extfieldsofstudy: Biology Medicine

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Spatial structure undermines parasite suppression by gene drive cargo

Gene drives may be used in two ways to curtail vectored diseases. Both involve engineering the drive to spread in the vector population. One approach uses the drive to directly depress vector numbers, possibly to extinction. The other approach leaves intact the vector population but suppresses the disease agent during its interaction with the vector. This second application may use a drive engineered to carry a genetic cargo that blocks the disease agent. An advantage of the second application is that it is far less likely to select vector resistance to block the drive, but the disease agent may instead evolve resistance to the inhibitory cargo. However, some gene drives are expected to spread so fast and attain such high coverage in the vector population that, if the disease agent can evolve resistance only gradually, disease eradication may be feasible. Here we use simple models to show that spatial structure in the vector population can greatly facilitate persistence and evolution of resistance by the disease agent. We suggest simple approaches to avoid some types of spatial structure, but others may be intrinsic to the populations being challenged and difficult to overcome.

INTRODUCTION
Genetic engineering has advanced to the point that it is not only possible to introduce arbitrary, massive changes into the genomes of countless organisms, but it is also possible to engineer changes that rapidly sweep throughout an entire species. The rapid sweeps are enabled by a class of genetic elements called gene drives that function on the principle of biasing transmission in gametes or in survival (Hamilton, 1967;Lyttle, 1977;Burt, 2003;Deredec, Burt & Godfray, 2008;Gould, 2008;Gould et al., 2008;Report, 2016). Perhaps the most powerful use of a gene drive is one that suppresses population numbers and even potentially drives the population extinct. A more benign form of gene drive is one that sweeps without causing much harm to its host. Any gene drive may be harnessed with additional genetic material (i.e., 'effector gene' or simply 'cargo') that is carried along with the drive as it spreads (Sandler & Novitski, 1957;Gould, 2008;Gould et al., 2008;Gantz et al., 2015). A harmless drive equipped with a cargo provides a fast and simple means of genetically transforming a population, potentially endowing that population with properties that meet social goals without harming the species.
The application of gene drives is limited in a few important ways. They require species with largely obligate sexual reproduction and moderate to high rates of outcrossing. Furthermore, drives that impair fitness are highly susceptible to evolution of resistance (Burt, 2003;Noble et al., 2017;Unckless, Clark & Messer, 2017;Bull, Remien & Krone, 2019). For these reasons, some applications are most amenable to species modification with harmless gene drives carrying genetic cargo. One such application is the use of gene drives to transform disease vectors so that the disease agent ('parasite' or 'pathogen') can no longer be transmitted: the pathogen cannot be targeted with a gene drive, but its vector can. Such a gene drive can be designed to have little effect on the vector yet completely block the pathogen (Sandler & Novitski, 1957;Burt, 2003;Gantz et al., 2015).
Multiple approaches to population replacement that involve gene drives have been proposed. Several of these have been implemented successfully in model systems, and important progress has been made with homing-based approaches. Current technology using CRISPR homing drives appears good enough to allow a gene drive to avoid resistance evolution and achieve wide coverage of a population (Kyrou et al., 2018;Champer et al., 2019c). Thus, an inhibitory cargo should also be able to achieve wide population coverage and thereby eradicate many types of parasite, provided that the cargo's suppression of the parasite cannot be overcome by single mutations. One potential limitation of this approach is that even slight fitness costs of cargo carriage will ultimately lead to a decay of cargo in the vector population, but the decay should often be slow enough to allow parasite suppression for tens to hundreds of generations (Beaghton et al., 2017)-still potentially enough for eradication.
Here we suggest another possible basis of cargo failure, spatial structure in the host population combined with imperfect gene drive coverage/expression. If parasite movement is limited, even small areas of incomplete suppression may allow parasite persistence that serve as nuclei for evolution of parasite resistance. We offer simple models of the sensitivity of parasite persistence and resistance evolution under spatial structure to gauge the plausibility of parasite escape from gene drive control. Our approach potentially applies to any widespread genetic modification of a population, not just gene drives.

Background
This section offers a biological framework for the problem addressed in the models section. This framework is easily explained at an intuitive level and helps anticipate the models. We henceforth use 'parasite' instead of 'pathogen', to avoid confusion as to the effect of the parasite-it will commonly be a pathogen of humans but not necessarily of the vector, where it is targeted. result from loss of the cargo from the gene drive or resistance to the gene drive itself, such that the host individual lacks the gene drive and its cargo altogether. The formulation of the model is trivially extended to multiple states of partial inhibition.
The model counts numbers of parasites in each type of host. Host type merely translates into parasite reproduction. Notation is x h : The relative frequency of hosts of type h b gh : The fecundity of a parasite genotype g in hosts of type h n gh : The current number of parasites of genotype g in hosts of type h N g : The current number of progeny produced by genotype g across all patches α: The fidelity of parasite reproduction to hosts of the same type, where parasite genotype g ∈ {0,1,...,M } and host type h ∈ {0,1,...,H }. Here, host type 0 indicates hosts with no cargo (hence no parasite suppression), and larger values of h correspond to increased levels of suppression, with H denoting the number of types of cargo-carrying hosts differing in some aspect of parasite suppression; parasite genotype 0 is the wild type (with no protection against the cargo), and larger values of g correspond to mutant strains with increased levels of resistance to the cargo.
To approximate the separation of phases between rapid gene drive spread and the subsequent effect of cargo on the parasite, we let the x h and b gh be constant in time, so the only changes are in parasite numbers. Time is discrete. For biological reasons, in the case of three host types, we also assume that the fecundity of parasite genotype 0 satisfies b 00 > 1 > b 01 > b 02 , which means that the parasite has negative growth in all host types except the one lacking cargo.
To set the stage for a structured host population, we suppose that hosts are clustered in patches of similar host types, patch types designated by subscript h ('Host' and 'patch' are used interchangeably below, but 'patch' helps convey structure). Patches could result, for example, from limited host migration and gene drive spread through the host population in a manner that follows host population structure. The clustering of hosts and the consequent movement of parasites between patches determines the extent to which structure is experienced by the parasites. To establish a mass-action baseline, adult parasites reproduce and release all progeny into a random pool, from which they settle into each of the H + 1 patch types at frequencies x 0 ,x 1 ,...,x H . Spatial structure is modeled indirectly by assuming that a fraction α of the progeny born in a patch type remains in the same patch type without entering the random pool; this 'fidelity' increases with the retention of progeny in their natal patch type. This process is fundamentally the same as migration in standard population genetics problems (Crow & Kimura, 1970). Our formulation is different in that α denotes a lack of movement from the natal site instead of movement between patches/populations; this formulation leads naturally to calculating the null case of mass action (α = 0), which is presumably the default expectation in gene drive applications. Note that fidelity to a patch type is imposed without inbreeding, so the numbers within a patch are assumed large enough that consanguinity can be ignored.
We first consider a simple system of linear dynamical equations. The overall progeny output of strain g across all environments is (1) Using primes to indicate one generation hence, The joint dynamics for genotype g across all host types can be written as a matrix projection recursion (Caswell, 2006). Dropping the genotype subscript g for ease of visualization, the recursion has form where n and n are (H + 1)-dimensional column vectors with hth element n h and n h , In words, matrix element M hi describes the rate that individuals who originate in patch type i contribute to the abundance in patch type h at the next time step. The densities of the genotype g at any time t , n(t ), can be computed as n(t ) = M t n(0) (Caswell, 2006). This model allows for a biological anomaly: when a genotype i is initially assigned to a patch (j), a sufficiently large combination of fecundity and fidelity (b ij α > 1) allows its numbers to persist even when the patch type is absent (even when x j = 0). This effect occurs because, once a genotype exists in a patch (by initial conditions), a portion of its growth comes from offspring who stay in the patch to reproduce. This effect is independent of patch size. Because we interpret x j as the fraction of hosts of type j, we require x j > 0 for any host type that harbors parasites. This requirement is further imposed when enforcing a carrying capacity (see below).
These equations assume fixed fecundities and thus allow unlimited population growth. They should be adequate to decide whether parasites persist or die out, because they can be applied to deterministic dynamics at low population densities to describe the direction of population growth, when fecundities are intrinsic and unaffected by densities. For dynamics and evolution in high-density populations, we use a related model that imposes density regulation. For simplicity, we limit our description of density regulation to the case of two patches and two strains.

Introducing density regulation
Density regulation may be important in the evolution of alternative genotypes in a persisting population, at least because a small portion of the environment with cargo-free hosts may be limited in how many parasites it can support-a small patch may allow parasite persistence but have little impact on parasite numbers across the entire environment. We thus introduce a simple form of patch-specific density regulation that will be used in some numerical trials of two patch types, 0 and 1.
Let K be the overall upper density limit for the environment and let K 0 = Kx 0 , K 1 = Kx 1 denote the ceilings for patch 0 and 1, respectively. Let 1 = min{n 01 b 01 +n 11 b 11 ,K 1 } be total parasite progeny production emanating from patch 1, and 0 = min{n 00 b 00 + n 10 b 10 ,K 0 } be the total parasite progeny production from patch 0, each limited locally, without respect to regulation in the other patch type. The extension to more than two patch types is straightforward.
To decide how overall parasite production in a patch is divided between two genotypes when progeny output is limited by carrying capacity, let p 11 = n 11 b 11 /(n 01 b 01 + n 11 b 11 ) denote the fraction of offspring that are strain 1 within patch 1, and p 01 = 1 − p 11 the fraction in patch 1 that are strain 0. Similarly, in patch 0 we have fractions p 10 = n 10 b 10 /(n 00 b 00 + n 10 b 10 ) and p 00 = 1 − p 10 . Accounting for mixing and assuming both genotypes are density-regulated the same, the total number of genotype 1 entering patch 1 is and the total number of genotype 0 entering patch 1 is Analogous equations apply to genotypes 0 and 1 entering patch 0. Note that mixing can lead to a temporary violation of local carrying capacities when patches are repopulated by adults, but the capacity limit is imposed again at the next round of reproduction.

Parasite persistence is facilitated by spatial structure
The growth or suppression of the parasite depends on whether the magnitude of the leading eigenvalue of M in Eq. (3) exceeds 1. Density dependence can be ignored when addressing persistence, but an eigenvalue exceeding 1 merely indicates that parasites have positive growth somewhere in the environment, perhaps in only a tiny locale, with negative growth everywhere else. Although the characteristic equation is easily found, the leading eigenvalue (λ max ) of a genotype is tractable for arbitrary H only for the extremes of mass action (α = 0), and complete parasite isolation among host types (α = 1). For mass action, As is well appreciated for mass action, parasite growth is just the weighted average fecundity across all host types. Small levels of weak suppression (i.e., low values of x 0 despite possibly high values of b 00 ) will not themselves enable parasite persistence except when parasite fecundity is extraordinarily high. When more than one genotype has an eigenvalue greater than 1, density dependence will determine which one prevails (see below).
With complete parasite separation across the host types (α = 1), (λ max is associated with patch type 0 because cargo-free hosts are assumed to offer the highest fecundity of all patches, regardless of genotype). Here, the parasites inhabiting each host type have their own eigenvalue, and any host type with b gh > 1 will allow parasites of genotype g to persist in that patch type (subject to competition among different parasite genotypes). In this extreme, the values of x h no longer matter: even a small fraction of permissive hosts will allow the parasite to persist. The question motivating our study is how sensitive parasite persistence is to fidelity α-reflecting spatial structure. As the eigenvalue for several patch/host types is unwieldy, we reduce the problem to just two patch types, h = 0 and 1 (fully permissive and fully blocking of wild-type); this reduction also simplifies patch type abundances: x 0 + x 1 = 1. For this case, the largest eigenvalue is Values of the eigenvalues for different fecundities in the two patch types are shown in Fig. 1, Figs. 1A-1D differing in fidelity (α) and cargo-free patch size (x 0 ) values. The graphs show four contour lines, but λ max = 1-the boundary between persistence and extinction-is thicker than the others. It is clear that persistence is enhanced by spatial structure, though typically a large cargo-free fecundity is required if the cargo is effective (b 00 must be well above 1 when α and b 01 are small). But for small cargo-free patch sizes (small x 0 ), parasite persistence becomes possible despite low fecundity values in cargo-free hosts when the cargo becomes moderately ineffective (when b 01 exceeds 1). In addition to the effect of fecundity, there are also effects of α and x 0 ; one interesting effect is that the isoclines are visually step-like except in Fig. 1A. For those cases, fecundity in cargo-bearing hosts has little effect on the parasite growth rate until b 01 exceeds λ max .
Most empirical interest is likely to be in the extreme case that the cargo completely suppresses wild-type parasite reproduction (b 01 = 0), as that would be the goal of the engineer. Indeed, any gene drive release could be avoided until such an appropriate inhibitor was found. This case corresponds to the the sliver defined by the vertical axes in Fig. 1. Furthermore, this case is highly tractable: Persistence in this case requires λ 0 = b 00 [x 0 + α(1 − x 0 )] ≥ 1. This implies, for example, that persistence of the parasite is assured even when completely inhibited by the cargo as long as fecundity in cargo-free hosts (b 00 ), exceeds [x 0 + α(1 − x 0 )] −1 . Consideration of this minimum cargo-free fecundity (Fig. 2) shows that spatial structuring with fidelity (α) well below 1 (e.g., 0.5) enables parasite persistence for even rare cargo-free hosts (small x 0 ), as long as the parasite can grow there moderately well (e.g., b 00 > 2). The results are largely insensitive to patch size x 0 when fidelity reaches 0.6.
It is also easy to see that, when cargo-free patch size x 0 is small, as it should be if the engineering worked as expected, the effect of increasing fidelity α is approximately the same as increasing the frequency of permissive hosts (x 0 )-both are mostly linear effects Each of (A)-(D) vary parasite fecundities in the two patch types (b 00 is cargo-free fecundity, b 01 is fecundity in cargo-bearing hosts). Parasite growth rates rise with increases in each fecundity, but the eigenvalues often show a step-like pattern in which fecundity increases in one host type have little effect until it reaches a threshold. (A)-(D) differ in parasite fidelity (α) and size of the cargo-free patches (x 0 ); λ max values are given adjacent to their respective contours. The wild-type genotype is represented as g = 0. Colors merely distinguish the regions bounded by the curves.
Full-size DOI: 10.7717/peerj.7921/ fig-1 and of the same magnitude. Whereas x 0 is somewhat under the control of the engineer, α is not and has the potential to thwart parasite eradication. Thus, a very small x 0 could easily cause parasite population collapse in the mass action case, but b 00 α > 1 is sufficient for persistence no matter how small x 0 . Persistence could be achieved by a cargo-free patch merely large enough that parasite progeny often did not disperse beyond the patch edges.

Ease of resistance evolution depends on ecology
At the population level, the evolution of resistance to a genetic cargo has many parallels with the evolution of resistance to antibiotics and pesticides. The latter problems are Minimum fecundity in cargo-free hosts required for parasite persistence when the cargo causes complete inhibition, (b 01 = 0, from Eq. (9)). Each curve represents a different size of cargo-free patch (x 0 ); the required cargo-free fecundity for parasite persistence (b 00 , vertical axis) decreases with the fidelity to patch type (α, horizontal axis). For α ≥ 0.6, there is little effect of patch size. The curves intersect α = 0 at b 00 = 1 x 0 . The horizontal line at b 00 = 1 indicates the minimum fecundity required for the parasite to persist in the absence of cargo, which all curves intersect at α = 1.
Full-size DOI: 10.7717/peerj.7921/ fig-2 thoroughly studied, and it is well appreciated that resistance is especially prone to evolve under intermediate levels of drug/chemical application (e.g., Gould & MacKenzie, 2002;Andersson & Hughes, 2012;Tabashnik & Gould, 2012;Neve et al., 2014;Gould, Brown & Kuzma, 2018). Inhibition by a gene drive cargo is different from chemicals in that the levels of inhibition are established at fixed, semi-permanent levels in the near term. They are also largely unchangeable, at least in the short term, should it be discovered that they are inadequate.
Of the many factors to consider, an important one is the mutational spectrum of resistance: a cargo for which simple, single mutations can allow parasite persistence seems doomed to fail, and intuition suffices for preliminary understanding, at least deterministically. Our interest instead lies in gradual evolution and the selection of weak resistance mutations. It might be hoped that cargo-based inhibitors can be found for which resistance mutations are impossible, but a more realistic hope is that inhibitors could be found for which resistance can evolve only gradually.
The evolution of resistance can be considered in two contexts. One is known as 'evolutionary rescue', whereby the population is in decline and a resistance mutation potentially reverses the decline (Martin et al., 2013;Uecker, Otto & Hermisson, 2014;Hufbauer et al., 2015;Gomulkiewicz, Krone & Remien, 2017). The other context, the one addressed here, is resistance evolution in a persisting population. For our application, we imagine that the parasite is persisting because of spatial structure and would go extinct under mass action-the large majority of hosts inhibit parasite reproduction because of the cargo. We address how selection acts on a weakly resistant mutation, a mutation that is not necessarily sufficient to provide positive parasite growth from inhibitory hosts alone.
It might seem valid to evaluate long term resistance evolution from a comparison of eigenvalues of wild-type and mutant growth, in which case the preceding figures could be used to infer evolution of alternative genotypes. However, we are considering resistance evolution in established populations at which density dependence is operating. When density dependence operates locally, as assumed here, it will have a different effect on parasite growth in cargo-free patches than in inhibitory patches. If the wild-type parasite cannot grow in inhibitory patches but the mutant can, the fecundities of both genotypes will be suppressed by density dependence in cargo-free patches but the mutant's fecundity in the inhibitory environment will be unaffected (at least while rare). Eigenvalue calculations do not include these fecundity modifications. Our model of resistance evolution uses the system of equations in (3) but with the density-dependent carrying capacity enforced as in Eqs. (4) and (5).
This model was evaluated numerically for different combinations of fidelity (α), cargofree patch size (x 0 ), and genotype fecundities (b ij ). We focused on the case of a wild-type (starting genotype) that was unable to grow in the inhibitory environment of cargo-bearing hosts (b 01 = 0), and for which the mutant could grow in the inhibitory environment at some reduction in its ability to grow in the cargo-free environment (i.e., a trade-off was imposed between growth in the two patch types). The patterns across trials are qualitatively similar and easily comprehended (Fig. 3). Resistance could invariably evolve if it did not incur too much of a cost to growth in the cargo-free hosts.
Some 'ecological' patterns are evident.
(1) A large effect of α-host patch fidelity-on evolution exists in some parameter ranges. Thus, to displace wild-type, the mutant is more sensitive to a reduction in cargo-free fecundity at larger α-until fecundity in cargo-bearing hosts is high enough that the mutant could sustain itself in those hosts alone (until b 11 exceeded 1). This effect is evident when comparing lower portions of Figs. 3B and 3D and was seen in other trials (not shown). We interpret this effect as that higher values of α increasingly partition growth in the two patch types, so that any fitness loss in the permissive patch-the one sustaining the population-is increasingly penalized, but only to the point that the mutant parasite can maintain itself in cargo-bearing hosts.
(2) Patch size x 0 also appears to have a large effect, but in the opposite direction as that of α: as x 0 increases, resistance evolves more easily (i.e., tolerates larger reductions in cargo-free fecundity, b 00 − b 10 ). This effect is seen by comparing Figs. 3A-3C which vary only x 0 . Although not shown, the trend does not continue at high values of x 0 , and the effect reverses. Indeed, mutants whose b 11 > 1 are not favored at high x 0 values if they suffer too much cost in b 10 . We do not pretend to grasp these patterns intuitively. One challenge in understanding these outcomes is that x 0 has effects at different steps of the life cycle: with higher x 0 , the cargo-free patch has an increased carrying capacity and thus produces more progeny; some of these progeny return to patch 0 and the others go to the random pool. But the increase in x 0 reduces the number of random-pool progeny that land in patch 1 and thereby reap the benefits of resistance. Patch-type fidelity is α, wild-type fecundity in cargo-free hosts is b 00 , and the patch size of cargo-free hosts is x 0 ; wild-type fecundity in cargo-bearing hosts (b 01 ) is zero in all panels. Each dot represents a different mutant allele whose fecundities in cargo-free hosts (b 10 ) and in cargo-bearing hosts (b 11 ) are given by its coordinates; axes in (A) are labeled to assist recollecting the b 1j . Green indicates that the wild-type was in a strong majority at the end of the trial, blue that the mutant was in a strong majority, and an intermediate color indicates that both alleles were moderately common. In the absence of competition from the mutant, the wild-type would persist for all conditions tested; its growth rate when rare is given as λ 0 in the panel, from Eq. (9). Trials were run for 1,000 generations in which both alleles started equally abundant in both patch types. Carrying capacity was 10 6 for all trials shown here. Full-size DOI: 10.7717/peerj.7921/ fig-3

DISCUSSION
Genetic engineering has brought us to the brink of being able to introduce selfish genetic elements (known as gene drives) in countless species (Esvelt et al., 2014;Report, 2016;Collins, 2018). Although safety concerns and regulatory hurdles, and to some extent technical hurdles, have so far prevented the release of engineered gene drives, it seems inevitable that they will be released and eventually become a standard intervention for pest management and disease control-if they work as expected. Much excitement is about using gene drives for population suppression (Hamilton, 1967;Lyttle, 1977;Burt, 2003;Godfray, North & Burt, 2017;Lambert et al., 2018), but an alternative approach that may encounter fewer regulatory obstacles is to use 'modification drives' for spreading genetic cargo through a population (Sandler & Novitski, 1957;Gould, 2008;Gantz et al., 2015;Beaghton et al., 2017). In this latter application, the gene drive is a rapid and potentially harmless means of genetically engineering an entire population to carry a novel gene of interest. Thus, a mosquito that transmits Plasmodium might be targeted for eradication by a gene drive (Kyrou et al., 2018) or be targeted with a modification drive to spread a cargo that expresses anti-Plasmodium antibodies that block Plasmodium reproduction in the mosquito (Gantz et al., 2015).
Our study addressed the latter type of application and even assumed that the gene drive cargo had already spread in the host (e.g., mosquito) population. If this inhibitor fully suppresses the parasite in any host individual expressing the cargo-if it operates as expected-how might parasites persist despite our efforts to eradicate? Our emphasis here is on the possible contribution of spatial structure in the host population to parasite escape. Even when the gene drive successfully spreads the cargo to most of the host population, spatial structure combined with imperfect gene drive spread may leave pockets of cargo-free mosquitoes that allow the parasite to persist locally.
Our findings suggest that spatial structure in the host population can contribute toindeed be sufficient to-enable parasite persistence against a cargo that would otherwise eradicate the parasite. Pockets of parasite persistence then foment the evolution of resistance to escape the cargo, unless resistance mutations cannot arise. The pockets need not be large, possibly representing a very small fraction of the range of the species targeted by the drive.
Our results follow work suggesting another reason that cargo-bearing gene drives may fail to eradicate parasites: the cargo frequency will begin to decline as soon as the gene drive carrier has reached its zenith in the host population (Beaghton et al., 2017). Thus, independent of spatial structure, any cost to carrying the gene drive element or cargo will select a loss of those elements once the gene drive spread has ended. An additional problem facing modification drives that use homing is that they may target genomic sequences that are not essential to the host and thus have few selective constraints. Weakly selected sequences can tolerate variation that would block a gene drive. A clever solution to this problem is to use drives that target highly conserved sequences; to avoid harmful effects, they carry an insensitive cargo that replaces the target gene (Burt, 2003;Champer et al., 2019c). They must then carry two cargos, one for the modification, one to replace the target gene. Alternatively, use of multiple targets with CRISPR homing (multiple gRNAs) may limit resistance evolution (Champer et al., 2019d).
There are two requirements for parasite escape under the models studied here: spatial structure and genetic variation in cargo presence/expression that coincides with the spatial structure. Observation of a spatially structured host population would indicate that the first requirement is met. Pre-existing genetic variation in the host population may even contribute to variation in cargo presence or expression, as when existing variation directly resists gene drive spread or affect cargo expression, and spatial structure of that variation would lead to both requirements being satisfied. But even if existing genetic variation does not affect gene drive evolution or cargo expression, any existing spatial structure may become a problem for genetic variation that evolves during gene drive spread. Gene drive spread can generate its own variation in cargo presence/expression by evolving as it follows demographic paths of reproduction in the host population (Beaghton et al., 2017). Such variation would be maintained by any spatial structure intrinsic to the host population.
The problem of parasite persistence and resistance evolution in response to a gene drive cargo has parallels with evolution of resistance in other contexts: antibiotic treatment of bacteria, use of chemical pesticides, and even genetically engineered 'Bt' crops. There is a widespread recognition that intermediate levels of pesticides and antibiotics favor the evolution of resistance (e.g., Gould & MacKenzie, 2002;Andersson & Hughes, 2012;Tabashnik & Gould, 2012;Neve et al., 2014;Gould, Brown & Kuzma, 2018). These problems are more often cast as stemming from temporal variation in dose rather than spatial variation, but the two types of heterogeneity are similar over the right time scales. In contrast, the practice of planting non-engineered strains among genetically engineered Bt crops to delay insect resistance evolution is explicitly one of destroying spatial structure (Tabashnik & Gould, 2012) and highlights the importance of spatial structure to the evolution of resistance. Gene drive cargo expression is presumably stable in time (for individual hosts and their dependants), at least for the short term, so parasite escape is primarily a problem of spatial variation rather than one of temporal variation.
The models leave many questions unanswered about the nature and magnitude of spatial structure required to enable parasite persistence. Indeed, it is the combination of spatial structure in conjunction with genetic variation that matters. The right kind of spatial structure might exist in one part of a species range, the appropriate genetic variation in another, yet the parasite be suppressed by the cargo throughout. Furthermore, the permanence of spatial structure in the host population will depend heavily on host dispersal patterns, and it is the combined movement over the the life cycle of the parasite that determines the relevant structure. For parasites that alternate between two host species (e.g., mosquito-borne diseases of humans), a highly structured mosquito species will not necessarily enable parasite persistence if the second host-humans-is sufficiently mobile on the right time scale. Issues such as longevity of the spatially structured patches, plus averages and variances of host dispersal distances may need to be explored in the context of specific applications before understanding the potential for parasite escape.
Evolution of resistance is perhaps the ultimate concern. If cargo can be engineered to be resistance-proof, the parasite persistence afforded by spatial structure will be only a temporary setback as additional interventions are implemented. Spatial structure will have the largest impact in facilitating evolution of resistance to cargo when resistance can evolve only gradually, in small steps. In this case, parasite eradication might well be achieved were it not for structure, but the structure provides the nucleus for gradual evolution of resistance that ultimately enables the parasite to maintain itself on cargo-bearing hosts. Stacking multiple inhibitors in the same host individual (as proposed for malaria (Gantz et al., 2015)) may, in the ideal case, prevent stepwise resistance evolution. Here the concern is an evolutionary loss of part of the cargo, so that only single inhibitors operate in some hosts; spatial structure would then contribute to evolution of resistance.
It has been convenient to focus on cargo-free patches as the type of reservoir enabling parasite escape. An alternative-or additional-type of refuge to consider is patches of intermediate cargo expression, enabling the parasite to persist at some level and directly favoring resistance. Intermediate patches may occur with many levels of expression and may arise because of genetic background effects in the host species or may arise by mutation during the spread of the gene drive itself. Thus, if cargo expression is costly to the host, drives with reduced expression will spread even faster than drives with the original engineering. These mutant-drive cargoes will form their own spatial structure as they spread, which may then be maintained by intrinsic host structure. (See Weinstein et al. (2017) for an interesting study on the evolution and dynamics of spatial structure in competing bacterial strains.) The engineering faces a delicate balance between cargo over-expression and cargo under-expression. Over-expression may impose a fitness cost that selects against the drive/cargo, whereas the under-expression risks facilitating parasite persistence and evolution of resistance. The effect of patch intermediacy on persistence may be evaluated for our 2-patch case in Fig. 1 merely by considering one of the two patches to be intermediate instead of extreme (e.g., cargo-free fecundity b 00 would be depressed or cargo-bearing fecundity b 01 would be greater than 0). In a sense, our two patch model describes a worst-case scenario for parasite persistence; we expect permissive conditions in the real world will be broader than our results suggest.
Subject to possible limitations of our analysis (see below), our findings can be tentatively used to inform implementation practices most likely to succeed. In any implementation, inhibitory cargo should be chosen so that resistance evolution is difficult (i.e., requires multiple steps) or impossible, as inhibitors that can be overcome with single mutations seemed assured of eventual failure. Anti-drive resistance evolution will also be a factor that should be considered and is likely to vary with design and even with host population characteristics (Champer et al., 2019a;Champer et al., 2019b), but that concern is not any more important for spatial structure than without. Beyond that, there are a few design features that may facilitate parasite suppression and work to limit evolution of resistance.
• Prevent emergence of spatially structured variation. Existing spatially structured genetic variation in a wild species may be difficult to change, although inundating small areas with release of lab-reared strains may offer a temporary solution-as underlies the sterile insect technique (Klassen & Curtis, 2005;Dyck, Hendrichs & Robinson, 2005). However, spatial variation that arises from gene drive spread and evolution (Beaghton et al., 2017) may be reduced by gene drive release at multiple sites (North et al., 2013), especially sites of import, such as population centers: the different waves of advance will collide with each other, reducing any spatial evolution from single release points. Releasing multiple, independent drives in the same population, as proposed to overcome resistance evolution within unstructured populations (Burt, 2003;Deredec, Godfray & Burt, 2011), may limit the extent to which any area is completely free of cargo from at least one drive. As pointed out by a reviewer, killer-rescue systems may be far more susceptible to the generation of spatial structured variation than are homing drives; indeed, that is one of their oft-cited advantages.
• Target areas of incomplete coverage. Following gene drive spread, areas can be assessed for cargo presence and expression. Regions identified as having inadequate coverage can be targeted for additional interventions to offset the limited effect on parasites.
• Consider gene knockouts as cargo. Quantitative variation in cargo expression may be structured, just as with cargo-free hosts. Partial expression of cargo may be even more conducive to parasite resistance evolution than is a complete absence of cargo (by selecting intermediates). A gene drive system that knocks out a non-essential host gene required for parasite reproduction/transmission may be less subject to intermediate expression than is a cargo transgene and thus less likely to select resistance. A possible downside is that ablation of a non-essential host gene may carry larger fitness defects than does expression of a foreign transgene, and thus select resistance to the drive.
Limitations of the models. The models analyzed here depicted spatial structure abstractly and used several other simplifications to achieve analytical tractability and comprehension: population regulation with a sharp threshold, deterministic dynamics, steady state analyses, and few host types. Parasite fecundity was abstracted to be as simple as possible. The models are best interpreted as augmenting intuition rather than formally capturing any natural process, as might be done with agent-based simulations (e.g. North et al., 2013;North, Burt & Godfray, 2019). The results may thus be seen as to invite more formal analyses that include, at a minimum, explicit spatial structure but also small population sizes that would accrue near extinction. Despite these limitations, the results unambiguously point toward spatial structure as seriously impeding gene drive implementations using cargo-an otherwise promising use of gene drives. From our results, we conjecture that spatial structure can be sufficient to enable parasite escape from inhibitory genetic cargo in the host population, but we equally suggest that there are likely to be many details affecting the ease of escape and evolution of resistance. Indeed, it will be desirable to study models specific to the biology of an implementation before making critical decisions about engineering and sites of release (e.g., Eckhoff et al., 2017;Lambert et al., 2018;North, Burt & Godfray, 2019).

CONCLUSIONS
Gene drives are being engineered for pest and disease control. They can spread rapidly and widely throughout a species, and they can be engineered either to disrupt existing genes or to carry an expressed genetic cargo. One compelling use of gene drives is to genetically convert a disease vector to be incapable of transmitting a parasite; the vector is not harmed in this approach, but the parasite is blocked. Using simple, deterministic mathematical models, our main result is that parasite persistence is greatly facilitated by small levels of incomplete parasite blocking combined with spatial structure of the parasite population-even though a well-mixed parasite population would cause parasite extinction. Gradual evolution of resistance to the block is also aided by spatial structure. We offer suggestions to minimize the deleterious impact of spatial structure on gene drive goals. An obvious next step is to develop explicit models of spatial structure with stochastic dynamics.

METHODS
Mathematica®(12.0.0.0) was used for some analyses and the generation of Fig. 1. R (3.6.1) was used for Figs. 2 and 3 (R Development Core Team, 2019). The code to generate the data used in Fig. 3 was written in C (code included as a Supplement).


s2fieldsofstudy: Biology Medicine

text:
MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common and complex inflammatory lung diseases. MicroRNAs (miRs) have emerged as novel gene regulatory molecules, serving a crucial role in a variety of complex diseases, including ALI. In the present study, the anti-inflammatory action of miR-223 on inflammation in ALI was demonstrated and the possible mechanism was further examined. In lipopoly-saccharide-induced ALI, the expression of miR-223 was reduced compared with that in the control normal group. An in vitro model was used to analyze the effect of miR-223 downregulation on an ALI model, which increased inflammation, and induced the activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome and Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway via rho-related GTP-binding protein RhoB (RHOB). In addition, the overexpression of miR-223 reduced inflammation and suppressed the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB in the in vitro model. Furthermore, TLR4 inhibitor or NLRP3 inhibitor reduced the pro-inflammatory effect of miR-223 downregulation in ALI. In conclusion, the results of the present study indicated that miR-223 functioned as a biological indicator by regulating inflammation in ALI, and may represent a novel potential therapeutic target and prognostic marker of ALI.

Introduction
Acute lung injury (ALI)/acute respiratory distress syndrome (ARdS) refers to the acute progressive respiratory insufficiency or respiratory failure caused by various factors, including trauma, blood transfusion and infection (1); this is a common respiratory crisis, pathologically characterized by pulmonary edema, pulmonary hemorrhage and severe respiratory impairment (1). The mortality rate of ALI/ARdS may be as high as 30-50% (2). Due to the undefined pathogenesis, there are no effective therapeutic drugs in clinical practice at present (2). Supportive treatment remains the principal treatment option (3). A series of ALI animal models have been established to simulate the status of ALI caused by diverse pathogeneses (3). Among them, the majority of studies have focused on lipopolysaccharide (LPS), one of the primary causative factors of ALI caused by bacterial infections, particularly Gram-negative bacteria (3). As the active component of bacterial endotoxin, LPS is an important pathogenic factor in ALI (4). In LPS-induced ALI animal models, the excessive inflammatory reaction of neutrophils leads to extensive damage and even mortality (4).
The NAcHT, LRR and PYd domains-containing protein 3 (NLRP3) inflammasome is a multiprotein large-cytoplasmic complex, which is composed of NLRP3, apoptosis-associated speck-like proteins and pro-caspase-1 (5). NLRP3 serves a critical role in regulating the relevant components of the inflammatory complex, including apoptosis-associated speck-like proteins and pro-caspase-1 (6). It has been reported that in acute injury, the stimulated intracellular reactive oxygen species may activate the NLRP3 inflammasome and caspase-1, and promote the cleavage and maturation of pro-interleukin (IL)-1β and pro-IL-18. As a result, inflammatory cytokines are released, resulting in damage to lung tissue (7,8).
Alterations in microRNA (miRNA/miR) expression are associated with immune responses, inflammatory signaling pathways and the pathogenesis of inflammatory lung diseases, including ALI. Therefore, miRNAs are novel promising therapeutic targets (9). miRNA-induced changes in gene expressions are generally modest (9). However, the results may affect the expression of a large number of subsequent genes, which

MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB
further affect multiple biological processes. Therefore, it is feasible to utilize miRNAs as markers (10). miRNA expression has been verified to be dynamic, which reflects alterations in the internal and external environments, and cell signals (10). Therefore, it is promising to use miRNAs as biomarkers to represent a specific disease state or the underlying pathophysiological processes at different stages. In addition, miRNAs may be detected in a variety of specimens, including tissue, blood or other body fluids (11). miRNAs are also fairly stable and are virtually unaffected by errors in sample handling, making miRNAs more attractive biomarkers (11). Intriguingly, miRNAs in plasma have been used as biomarkers to diagnose and monitor a variety of inflammatory lung diseases, including ALI. Neudecker et al (12) suggested that miRNA-223 deficiency was associated with severe lung inflammation. In the present study, the anti-inflammatory effect of miRNA-223 on inflammation in ALI, and the possible mechanism, was demonstrated.

Materials and methods
Mice and histopathological assay. Male c57BL/6 mice (5-6 weeks; 18-20 g) were obtained from Shandong University Laboratory Animal center (Jinan, china). All mice were housed at 22-23˚C, 55-60% humidity, on a 12-h light/dark cycle with free access to food/water. All mice were randomly assigned to two groups: control and ALI mice. All ALI model mice were injected with 35 mg/kg pentobarbital sodium [intraperitoneal (i.p.)] and injected with LPS at 5 mg/kg (Sigma-Aldrich; Merck KGaA, darmstadt, Germany) into the chest. After 1 day, all mice were injected with 35 mg/kg pentobarbital sodium and sacrificed via decollation. Lung tissue was acquired and washed with PBS, and fixed with 4% paraformaldehyde for 24 h at room temperature. The lung tissue was dehydrated using 100-75% ethyl alcohol for 5 min at 4˚C, and cut into 5-µM sections. Lung tissue sections were stained with hematoxylin and eosin (HE) for 5 min at room temperature, and were finally examined under a light microscope (Nikon Eclipse TE2000-U; Nikon corporation, Tokyo, Japan) at x100 magnification. The experimental procedures in the present study were performed with the approval of Binzhou Medical University Hospital (Liaocheng, china).
Cytokine detection. Serum samples were centrifuged at 1,000 x g for 10 min and used to measure TNF-α (cat. no. H052), IL-1β (cat. no. H002), IL-6 (cat. no. H007) and IL-18 (cat. no. H0015) levels using ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, china). cells were lysed with radioimmunoprecipitation assay buffer for 15 min and protein concentrations in the extracts were measured by bicinchoninic acid assay. Proteins (10 µg) were centrifuged at 1,000 x g for 10 min and collected to measure TNF-α, IL-1β, IL-6 and IL-18 levels using ELISA kits. Statistical analysis. All the experiments were performed at least three times independently (n=3) using SPSS 17.0 (SPSS, Inc., chicago, IL, USA). A Student's t-test was used to determine statistical significance between two groups. One-way analysis of variance and Tukey's post hoc test were performed for parametric multivariable analysis. P<0.05 was considered to indicate a statistically significant difference.

miRNA-223 expression in ALI mice.
To investigate the function of miR-223 in ALI mice, the alterations in miR-223 was analyzed in ALI mice. As presented in Fig. 1A, HE staining indicated that pulmonary alveoli were damaged in ALI mice compared with control mice. The levels of TNF-α, IL-1β, IL-6 and IL-18 were increased in ALI mice, in comparison with the control group (Fig. 1B-E). A gene chip assay was used to analyze the alterations in miRNAs, which revealed that miR-223 expression was decreased in ALI mice compared with the control group (Fig. 1F). The RT-qPcR assay demonstrated that miR-223 expression was decreased in ALI mice compared with the control group (Fig. 1G). Therefore, miR-223 expression may be associated with inflammation in ALI.

miR-223 expression in ALI in vitro.
Moreover, the levels of IL-1β and IL-18 were increased in A549 cells following treatment with LPS, compared with the control group ( Fig. 2A  and B). miR-223 expression was reduced in A549 cells following treatment with LPS, compared with the control group (Fig. 2c). To analyze the function of miR-223 in ALI, miR-223 and anti-miR-223 mimics were used to increase or inhibit the expression of miR-223 in A549 cells treated with LPS, compared with a negative control group (Fig. 2d and E).
Overexpression of miR-223 reduced the levels of IL-1β and IL-18 in A549 cells following treatment with LPS, compared with the negative control group (Fig. 2F and G). downregulation of miR-223 promoted the levels of IL-1β and IL-18 in A549 cells treated with LPS, compared with the negative control group (Fig. 2H and I). Together, these results demonstrated that miR-223 regulated inflammation in ALI, while its mechanism required investigation.

miRNA-223 regulates NLRP3 protein expression in ALI in vitro.
In order to investigate the mechanism of action of miR-223 in ALI, a gene chip assay was used to measure the alterations in miR-223 in ALI in vitro following inhibition of miR-223. consequently, inhibition of miR-223 induced the gene expression of TLR4, NF-κB and NLRP3 in ALI in vitro compared with the negative control (Fig. 3A). downregulation of miR-223 also increased the mRNA expression of TLR4, NF-κB and NLRP3 in ALI in vitro, compared with the negative control group (Fig. 3B-d). The 3'-UTR of NLRP3 mRNA is a target site of miR-223 in various species (Fig. 3E). The relative luciferase activity of the wild-type 3'-UTR following downregulation of miR-223 was increased, in comparison with the control group (Fig. 3F). As presented in Fig. 3G, immunofluorescence demonstrated that the downregulation of miR-223 induced NLRP3 protein expression compared with the control group. These results indicated that miR-223 may regulate pro-inflammatory cytokine expression by targeting NLRP3.
miRNA-223 regulates the TLR4 and NLRP3 signaling pathway in ALI in vitro. downregulation of miR-223 induced the protein expression of TLR4, NF-κB, NLRP3 and caspase-1 in ALI in vitro, compared with the negative control group (Fig. 4A-E). Overexpression of miR-223 suppressed the protein expression of TLR4, NF-κB, NLRP3 and caspase-1 in ALI in vitro, compared with the negative control group (Fig. 4F-J).
Taken together, these results demonstrated that miR-223 may regulate the TLR4 and NLRP3 signaling pathway in ALI in vitro.

Inhibition of TLR4 reduces the pro-inflammatory effect of anti-miR-223 in ALI in vitro.
To elucidate the mechanism of action of miR-223 in ALI in vitro, a TLR4 inhibitor was used to decrease the protein expression of TLR4 in ALI in vitro. The inhibition of TLR4 did not affect the protein expression of NLRP3 and caspase-1, but suppressed that of TLR4 and NF-κB in ALI in vitro following miR-223 downregulation, compared with miR-223 downregulation alone (Fig. 5A-E). The inhibition of TLR4 reduced the levels of IL-1β and IL-18 in ALI in vitro following miR-223 downregulation, in comparison with miR-223 downregulation alone ( Fig. 5F and G). Taken together, these results demonstrated that TLR4 is required to maintain the proinflammatory effect of anti-miR-223 in ALI.
Inhibition of NLRP3 reduces the pro-inflammatory effect of anti-miR-223 in ALI in vitro. To further elucidate the role   of NLRP3 in the pro-inflammatory effect of anti-miR-223 in ALI in vitro, an NLRP3 inhibitor was used to decrease the protein expression of NLRP3. As a result, the inhibition of NLRP3 suppressed the protein expression of NLRP3 and caspase-1 in ALI in vitro following treatment with anti-miR-223, compared with treatment with anti-miR-223 alone (Fig. 6A-c). The inhibition of NLRP3 reduced the levels of IL-1β and IL-18 in ALI in vitro following treatment with anti-miR-223, compared with treatment with anti-miR-223 alone (Fig. 6d and E). Taken together, these results demonstrated that NLRP3 is required to maintain the proinflammatory effect of anti-miR-223 in ALI, mediated by IL-1β and IL-18.
miRNA-223 regulates the TLR4 signaling pathway in ALI in vitro via RHOB. Prior to determining the role of miR-223 in RHOB-activated inflammation, the 3'-UTR of RHOB mRNA was revealed to be a target site of miR-223 in various species (Fig. 7A). The relative luciferase activity of the wild-type 3'UTR in miR-223 was decreased compared with the control group (Fig. 7B). Overexpression of miR-223 suppressed the protein expression of RHOB, while downregulation of miR-223 induced RHOB protein expression in ALI in vitro, compared with the negative control group (Fig. 7c-F). A RHOB plasmid was utilized to induce the protein expression of RHOB, which did not affect TLR4 protein expression in ALI in vitro following treatment with anti-miR-223, compared with treatment with anti-miR-223 alone (Fig. 7G-I). The activation of RHOB reduced the pro-inflammatory effect of anti-miR-223 on the levels of IL-1β and IL-18 in ALI in vitro following treatment with anti-miR-223, compared with treatment with anti-miR-223 alone ( Fig. 7J and K).

Activation of RHOB reduces the effects of miR-223 on inflammation in ALI in vitro.
To further study the role of RHOB in the effects of miR-223 on inflammation in ALI in vitro, RHOB plasmid was used to induce RHOB protein expression in ALI following overexpression of miR-223. RHOB plasmid did not affect miR-223 expression in the ALI model, compared with the negative group (Fig. 8A). As presented in Fig. 8B-G, RHOB plasmid induced RHOB, TLR4 and NF-κB protein expression, and did not affect NLRP3 and caspase-1 protein expression in ALI following overexpression of miR-223, compared with overexpression of miR-223 alone.

Discussion
ALI/ARdS is a common and important rapidly progressive inflammatory lung disease. It is estimated that there are 190,000 new ALI cases in the USA annually, with approxi-  mately 75,000 mortalities (2). In china, the incidence of ALI/ARdS is even higher, with a mortality rate of 52% (14). In recent years, scientists have noticed that miRNAs serve vital roles in multiple biological processes and signal transduction pathways of ALI/ARdS (14). In the present study, it was identified that miRNA-223 expression was decreased in ALI mice. Neudecker et al (12) suggested that miRNA-223 deficiency was associated with severe lung inflammation.
ALI/ARdS is damage to alveolar epithelial cells and capillary endothelial cells due to a variety of direct causes (including pneumonia and pulmonary contusion) and indirect causes (including adenosis and trauma) (15). This leads to diffuse edema of the pulmonary interstitium and alveoli, subsequently triggering acute hypoxemia. ALI/ARdS is not considered a simple lung disease or inflammatory disease (15,16). Instead, it is a systemic inflammatory response in the lung tissue, according to current opinion (17). In cases of ALI/ARdS, neutrophils, macrophages and endothelial cells release a large quantity of proinflammatory cytokines, chemokines and inflammatory mediators. This leads to damage to the lung epithelial cells and pulmonary vascular endothelial cells, increased alveolar permeability, and exudation of inflammatory cells, proteins and water. Additionally, this results in reduced lung tissue volume, decreased pulmonary compliance, disorders of gas diffusion, exchange and metabolism, and eventual mortality attributed to respiratory failure. The present study demonstrated that the downregulation of miRNA-223 promoted IL-1β and IL-18 levels in A549 cells treated with LPS, and the downregulation of miRNA-223 promoted TLR4 and NF-κB protein expression in A549 cells treated with LPS. Wang et al (18) indicated that miRNA-223 negatively regulated the activation of the TLR4/NF-κB pathway in macrophages.
In the occurrence and development of ALI, the release of proinflammatory mediators and apoptosis serve an important role (19). Disorders of the secretion of cytokines, inflammatory chemokines and other associated proteins promote the inflammatory response and accelerate the development of lung injury (20). Previous studies have confirmed that the NLRP3 inflammasome serves an important role in this process (21,22). It not only activates caspase-1 and inflammatory factors, but also promotes the maturation and secretion of inflammasomes (21,22). Therefore, the regulatory approach targeting NLRP3 inflammasomes may be of great importance for the treatment of ALI. The NLRP3 inflammasome is a multiprotein large-cytoplasmic complex that is composed of NLRP3, apoptosis-related spot-like proteins and pro-caspase-1 (23). NLRP3 serves a critical role in regulating the relevant components of the inflammation complex, including apoptosis-related point-like proteins and pro-caspase-1 (23). Recent studies have confirmed that activation of the inflammasome induces the division of pro-IL-1B and pro-IL-18 (21,22). Furthermore, it results in increased alveolar permeability, subsequently leading to pulmonary edema and lung injury (21,22). In the present study, miRNA-223 negatively regulated the TLR4/ NF-κB signaling pathway via RHOB in ALI. downregulation of miRNA-223 induced TLR4, NF-κB, NLRP3 and caspase-1 protein expression in ALI in vitro. Yang et al (24) reported that miRNA-223 regulates inflammation and brain injury via feedback to NLRP3 inflammasome after intracerebral hemor-rhage. Zeng et al (25) demonstrated that miRNA-223 attenuates hypoxia-induced vascular remodeling through RHOB/myosin light chain 9 in pulmonary arterial smooth muscle.
In conclusion, the present study demonstrated that miRNA-223 reduced inflammation, suppressed the NLRP3 inflammasome, and negatively regulated the TLR4/NF-κB signaling pathway via RHOB in ALI (Fig. 9). miRNA-223 is involved in the activation of the TLR4/NF-κB signaling pathway via RHOB, and the NLRP3 inflammasome may be a therapeutic target for the treatment of inflammation in ALI. Therefore, miRNA-223 serves as a potent positive regulator of inflammation in ALI.


extfieldsofstudy: Medicine Chemistry

text:
Arabidopsis JANUS Regulates Embryonic Pattern Formation through Pol II-Mediated Transcription of WOX2 and PIN7

Summary Embryonic pattern formation relies on positional coordination of cell division and specification. Early axis formation during Arabidopsis embryogenesis requires WUSCHEL RELATED HOMEOBOX (WOX)-mediated transcription activation and PIN-FORMED7 (PIN7)-mediated auxin asymmetry. How these events are regulated is obscure. We report that Arabidopsis JANUS, a putative subunit of spliceosome, is essential for embryonic pattern formation. Significantly reduced transcription but not mRNA processing of WOX2 and PIN7 in janus suggested its role in transcriptional regulation. JANUS interacts with RNA polymerase II (Pol II) through a region outside of its spliceosome-association domain. We further show that Pol II mediates the transcription of WOX2 and PIN7 in a JANUS-dependent way and is essential for embryonic pattern formation. These findings reveal that JANUS recruits Pol II for the activation of two parallel pathways to ensure proper pattern formation during embryogenesis.

INTRODUCTION
Development of a specific body plan during embryogenesis requires precise cell fate determination based on the position of cells along the embryo axes. In Arabidopsis, the crucial cell types are established extremely early during embryogenesis as reflected by the stereotypic sequence of oriented cell divisions (Lau et al., 2012;ten Hove et al., 2015). Two pathways have been linked to the establishment of apical-basal axis and cell specification after zygotic division. One involves the transcription factors WUSCHEL RELATED HOMEOBOX2 (WOX2), WOX8, and WOX9, whereas the other depends on auxin, whose asymmetry is maintained by auxin efflux carrier PIN-FORMED7 (PIN7) (Lau et al., 2012). WOX2 and WOX8 are initially co-expressed in the zygote (Haecker et al., 2004). After zygotic division, WOX2 and WOX8 are restricted to the apical and basal cell lineage to control the following cell specification, respectively (Breuninger et al., 2008;Haecker et al., 2004). PIN7 is polarly localized to the apical plasma membrane (PM) of the basal cell, where it provides maternal auxin to the apical cell (Friml et al., 2003;Robert et al., 2018). The polar distribution of PIN7 ensures auxin maximum in the apical cell, which generates the proembryo and all apical structures of the plant. Functional loss of WOX2 or PIN7 compromised the formation of apical-basal axis during early embryogenesis. However, their defects at early embryonic pattern formation are later recovered (Friml et al., 2003;Robert et al., 2018). Whether these two pathways play redundant roles in embryogenesis and how their specific expression is controlled are unclear.
RNA polymerase II (Pol II) plays a pivotal role in regulating gene expression (Thomas and Chiang, 2006). Pol II in Arabidopsis consists of 12 core subunits (Ream et al., 2009), in which Nuclear RNA Polymerase B1 (NRPB1) and NRPB2 interact to form the catalytic center for RNA synthesis, whereas other subunits play structural and regulatory roles in transcription initiation, elongation, termination, or RNA processing (Cramer et al., 2008;Werner and Grohmann, 2011). Functional studies of genes encoding for Pol II subunits suggested its role in embryogenesis such that no homozygous mutants could be obtained for functional loss of NRPB2, as well as that of NRPB9 and NRPB11, genes encoding for the noncatalytic subunits of Pol II (Onodera et al., 2008;Ream et al., 2009;Tan et al., 2012). However, whether and how functional loss of Pol II affects embryogenesis is unclear.
Spliceosomes are large RNA-protein complexes mainly involved in pre-mRNA splicing. Nevertheless, subunits of spliceosomes also participate in many other processes, including mRNA export (Howard and Sanford, 2015;Muller-McNicoll et al., 2016), the maintenance of genome stability (Li and Manley, 2005;Xiao et al., 2007), and microRNA processing (Ben Chaabane et al., 2013;Wu et al., 2010). Interestingly, reports both in metazoans and in plants showed that a subunit of spliceosome, Serine/arginine-rich splicing factor 35 (SC35), interacts with a Pol II subunit and is required for the transcription of genes, in addition to its role in pre-mRNA splicing (Lin et al., 2008;Yan et al., 2017). A role of the spliceosome subunit in regulating Pol II-mediated transcription was proposed (Yan et al., 2017).
In this study, we report that the Arabidopsis homolog of human SPLICEOSOME-ASSOCIATED PROTEIN 49 (SAP49) and yeast Hsh49p, a subunit of spliceosome, is essential for pattern formation during early embryogenesis. We named it JANUS as it represents the god of new beginnings in ancient Rome and associates with the first steps of a journey. Functional loss of JANUS resulted in complete embryo lethality due to abnormal cell division immediately after the first zygotic division. The specific expression of PIN7 was disrupted in janus, resulting in defective auxin signaling. On the other hand, WOX2 was also transcriptionally downregulated in janus. Consistently, the disruption of both WOX2and PIN7-dependent pathways resembled pattern formation defects of janus during early embryogenesis. We further showed that JANUS interacts with Pol II subunits independent of its role as a splicing factor and is required for Pol II-dependent transcription of WOX2 and PIN7. Indeed, functional loss of Pol II subunits mimicked embryonic defects of janus. Taken together, our findings demonstrate that JANUS recruits Pol II to transcriptionally activate WOX2-and PIN7-mediated pathways for pattern formation during early embryogenesis.

JANUS Is Essential for Pattern Formation during Embryogenesis
JANUS was isolated for characterization because of the complete embryo lethality of its mutant emb2444 (Meinke et al., 2008). JANUS contains two RNA recognition motifs (RRMs) and is homologous to a subunit of the splicesome (Figures S1A, S1E, and S1F). Segregation ratio from reciprocal crosses between wild-type and janus/+ indicated that gametophytic transmission of the mutant was not affected (Table S1). The heterozygous mutant showed one-fourth of seed abortion ( Figure 1A). Transcript abundance of JANUS was significantly reduced in janus/+ compared with that in the wild-type ( Figure S1). In addition, the genomic fragment of JANUS with a GFP reporter gene in the control of its native promoter was introduced into janus/+. Transgenic lines of JANUSg-GFP;janus were obtained, and all showed no seed abortion ( Figures  1A and S1), indicating that JANUS is the causal gene for seed abortion of janus. Results are means G standard deviation (SD, n = 8). Seed set of janus/+ is significantly different from others (Tukey's multiple comparison test, p < 0.05). Scale bars, 1 mm. (B and C) Wild-type (WT) and janus embryo development by ovule clearing. DAP indicates days after pollination. Because janus embryos are much delayed in development, wild-type embryos and janus embryos are shown in pairs according to their developmental stages but not to the same DAP. Dotted lines in (C) indicate division planes. Scale bars, 20 mM. (D and E) Confocal laser scanning microscopy (CLSM) of a Pro WOX5 :GFP embryo (D) or a Pro WOX5 :GFP;janus embryo (E). Images shown are merges of the GFP channel and RFP channel (propidium iodide [PI] staining in magenta). (F) Schematic illustration of wild-type or janus embryogenesis. Arrowheads point at aborted seeds in (A). The arrowhead points at the Quiescent Center labeled by GFP in (D) but its absence in (E).
To determine at which stage developing seeds started to show defects in janus, we examined self-fertilized janus/+ by whole-mount clearing. Embryos developing within a single silique are approximately at the same developmental stage (Breuninger et al., 2008), which enabled an estimate of janus embryos, which are much delayed compared with their wild-type siblings. After the first zygotic division, one-fourth of embryos from janus/+ plants displayed an asymmetric oblique division ( Figures 1C and 1F) rather than symmetric vertical division as observed in three-fourth of the other embryos as well as in embryos of the wild-type ( Figures 1B  and 1F). The division pattern followed was impaired in these presumably janus embryos ( Figures 1C and 1F), which showed severe morphological defects at the early globular stage and were eventually arrested at the late globular stage ( Figures 1C and 1F). In the arrested embryos, the outer walls of protoderm cells were distended, producing an uneven surface on the embryo proper ( Figures 1C and 1F). Abnormal divisions occurred both in the apical and the basal lineages ( Figures 1C and 1F). Furthermore, the formation and specification of quiescent center (QC) was also compromised judged by the irregularly oblique divisions in hypophysis and by the absence of GFP signals in Pro WOX5 :GFP;janus ( Figures 1D and 1E), which specifies the QC (Blilou et al., 2005). These results demonstrated that JANUS is an essential gene for early embryonic pattern formation and cell fate specification. Consistent with its role in embryogenesis, JANUS is highly expressed in developing embryos from the zygotic stage to the cotyledon stage ( Figure S1).

JANUS Mediates the Expression of WOX2 and PIN7
JANUS is nuclear localized ( Figure S1), suggesting a potential role in gene expression. We thus tested whether the two major pathways in controlling early embryogenesis, i.e., WOX-mediated transcriptional pathway and PIN7-mediated auxin signaling pathway, were affected in janus. First, we analyzed the transcription activity of WOX2 and WOX8 by introducing Pro WOX2 :DsRed2/Pro WOX8gD :NLS-vYFP 3 (Yu et al., 2016) in janus/+. WOX2 and WOX8 were transcriptionally activated in the apical and basal cells after the zygotic division in wild-type, respectively (Figure 2A), as reported (Breuninger et al., 2008;Haecker et al., 2004). By contrast, WOX2 showed a substantially reduced transcriptional activity in the apical cell lineage of one-fourth embryos in janus/+, whereas WOX8 was not affected (Figures 2A and 2B). The reduced transcription of WOX2 but not WOX8 was confirmed by additional reporter line ( Figure S2) or native transcript levels ( Figure S3). These results suggested that the transcription activity of WOX2 but not WOX8 in early embryogenesis depended on JANUS.
Second, we examined whether PIN7 was transcriptionally affected by functional loss of JANUS. PIN7 (Blilou et al., 2005) was polarly localized to the apical PM of the basal cells in wild-type globular embryos (Figure 2C), as reported (Friml et al., 2003). In one-fourth embryos from PIN7:GFP;janus/+, presumably of the janus genotype, GFP signals were significantly reduced or even undetectable ( Figure 2C). Because JANUS is homologous to spliceosome subunits ( Figure S1), we examined the transcription activity of PIN7 in Pro PIN7 :NLS-YFP;janus/+, which was reflected by fluorescence intensity, to exclude the possibility that mRNA splicing of PIN7 contributed to the expression difference. Consistent with the results obtained from PIN7:GFP, YFP signals were dramatically reduced or undetectable in the basal cells of one-fourth embryos in Pro PIN7 :NLS-YFP;janus/+ ( Figure 2D). These results implied that JANUS is crucial for the transcription of PIN7 during early embryogenesis. Indeed, no splicing defects of PIN7 as well as of WOX2 were detected in the siliques of janus/+ ( Figure S2), confirming a role of JANUS in the transcription rather than RNA processing of the two genes. Because PIN7 is critical for auxin polar transport during early embryogenesis (Friml et al., 2003), the reduced PIN7 in janus would have severely compromised auxin signaling. It was indeed the case. By examining DR5:GFP, a synthetic auxin maximum reporter (Friml et al., 2003), we confirmed that GFP was accumulated in the apical cell lineage immediately after zygotic division and then restricted to the hypophysis of the globular embryos in wild-type plants ( Figure 2E), as reported (Friml et al., 2003). By contrast, GFP signals were restricted to the basal cells and failed to establish an apical-basal gradient in one-fourth of embryos in DR5:GFP;janus/+ ( Figure 2E). Similar defects were reported in embryos defective in PIN7 or treated with auxin efflux inhibitors (Friml et al., 2003).
Finally, we generated a double mutant of WOX2 and PIN7 under the rationale that if JANUS mediates the transcriptional activity of WOX2 and PIN7 during early embryogenesis, functional loss of both pathways would at least partially mimic the defects of janus. Compared with wild-type, the single mutant of PIN7 or WOX2, i.e., pin7-1 or wox2-4 (Friml et al., 2003;Zhang et al., 2017), showed a slight defect of apicalaxis patterning in early stages during embryogenesis but finally recovered at the globular stage (Figures 2G and 2H), consistent with previous reports (Breuninger et al., 2008;Friml et al., 2003;Haecker et al., 2004). In comparison, the proportion of wox2-4;pin7-1 embryos with abnormal asymmetric division in early stages substantially increased ( Figure 2I). In the wild-type, the first division of the apical cell was vertical and symmetric ( Figure 2F), as reported (Breuninger et al., 2008). By contrast, in wox2-4;pin7-1, the divisions were asymmetric, either oblique or horizontal in both embryo proper and hypophysis ( Figure 2I), which largely resembled those of janus during early embryogenesis ( Figure 2J). Instead of a full recovery at late stages as seen in each single mutant (Breuninger et al., 2008;Friml et al., 2003;Haecker et al., 2004), over 30% embryos of wox2-4;pin7-1 arrested immediately after the first zygotic division ( Figure 2I). By contrast, enhanced expression of PIN7 partially complemented the defects of janus such that early embryonic pattern formation was largely normal in Pro UBQ10 :PIN7;janus/+ ( Figure 2K). These results suggested that both WOX2-and PIN7-dependent pathways may be transcriptionally regulated by JANUS and play redundant roles in apical-basal patterning during early embryogenesis.

JANUS Interacts with Pol II Whose Functional Loss Resulted in Embryo Lethality
To determine how JANUS affected the transcription activity of WOX2 and PIN7 during embryonic pattern formation, we tested whether JANUS interacted with components of Pol II since Pol II is responsible for the transcription of most mRNAs in eukaryotes (Thomas and Chiang, 2006) and was shown to interact with other components of the spliceosome (Yan et al., 2017). Of 11 Pol II subunits tested, NRPB7, NRPB10, and NRPB11 showed interaction with JANUS by yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays ( Figures 3A-3C and S4). Interestingly, the interaction is evolutionarily conserved: JANUS homolog also interacts with Pol II components in mice ( Figure S4).
The physical interaction between JANUS and Pol II suggested that they might function in the same pathway. Indeed, all three genes are highly expressed during embryogenesis based on reporter analysis ( Figure S4). We thus hypothesized that functional loss of Pol II would also result in defective embryonic pattern formation. To test this hypothesis, we characterized mutants of NRPB10 and NRPB11, both of which interact with JANUS ( Figure S4). Although NRPB7 also interacts with JANUS, the lack of its mutants in all stock centers prevented further analysis. Likely due to the redundancy with NRPB10-like genes , functional loss of NRPB10 did not show seed set reduction and embryo lethality ( Figure S4). In comparison, functional loss of NRPB11, a single gene in Arabidopsis , resulted in embryo lethality ( Figure S4), similar to nrpb2 (Onodera et al., 2008). Because both genes are required for Pol II activity (Kershnar et al., 1998;  Ream et al., 2009), these results hinted at an essential role of Pol II in embryogenesis. To identify the defects of embryogenesis due to Pol II loss of function, we examined developing embryos in nrpb2-1/+, nrpb2-2/+, and nrpb11-1/+. The apical-basal patterning during early embryogenesis was significantly affected in one-fourth of nrpb11-1/+ as well as in 29% of the nrpb2/+ mutants (Figures 3D and 3E). Similar to that in janus, the first division of the apical cell, i.e., symmetric and horizontal in wild-type, was asymmetric and oblique in nrpb11 and nrpb2 (Figures 3D and 3E). Irregular division patterns resulted in abnormal formation of embryo proper and hypophysis ( Figures 3D and 3E). In most severe cases, embryos were arrested at as early as the two-cell stage ( Figures 3D and 3E). Thus, these results demonstrated that Pol II is essential for early embryonic pattern formation and likely functions in the same genetic pathway as JANUS.
Pol II-Mediated WOX2 and PIN7 Transcription Depends on JANUS That JANUS interacts with Pol II and affects the transcription activity of WOX2 and PIN7 suggested that Pol II might mediate WOX2 and PIN7 transcription during embryonic pattern formation in a JANUS-dependent way. We thus hypothesized that functional loss of Pol II would compromise WOX2 and PIN7 expression during early embryonic pattern formation. To test this hypothesis, we introduced Pro WOX2 :GFP and PIN7:GFP into nrpb2-1/+ and examined the fluorescence distribution during embryogenesis. Indeed, the expression of WOX2 and PIN7 was severely reduced or even undetectable in one-fourth embryos of the nrpb2-1/+ plants ( Figures 4A and 4B), reminiscent of that in janus/+. Auxin signaling was also compromised during early embryogenesis by functional loss of NRPB2 such that ectopic GFP signals were detected in the basal cells of one-fourth DR5:GFP;nrpb2-1/+ embryos ( Figure 4C), similar to that by functional loss of JANUS. These results suggested that Pol II mediates the transcription of WOX2 and PIN7 during early embryonic pattern formation.
To test whether Pol II-mediated transcription of WOX2 and PIN7 depended on JANUS, we examined Pol II occupancy at the promoter regions of WOX2 and PIN7 by chromatin immunoprecipitation (ChIP) using an antibody against NRPB2 (Yan et al., 2017). Because no homozygous janus plants could be obtained, we generated Pro 35S :JANUS-RNAi transgenic plants. Transcript analysis as well as fluorescence quantification supported a significant reduction of JANUS by RNAi in Pro 35S :JANUS-RNAi transgenic plants ( Figure S5). ChIP assays indicated that Pol II occupancy was significantly reduced at the promoter regions of PIN7 in Pro 35S :JANUS-RNAi lines ( Figures 4F and 4G) as compared with that in the wild-type ( Figures 4F and 4G). Consistent with the reduced Pol II occupancy, PIN7 was transcriptionally downregulated in Pro 35S :JANUS-RNAi lines, either by quantitative PCRs for the endogenous genes ( Figures 4J and 4K) or by fluorescence quantification of the PIN7:GFP transgenic plants ( Figures 4D, 4E, 4H, and 4I). Except for embryos, WOX2 was weakly expressed in a few cells at root maturation zone ( Figure 4D), which hindered the application of ChIP assays on its promoter regions. However, by analyzing the fluorescence in the Pro 35S :JANUS-RNAi;Pro WOX2 :H2B-GFP or Pro WOX2 :DsRed2;janus/+ plants, we confirmed that JANUS-RNAi or janus significantly reduced the transcription activity of Pro WOX2 (Figures 2A and 4D). These results suggested that Pol II-mediated transcription of WOX2 and PIN7 depends on JANUS. By contrast, the transcription of WOX8 and SCARECROW (SCR), a critical embryonic gene (Wysocka-Diller et al., 2000), depends on Pol II but not JANUS ( Figure S3), suggesting that JANUS did not affect the transcriptional activity of Pol II in general.

DISCUSSION
Early embryogenesis is the critical developmental phase during which the basic features of the plant body are established. Although distinct expression domains of WOX family transcription factors as well as directional auxin transport are known to be involved in early apical-basal patterning, their upstream regulators and potential interactions were obscure. Our results demonstrated that the two pathways control early embryonic pattern formation in a parallel way and both mediated by JANUS. First, functional loss of WOX2 and PIN7 caused a severe defect immediately after the zygotic division, which cannot be restored at late stages, unlike the disruption of each pathway. Second, both WOX2 and PIN7 were transcriptionally downregulated by JANUS loss of function. The effect of JANUS on PIN7 and WOX2 is specific because WOX8, another embryonic gene, requires RNA Pol II but not JANUS for its expression during early embryogenesis. Third, wox2-4;pin7-1 resembled janus in early embryogenesis. Finally, the accumulation of Pol II at PIN7 was significantly reduced by JANUS-RNAi, suggesting JANUS-dependent recruitment of the transcriptional machinery at PIN7. with transcription machinery to directly influence gene expression in metazoans (Braunschweig et al., 2013;Das et al., 2007). Although we cannot exclude a role of JANUS in RNA splicing owing to its homology to the component of the spliceosome during embryogenesis, results presented here strongly suggested its role through transcriptional regulation. First, JANUS is critical for the transcription but not RNA splicing of (G) Quantification data of the ChIP results. ChIP-PCRs were used to analyze the Pol II enrichment at PIN7, which is presented as ratio of (Pol II PIN7/input PIN7) to (Pol II Actin/input Actin). Results are means G standard error (SEM) from three technical repeats. ChIP assays were repeated three times with similar results. Asterisks indicate significant difference (t test, p < 0.05). WOX2 and PIN7, two genes critical for early embryonic pattern formation. Second, the RRM1 domain of yeast SAP49 or metazoan Hsh49p, homolog of Arabidopsis JANUS, is responsible for RNA binding and spliceosome association and is essential for spliceosome-mediated RNA splicing (Igel et al., 1998;Kuwasako et al., 2017;Pauling et al., 2000;van Roon et al., 2017). However, we found that JANUS directly interacts with Pol II through its RRM2 but not RRM1 domain, suggesting that its interaction with Pol II does not require its association with the spliceosome. Third, yeast and metazoan homologs of JANUS are part of an SF3b complex within the spliceosome (Kuwasako et al., 2017;van Roon et al., 2017). However, although other components of the SF3b complex in Arabidopsis have been functionally characterized (Aki et al., 2011;Wang and Brendel, 2006), none of the related mutants showed embryo lethality.
Eukaryotes decode their genomes using three essential nuclear DNA-dependent RNA polymerases (Cramer et al., 2008;Werner and Grohmann, 2011). In Arabidopsis, several subunits of Pol II, including NRPB2, NRPB5, NRPB9, and NRPB11, have been reported to affect plant viability (Onodera et al., 2008;Ream et al., 2009;Tan et al., 2012). However, whether and how Pol II regulates embryogenesis was unclear. We showed here that mutations at a few Pol II components resulted in complete embryo lethality ( Figures  3D and S3I). A few components of Pol II are shared by two plant-specific RNA polymerase complexes, Pol IV and Pol V (Ream et al., 2009). However, we believe that Pol II is key for JANUS-mediated transcription of WOX2 and PIN7 because the JANUS-interacting component NRPB7 is specific for Pol II (Figures 3A and  S3B) and only Pol II was reported to be essential for viability (Herr et al., 2005;Kanno et al., 2005;Onodera et al., 2005;Pontier et al., 2005;Ream et al., 2009).
Data presented here suggested that JANUS is important for the Pol II-mediated transcription of WOX2 and PIN7 during early embryogenesis. However, JANUS has no recognizable DNA-binding domains. It is unclear how JANUS determines the selectivity of Pol II on target genes such as WOX2 and PIN7. In eukaryotes, mediators, multi-subunit complexes, bridge transcription activators with Pol II at specific cis-elements for transcription initiation (Dolan and Chapple, 2017). Whether JANUS fulfills a role of mediator, recruiting DNA-binding transcription factors together with Pol II, to the promoter regions of WOX2 and PIN7, is an interesting scenario worthy of further investigation.

Limitations of the Study
We would like to note that, because both janus and Pol II mutants are embryo lethal, some molecular and biochemical experiments can only be carried out in Pro 35S :JANUS-RNAi plants. Future efforts will be dedicated to generate weak mutant alleles of JANUS, for which homozygous plants can be used for further mechanistic analysis. We also would like to point out that, although JANUS is important for Pol II-mediated transcription of WOX2 and PIN7 during early embryogenesis, it is unclear how JANUS, a protein containing no DNA-binding domains, determines target selectivity for Pol II. Whether JANUS contains untraditional DNA-binding domains or interacts with transcription factors for its function will be interesting scenario for future studies.

METHODS
All methods can be found in the accompanying Transparent Methods supplemental file.  (G) Intron retention that was detected via RT-PCRs shows no significant difference between WT, janus/+ and RNAi-1 siliques. (H) Splicing efficiency of PIN7 and WOX2 was unaffected in janus/+ and RNAi-1 siliques. Splicing efficiency was calculated as the ratio of spliced to total (spliced + unspliced) transcripts for two representative PIN7 introns and WOX2 intron. Results are means ± SE from three experiments. ns indicates no significant difference (t-test, P>0.05). Related to Figure 2.    WT : janus/+: janus 1 : 2 : 1 53 : 113 b a Not significantly different from the segregation ratio 1:1 ( 2 , P>0.05). b Significantly different from the segregation ratio 1:2:1 ( 2 , P>0.05). Related to Figure 1.  Figure 3.