Planar cell polarity: intracellular asymmetry and supracellular gradients of Frizzled.

Planar cell polarity (PCP), the coordinated orientation of structures such as cilia, mammalian hairs or insect bristles, depends on at least two molecular systems. We have argued that these two systems use similar mechanisms; each depending on a supracellular gradient of concentration that spans a field of cells. In a linked paper, we studied the Dachsous/Fat system. We found a graded distribution of Dachsous in vivo in a segment of the pupal epidermis in the abdomen of Drosophila. Here we report a similar study of the key molecule for the Starry Night/Frizzled or 'core' system. We measure the distribution of the receptor Frizzled on the cell membranes of all cells of one segment in the living pupal abdomen of Drosophila. We find a supracellular gradient that falls about 17% in concentration from the front to the rear of the segment. We present some evidence that the gradient then resets in the most anterior cells of the next segment back. We find an intracellular asymmetry in all the cells, the posterior membrane of each cell carrying about 22% more Frizzled than the anterior membrane. These direct molecular measurements add to earlier evidence that the two systems of PCP act independently.

Planar cell polarity: intracellular asymmetry and supracellular gradients of Dachsous.

The slope of a supracellular molecular gradient has long been thought to orient and coordinate planar cell polarity (PCP). Here we demonstrate and measure that gradient. Dachsous (Ds) is a conserved and elemental molecule of PCP; Ds forms intercellular bridges with another cadherin molecule, Fat (Ft), an interaction modulated by the Golgi protein Four-jointed (Fj). Using genetic mosaics and tagged Ds, we measure Ds in vivo in membranes of individual cells over a whole metamere of the Drosophila abdomen. We find as follows. (i) A supracellular gradient rises from head to tail in the anterior compartment (A) and then falls in the posterior compartment (P). (ii) There is more Ds in the front than the rear membranes of all cells in the A compartment, except that compartment's most anterior and most posterior cells. There is more Ds in the rear than in the front membranes of all cells of the P compartment. (iii) The loss of Fj removes intracellular asymmetry anteriorly in the segment and reduces it elsewhere. Additional experiments show that Fj makes PCP more robust. Using Dachs (D) as a molecular indicator of polarity, we confirm that opposing gradients of PCP meet slightly out of register with compartment boundaries.

Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules.

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous-Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.

A refutation to 'A new A-P compartment boundary and organizer in holometabolous insect wings'.

We respond to a recent report by Abbasi and Marcus who present two main findings: first they argue that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, they present evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a "far posterior" compartment. Clones of cells were marked with the yellow mutation and they reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In our hands yellow proved an unusable marker for clones in the wing blade and therefore we reexamined the matter. We marked clones of cells with multiple wing hairs or forked and found a substantial proportion of these clones cross the proposed lineage boundary near vein 5, in conflict with their findings and conclusion. As internal controls we showed that these same clones respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports. We question the validity of Abbasi and Marcus' conclusions regarding the butterfly wing but present no new data.Arising from: R. Abbasi and J. M. Marcus Sci. Rep. 7, 16337 (2017); https://doi.org/10.1038/s41598-017-16553-5 .

Planar cell polarity: two genetic systems use one mechanism to read gradients.

Our aim in this short Primer is to explain the principles of planar cell polarity (PCP) in animal development. The literature in this small field is complex and specialized, but we have extracted a simple and central story from it. We explain our hypothesis that polarity, initially cued by the direction of slope of a multicellular gradient, is interpreted at the cellular level so that each cell becomes molecularly polarised. The mechanism involves a comparison between a cell and its neighbours. To achieve this comparison there are (at least) two disparate and independent molecular systems, each depending on molecular bridges that span between neighbouring cells. Even though the two systems are made up of different molecules, we argue that both systems function in a logically equivalent way.

Planar cell polarity: the prickle gene acts independently on both the Ds/Ft and the Stan/Fz systems.

Epithelial cells are polarised within the plane of the epithelium, forming oriented structures that have a coordinated and consistent polarity (planar cell polarity, PCP). In Drosophila, at least two separate molecular systems generate and interpret intercellular polarity signals: Dachsous/Fat, and the 'core' or Starry night/Frizzled system. Here, we study the prickle gene and its protein products Prickle and Spiny leg. Much research on PCP has focused on the asymmetric localisation of core proteins in the cell and as a result prickle was placed in the heart of the Starry night/Frizzled system. We investigate whether this view is correct and how the prickle gene relates to the two systems. We find that prickle can affect, separately, both systems; however, neither Prickle nor Spiny leg are essential components of the Dachsous/Fat or the Starry night/Frizzled system, nor do they act as a functional link between the two systems.

Francis Crick: A Singular Approach to Scientific Discovery.

Francis' office window (at the Salk) commanded a panorama of the Pacific. "This grand natural scene was a physical correlate of Francis's intellectual world: wide-ranging, brilliantly lit, a little overawing, but also immensely inviting and above all an exciting place to be." (Mitchison, 2004).

Planar cell polarity: the Dachsous/Fat system contributes differently to the embryonic and larval stages of Drosophila.

The epidermal patterns of all three larval instars (L1-L3) ofDrosophilaare made by one unchanging set of cells. The seven rows of cuticular denticles of all larval stages are consistently planar polarised, some pointing forwards, others backwards. In L1 all the predenticles originate at the back of the cells but, in L2 and L3, they form at the front or the back of the cell depending on the polarity of the forthcoming denticles. We find that, to polarise all rows, the Dachsous/Fat system is differentially utilised; in L1 it is active in the placement of the actin-based predenticles but is not crucial for the final orientation of the cuticular denticles, in L2 and L3 it is needed for placement and polarity. We find Four-jointed to be strongly expressed in the tendon cells and show how this might explain the orientation of all seven rows. Unexpectedly, we find that L3 that lack Dachsous differ from larvae lacking Fat and we present evidence that this is due to differently mislocalised Dachs. We make some progress in understanding how Dachs contributes to phenotypes of wildtype and mutant larvae and adults.

The Last 50 Years: Mismeasurement and Mismanagement Are Impeding Scientific Research.

In the last 50 years, there have been many changes to the substance, conduct, and style of research. Many of these changes have proved disastrous to the life of scientists and to science itself. As a consequence, the near-romantic spirit of adventure and exploration that inspired young scientists of my own and earlier generations has become tarnished. Now, many of us feel beleaguered by bureaucrats and by politicians: they affect our lives profoundly, apparently without an understanding of the way discoveries are made or of the nature of science itself. The core purposes of universities, teaching and research, are being eroded by excessive administration. The number and locations of our publications are counted up like beans and the outcomes are used to rank us, one against another; a process of evaluation that has recast the purposes of publication. Applying for grants takes far too much time from a young scientist's life.

Regions within a single epidermal cell of Drosophila can be planar polarised independently.

Planar cell polarity (PCP), the coordinated and consistent orientation of cells in the plane of epithelial sheets, is a fundamental and conserved property of animals and plants. Up to now, the smallest unit expressing PCP has been considered to be an entire single cell. We report that, in the larval epidermis of Drosophila, different subdomains of one cell can have opposite polarities. In larvae, PCP is driven by the Dachsous/Fat system; we show that the polarity of a subdomain within one cell is its response to levels of Dachsous/Fat in the membranes of contacting cells. During larval development, cells rearrange (Saavedra et al., 2014) and when two subdomains of a single cell have different types of neighbouring cells, then these subdomains can become polarised in opposite directions. We conclude that polarisation depends on a local comparison of the amounts of Dachsous and Fat within opposing regions of a cell's membrane.

Plasticity of both planar cell polarity and cell identity during the development of Drosophila.

Drosophila has helped us understand the genetic mechanisms of pattern formation. Particularly useful have been those organs in which different cell identities and polarities are displayed cell by cell in the cuticle and epidermis (Lawrence, 1992; Bejsovec and Wieschaus, 1993; Freeman, 1997). Here we use the pattern of larval denticles and muscle attachments and ask how this pattern is maintained and renewed over the larval moult cycles. During larval growth each epidermal cell increases manyfold in size but neither divides nor dies. We follow individuals from moult to moult, tracking marked cells and find that, as cells are repositioned and alter their neighbours, their identities change to compensate and the pattern is conserved. Single cells adopting a new fate may even acquire a new polarity: an identified cell that makes a forward-pointing denticle in the first larval stage may make a backward-pointing denticle in the second and third larval stages. DOI: http://dx.doi.org/10.7554/eLife.01569.001.

The mechanisms of planar cell polarity, growth and the Hippo pathway: some known unknowns.

Planar cell polarity (PCP) is a small but important area of research. In this review we discuss a limited number of topics within the PCP field, chosen because they are difficult, unsolved, controversial or just because we find them interesting. Because Drosophila is the best studied and technically most amenable system we have concentrated on it, but also consider some examples from work on vertebrates. Topics discussed include the number of genetic pathways involved in PCP, as well as the causal relationship between embryonic axes, gradients of morphogens and PCP itself. We consider the vexed question of the roles of the Wnt genes in PCP in both vertebrates and Drosophila. We discuss whether the proteins involved in PCP need to be localised asymmetrically in cells in order to function. We criticise the way the Hippo pathway is described in the literature and ask what its wildtype function is. We explore afresh how the Hippo pathway might be linked both to growth and to PCP through the gigantic cadherin molecule Fat. We offer some new ways of making sense of published results, particularly those relating to the Frizzled/Starry night and Dachsous/Fat systems of PCP.

Substrate-borne vibratory communication during courtship in Drosophila melanogaster.

Courtship in Drosophila melanogaster has become an iconic example of an innate and interactive series of behaviors. The female signals her acceptance of copulation by becoming immobile in response to a male's display of stereotyped actions. The male and female communicate via vision, air-borne sounds, and pheromones, but what triggers the female's immobility is undetermined. Here, we describe an overlooked and important component of Drosophila courtship. Video recordings and laser vibrometry show that the male abdomen shakes ("quivers"), generating substrate-borne vibrations at about six pulses per second. We present evidence that the female becomes receptive and stops walking because she senses these vibrations, rather than as a response to air-borne songs produced by the male fluttering the wings. We also present evidence that the neural circuits expressing the sex-determination genes fruitless and doublesex drive quivering behavior. These abdominal quivers and associated vibrations, as well as their effect on female receptivity, are conserved in other Drosophila species. Substrate-borne vibrations are an ancient form of communication that is widespread in animals. Our findings in Drosophila open a door to study the neuromuscular circuitry responsible for these signals and the sensory systems needed for their reception.

Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity.

Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). Here, using an in vivo assay for function, we show that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, we show that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. We deduce that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. We propose that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction.

The muscle pattern of the Drosophila abdomen depends on a subdivision of the anterior compartment of each segment.

In the past, segments were defined by landmarks such as muscle attachments, notably by Snodgrass, the king of insect anatomists. Here, we show how an objective definition of a segment, based on developmental compartments, can help explain the dorsal abdomen of adult Drosophila. The anterior (A) compartment of each segment is subdivided into two domains of cells, each responding differently to Hedgehog. The anterior of these domains is non-neurogenic and clones lacking Notch develop normally; this domain can express stripe and form muscle attachments. The posterior domain is neurogenic and clones lacking Notch do not form cuticle; this domain is unable to express stripe or form muscle attachments. The posterior (P) compartment does not form muscle attachments. Our in vivo films indicate that early in the pupa the anterior domain of the A compartment expresses stripe in a narrowing zone that attracts the extending myotubes and resolves into the attachment sites for the dorsal abdominal muscles. We map the tendon cells precisely and show that all are confined to the anterior domain of A. It follows that the dorsal abdominal muscles are intersegmental, spanning from one anterior domain to the next. This view is tested and supported by clones that change cell identity or express stripe ectopically. It seems that growing myotubes originate in posterior A and extend forwards and backwards until they encounter and attach to anterior A cells. The dorsal adult muscles are polarised in the anteroposterior axis: we disprove the hypothesis that muscle orientation depends on genes that define planar cell polarity in the epidermis.

Planar cell polarity: fashioning solutions.

Scientists like to consider themselves as especially objective, but, however hard we try we cannot be very different from everyone else. Like them we helplessly absorb our knowledge, our perspectives, our valuation of whether something is exciting or boring from those around us. In this "extra view" I reflect on fashion, illustrating by a small discovery of ours, and discussing why it was not made before.

Planar cell polarity: the orientation of larval denticles in Drosophila appears to depend on gradients of Dachsous and Fat.

The larval ventral belts of Drosophila consist of six to seven rows of denticles that are oriented, some pointing forwards, some backwards. We present evidence that denticle orientation is determined almost entirely by Dachsous and Fat, one of two planar cell polarity systems. If we change the distribution of Dachsous we can alter the polarity of denticles. We suggest that the orientation of the individual denticle rows, in both the anterior compartment (which mostly point backwards) and the posterior compartment (which point forwards), is determined by the opposing slopes of a Dachsous/Fat gradient. We show, by altering the concentration gradients of Dachsous during development, that we can change the polarity of the denticles made by larval cells as they progress between the first and third larval instars without mitosis.

Mechanosensilla in the adult abdomen of Drosophila: engrailed and slit help to corral the peripheral sensory axons into segmental bundles.

The abdomen of adult Drosophila bears mechanosensory bristles with axons that connect directly to the CNS, each hemisegment contributing a separate nerve bundle. Here, we alter the amount of Engrailed protein and manipulate the Hedgehog signalling pathway in clones of cells to study their effects on nerve pathfinding within the peripheral nervous system. We find that high levels of Engrailed make the epidermal cells inhospitable to bristle neurons; sensory axons that are too near these cells are either deflected or fail to extend properly or at all. We then searched for the engrailed-dependent agent responsible for these repellent properties. We found slit to be expressed in the P compartment and, using genetic mosaics, present evidence that Slit is the responsible molecule. Blocking the activity of the three Robo genes (putative receptors for Slit) with RNAi supported this hypothesis. We conclude that, during normal development, gradients of Slit protein repel axons away from compartment boundaries - in consequence, the bristles from each segment send their nerves to the CNS in separated sets.

Four-jointed modulates growth and planar polarity by reducing the affinity of dachsous for fat.

The Drosophila genes fat (ft) and dachsous (ds) encode large atypical cadherins that collaborate to coordinately polarize cells in the plane of the epithelium (planar cell polarity) and to affect growth via the Warts/Hippo pathway. Ft and Ds form heterodimeric bridges that convey polarity information from cell to cell. four-jointed (fj) is a modulator of Ft/Ds activity that acts in a graded fashion in the abdomen, eye, and wing. Genetic evidence indicates that Fj acts via Ds and/or Ft, and here we demonstrate that Fj can act independently on Ds and on Ft. It has been reported that Fj has kinase activity and can phosphorylate a subset of cadherin domains of both Ft and Ds in vitro. We have used both cell and in vitro assays to measure binding between Ft and Ds. We find that phosphorylation of Ds reduces its affinity for Ft in both of these assays. By expressing forms of Ds that lack the defined phosphorylation sites or have phosphomimetic amino acids at these positions, we demonstrate that effects of Fj on wing size and planar polarity can be explained by Fj phosphorylating these sites.