When reverse genetics meets physiology: the use of site-specific recombinases in mice

The use of site-specific recombinases enables the precise introduction of defined genetic mutations into the mouse genome. In theory, any deletion, point mutation, inversion or translocation can be modeled in mice. Because gene targeting is controlled both spatially and temporally, the function of a given gene can be studied in the desired cell types and at a specific time point. This 'genetic dissection' allows to define gene function in development, physiology or behavior. In this review, we focus on the technical possibilities of Cre and other site-specific recombinases but also discuss their limitations.     

Temporally- and spatially regulated generation of distinct descendants by sonic hedgehog-expressing progenitors in the forebrain

The generation of distinct neural subtypes depends on the activities of cell-extrinsic and -intrinsic factors during the development of the vertebrate CNS. Previous studies have provided a molecular basis for how neural progenitors are patterned and generate distinct descendants that are spatially and temporally regulated by inductive signals secreted by polarized sources. However, it still remains unknown how the generation of neural descendants by progenitors located at polarized sources of inductive signals is controlled. Sonic hedgehog (Shh), which is expressed at the ventral midline in the forebrain, has been shown to play a critical role for the patterning and specification of distinct neural subtypes in the forebrain. Here, we analyzed the identities and distributions of Shh-descendants generated at discrete time points in the forebrain by using a ShhcreER(T2) mouse driver line in which a tamoxifen-inducible Cre cassette was inserted into the Shh locus together with a Z/EG mouse reporter line. Our results showed that Shh-expressing neural progenitors generated neuronal and glial descendants distributed throughout the telencephalon and diencephalon in a temporally distinct manner. Furthermore, our results showed that Shh-progenitors are located at two spatially distinct sub-domains that can be characterized by their temporally distinct patterns of Shh expression. These results suggest that temporally- and spatially controlled mechanisms that specify neural subtypes operate in the Shh-expressing progenitor domain, and raise the possibility that the distinct temporal gradient of Shh activity might be responsible for the generation of distinct neural subtypes in the telencephalon.     

Cardiac neuronal hierarchy in health and disease

The cardiac neuronal hierarchy can be represented as a redundant control system made up of spatially distributed cell stations comprising afferent, efferent, and interconnecting neurons. Its peripheral and central neurons are in constant communication with one another such that, for the most part, it behaves as a stochastic control system. Neurons distributed throughout this hierarchy interconnect via specific linkages such that each neuronal cell station is involved in temporally dependent cardio-cardiac reflexes that control overlapping, spatially organized cardiac regions. Its function depends primarily, but not exclusively, on inputs arising from afferent neurons transducing the cardiovascular milieu to directly or indirectly (via interconnecting neurons) modify cardiac motor neurons coordinating regional cardiac behavior. As the function of the whole is greater than that of its individual parts, stable cardiac control occurs most of the time in the absence of direct cause and effect. During altered cardiac status, its redundancy normally represents a stabilizing feature. However, in the presence of regional myocardial ischemia, components within the intrinsic cardiac nervous system undergo pathological change. That, along with any consequent remodeling of the cardiac neuronal hierarchy, alters its spatially and temporally organized reflexes such that populations of neurons, acting in isolation, may destabilize efferent neuronal control of regional cardiac electrical and/or mechanical events.     

Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons. When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C. This temperature-induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness. These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals.     

Stochasticity in reproductive opportunity and the evolution of egg limitation in insects

Is reproduction by adult female insects limited by the finite time available to locate hosts (time limitation) or by the finite supply of eggs (egg limitation)? An influential model predicted that stochasticity in reproductive opportunity favors elevated fecundity, rendering egg limitation sufficiently rare that its importance would be greatly diminished. Here, I use models to explore how stochasticity shapes fecundity, the likelihood of egg limitation, and the ecological importance of egg limitation. The models make three predictions. First, whereas spatially stochastic environments favor increased fecundity, temporally stochastic environments favor increases, decreases, or intermediate maxima in fecundity, depending on egg costs. Second, even when spatially or temporally stochastic environments favor life histories with less‐frequent egg limitation, stochasticity still increases the proportion of all eggs laid in the population that is laid by females destined to become egg limited. This counterintuitive result is explained by noting that stochasticity concentrates reproduction in the hands of a few females that are likely to become egg limited. Third, spatially or temporally stochastic environments amplify the constraints imposed by time and eggs on total reproduction by the population. I conclude that both egg and time constraints are fundamental in shaping insect reproductive behavior and population dynamics in stochastic environments.     

Light-mediated remote control of signaling pathways

Cell signaling networks display an extraordinary range of temporal and spatial plasticity. Our programmatic approach focuses on the construction of intracellular probes, including sensors, inhibitors, and functionally unique proteins that can be temporally and spatially controlled by the investigator even after they have entered the cell. We have designed and evaluated protein kinase sensors that furnish a fluorescent readout upon phosphorylation. In addition, since the sensors are inert (i.e., cannot be phosphorylated) until activated by light, they can be carried through the various stages of any given cell-based behavior without being consumed. Using this strategy, we have shown that PKCβ is essential for nuclear envelope breakdown and thus the transition from prophase to metaphase in actively dividing cells. Photoactivatable proteins furnish the means to initiate cellular signaling pathways with a high degree of spatial and temporal control. We have used this approach to demonstrate that cofilin serves as a component of the steering apparatus of the cell. Finally, inhibitors are commonly used to assess the participation of specific enzymes in signaling pathways that control cellular behavior. We have constructed a photo-deactivatable inhibitor, an inhibitory species that can be switched off with light. In the absence of light, the target enzyme is inactive due to the presence of the potent inhibitory molecule. Upon photolysis, the inhibitory molecule is destroyed and enzymatic activity is released.     

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.     

Environmentally induced dispersal under heterogeneous logistic growth

We consider a single-species model which is composed of several habitats connected by linear migration rates and having logistic growth. A spatially varying, temporally constant environment is introduced by the non-homogeneity of its carrying capacity. Under this condition any type of purely diffusive behavior, characterized in our model by symmetric migration rates, produces an unbalanced population distribution, i.e. some locations receive more individuals than can be supported by the environmental carrying capacity, while others receive less. Using an evolutionarily stable strategy (ESS) approach we show that an asymmetric migration mechanism, induced by the heterogeneous carrying capacity of the environment, will be selected. This strategy balances the inflow and outflow of individuals in each habitat (balanced dispersal), as well as 'balancing' the spatial distribution relative to variation in carrying capacity (the Ideal Free Distribution from habitat selection theory). We show that several quantities are maximized or minimized by the evolutionarily stable dispersal strategy.     

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits^1-6. Application of optogenetics^7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways^9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system^14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin^16,17 or halorhodopsin^18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.     

Developmental expression of alcohol dehydrogenases in maize

Alcohol dehydrogenase (ADH) in Zea mays exists in five distinct electrophoretic forms (isozymes), ADH-1, ADH-2, ADH-3, ADH-4, and ADH-T. The mode of inheritance of ADH-1 and ADH-2 has been previously reported; preliminary data suggest that ADH-3 is controlled by a different locus than ADH-2; no genetic analysis has yet been made for ADH-4 and ADH-T. Analyses at different stages of ontogenesis and of different organs have shown that the ADH isozyme pattern fluctuates qualitatively and quantitatively during the course of development and differentiation of the maize plant. ADH-T is controlled spatially and temporally in a very strict manner, being present only in extracts from the pericarp of 19- to 40-day-old kernels. ADH-3 and ADH-4 are present in the scutella of mature kernels and during early sporophytic development. ADH-1 and ADH-2 are the most common isozymes in all tissues examined, but ADH-1 is not found in endosperm of mature kernels or during germination. None of the isozymes have been found to be associated with any particulate cellular component at any stage of development. These findings are discussed with respect to differential gene expression, physiology, and cellular metabolism.     

Evolution of unconditional dispersal in periodic environments

Organisms modulate their fitness in heterogeneous environments by dispersing. Prior work shows that there is selection against 'unconditional' dispersal in spatially heterogeneous environments. 'Unconditional' means individuals disperse at a rate independent of their location. We prove that if within-patch fitness varies spatially and between two values temporally, then there is selection for unconditional dispersal: any evolutionarily stable strategy (ESS) or evolutionarily stable coalition (ESC) includes a dispersive phenotype. Moreover, at this ESS or ESC, there is at least one sink patch (i.e. geometric mean of fitness less than one) and no sources patches (i.e. geometric mean of fitness greater than one). These results coupled with simulations suggest that spatial-temporal heterogeneity is due to abiotic forcing result in either an ESS with a dispersive phenotype or an ESC with sedentary and dispersive phenotypes. In contrast, the spatial-temporal heterogeneity due to biotic interactions can select for higher dispersal rates that ultimately spatially synchronize population dynamics.     

Foraging responses ofFormica truncorum (Hymenoptera; Formicidae); exploiting stable vs spatially and temporally variable resources

Summary Social organization allows a division of labor between reproduction and foraging, as well as a task allocation among foraging individuals. Therefore, a colony simultaneously exploiting various resources can use different ways of getting them. This work analyzes wood ant foraging tactics both at the collective and the individual level. The main issues are: (1) How does a wood ant colony respond to stable vs temporally and spatially variable resources in terms of worker force allocation? (2) How is retrieval of ephemeral but rich resources effected at the individual level? The results show a correspondence between resource stability and behavior. Ants visited stable resources continually and recruited to ephemeral ones. They also visited empty food sites at a low frequency suggesting territoriality and constant scanning. The results suggest that recruitment may involve defence of the resource, since only a small fraction of all the workers available carry the food home. Additional ants arriving at the resource are recruited from inside the nest, not by reallocating outdoor workers. The foragers also form two separate groups, one site-persistent and another site-flexible. These two forager groups may reflect specialization on two different kinds of diet: honeydew or insect prey (spatially and temporally stable vs unstable resources, respectively).Formica truncorum is thus an example of how sociality allows the use of two different foraging strategies simultaneously: one that is based on recruitment to ephemeral resources and another that is based on search persistance and memory.     

Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.     

A computer simulation of pulsatile aggregation in Dictyostelium discoideum.

A computer simulation is used to examine how the observed individual features of cellular behavior can be combined into a quantitatively consistent representation of the spatially and temporally periodic aggregative pattern found in Dictyostelium. The (one-dimensional) simulation incorporates such features as secretion delays and refractory periods, movement steps generated by superthresholdattractant derivatives sensed by extending pseudopods, and attractant diffusion and hydrolysis. Parameters are taken as far as possible from the experimental literature, but there are a number of uncertain areas where various alternatives are tried. Among the principal issues discussed are the length of secretion and the duration of the chemotactic signal.     

Phytophagous arthropods and a pathogen sharing a host plant: evidence for indirect plant-mediated interactions

In ecological systems, indirect interactions between plant pathogens and phytophagous arthropods can arise when infestation by a first attacker alters the common host plant so that although a second attacker could be spatially or temporally separated from the first one, the former could be affected. The induction of plant defense reactions leading to the production of secondary metabolites is thought to have an important role since it involves antagonistic and/or synergistic cross-talks that may determine the outcome of such interactions. We carried out experiments under controlled conditions on young rose plants in order to assess the impact of these indirect interactions on life history traits of three pests: the necrotrophic fungus Botrytis cinerea Pers.: Fr. (Helotiales: Sclerotiniaceae), the aphid Rhodobium porosum Sanderson (Hemiptera: Aphididae) and the thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae). Our results indicated (i) a bi-directional negative interaction between B. cinerea and R. porosum, which is conveyed by decreased aphid growth rate and reduced fungal lesion area, as well as (ii) an indirect negative effect of B. cinerea on insect behavior. No indirect effect was observed between thrips and aphids. This research highlights several complex interactions that may be involved in structuring herbivore and plant pathogen communities within natural and managed ecosystems.     

CDPK-mediated signalling pathways: specificity and cross-talk

Plants are constantly exposed to environmental changes and have to integrate a variety of biotic and abiotic stress stimuli. Calcium-dependent protein kinases (CDPKs) are implicated as important sensors of Ca2+ flux in plants in response to these stresses. CDPKs are encoded by multigene families, and expression levels of these genes are spatially and temporally controlled throughout development. In addition, a subset of CDPK genes responds to external stimuli. Biochemical evidence supports the idea that CDPKs are involved in signal transduction during stress conditions. Furthermore, loss-of-function and gain-of-function studies revealed that signalling pathways leading to cold, salt, drought or pathogen resistance are mediated by specific CDPK isoforms