Ciliate Genome Sequence Reveals Unique Features of a Model Eukaryote Richard Robinson | DOI: 10.1371/Journal.Pbio.0040304

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Synopses of Research Articles Ciliate Genome Sequence Reveals Unique Features of a Model Eukaryote Richard Robinson | DOI: 10.1371/journal.pbio.0040304

One reason to sequence the genomes from what were once free-living of non-human organisms is to better cyanobacteria; typically, many of the understand our similarities and genes of such an endosymbiont are differences. And, at fi rst sight, it is hard shifted into the host nucleus, as they to imagine a eukaryote more different have been in the apicomplexans and from humans than Tetrahymena dinofl agellates. T. thermophila has no thermophila. A relative of Paramecium, plastids, but it has been suggested this single-celled creature has a strong that its ancestors did. The authors but fl exible exterior covered with discovered no remnants of plastid rows of cilia; but it is inside where genes within T. thermophila, strongly things seem to get really alien. Each suggesting that plastid acquisition cell contains not one but two nuclei: a occurred after the other two groups micronucleus, which contains only fi ve split off from the ciliates. chromosomes, and a macronucleus, All told, the genome contains over which has more than 200. 27,000 protein-coding genes, more Biologists have long known that than naively expected for a single- the micronucleus contains the DNA celled species and comparable to the reserved for reproduction, and that number in humans. Certain gene the macronucleus arises from the families appear to have expanded micronucleus and controls the cell’s signifi cantly in T. thermophila, indicating other functions. During macronucleus the likely importance of the processes formation (which happens each carried out by the proteins each time the cells mate), each of the fi ve family encodes. An example is the chromosomes splinters into multiple DOI: 10.1371/journal.pbio.0040304.g001 presence of over 300 genes for voltage- fragments, which in turn replicate The genome sequence of the single-celled gated ion channels, which control to form many copies of the resulting ciliate Tetrahymena thermophila sheds light membrane transport, a key function of smaller chromosomes. In a new study, on early eukaryotic evolution. this free-living, single-celled creature. Jonathan Eisen and a team of over 50 Previous analysis of gene structure scientists report the full sequence of contain much less repetitive DNA showed that T. thermophila uses only the macronuclear genome. than most other eukaryotes—about one stop codon (UGA) during protein The authors began by isolating 2% of the total DNA, versus over synthesis, compared to the three that DNA from purifi ed macronuclei (no 50% in humans—partly because most are standard in most eukaryotes; the mean feat in itself), and performed a repetitive DNA is jettisoned during unused ones instead encode glutamine. “shotgun” sequence, splitting the DNA the formation of the macronucleus, As in many other organisms, UGA into millions of fragments, sequencing when about 15% of micronuclear itself is also used in some genes to each of these, and then reconstructing genomic DNA is excised. The authors encode the amino acid selenocysteine, the whole by using computers to match provide evidence that excision targets making T. thermophila the only known overlaps. They estimate that they not only repeated elements per se but organism to translate all 64 codons. have captured more than 95% of the also foreign DNA (such as “selfi sh” The authors also wish to sequence genome, and conclude it is 105 million mobile DNA transposons) in particular, the micronucleus genome, which base pairs in length. The exact number indicating the importance of this should provide insights into T. of chromosomes is still at issue, though process in maintaining the integrity thermophila biology that is unavailable the authors present evidence that it lies of the expressed genome from such from the macronucleus alone. A between 185 and 287, and, based on outside invasions. key component of the project is the number of telomeres, is probably Sequencing the genome also that all of the data have been made about 225. allowed the authors to address a publicly available without restrictions T. thermophila macronuclear nagging evolutionary question, namely throughout the project, allowing the chromosomes, unlike those in the the timing of plastid acquisition scientifi c community to freely analyze micronucleus and other species, are in the alveolates, a group of three the genome of this organism even prior highly unusual because they appear related phyla: the ciliates (including to this publication. to lack centromeres, the regions that Tetrahymena), the apicomplexans link chromosomal replicants and then (parasites that cause malaria, among Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan guide their separation during mitosis other diseases), and the dinofl agellates M, et al. (2006) Macronuclear genome and meiosis. This makes some sense, (ocean-dwelling photosynthetic sequence of the ciliate Tetrahymena since the macronucleus undergoes protozoans). Plastids, such as the thermophila, a model eukaryote. DOI: neither process. Furthermore, they chloroplast, are organelles descended 10.1371/journal.pbio.0040286

The tiny nematode Caenorhabditis high-oxygen avoidance, and that these elegans spends most of its life in the neurons can be suppressed in some soil, searching for an abundance cases by other neurons that provide of food and just the right amount information about food availability. of oxygen. But what happens when The result is an integrated system optimal oxygen and food supplies can’t that allows this simple organism to be found in the same place? More respond to its complex environment generally, how does the organization in an equally complex manner. of an animal’s neural networks help Electrophysiological examination of it produce the right behaviors in other “simple” systems, like motor competing contexts? circuits in the leech and the lobster, With only 302 neurons, and has demonstrated comparable powerful genetic tools available to the complexity in well-defi ned neural DOI: 10.1371/journal.pbio.0040306.g001 researchers who study it, C. elegans is networks, with context-dependent a valuable subject for exploring the Wild-type C. elegans prefers 7%–14% neuronal participation in a particular neural control of behavior. Previous oxygen when placed in an oxygen gradient. behavior. The principles uncovered work has identifi ed just three kinds of in these systems are likely to be neurons as important for sensing and neurons URX, AQR, and PQR suppress applicable to even more complex responding to oxygen. These neurons npr-1(215V)’s locomotor response to brains, whose neuronal circuits are not express a family of genes that appear oxygen. In this study, the researchers amenable to comparable dissection. to encode enzymes called soluble found another set of neurons—SDQ, guanylate cyclases (sGCs). C. elegans ALN, and PLN—expressing sGCs Chang AJ, Chronis N, Karow DS, Marletta sGCs bind oxygen and initiate signaling that were able to process information MA, Bargmann CI (2006) A distributed cascades within the neurons. Animals about ambient oxygen levels. They also chemosensory circuit for oxygen preference lacking certain members of this gene found that the ion channels OSM-9 and in C. elegans. DOI: 10.1371/journal. family no longer respond normally to OCR-2 in yet another set of neurons pbio.0040274 oxygen. But, since other neurons also (ADF and ASH) promote high-oxygen express sGCs, these neurons could play avoidance. The researchers concluded a role in oxygen sensing as well. that these neurons interact with sGC C. elegans’ response to high ambient neurons to produce high-oxygen oxygen (above 14%) in the presence avoidance and modulation of this Bacterial Fimbriae of food depends on the activity of a response by food. Designed to Stay with neuropeptide receptor called NPR- Another aggregating strain of C. 1. Naturally occurring npr-1(215F) elegans, daf-7, gave the researchers yet the Flow nematode strains and laboratory- another angle to explore. In crowded, Liza Gross induced npr-1(lf) strains avoid high low-food conditions, the developmental DOI: 10.1371/journal.pbio.0040314 oxygen whether or not food is present gene daf-7 shows low activity and the and aggregate in the presence of food. nematode enters an alternative larva The human digestive system Another naturally occurring strain, npr- stage called a dauer. The researchers houses a diverse colony of benefi cial 1(215V), avoids high oxygen only when found that daf-7 mutants avoided high bacteria, but one species—E. coli— food is absent. How does npr-1(215V) oxygen with or without food, suggesting can wreak havoc when it colonizes integrate the information about the that daf-7, like npr-1(215V), is involved mucous membranes that normally two stimuli? To learn the answer, in suppressing high-oxygen avoidance exist unmolested (for example, Andy Chang, Cornelia Bargmann, and in the presence of food. Further in the urinary tract). To latch on colleagues systematically assessed the studies suggested that food might be to cells and establish infection, possible role of a number of neurons exerting its infl uence in part by altering E. coli uses fi mbriae—long, hairlike and genes using mutation and selective daf-7 expression in ASI neurons. The organelles that project from the gene replacement. Their experiments researchers also found that daf-7 bacterium’s surface. Fimbriae involved fi rst removing the function mutants expressed higher levels of a consist of interlinking subunits of a particular gene (for example, an gene involved in serotonin synthesis of a single protein called pilin that sGC), then assessing the change in in ADF neurons, suggesting that ADF forms a rigid, coiled helix-shaped response to oxygen (by looking for may represent a convergence point rod. Sticky proteins called adhesins changes in the typical distribution of for networks that promote response to cap the tip of the rod and bind to animals along an oxygen gradient), and high oxygen and those that suppress it. carbohydrate receptors on their then fi nally replacing that gene in only The researchers concluded that host, thus securing bacteria on the one kind of neuron to see if normal at least four sets of sensory neurons host cells as extracellular fl uids swirl function returns. (some or all of URX, AQR, and PQR; around them. Their results revealed some surprises. some or all of SDQ, ALN, and PLN; A previous study led by Evgeni Previous studies showed that the ADF; and ASH) in C. elegans promote Sokurenko and Viola Vogel

PLoS Biology | www.plosbiology.org 1474 September 2006 | Volume 4 | Issue 9 | e306 | e314 investigated the most common type was affi xed to a glass surface. After of E. coli fi mbriae. The sticky protein mannose bound to the fi mbrial FimH, at the tip of these fi mbriae is called the cantilever retreated from the FimH and binds to a carbohydrate bacterium at a constant velocity. The called mannose. They showed that researchers determined that, instead of powerful drag forces created by the the FimH–mannose bond breaking, the extracellular fl uids don’t carry the fi mbriae stretched out far beyond their bound bacteria away, as one might original length. expect, but instead strengthen One reason that fi mbriae extend their adhesion to their host. The could be that the individual pilin researchers attributed this increased subunits of the fi mbrial rod are binding to a biphasic “catch bond” uncoiling. The researchers tested this mechanism whereby increased drag hypothesis by applying a constant forces cause the FimH at the tip of force between the cantilever and the fi mbria to switch from a form that fi mbria—under which fi mbrial binds mannose weakly to a form that length changes slowly. They observed binds strongly. Because of this, the that stepwise jumps in distance bacteria bind best at an optimal force corresponded to the expected length that is high enough to switch FimH to of individual subunits unwinding from DOI: 10.1371/journal.pbio.0040314.g001 strong binding but not so high that the coiled shaft one at a time. Thus, it breaks the strong FimH–mannose the researchers concluded, fi mbrial The long, sticky fi laments covering E. coli bacteria uncoil under force, apparently bond. elongation proceeds as subunits improving the binding of the terminal And now, in a new study, the same uncoil one after another. This was adhesive unit in the presence of forces group of researchers (including also supported by a mathematical generated by fl uid fl ow. (Image: Manu fi rst author Manu Forero) set out to model developed by the researchers Forero) determine whether the coiled rod to quantify the biophysical forces structure of fi mbriae affects how the governing the dynamics of fi mbrial sticky FimH at the tip binds. It had uncoiling. exceed the catch-bond threshold, been assumed that fi mbrial rods play a Forero et al. also detected that, shortening the life of the bond. largely static structural role, either by after uncoiling at increasing force, Because E. coli living in the gut or extending the tip adhesins’ reach or by the stretched fi mbriae re-coil if the other mucosal surfaces experience resisting electrostatic repulsive forces pulling force drops. Importantly, while constantly changing fl ow rates and between bacteria and cell surfaces. fi mbrial uncoiling under high force forces, these adjustments should But Forero et al. show that the rods decreases the tension within the rod, enhance fi mbrial attachment under a function more dynamically, using their re-coiling under low force increases diverse range of fl uid conditions. The mechanical properties to help stabilize the tension. Thus, the tensile force correspondence of forces suggests the FimH–mannose bond against a within the rod stays within some that the mechanical properties of the turbulent background. intermediate level when fi mbrial fi mbrial rod and the FimH–mannose Fimbriae-mediated adhesion was length is stable. complex co-evolved to optimize investigated with an atomic force The researchers found that the adhesive stability in fl uids. microscope, which uses a cantilever to intermediate force range corresponds apply (and measure) forces between its to the force level where the FimH– Forero M, Yakovenko O, Sokurenko EV, tip and the sample under investigation. mannose bonds last longest. Lower, Thomas WE, Vogel V (2006) Uncoiling Forero et al. outfi tted the cantilever coiling forces are too weak to switch mechanics of Escherichia coli type I fi mbriae tip with mannose, and then used this bonds to a long-lived state before are optimized for catch bonds. DOI: 10.1371/ to touch a fi mbriated E. coli cell that breaking, and higher, uncoiling forces journal.pbio.0040298

A Bacterial Protein Puts a New Twist on DNA Transcription Mason Inman | DOI: 10.1371/journal.pbio.0040294

For organisms to adapt, develop, and simply live, they must transcription by facilitating DNA strand separation, which regulate hundreds to thousands of genes, making fi ne- must occur before RNA polymerase can begin copying the tuned, precisely timed adjustments to produce the specifi c DNA code. Once transcription begins, the sigma factor complement of proteins required for the occasion. For disengages from the RNA polymerase, becoming available for bacteria, this task falls largely to proteins called sigma factors. new joint ventures with different RNA polymerases. These small proteins associate with RNA polymerase, the A single sigma factor can control the expression of enzyme that mediates gene transcription, to form a complex hundreds of genes through these partnerships, carrying out called the holoenzyme. The holoenzyme, guided by the sigma everything from basic metabolic activities to physiological factor, recognizes promoter regions, which are specifi c DNA responses to environmental stress (which, for bacteria, might sequences that precede protein-coding sequences and mark include antibiotic therapy). Knowing how sigma factors bind the transcription start site. Sigma factors also contribute to to DNA is an important step in understanding how they

PLoS Biology | www.plosbiology.org 1475 September 2006 | Volume 4 | Issue 9 | e314 | e294 promoter sequences, comprising conserved sequence elements centered about 10 and 35 base pairs upstream of the transcription start site. These so-called –10 and –35 elements are recognized by distinct structural domains of the sigma factor. Structures of one of the most studied sigma factors, a primary sigma factor called sigma-A, have been solved in previous studies. Here, Lane and Darst analyzed the –35- element-binding domain (domain 4) of an alternative Group IV sigma factor found in Escherichia coli, called sigma E4. Group IV sigma factors comprise the largest and most diverse set of sigma factors. Both sigma-A4 and sigma-E4 allow RNA polymerase to bind to the –35 promoter element, but in each case the sequence is very different. In the case of sigma-E4, the sequence is GGAACTT (and others that resemble it). Previous studies showed that sigma-A4 recognizes its consensus sequence, TTGACA, through direct interactions with these six nucleotide bases. It was tempting to assume that sigma-E4 would operate in a similar manner, since the two sigma factors are similar in structure. But, using X-ray crystallography, Lane and Darst showed DOI: 10.1371/journal.pbio.0040294.g001 that sigma-E4 binds its consensus sequence using a more subtle method. By determining the structure of the sigma A crystal structure of domain 4 of an E. coli Group IV sigma factor bound to its consensus sequence, they found that factor bound to –35 element DNA identifi es a unique interaction mechanism that relies on the rigid conformation of the DNA sigma-E4 doesn’t recognize the identity of the sequences per consensus sequence. se but the shape of the DNA helix at those sequences. While one region of the sigma factor sits deep within a groove along the double helix’s side, another region holds the promoter mediate their cosmopolitan regulatory duties. Structural –35 sequence straight. The AA in the center of sigma-E4’s studies provide important clues to the nature and function consensus sequence, the researchers believe, is required for of associations between sigma factors and DNA. In a new the DNA to assume this shape. study, William Lane and Seth Darst used structural analysis Because evolution has conserved the site in these proteins techniques to determine the detailed shape of one type of that sits alongside the AA of the consensus sequence, Lane sigma factor. They show that it binds to short DNA sequences and Darst propose that this method of recognizing –35 using a molecular recognition method that has not been seen promoter sequences may be common across the Group IV before in sigma factors. sigma factors. With further studies of the structures of sigma Sigma factors come in two structurally unrelated families: factors and their means of recognizing specifi c promoters— sigma 54 and sigma 70. The sigma 54 family is associated and thus activating specifi c genes—researchers can better with a diverse range of metabolic processes. The much predict the full complement of genes a given promoter larger sigma 70 family encompasses four groups: the Group will regulate, and in turn gain insight into the diverse I “primary” sigma factors facilitate metabolic and growth physiological responses they help mediate. processes; the Group II–IV “alternative” sigma factors mediate specialized processes like sporulation and the Lane WJ, Darst SA (2006) The structural basis for promoter −35 environmental stress response. The sigma 70-type sigma element recognition by the group IV s factors. DOI: 10.1371/journal. factors recruit the RNA polymerase holoenzyme to bipartite pbio.0040269

Anatomy of a Fever Liza Gross | DOI: 10.1371/journal.pbio.0040305

Many parents experience fear and anxiety when their child While it’s well established that PGE2 originating in brain cells comes down with a fever, unaware that fever is an ancient, causes the second and later phases, the initial phase of fever often benefi cial, response to infection. The fever response is has proven diffi cult to characterize. Of particular interest is conserved across all mammals and many vertebrate classes. whether fever onset is triggered by PGE2 that originates inside (Even reptiles and other cold-blooded animals fare better or outside the brain—a question that has dogged researchers against infection when they develop fever by soaking up for nearly three decades. Now, Alexandre Steiner, Andrej the sun’s heat.) Among other potential adaptive benefi ts, a Romanovsky, and colleagues provide evidence that PGE2 higher temperature can inhibit the growth of bacterial strains synthesis doesn’t begin in the brain as previously thought, that lack sophisticated mechanisms for coping with heat but in the lungs and liver. They also describe the molecular shock. mechanisms that produce PGE2 in these organs. Fever, which is mediated by a lipid called prostaglandin Many of the mechanisms of fever have been established

E2 (PGE2), can pass through multiple temperature phases. by exposing rodents to bacterial endotoxins called

an IV albumin solution. After conﬁ rming that the PGE2 infusion induced fever, the researchers collected venous and

arterial blood from LPS-treated rats. (PGE2 synthesized in

tissues amasses in venous blood; arterial blood delivers PGE2

to the brain.) At the onset of fever, PGE2 was signiﬁ cantly elevated both in the venous and arterial blood.

To investigate the origin of fever-inducing PGE2, Steiner et al. used an antibody-based technique. (Antibodies are too large to cross the blood–brain barrier.) Rats were pretreated

with IV anti-PGE2 antibody serum (controls received normal serum) and then injected with IV LPS. The ﬁ rst phase of LPS fever was signiﬁ cantly attenuated by the antibody (but not the serum), which was found in the blood but not in the brain. These results show that LPS-induced fever is triggered by

circulating PGE2. A previous study from the Romanovsky laboratory reported that onset of fever is accompanied by signifi cantly increased transcription of COX-2 and mPGES-1 in the lung and liver, and a moderate increase of the COX-2 transcript in the hypothalamus (the body’s “fever center”). In this study, they examined the protein distribution of these enzymes in all DOI: 10.1371/journal.pbio.0040305.g001 three tissues. After LPS exposure, the content of neither protein was increased in the hypothalamus. But the lung Cyclooxygenase-2 (green immunofl uorescence) and the and liver did show increased COX-2 expression—primarily macrophage marker ED2 (red immunofl uorescence) in rat lung at in macrophages, which play a role in the infl ammatory the onset of bacterial lipopolysaccharide-induced fever (photo: Jordi Serrats). response—along with activation of an enzyme (cytosolic phospholipase A2) involved in the early steps of PGE2 lipopolysaccharides (LPS). The fi rst phase of LPS-induced synthesis. These organs, but not the hypothalamus, also fever starts within 30 minutes after exposure to the pyrogen. showed signs of infl ammatory signaling. Interestingly, the During the second and subsequent phases—between 90 researchers did not fi nd increased levels of mPGES-1, which minutes and 12 hours after LPS administration—brain facilitates the fi nal step of PGE2 synthesis, suggesting that the cells increase production (called upregulation) of enzymes cell’s normal supply of this enzyme manages the task. involved in PGE2 synthesis. Thus, fever starts about an hour Altogether, these results provide a cellular and molecular before the PGE2-synthesizing enzymes—cyclooxygenase-2 portrait of the fi rst phase of fever and show that it depends, at

(COX-2) and microsomal PGE synthase-1 (mPGES-1)—are least in part, on PGE2 that originates in peripheral tissues. upregulated in the brain, suggesting that fever must be triggered by PGE2 produced peripherally, outside the brain. Steiner AA, Ivanov AI, Serrats J, Hosokawa H, Phayre AN, et al. (2006) To test this hypothesis, Steiner et al. gave rats an Cellular and molecular bases of the initiation of fever. DOI: 10.1371/ intravenous (IV) solution of PGE2 bound to albumin, the journal.pbio.0040284

Master Proteins Dictate Retinal Diﬀ erentiation Timetable Liza Gross | DOI: 10.1371/journal.pbio.0040293

The embryonic construction of show that three homeobox genes signals to the brain, are the ﬁ rst-born the vertebrate retina is a highly work in conjunction with a cellular cells—that is, the ﬁ rst to exit the cell ordered affair. Following a precise timepiece that determines the cycle and stop dividing. Though their timetable, six different specialized sequential emergence of distinct cell birthdays vary somewhat by species, the cell types emerge from a mass of types. Surprisingly, the schedule of horizontal, cone, and amacrine cells identical, proliferating cells. The both homeobox gene expression and come next, then the rod and bipolar process of retinal cell differentiation, retinal cell differentiation is controlled cells. when so-called progenitor cells by the translation, rather than by the Decembrini et al. suspected that stop dividing and choose among transcription, of the genes. cell identity may be tied to cell cycle the six fates, depends primarily on Retinal cells transform light progression because different retinal homeobox genes, major regulators signals into visual information for cell types are produced when cell of embryonic patterning. How these further processing in the brain. After cycle length is manipulated. To genes control the timing of retinal cell light stimulates the rod and cone test this hypothesis, they studied a differentiation has remained an open photoreceptors, visual signals travel subset of homeobox genes, including question—until now. to horizontal and bipolar cells, which otx5, which supports photoreceptor In a new study, Sarah Decembrini, in turn interface with amacrine cells. differentiation, and vsx1 and otx2, Federico Cremisi, and colleagues Ganglion cells, which then relay these which promote bipolar differentiation.

PLoS Biology | www.plosbiology.org 1477 September 2006 | Volume 4 | Issue 9 | e305 | e293 of gene-repressing RNAs that bind to inhibition, they found, reduced the complementary sequences of RNA and number of photoreceptor and bipolar mediate mRNA destruction. (Future cells—an effect that was reversed when experiments must confi rm whether the proteins were overexpressed, these sequences do in fact interact.) supporting the connection between The GFP sensors were detected at the protein expression and cell identity. same stages as their corresponding Altogether, these results indicate homeobox proteins had been in the that a post-transcriptional mechanism previous experiments. This timing, regulates when these proteins are it turned out, coincided with the expressed and in which cells. This birthdates of the photoreceptors and mechanism operates in synch with a bipolar cells. cellular clock that measures cell cycle The correlation between the length to generate the later developing timing of protein expression and photoreceptors and bipolar cells. The DOI: 10.1371/journal.pbio.0040293.g001 the photoreceptor and bipolar cell next step will be to determine how birthdates prompted the researchers these fi ndings apply to other genes Green fl uorescent protein traces different to examine the effect of cell cycle controlling retinal cell fate, and then types of lipofected cells in the neural progression on protein translation. By to identify the molecular mechanisms retina of a Xenopus tadpole, some of which (ganglion cells) generate fi bers of the optic blocking cell cycle progression with driving translational inhibition. nerve. drugs that inhibit DNA replication, they found that Xotx5b, Xvsx1, and Decembrini S, Andreazzoli M, Vignali R, Working with Xenopus frogs, a classic Xotr2 require progressively longer cell Barsacchi G, Cremisi F (2006) Timing the developmental biology model, they cycles for effi cient translation. And generation of distinct retinal cells by found that each of the homeobox the attenuated production of Xotx5b homeobox proteins. DOI: 10.1371/journal. genes was expressed in sequence, in and Xvsx1 proteins after cell cycle pbio.0040272 different cells. By mid-stage retinal development (stage 34), the messenger RNA (mRNA) transcripts of all three genes were expressed, but only Xotx5 proteins were detected. Xvsx1 and Your Synapse or Mine? Promiscuous Protein Xotx2 were detected at stages 37 and 38-39, respectively. By stage 42, Xotx2 Exchanges between Neighboring Synapses and Xvsx1 proteins were observed in Françoise Chanut | DOI: 10.1371/journal.pbio.0040297 bipolar cells, while Xotx5b was detected only in photoreceptors. These results Synapses, the connections that link indicated that the genes had been neurons into circuits, can be plastic or regulated after transcription and were stable in the mammalian brain. Right expressed as proteins after cells exited after birth, synapses form and dissolve the cell cycle. among nascent neurons at breakneck What controlled the genes’ speed as the animal adapts to its new translation into protein? To fi nd surroundings. But, over time, while out, the researchers linked a specifi c some plasticity remains and allows for sequence of each homeobox gene— learning, most synapses stabilize and called the three prime untranslated some may last a lifetime. How synapses region (3' UTR)—with the gene are maintained over such long periods encoding green fl uorescent protein is somewhat of a mystery, especially (GFP). These GFP sensors indicated in light of the fact that structural DOI: 10.1371/journal.pbio.0040297.g001 how the 3' UTR affects expression of proteins constantly move in and out of Photoactivation of synapses on one the gene. They delivered the DNA of synapses. In theory, the active turnover side of a dendritic segment (orange) is sensors into embryos at an early stage of synaptic components might simply followed by migration and incorporation of retinal development (stage 17-18), refl ect the balance between protein of photoactivated PA-GFP-tagged ProSAP2 using a technique called lipofection. synthesis and degradation. But, in a (blue) into neighboring synapses. GFP proteins were detected only in recent study, Shlomo Tsuriel, Ran photoreceptors (the Xotx5b sensor) Geva, Noam Ziv, and their colleagues Synapses are specialized devices that and bipolar cells (Xvsx1 and Xotx2 fi nd that two prominent synaptic serve to transfer electrical impulses sensors). Thus, the 3' UTRs of these proteins, Synapsin I and ProSAP2, turn between neurons. They form at discrete genes had blocked GFP translation over primarily through rapid exchanges contact points between the neuron’s into protein in all but late-developing between neighboring synapses, rather main branch (the axon) and the retinal cells. The 3' UTRs were able to than via synthesis and degradation. complex arborizations (dendrites) do this because they contain sequences These observations add an interesting that sprout from its target neuron’s (called cis-regulatory sequences) that twist to the already complex picture of cell body. A number of specialized can interact with microRNAs—a class synapse biology. structures and molecules accumulate

PLoS Biology | www.plosbiology.org 1478 September 2006 | Volume 4 | Issue 9 | e293 | e297 at synapses, including synaptic vesicles beam. Over time, a fl uorescent signal bodies, where most synthesis typically chock-full of neurotransmitters on reappeared at the bleached synapses, occurs, into dendrites and axons, the the axonal (presynaptic) side, and indicating that bleached proteins were researchers determined that newly neurotransmitter receptors on the replaced with tagged proteins from synthesized Synapsin I and ProSAP2 dendritic (postsynaptic) side. Synapsin unbleached areas. Tagged Synapsin moved too slowly to explain the rapid I and ProSAP2 play important replenished bleached synapses in about replenishment of bleached synapses. In structural roles: Synapsin I tethers 40 minutes, and tagged ProSAP2 in two addition, inhibitors of protein synthesis synaptic vesicles underneath the to four hours. and degradation did not signifi cantly presynaptic lipid membrane and But these experiments did not affect the synapses’ replenishment rates, ProSAP2 organizes the postsynaptic show where the replenishing proteins confi rming that the high turnover rate architecture. came from. To answer this question, of Synapsin I and ProSAP2 owes mostly To follow the whereabouts the researchers took advantage of a to local exchanges among neighboring of Synapsin I and ProSAP2, the second dye, photoactivatable variant synapses. researchers tagged each protein with of GFP (PA-GFP), whose fl uorescence How the promiscuous exchange of fl uorescent dyes and coaxed cultured is activated, rather than extinguished, structural proteins such as Synapsin I neurons from the hippocampus (a with intense illumination. The and ProSAP2 affects synaptic stability brain region involved in learning) researchers photoactivated PA- is still unclear. Competition for a local of newborn rats to synthesize these GFP-tagged Synapsin I or ProSAP2 pool of synaptic components could fl uorescently tagged proteins. As over small portions of dendrites or eventually determine which synapse is the neurons grew in culture, they axons. Over the course of 10 to 40 stabilized. Curiously, synaptic signaling established synapses that incorporated minutes, fl uorescence gradually may be a destabilizing factor in the the tagged Synapsin I or ProSAP2. The declined at the illuminated synapses, young hippocampal neurons, as electric synapses were easily visualized as bright and concomitantly increased in stimulations to the cultures greatly fl uorescent spots studding dendrites neighboring synapses. These results increased Synapsin I and ProSAP2 and axon branches. The fi rst dye, indicate that pre- and postsynaptic traffi cking. Whether local promiscuity called green fl uorescent protein (GFP, proteins routinely hop from one is a characteristic of youthful synapses a small protein that was originally synapse to the next with timescales of or also holds true for more mature isolated from jellyfi sh), fl uoresces tens of minutes, a behavior that might ones remains to be seen. readily but can be extinguished with account for the rapid replenishment of intense illumination, a phenomenon photobleached synapses. Tsuriel S, Geva R, Zamorano P, Dresbach T, called photobleaching. The researchers Still, some of the replenishing Boeckers T, et al. (2006) Local sharing as a photobleached individual synapses material could also have come from predominant determinant of synaptic matrix containing GFP-tagged Synapsin I new protein synthesis. By tracking molecular dynamics. DOI: 10.1371/journal. or ProSAP2 with an intense laser PA-GFP-tagged proteins from cell pbio.0040271

Conﬂ ict within the Genome: Evolving Defenses to Suppress the Male Killers Liza Gross | DOI: 10.1371/journal.pbio.0040308

In the game of survival, anything goes—even the selective extermination of males. Male killing is the preferred strategy for a diverse group of bacteria that infect insects and other arthropods. Aside from its tabloid appeal, male killing offers biologists a platform for investigating genetic confl ict—evolutionary battles between competing elements within the same genome. Male-killing bacteria are passed from mother to offspring, but only males die from infection, suggesting that males harbor genetic elements that allow them to succumb to infection. In keeping with evolutionary theory, these selfi sh genetic elements, which spread at the expense of the organism, should engender counteracting elements that promote male survival. Yet scant evidence has linked the evolution of host suppressors to selfi sh elements that mediate male killing. But now, Emily Hornett, Gregory Hurst, and colleagues report the fi rst case of total suppression of male killing in a butterfl y, Hypolimnas bolina, infected with the wBol1 strain of the male-killing bacterium Wolbachia. They attribute survival to genetic elements expressed in the male embryo, an effect DOI: 10.1371/journal.pbio.0040308.g001 called zygotic suppression. Because this mechanism of suppression can inactivate male killers—which lie dormant A male nymphalid tropical butterfl y Hypolimnas bolina, otherwise until presented with a novel, vulnerable host—it’s possible known as the Common Eggfl y. (Photo: Sylvain Charlat)

PLoS Biology | www.plosbiology.org 1479 September 2006 | Volume 4 | Issue 9 | e297 | e308 that insects that don’t succumb to male killing today may have genetic background produced high hatch rates and a in fact evolved the means to counteract lethal infection. normal sex ratio. H. bolina is found throughout the Indo-Pacifi c. Because Infected Thai and Philippine females were serially mated wBol1 infection kills males in Polynesia but not in Southeast with Moorean males, thus progressively increasing the Asia, breeding individuals from each region could reveal proportion of Moorean genetic material. By the second genetic elements in the different populations that favor life generation, some male killing occurred, and by the third over death. And because infected females transmit infection generation, males were killed in fi ve out of 13 crosses. By the directly to offspring, breeding could also introduce wBol1 fi fth generation, no males survived. genes (and infection) onto the butterfl y genetic background From these results, the researchers concluded that (a technique called introgression). suppression occurs in the embryo, because male offspring The breeding experiments tested two questions: would of Moorean females crossed with the Southeast Asian males male-killing wBol1 taken from Moorea in Polynesia lose that survived even though the mother’s genetic profi le allows ability against Southeast Asian males with a Thai or Philippine killing. The fact that fi rst-generation hybrids survived at genetic background, and would benign wBol1 from Thailand nearly the same ratio as seen in wild Southeast Asian males, or the Philippines turn lethal against males with a Moorean they explain, suggests the effect is dominant (requires genetic background? just one copy of the gene) and is at high frequency in the To fi nd out, Hornett et al. mated infected Moorean population. A dominant effect also explains why male killing females with Thai and Philippine males, and mated infected didn’t occur in fi rst-generation crosses between Southeast Thai and Philippine females with Moorean males. As a Asian females and Moorean males—the suppressor elements control, wBol1-infected females from both regions were also had not been segregated out of the population yet. crossed with males from their native populations. Crossing Through simulations, the researchers show that the the Moorean and Southeast Asian populations suppressed suppressor could spread through the population in just 100 the male-killing effects of wBol1 from Moorea in just a generations, suggesting that male killing could disappear single generation—in stark contrast to the control crosses relatively quickly after a suppressor mutation occurs. Thus, (Moorean females mated with Moorean males), which genetic confl ict between killing abettors and suppressors may yielded no males at all. be far more widespread than once thought, but has simply But when Moorean wBol1 infection was reintroduced eluded detection. Given the diversity of species affl icted by to its native host background—by backcrossing fi rst- male-killing bacteria, researchers will have plenty of options generation hybrid Moorean/Southeast Asian females with for testing this possibility. wild Moorean males—it became male-lethal again. Egg hatch rates decreased dramatically and just a fraction of Hornett EA, Charlat S, Duplouy AMR, Davies N, Roderick GK, et al. males survived. In contrast, continued introduction of (2006) Evolution of male-killer suppression in a natural population. Moorean wBol1 infection onto the Southeast Asian male DOI: 10.1371/journal.pbio.0040283

In Drosophila Hair Development, Shavenbaby Is at the Beginning of the End Richard Robinson | DOI: 10.1371/journal.pbio.0040310

In the past two decades, the grand known as trichomes. Called denticles strategy of animal development on the ventral surface and hairs on has become clear: initial sets of the dorsal surface, trichomes arise transcription factors turn on some from extensions of the cytoplasm of genes and turn off others, creating individual cells, and are fi lled with the new sets in turn, at each step defi ning cytoskeletal protein actin. Previous the fate of cells ever more precisely work has shown the importance of as embryonic development proceeds. several transcription factors in trichome At the end of this process, a fully formation, converging on Shavenbaby differentiated cell with a characteristic (Svb), the most “downstream” regulator shape emerges, but the signals that of trichomes yet identifi ed (svb mutants bring about these fi nal steps have do not form trichomes, giving the often remained elusive. A new study embryos a “shaven” look). by Hélène Chanut-Delalande, Serge To fi nd the downstream targets of Plaza, and colleagues deciphers these Shavenbaby, the authors examined DOI: 10.1371/journal.pbio.0040310.g001 signals for epidermal hair formation gene expression patterns in Drosophila Cell shape remodeling: analyzing the in Drosophila, illuminating the link epidermis, looking for genes whose function of genes specifi cally expressed in between the cascade of transcription expression correlated in space and cellular extensions of Drosophila embryonic factors and the production of a specifi c time with that of trichome formation. epidermis (green stain of a protein called cell shape in this model animal. A gene called miniature matched the Miniature). The embryo of the fruit fl y is divided pattern closely. They showed that into parallel segments. The epidermal svb mutation abolished miniature where it is normally silent, miniature was surface of each segment may be expression, as did a repressor of svb also expressed. And in a species with smooth, or studded with projections activity. When they expressed svb in cells restricted svb expression, the pattern

PLoS Biology | www.plosbiology.org 1480 September 2006 | Volume 4 | Issue 9 | e308 | e310 of restriction was matched by restricted reorganization in the epidermis. a “morphological module” that miniature expression as well. The sine No single gene mutation abolished directly infl uences epidermal form. qua non of a transcription factor is its denticle formation, but if all were They note that Shavenbaby’s role in ability to directly interact with its target mutated, denticles (or dorsal hairs) forming both denticles and dorsal DNA. The authors showed that the Svb were either tiny and misshapen, or hairs, which have different shapes, protein was indeed able to bind with altogether absent, suggesting that indicates the fl exibility of the module, a small region of the miniature gene, collectively, the identifi ed genes were suggesting the module may be used infl uencing its transcription. in charge of trichome formation. elsewhere as well, and that the fi ne But is miniature the only gene miniature, their experiments showed, tuning that produces one or another Shavenbaby controls in trichome does not control actin dynamics, but type of trichome is likely done by formation? When deleted, denticles acts at the epidermal cell membrane to elements both up- and downstream still form, but are misshapen, and when regulate the interaction of cytoskeletal from Shavenbaby. It is likely that other expressed where it is normally silent, it elements with the overlying hard genes, yet to be identifi ed, are also was not suffi cient to form denticles by cuticle layer. Thus, Shavenbaby regulated by Shavenbaby, and that itself. From this, the authors deduced controls both actin-related genes and some or most of these may be involved that Shavenbaby must have other at least one other gene critical for in actin remodeling or other aspects of targets besides miniature that control formation of the fi nal shape of the epidermal shape determination. And denticle formation. Beginning with epidermal cell. Finally, the authors now, researchers have a platform for database searches and continuing showed that Shavenbaby also helps investigating these questions. with molecular analysis, they found control pigmentation of denticle cells, a small handful of genes specifi cally through regulation of a gene in the Chanut-Delalande H, Fernandes I, Roch F, activated by Shavenbaby and involved pigment synthesis pathway. Payre F, Plaza S (2006) Shavenbaby couples in formation of denticles, each of From these results, the authors patterning to epidermal cell shape control. which helps control dynamics of actin propose that Shavenbaby regulates DOI: 10.1371/journal.pbio.0040290

Sharing Responsibility for Clathrin Coat Assembly Liza Gross | DOI: 10.1371/journal.pbio.0040301

Membranes protect cells from extracellular insults, but in so doing also block entry to nutrients and other essential molecules. One way cells circumvent this problem is by selectively binding such molecules to receptors on the membrane, then pulling the whole lot into the cell and packaging them into vesicles. Clathrin molecules—three- pronged pinwheel-shaped proteins—form an elaborate lattice coat around the vesicles, which ultimately bud off from the membrane and transport their cargo to their cellular destination. This highly complex process, called clathrin-mediated endocytosis, requires a constellation of accessory proteins that interact with key protein hubs. Vesicle formation has traditionally been described as a linear process with the core proteins being clathrin and adaptor protein (AP) complexes. In a previous paper, Harvey McMahon and colleagues DOI: 10.1371/journal.pbio.0040301.g001 suggested that the process can be viewed as a network of Painting by Alex McCuish (Hills Road Sixth Form College, protein interactions with clathrin and APs forming the two Cambridge, United Kingdom) illustrating the protein interaction main hubs of the network. In a new study, Eva Schmid, network underlying nerve function. Marijn Ford, McMahon, and colleagues use an impressive array of tools—biophysical, biochemical, structural, and cell biological—to shed light on the network dynamics of this two fl anking appendages forming his ears, but mounting “endocytic interactome.” APs orchestrate the process of cargo evidence suggests the British children’s book character Mr. recruitment and assembly of the nascent vesicle and are the Tickle—a circular blob with gangly, elastic arms and little fi rst hub of the endocytic network. They found that clathrin hands—may be a more apt comparison. Mr. Tickle’s body is takes over from adaptors as a hub as clathrin assembles into the core, his arms are the two fl exible hinge domains, and a coat. This shift requires collaboration between the hubs, his hands are the two appendages, β-appendage and α- which operate within a dynamic network that performs appendage. Whichever character you prefer, the core anchors multiple tasks simultaneously. the complex to the membrane and interacts with cargo Of four AP complexes involved in cellular transport, molecules, and the appendages recruit accessory proteins for AP2 fi gures mostly in plasma membrane endocytosis. The vesicle formation. AP2 structure has long been likened to Mickey Mouse, with In their previous study, McMahon and colleagues found the four-subunit core representing Mickey’s body and the that α-appendages have two distinct interaction sites,

PLoS Biology | www.plosbiology.org 1481 September 2006 | Volume 4 | Issue 9 | e310 | e301 allowing for clustered adaptor proteins to interact with many it’s likely that the appendages collaborate to mediate these accessory proteins simultaneously. The AP2 α-appendage interactions. becomes a hub for protein interactions only in the initial Clathrin coat formation, Schmid et al. propose, is an stages of assembly. In this study, they focused on the β- outgrowth of increasingly stable interactions among a appendage. shifting network of proteins. Rapidly shifting interactions First, Schmid et al. determined the interaction partners between isolated proteins give rise to coordinated, dynamic of both appendages by removing the bound partners from interactions between a network of proteins centered around cell extracts then analyzing them with mass spectrometry. the membrane, then to increasingly stable interactions as the They found a number of previously unidentiﬁ ed interaction coat assembles. The presence of both activated cargo receptors partners for the β-appendage (and a few more for the α- and lipid signaling molecules (phosphoinositides) in the appendage). Some interact only with the β-appendage, but membrane trigger the accumulation of adaptor complexes, many also interact with the α-appendage. which rapidly stabilize with the help of accessory proteins with To understand the molecular details of the interactions, multiple sites for AP2 appendage interactions. The accessory the researchers mutated key regions of the β-appendage proteins recruit clathrin, which interacts with β-appendages interaction sites (the β-appendage also has a top and side and displaces accessory proteins as it accumulates and self- site) then assessed the impact on their binding partners. assembles during coat formation. Accessory proteins that They found that the top site mediates most interactions interact only with appendages are shunted to the side, where for the α-appendage and the side site does the same for clathrin polymers have not yet formed, while accessory the β-appendage. With this setup, accessory proteins that proteins that can interact with clathrin are maintained. Having bind to the α-appendage’s top site can also bind to the β- demonstrated the power of using a multidisciplinary approach appendage’s side site, leaving the appendages’ other sites to study the endocytic interactome, the researchers believe that free to interact with still more proteins. Interactors can bind the principles uncovered will apply to other protein networks. to multiple appendages, allowing APs to serve as scaffolds for protein assembly. These results do not fully explain why Schmid EM, Ford MGJ, Burtey A, Praefcke GJK, Peak-Chew SY, et al. two appendages exist, the researchers acknowledge, but (2006) Role of the AP2 β-appendage hub in recruiting partners for because the same proteins interact with the top and side sites, clathrin-coated vesicle assembly. DOI: 10.1371/journal.pbio.0040262

Unique Development in Hemichordates Suggests Some Unique Features of Chordates Richard Robinson | DOI: 10.1371/journal.pbio.0040288

Underlying all the rich variety of In both chordates and Drosophila, form among chordates, from snakes the canonical arthropod of the world to humans, are several invariant of research, the dorsal–ventral axis characteristics in body plan. One develops in response to opposing of the most fundamental of these is gradients of two sets of proteins, the front-to-back, or dorsal–ventral, Chordin and Bmp. In the embryo, axis. Our nerve chords run dorsally; where Chordin is high and Bmp is low, our mouths project ventrally. This the nervous system develops (on the three-dimensional pattern in the dorsal side for chordates; on the ventral adult is created by a four-dimensional side for arthropods). Nervous system pattern of gene expression during development proceeds in two phases, development, as transcription factors both in response to Bmp gradients. First, turn on and turn off suites of genes in the ectoderm (one of the three basic concert. tissue layers in the embryo) segregates Many of these transcription factors DOI: 10.1371/journal.pbio.0040288.g001 into epidermis (high Bmp) and neural are even more ancient than the origin tissue (low Bmp). Then, within the of our body plan, and are shared with The embryo of the hemichordate neural tissue, regions of high Bmp give Saccoglossus kowalevskii expresses the other creatures, including arthropods, rise to sensory neurons, while areas of chordin gene on the ventral midline and which also have bilateral symmetry the bmp2/4 gene on the dorsal midline. All low Bmp give rise to motor neurons and a central nervous system. In a new dorsoventral development depends on this and interneurons. (Bmp gradients also study, Christopher Lowe, John Gerhart, Bmp–Chordin polarity. inﬂ uence development of other organ Marc Kirschner and colleagues show systems in the other tissue layers.) that many of these same signals are The acorn worm, Saccoglossus employed by the hemichordates, these signals in some ways that are kowalevskii, is a hemichordate that which are the phylum of bilaterally signiﬁ cantly different both from lives in intertidal zones and grows to symmetrical adults closest to chordates chordates, which they are more closely about 8 inches long. It is dorsoventrally but surprisingly do not have a related to, and arthropods, with which polarized in the development and central nervous system. However, the they nonetheless share some important location of its organs, such as the developing hemichordate interprets features. gill slits, the gonads, and the heart/

PLoS Biology | www.plosbiology.org 1482 September 2006 | Volume 4 | Issue 9 | e301 | e288 kidney complex. It has a nerve net and Unlike in either chordates in comparative evolutionary anatomy. axon tracts, but no central nervous or Drosophila, the fi rst phase of While high Chordin and low Bmp system—there is no brain-like mass of neurogenesis in the hemichordate characterize the dorsal side of the neural ganglia—and, unlike chordates embryo did not respond to changes chordate embryo, this pattern is and arthropods, its nerve cells and in Bmp–Chordin concentration— associated with the ventral (mouth) epidermal cells are fi nely intermixed. there was no alteration in the side of the Drosophila embryo, The authors showed that Bmp and neural ectoderm-versus-epidermis as well as in all other bilaterally Chordin act within the hemichordate differentiation as Bmp was increased. symmetric animals, including the embryo to establish the dorsal–ventral These results suggest that the Bmp– hemichordates. This suggests that axis, and they identifi ed multiple Chordin-mediated centralization during early chordate evolution there genes along this axis whose expression of the nervous system, occurring was an inversion in the Bmp–Chordin was infl uenced by these two proteins. in both arthropods and chordates, developmental axis relative to the When they supplied an excess of arose independently in the two mouth, by movement of the mouth Bmp, embryos became excessively groups after they diverged. The or axis, resulting in the chordate’s dorsalized in their expression domains Bmp–Chordin gradient did affect unique pattern of response to these and anatomical features; the opposite differentiation of neuronal cell types in ubiquitous molecular determinants. occurred when Bmp was diminished. the hemichordate embryo, analogous Before or after this inversion, the In this respect, the pattern was similar to the second phase of patterning in nervous system was centralized to to arthropods and chordates. (Along chordates and Drosophila, although the Chordin (low Bmp) side in the with its theoretical importance, the details differed signifi cantly. chordate lineage. Further study is the identifi cation of this molecular These differences suggest that much needed to answer these questions. determinant of anatomy aids the of the “regulatory architecture” of the practical study of the creature as developing nervous system evolved well, since its anatomy, plus the fact after the chordate–hemichordate Lowe CJ, Terasaki M, Wu M, Freeman RM Jr, that it spends most of its time in the split. The full implications of these Runft L, et al. (2006) Dorsoventral patterning vertical position, has made it diffi cult differences remain to be worked out. in hemichordates: Insights into early to unambiguously identify a dorsal or Finally, the results from this study chordate evolution. DOI: 10.1371/journal. ventral side.) shed light on an important question pbio.0040291

The Path to Digestion Is Paved with Repair Jason Underwood | DOI: 10.1371/journal.pbio.0040307

During the normal course of digesting a human meal, the stomach and subsequent meters of intestinal lining can sustain scratches and physical stresses as food winds through the coiled path. Abrasions are kept to a minimum through the activity of specialized cells that secrete mucus to lubricate the lining. Now, a study by Katsuya Miyake, Toru Tanaka, and Paul McNeil suggests that the digestive track responds to stresses with a local lubrication response. They used a variety of mucus-producing rodent cells and tissues in combination with several damaging treatment methods to demonstrate that mucus is secreted at the site of injury. At the same time, cells repair their own damaged outer membrane by depositing a “patch” on the injury. The authors used a simple, yet powerful approach to visualize mucus secretion. Mucus contains glycoproteins, which are modifi ed protein–carbohydrate complexes. Glycoproteins can be monitored using fl uorescent versions of proteins called lectins. Since these proteins bind tightly to carbohydrates, the location and intensity of the mucus can be inferred by monitoring the fl uorescent glow under a microscope. They also developed an assay to carefully quantify how much mucus was secreted. DOI: 10.1371/journal.pbio.0040307.g001 Miyake et al. grew gastric surface cells from a rat in culture A surface mucous cell bordering on the stomach lumen secretes and subjected them to a variety of stresses. As a general stress, mucus (pink stain). they pushed the cells through a thin syringe needle multiple times, creating perforations in the plasma membrane. The number of syringe strokes. Interestingly, without extracellular assay revealed that the amount of mucus in the extracellular calcium, mucus secretion was absent. This hinted that the space increased in a remarkably linear fashion with the mucus response requires some form of calcium signaling.

PLoS Biology | www.plosbiology.org 1483 September 2006 | Volume 4 | Issue 9 | e288 | e307 These observations led to an intriguing question: do cells approximated the real situation with a different method: respond to injury by switching on generalized secretion and using a needle to scratch monolayer-cultured cells or repair or instead have a more specialized mechanism for segments of rodent colon. The same localized response localizing the mucus response and repairing the wound? To was observed; mucus was secreted at the site of injury and address this question, the researchers used a laser to cause resealing occurred. Again, calcium played a key role in targeted injuries to cells. The response was then visualized both processes. When the authors examined the injury with with a fl uorescent lectin to monitor mucus levels while a electron microscopy, they could see that the extracellular special dye in the media monitored the repair response. membrane architecture changes drastically near the damaged Without a hole in the cell, the dye is found only on the site. Cells form new projections, as if a rough scab is laid outside of the cell. If a hole is formed by the laser and is not down after injury. Again, they uncovered calcium as an resealed, the dye can leak through the wound, resulting in a essential partner to the repair machinery. bright intracellular glow. These results show that the cells of the stomach and When the experiment was performed with calcium intestine have an effi cient mechanism for repairing ongoing present, the laser insult resulted in a fast, potent response to assaults on the digestive tract. The injury itself acts as a signal the injury site. Mucus is preferentially secreted on the side for both mucus release and an emergency patch response. of the cell where the injury occurred. Also, with calcium This adds another checkmark on the growing list of cellular present, very little dye accumulates inside the cell during the events that function through calcium signaling. With this experimental time course, indicating that the hole is quickly elegant mechanism in hand, the road ahead is full of patched. Without extracellular calcium, mucus secretion is important questions: how might one’s food intake, genetic absent and the inside of the cell quickly fi lls with the dye. disposition or an illness tweak the repair process? And what The researchers’ time-lapse movies of these events (see DOI: cellular proteins act as gatekeepers for this process? In any 10.1371/journal.pbio.0040276.sv001 and DOI: 10.1371/ case, the normally dark digestive system has seen a new light. journal.pbio.0040276.sv002) dramatically illustrate this point. Of course, injuries to the mammalian intestinal lining Miyake K, Tanaka T, McNeil PL (2006) Disruption-induced mucus do not result from shear force or lasers, so Miyake et al. secretion: Repair and protection. DOI: 10.1371/journal.pbio.0040276

Evolution of Neonatal Imitation Liza Gross | DOI: 10.1371/journal.pbio.0040311

Humans do it. Chimps do it. Why shouldn’t monkeys do it, too? Mimicry exists throughout the animal kingdom, but imitation with a purpose— matching one’s behavior to others’ as a form of social learning—has been seen only in great apes. (Mockingbirds can imitate an impressive number of other birds’ songs, but they can’t mimic you sticking out your tongue like a chimp can.) This matching behavior likely helps individuals conform to social norms and perform actions in the DOI: 10.1371/journal.pbio.0040311.g001 proper context. It’s generally believed A newborn macaque imitates tongue protrusion. that monkeys do not imitate in this way. However, the discovery that rhesus They later found similar results in ape lineage, their study suggests that the monkeys have “mirror neurons”— newborns, demonstrating that imitation trait is not unique to great apes after all. neurons that fi re both when monkeys is innate, not learned. A handful of Ferrari et al. tested 21 baby watch another animal perform an studies on newborn chimps found a rhesus monkeys’ response to various action and when they perform the similar capacity for imitating human experimental conditions at different same action—suggests they possess facial gestures. In a new study, Pier ages (one, three, seven, and 14 days the common neural framework for Ferrari, Stephen Suomi, and colleagues old). Infants were held in front of a perception and action that is associated explored the possibility that imitation researcher who began with a passive with imitation. evolved earlier in the primate tree by expression (the baseline condition) Most studies exploring the early signs studying neonatal imitation in rhesus and then made one of several gestures, of matching behavior have focused monkeys, which split from the human including tongue protrusion, mouth on humans. A landmark 1977 study lineage about 25 million years ago. opening, lip smacking, and hand by Andrew Meltzoff and Keith Moore They found that rhesus infants can opening. showed that 12- to 21-day-old infants indeed imitate a subset of human facial Day-old infants rarely displayed could imitate adults who pursed their gestures—gestures the monkeys use to mouth opening behavior, but lips, stuck out their tongue, opened communicate. The fi rst investigation smacked their lips frequently. When their mouth, and extended their fi ngers. of neonatal imitation outside the great experimenters performed the mouth

PLoS Biology | www.plosbiology.org 1484 September 2006 | Volume 4 | Issue 9 | e307 | e311 opening gesture, infants responded It’s possible that rhesus babies show individuals. For rhesus monkeys, lip with increased lip smacking but did more varied and prolonged imitative smacking (which often alternates not increase any other behavior. behavior in response to mom or with tongue protrusion) accompanies None of the other stimuli produced other monkeys than to human grooming sessions and signals signifi cant responses. But by day 3, experimenters, who may not provide affi liation—an important social cue matched behaviors emerged: infants the most relevant biological cues. But for a species that is often described as stuck out their tongues far more this narrow window does comport with “despotic and nepotistic.” Picking up often in response to researchers’ the development schedule of rhesus these social gestures early in life may tongue protrusions compared with monkeys, which is much shorter than well facilitate the animal’s early social control conditions, and smacked their that of humans and chimps. relations (primarily with the mother) lips far more often while watching Many questions remain about and assimilation into the social fabric researchers smacking theirs. (Watch the neural mechanisms of neonatal of the group, providing a mechanism an infant imitating mouth opening at imitation. The researchers argue that for distinguishing friend from foe. It DOI: 10.1371/journal.pbio.0040302. their results support a resonance will be interesting to test the extent sv001.) By day 7, the monkeys tended mechanism linked to mirror neurons, of imitation in monkeys with more to decrease lip smacking when humans which have recently been identifi ed complex social dynamics. While the performed the gesture, and by two while monkeys observe others’ lip social life of rhesus monkeys may weeks, all imitative behavior stopped. smacking and tongue protrusion. In not demand the more sophisticated Infant rhesus monkeys, these results this model, observing human mouth repertoire of behaviors seen in great suggest, have a narrow imitation gestures directly activates mirror apes, they seem to be hard-wired for window that opens three days after neurons in the monkeys’ brain, imitation just like apes. birth, when they can reproduce human ultimately leading to a replication of tongue protrusion and lip smacking. the gesture. Ferrari PF, Visalberghi E, Paukner A, Fogassi L, This imitation period is much longer Human babies can imitate an Ruggiero A, et al. (2006) Neonatal imitation in humans (two to three months) adult’s facial gesture a day after seeing in rhesus macaques. DOI: 10.1371/journal. and chimps (about two months). it, which may help them identify pbio.0040302

PLoS Biology | www.plosbiology.org 1485 September 2006 | Volume 4 | Issue 9 | e311