Multiple Levels Of Control Research Articles

Abstract IA31: Control of TGF-β responsiveness and epithelial plasticity

Abstract TGF-β signaling inhibits cell growth in epithelial cells and thus acts as tumor suppressor, yet increased TGF-β signaling often promotes cancer progression through effects on the tumor micro-environment and effects on the carcinoma cells that can lead to epithelial plasticity responses and increased invasion. The responsive epithelial or carcinoma cells can regulate their cell surface levels of TGF-β receptors, and thus enhance their responses to TGF-β. In addition to transcription control of TGF-β receptor expression, the cells can rapidly mobilize TβRII and TβRI from a large intracellular receptor pool to the cell surface, and thus enhance their TGF-β responsiveness. For example, high glucose, as seen in hyperglycemia, rapidly induces high levels of cell surface receptors that increase the cell’s TGF-β responsiveness, resulting in extracellular matrix production and increased cell size. Similarly, increased cell surface TGF-β receptor levels and TGF-β responsiveness also occur in response to insulin, which is therapeutically used against diabetes-associated hyperglycemia. The insulin-induced increase in cell surface levels requires activation of Akt, which then phosphorylates the membrane-associated RabGAP AS160, thus alleviating AS160-mediated retention of vesicles containing TβRII and TβRI, and allowing them to reach the cell surface. Additionally, the levels of functional TGF-β receptor complexes at the cell surface is controlled by TACE-mediated ectodomain shedding, which is activated in response to Erk and p38 MAPK activation. We will discuss the physiological implications of these multiple levels of control of TGF-β receptor cell surface presentation and TGF-β responsiveness in cancer cell behavior and cancer progression. Finally, the epithelial plasticity response to TGF-β is also controlled by the direct interaction of activated Smad3 with a methyltransferase, which consequently controls the histone modifications of the gene encoding the transcription factor Snail, a master regulator that drives epithelial-mesenchymal transition. Thus, Smad-mediated changes of histone methylation complement direct Smad-mediated transcription effects in the control of epithelial-mesenchymal transition. Citation Format: Rik Derynck, Erine Budi, Baby-Periyanayaki Muthusamy, Yoko Katsuno, Dan Du. Control of TGF-β responsiveness and epithelial plasticity. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA31.

Neural Control of Vascular Function in Skeletal Muscle.

The sympathetic nervous system represents a fundamental homeostatic system that exerts considerable control over blood pressure and the distribution of blood flow. This process has been referred to as neurovascular control. Overall, the concept of neurovascular control includes the following elements: efferent postganglionic sympathetic nerve activity, neurotransmitter release, and the end organ response. Each of these elements reflects multiple levels of control that, in turn, affect complex patterns of change in vascular contractile state. Primarily, this review discusses several of these control layers that combine to produce the integrative physiology of reflex vascular control observed in skeletal muscle. Beginning with three reflexes that provide somewhat dissimilar vascular patterns of response despite similar changes in efferent sympathetic nerve activity, namely, the baroreflex, chemoreflex, and muscle metaboreflex, the article discusses the anatomical and physiological bases of postganglionic sympathetic discharge patterns and recruitment, neurotransmitter release and management, and details of regional variations of receptor density and responses within the microvascular bed. Challenges are addressed regarding the fundamentals of measurement and how conclusions from one response or vascular segment should not be used as an indication of neurovascular control as a generalized physiological dogma. Whereas the bulk of the article focuses on the vasoconstrictor function of sympathetic neurovascular integration, attention is also given to the issues of sympathetic vasodilation as well as the impact of chronic changes in sympathetic activation and innervation on vascular health. © 2016 American Physiological Society.

Putting vaginal fistulas on the international social work map: A critical perspective

Why should social workers care about vaginal fistulas? Why should we turn our attention to a health problem mostly experienced by materially impoverished young Black women, especially those in Africa? Using a critical perspective, which we define, we argue that vaginal fistulas are much more than a gynaecological health issue but symptomatic of the gender-, race-, class- and age-based oppressions many young Black women in the ‘Third World’ suffer. Affecting very few White western women but plenty of subjugated Black women forced to live in chronic poverty, and exposed to multiple levels of control from early in their lives, vaginal fistula sufferers often have little, if any, access to adequate health services. Our aim is to put vaginal fistulas on the global social work agenda. It is a call to work towards ending a problem that produces much shame and suffering for far too many women.

Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB.

Ribonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in KD (ΔΔG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128′A) causes an 83-fold increase in KD (ΔΔG = +2.7 kcal/mol). The greater effect of the D128A/D128′A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Channeling satiation: A primer on the role of TRP channels in the control of glutamate release from vagal afferent neurons

Obesity results from the chronic imbalance between food intake and energy expenditure. To maintain homeostasis, the brainstem nucleus of the solitary tract (NTS) integrates peripheral information from visceral organs and initiates reflex pathways that control food intake and other autonomic functions. This peripheral-to-central neural communication occurs through activation of vagal afferent neurons which converge to form the solitary tract (ST) and synapse with strong glutamatergic contacts onto NTS neurons. Vagal afferents release glutamate containing vesicles via three distinct pathways (synchronous, asynchronous, and spontaneous) providing multiple levels of control through fast synaptic neurotransmission at ST–NTS synapses. While temperature at the NTS is relatively constant, vagal afferent neurons express an array of thermosensitive ion channels named transient receptor potential (TRP) channels. Here we review the evidence that TRP channels pre-synaptically control quantal glutamate release and examine the potential roles of TRP channels in vagally mediated satiety signaling. We summarize the current literature that TRP channels contribute to asynchronous and spontaneous release of glutamate which can distinctly influence the transfer of information across the ST–NTS synapse. In other words, multiple glutamate vesicle release pathways, guided by afferent TRP channels, provide for robust while adaptive neurotransmission and expand our understanding of vagal afferent signaling.

Branching out: origins of the sea urchin larval skeleton in development and evolution.

It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production.

The CALMODULIN-BINDING PROTEIN60 Family Includes Both Negative and Positive Regulators of Plant Immunity

Two members of the eight-member CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1), encode positive regulators of plant immunity that promote the production of salicylic acid (SA) and affect the expression of SA-dependent and SA-independent defense genes. Here, we investigated the other six family members in Arabidopsis (Arabidopsis thaliana). Only cbp60a mutations affected growth of the bacterial pathogen Pseudomonas syringae pv maculicola ES4326. In contrast to cbp60g and sard1 mutations, cbp60a mutations reduced pathogen growth, indicating that CBP60a is a negative regulator of immunity. Bacterial growth was increased by cbp60g only in the presence of CBP60a, while the increase in growth due to sard1 was independent of CBP60a, suggesting that the primary function of CBP60g may be to counter the repressive effect of CBP60a. In the absence of pathogen, levels of SA as well as of several SA-dependent and SA-independent pathogen-inducible genes were higher in cbp60a plants than in the wild type, suggesting that the enhanced resistance of cbp60a plants may result from the activation of immune responses prior to pathogen attack. CBP60a bound calmodulin, and the calmodulin-binding domain was defined at the C-terminal end of the protein. Transgenes encoding mutant versions of CBP60a lacking the ability to bind calmodulin failed to complement null cbp60a mutations, indicating that calmodulin-binding ability is required for the immunity-repressing function of CBP60a. Regulation at the CBP60 node involves negative regulation by CBP60a as well as positive regulation by CBP60g and SARD1, providing multiple levels of control over the activation of immune responses.

Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

Hypothalamic gonadotropin-inhibitory hormone precursor mRNA is increased during depressed food intake in heat-exposed chicks

The regulation of food intake in chickens (Gallus gallus domesticus) represents a complex homeostatic mechanism involving multiple levels of control, and regulation during high ambient temperatures (HT) is poorly understood. In this study, we examined hypothalamic mRNA expression of gonadotropin-inhibitory hormone (GnIH) to understand the effect of HT on an orexigenic neuropeptide. We examined the effects of HT (35°C ambient temperature for 1, 24 or 48h) on 14-day old chicks. HT significantly increased rectal temperature and suppressed food intake, and also influenced plasma metabolites. The expression of GnIH precursor mRNA in the diencephalon was significantly increased in chicks at 24-and 48h of HT when food intake was suppressed significantly, whilst no change was observed for GnIH precursor mRNA and food intake at 1h of HT. In situ hybridization and immunocytochemistry further revealed the cellular localization of chicken GnIH precursor mRNA and its peptide in the paraventricular nucleus (PVN) in the chick hypothalamus. We examined plasma metabolites in chicks exposed to HT for 1 or 48h and found that triacylglycerol concentration was significantly higher in HT than control chicks at 1h. Total protein, uric acid and calcium were significantly lower in HT chicks than control chicks at 48h. These results indicate that not only a reduction in food intake and alteration in plasma metabolites but also the PVN-specific expression of GnIH, an orexigenic agent, may be induced by HT. The reduced food intake at the same time as GnIH expression was increased during HT suggests that HT-induced GnIH expression may oppose HT-induced feeding suppression, rather than promote it. We suggest that the increased GnIH expression could be a consequence of the reduced food intake, and would not be a direct response to HT.

Strand swapping regulates the iron-sulfur cluster in the diabetes drug target mitoNEET

MitoNEET is a recently identified diabetes drug target that coordinates a transferable 2Fe-2S cluster, and additionally contains an unusual strand swap. In this manuscript, we use a dual basin structure-based model to predict and characterize the folding and functionality of strand swapping in mitoNEET. We demonstrate that a strand unswapped conformation is kinetically accessible and that multiple levels of control are employed to regulate the conformational dynamics of the system. Environmental factors such as temperature can shift route preference toward the unswapped pathway. Additionally we see that a region recently identified as contributing to frustration in folding acts as a regulatory hinge loop that modulates conformational balance. Interestingly, strand unswapping transfers strain specifically to cluster-coordinating residues, opening the cluster-coordinating pocket. Strengthening contacts within the cluster-coordinating pocket opens a new pathway between the swapped and unswapped conformation that utilizes cracking to bypass the unfolded basin. These results suggest that local control within distinct regions affect motions important in regulating mitoNEET's 2Fe-2S clusters.

Moving Towards Self-Assembling Machines: Harnessing Spindle Components for Biosynthetic Devices

The remarkably dynamic and complex manufacturing environment of the eukaryotic cell is currently unrivaled by manmade systems. Through dissection and harnessing of biological machines and underlying processes, and merger with synthetic materials, we hope to scale manufacturing into realms currently restricted from physical manipulation. Spindle assembly is the first critical step in chromosome segregation and a primary target for anti-cancer therapeutics. An assemblage of nanoscale components and communication networks must be integrated for spindle formation and function. We are applying nanotechnology in parallel with traditional methods of yeast genetics, timelapse microscopy, AFM and biochemistry. Dramatic reorganization of interphase microtubules into a bipolar organization for chromosome segregation requires focused nucleation from spindle pole body microtubule organizing centers (MTOCs). The γ-tubulin small complex (γ-TuSC) MTOC associates with growing microtubules and is part of a larger ring complex (γ-TuRC). The complex controls microtubule nucleation, organization and dynamics. Three conserved kinesin-like protein (Klp) families in fission yeast contribute in critical roles in spindle assembly. Kinesin-14 Pkl1 and Kinesin-5 associate with γ-TuSC at poles and provide counterbalanced roles in microtubule nucleation and organization, while Kinesin-6 acts on overlapping anti-parallel microtubules. The coupled action of the MTOC and Klps enables multiple levels of control. By purifying the γ-TuSC and γ-TuRC complexes from human and fission yeast cells using superose 6 FPLC chromatography, co-immunoprecipitation with magnetic beads and western analysis and attachment of these complexes to functionalized surfaces we are analyzing minimal components and frameworks for generating anti-parallel, bipolar and more complex microtubule array structures in the presence of multiple Klps. Our goal is to incorporate the MTOC, tubulin and multiple Klp families in different biosynthetic platforms to better understand self-assembling machines and transitional dynamic architectures thereby refining both in vivo models and in vitro advanced applications.

Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions.

To provide insight as to how cells receive information from their external surroundings, synthetic hydrogels have emerged as systems for assaying cell function in well-defined microenvironments where single cues can be introduced and subsequent effects individually elucidated. However, as the field seeks to answer more complex biological questions, advanced material systems are needed that allow dynamic alteration of the 3D cellular environment with orthogonal reactions that enable multiple levels of control of biochemical and biomechanical signals. Here, we sought to synthesize one such 3D culture system using cytocompatible and wavelength-specific photochemical reactions to create hydrogels that allow orthogonal and dynamic control of the material properties through independent spatiotemporally-regulated photocleavage of crosslinks and photoconjugation of pendant functionalities. Results demonstrate the versatile nature of the chemistry to create programmable niches to study and direct cell function by modifying the local hydrogel environment.

ISPs & Rowdy Web Sites Before the Law: Should We Change Today’s Safe Harbour Clauses?

The paper examines today’s debate on the new responsibilities of Internet service providers (ISPs) in connection with legal problems concerning jurisdiction, data processing, people’s privacy and education. The focus is foremost on the default rules and safe harbour clauses for ISPs liability, set up by the US and European legal systems. This framework is deepened in light of the different functions of the services provided on the Internet so as to highlight multiple levels of control over information and, correspondingly, different types of liability. The new responsibilities of ISPs concern the original “end-to-end” architecture of the medium and policies on design rather than responsibility for user content and individual messages.

Release mode of large and small dense-core vesicles specified by different synaptotagmin isoforms in PC12 cells

Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca(2+) sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca(2+) sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.

Regulation of Neutrophil Survival/Apoptosis by Mcl-1

Neutrophil granulocytes have the shortest lifespan among leukocytes in the circulation and die via apoptosis. At sites of infection or tissue injury, prolongation of neutrophil lifespan is critical for effective host defense. Apoptosis of inflammatory neutrophils and their clearance are critical control points for termination of the inflammatory response. Evasion of neutrophil apoptosis aggravates local injury and leads to persistent tissue damage. The short-lived prosurvival Bcl-2 family protein, Mcl-1 (myeloid cell leukemia-1), is instrumental in controlling apoptosis and consequently neutrophil lifespan in response to rapidly changing environmental cues during inflammation. This paper will focus on multiple levels of control of Mcl-1 expression and function and will discuss targeting Mcl-1 as a potential therapeutic strategy to enhance the resolution of inflammation through accelerating neutrophil apoptosis.

Hypothalamic Melanocortin System on Feeding Regulation in Birds: A Review

Regulation of feed intake in chickens represents a complex homeostatic mechanism involving multiple levels of control. Understanding the regulation of feeding behavior can be a very important theme in animal production. Recently, a close evolutionary relationship between the peripheral and hypothalamic neuropeptides has become apparent. In the infundibular nucleus (the avian equivalent of the mammalian arcuate nucleus), the melanocortin system, which contains neuroendocrine neurons that regulate endocrine secretions by releasing substances, is an essential site in the brain for signals about the status of peripheral energy balance. The structure and function of many hypothalamic neuropeptides, melanocortins, neuropeptide-Y (NPY) and agouti-related protein (AGRP) have been characterized. This review provides as overview of the various effects and interrelationship of these central and peripheral neuropeptides, and summarizes the role of the melanocortin system on feeding regulation in chicks.

A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation

Multiple levels of control are in play to regulate pluripotency and differentiation in human embryonic stem cells (hESCs). At the transcriptional level, the core factors OCT4, NANOG and SOX2 form a positive autoregulatory loop that is pivotal for maintaining the undifferentiated state. At the post-transcriptional level, microRNAs (miRNAs) belonging to the miR-302 family are emerging as key players in the control of proliferation and cell fate determination during differentiation. Here, we show that the transcriptional factors OCT4 and NR2F2 (COUP-TFII) and the miRNA miR-302 are linked in a regulatory circuitry that critically regulate both pluripotency and differentiation in hESCs. In the undifferentiated state, both OCT4 and the OCT4-induced miR-302 directly repress NR2F2 at the transcriptional and post-transcriptional level, respectively. Conversely, NR2F2 directly inhibits OCT4 during differentiation, triggering a positive feedback loop for its own expression. In addition, we show that regulation of NR2F2 activity itself relies on alternative splicing and transcriptional start site choice to generate a full-length transcriptionally active isoform and shorter variants, which enhance the activity of the long isoform. During hESC differentiation, NR2F2 is first detected at the earliest steps of neural induction and thus is among the earliest human embryonic neural markers. Finally, our functional analysis points to a crucial role for NR2F2 in the activation of neural genes during early differentiation in humans. These findings introduce a new molecular player in the context of early embryonic stem cell state and cell fate determination in humans.

The effect of hepatocyte growth factor on the initial stages of mouse follicle development

Interactions between theca and granulosa cells of the follicle are critical for the coordination of ovarian follicle development. The cell-cell interactions are mediated through the local production and actions of a variety of factors. The current study is designed to investigate the expression of Hgf and its receptor, c-Met, in the mouse ovary during in vivo folliculogenesis. We found that Hgf and c-Met mRNAs were already expressed in 2-day-old ovaries, and that, while c-Met levels remained constant until 22-day-old, Hgf levels slightly but not significantly increased with age. The expression of Hgf mRNA in theca/interstitial cells was higher than in granulosa cells in 22-day-old ovaries. Immunohistochemistry analysis confirmed the expression pattern demonstrated by RT-PCR. We investigated the role of hepatocyte growth factor (HGF) at the beginning of mouse folliculogenesis and its possible interaction with kit ligand (KL). Interestingly, both KL and HGF were able to increase the expression of each other, creating a positive feedback loop. In the presence of HGF, we observed an increase of granulosa cell proliferation and an increase in the number of pre-antral and early antral follicles in ovary organ cultures. We also observed a significant increase in the diameters of follicles in individual follicle cultures. Moreover, HGF stimulated the expression of the FSH receptors, both in the whole ovary and in isolated pre-antral follicle cultures. Based on the data presented, we concluded that HGF exerts multiple levels of control over follicular cell functions, which collectively enable the progression of follicular development.

Protein Transport into Chloroplasts

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized as precursors with N-terminal targeting signals called transit peptides. Novel machinery has evolved to specifically import these proteins from the cytosol into chloroplasts. This machinery consists of more than a dozen components located in and around the chloroplast envelope, including a pair of GTPase receptors, a beta-barrel-type channel across the outer membrane, and an AAA(+)-type motor in the stroma. How individual components assemble into functional subcomplexes and the sequential steps of the translocation process are being mapped out. An increasing number of noncanonical import pathways, including a pathway with initial transport through the endomembrane system, is being revealed. Multiple levels of control on protein transport into chloroplasts have evolved, including the development of two receptor subfamilies, one for photosynthetic proteins and one for housekeeping proteins. The functions or expression levels of some translocon components are further adjusted according to plastid type, developmental stage, and metabolic conditions.

A positive approach to branching

Plants regulate the development of branches in response to environmental and developmental signals in order to maximise reproductive success. A number of hormone signals are involved in the regulation of branching and both their production and transmission affect axillary meristem outgrowth. With the identification of strigolactones as root-derived branch inhibitors it seems likely that a biochemical pathway starting from a carotenoid and resulting in production of a strigolactone hormone is present in most plants. Our observation that loss of CCD7 or CCD8 also results in production of a promoter of branching from roots shows the branching pathway has multiple levels of control which allows a high degree of sensitivity to subtle differences in environmental and developmental signals.