. To date, there are five family 4 Ludwig Institute for Cancer Research members described as full length protein sequences S75124 Uppsala, Sweden in human, mouse, and/or Xenopus. Because of their 5 Department of Oncology diversity and simultaneous identification in different labJohn Hopkins School of Medicine oratories, the Mad-related products in vertebrates have Baltimore, Maryland 21205-2196 received different names. In order to facilitate future 6 Department of Cell Biology work and the dissemination of information in this area, Memorial Sloan-Kettering Cancer Center we propose to unify the nomenclature of the vertebrate New York, New York 10021 genes and their products by referring to them as 7 Howard Hughes Medical Institute “Smad.” This term, a merger of Sma and Mad, differenti8 Developmental Biology Programme ates these proteins from unrelated gene products preEuropean Molecular Biology Laboratory viously called Mad. We propose that each individual 69117 Heidelberg, Germany family member be designated as follows. 9 Waksman Institute Department of Molecular Biology and Biochemistry Smad1 and Cancer Institute of New Jersey GenBank accession numbers are U54826, U57456, Rutgers University U58992, U59912, U59423, U58834, and L77888. Smad1 Piscataway, New Jersey 08855-0759 has been previously referred to as Madr1, bsp1, 10 National Cancer Institute Dwarfin-A, Xmad, Xmad1, and JV4-1. National Institute of Health Bethesda, Maryland 20892-5055 Smad2 11 National Institute for Medical Research GenBank accession numbers are U59911, U60530, The Ridgeway, Mill Hille U65019, U68018 and L77885. Smad2 has been preLondon NW7 1AA, England viously referred to as Madr2, hMAD-2, Xmad2, and 12 The Johns Hopkins Oncology Center JV18-1. Baltimore, Maryland 21231 13 Department of Pharmacology Duke University Medical Center Smad3 Durham, North Carolina 27710 GenBank accession number isU68019. Smad3 has been 14 Department of Biochemistry and Cell Biology previously referred to as hMAD-3. Institute for Cell and Developmental Biology State University of New York Smad4 also referred to in the human as DPC4 (deleted Stony Brook, New York 11794-5215 in pancreatic carcinoma). GenBank accession number isU44378. Smad4 has been References previously referred to as Xmad4.

Transforming growth factor β signaling mediators and modulators

Abstract Transforming growth factor β is a multi-functional growth and differentiation factor responsible for regulating many diverse biological processes in both vertebrate and invertebrate species. Among the most dramatic of TGFβs effects are those associated with specification of cell fates during development and inhibition of cell cycle progression. The core TGFβ signaling pathway has now been described using a synergistic combination of genetic and biochemical approaches. Transmembrane receptors with intrinsic protein serine kinase activity bind ligand in the extracellular milieu and then phosphorylate intracellular proteins known as Smads. Phosphorylated Smads form heterooligomers and translocate into the nucleus where they can modulate transcriptional responses. More recent studies indicate that many other proteins serve as modulators of Smad activity, and utimately define specific cellular responses to TGFβ. Here we describe both the simplistic core TGFβ signaling pathway and the growing number of proteins that impinge on this pathway at the level of Smad function to either enhance or inhibit TGFβ responses

Modulated microRNA expression during adult lifespan in Caenorhabditis elegans

MicroRNAs (miRNAs) are small, abundant transcripts that can bind partially homologous target messages to inhibit their translation in animal cells. miRNAs have been shown to affect a broad spectrum of biological activities, including developmental fate determination, cell signaling and oncogenesis. Little is known, however, of miRNA contributions to aging. We examined the expression of 114 identified Caenorhabditis elegans miRNAs during the adult lifespan and find that 34 miRNAs exhibit changes in expression during adulthood (P≤ 0.05), 31 with more than a twofold level change. The majority of age‐regulated miRNAs decline in relative abundance as animals grow older. Expression profiles of developmental timing regulators lin‐4 and let‐7 miRNAs, as well as conserved muscle miRNA miR‐1, show regulation during adulthood. We also used bioinformatic approaches to predict miRNA targets encoded in the C. elegans genome and we highlight candidate miRNA‐regulated genes among C. elegans genes previously shown to affect longevity, genes encoding insulin‐like ligands, and genes preferentially expressed in C. elegans muscle. Our observations identify miRNAs as potential modulators of age‐related decline and suggest a general reduction of message‐specific translational inhibition during aging, a previously undescribed feature of C. elegans aging. Since many C. elegans age‐regulated miRNAs are conserved across species, our observations identify candidate age‐regulating miRNAs in both nematodes and humans.

TGF-β signaling, Smads, and tumor suppressors

The transforming growth factor‐β (TGF‐β) superfamily is used throughout animal development for regulating the growth and patterning of many tissue types. During the past few years, rapid progress has been made in deciphering how TGF‐β signals are transduced from outside the cell to the nucleus. This progress is based on biochemical studies in vertebrate systems and a combination of genetic studies in Drosophila and Caenorhabditis elegans. These studies have identified a novel family of signaling proteins, the Smad family. Smads can act positively and be phosphorylated by TGF‐β‐like receptors or can act negatively and prevent activation of the positively acting group. The positively acting Smads translocate to the nucleus, bind DNA, and act as transcriptional activators. Thus, genetic and biochemical studies suggest a very simple signaling pathway, in which Smads are the primary downstream participant. BioEssays 20:382–390, 1998.

Circadian regulation of a limited set of conserved microRNAs in Drosophila

BackgroundMicroRNAs (miRNAs) are short non-coding RNA molecules that target mRNAs to control gene expression by attenuating the translational efficiency and stability of transcripts. They are found in a wide variety of organisms, from plants to insects and humans. Here, we use Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs.ResultsWe used a microarray platform to survey the daily levels of D. melanogaster miRNAs in adult heads of wildtype flies and the arrhythmic clock mutant cyc01. We find two miRNAs (dme-miR-263a and -263b) that exhibit robust daily changes in abundance in wildtype flies that are abolished in the cyc01 mutant. dme-miR-263a and -263b reach trough levels during the daytime, peak during the night and their levels are constitutively elevated in cyc01 flies. A similar pattern of cycling is also observed in complete darkness, further supporting circadian regulation. In addition, we identified several miRNAs that appear to be constitutively expressed but nevertheless differ in overall daily levels between control and cyc01 flies.ConclusionThe circadian clock regulates miRNA expression in Drosophila, although this appears to be highly restricted to a small number of miRNAs. A common mechanism likely underlies daily changes in the levels of dme-miR-263a and -263b. Our results suggest that cycling miRNAs contribute to daily changes in mRNA and/or protein levels in Drosophila. Intriguingly, the mature forms of dme-miR-263a and -263b are very similar in sequence to several miRNAs recently shown to be under circadian regulation in the mouse retina, suggesting conserved functions.

Rational Probe Optimization and Enhanced Detection Strategy for MicroRNAs Using Microarrays

Loyal A. Goff, Maocheng Yang, Jessica Bowers, Robert C. Getts, Richard W. Padgett and Ronald P. Hart

Glypican LON-2 Is a Conserved Negative Regulator of BMP-like Signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.

BMP signaling requires retromer-dependent recycling of the type I receptor

Significance The mechanisms that mediate bone morphogenetic protein (BMP) receptor recycling, and the importance of such recycling for signaling in vivo, have remained poorly understood. We find that the retromer complex functions as a linchpin in the recycling of the BMP type I receptor SMA-6 (small-6). In the absence of retromer-dependent recycling, retromer mutants result in the missorting of SMA-6 to lysosomes and a loss of BMP-mediated signaling. Surprisingly, we find that the BMP type II receptor, DAF-4 (dauer formation-defective-4), recycles through a distinct recycling pathway. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation. The transforming growth factor β (TGFβ) superfamily of signaling pathways, including the bone morphogenetic protein (BMP) subfamily of ligands and receptors, controls a myriad of developmental processes across metazoan biology. Transport of the receptors from the plasma membrane to endosomes has been proposed to promote TGFβ signal transduction and shape BMP-signaling gradients throughout development. However, how postendocytic trafficking of BMP receptors contributes to the regulation of signal transduction has remained enigmatic. Here we report that the intracellular domain of Caenorhabditis elegans BMP type I receptor SMA-6 (small-6) binds to the retromer complex, and in retromer mutants, SMA-6 is degraded because of its missorting to lysosomes. Surprisingly, we find that the type II BMP receptor, DAF-4 (dauer formation-defective-4), is retromer-independent and recycles via a distinct pathway mediated by ARF-6 (ADP-ribosylation factor-6). Importantly, we find that loss of retromer blocks BMP signaling in multiple tissues. Taken together, our results indicate a mechanism that separates the type I and type II receptors during receptor recycling, potentially terminating signaling while preserving both receptors for further rounds of activation.

Drosophila dSmad2 and Atr-I transmit activin/TGFbeta signals.

Much is known about the three subfamilies of the TGFβ superfamily in vertebrates—the TGFβs, dpp/BMPs, and activins. Signalling in each subfamily is dependent on both shared and unique cell surface receptors and Smads. In invertebrates, mutants for BMP pathway components have been extensively characterized, but thus far, evidence for an activin‐ or TGFβ‐like pathway has been lacking, preventing the use of the extensive genetic tools available for studying several key issues of TGFβ signalling.