A receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors.Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABADOI:http://dx.doi.org/10.7554/eLife.01936.001 The electrical signals that are fired along neurons cannot be transmitted across the small gaps, called synapses that are found between neurons. Instead, the neuron sending the signal releases chemicals called neurotransmitters into the synapse. These neurotransmitters bind to receptor proteins on the surface of the second neuron and control how it fires.A neurotransmitter called dopamine plays a key role in the circuits of the brain that control how we learn certain tasks involving movement. In particular, two populations of neurons from the midbrain that release dopamine target the striatum, an area of the brain that is responsible for motor control. These neurons also release other neurotransmitters, but the identity of these other chemicals is not known, and the details of the interaction between the neurons and the striatum are poorly understood.Previous research showed that some of the midbrain neurons activate receptors that normally respond to a neurotransmitter called gamma-aminobutyric acid (GABA). However, several different chemicals can trigger this receptor. Using a range of techniques, Tritsch et al. now confirm that dopamine neurons release GABA alongside dopamine, and that this applies to both sets of the dopamine-producing neurons that feed into the striatum.Some neurons can manufacture GABA from amino acids found in their internal fluid. However, Tritsch et al. could not detect the enzymes needed for this reaction in dopamine-producing neurons. Instead, these neurons contain proteins that can transport GABA across the cell membrane, which suggests that the neurons collect GABA from the extracellular fluid that surrounds them.DOI:http://dx.doi.org/10.7554/eLife.01936.002 Dopamine (DA)-releasing neurons in the mammalian midbrain play an important role in fundamental behaviors, including motivation, reinforcement learning and motor control, and their dysfunction is associated with a wide range of neuropsychiatric disorders . The majA receptors  in SPNs  transgenes to permit distinction between direct- and indirect-pathway SPNs, respectively (B receptors (CGP55845) to prevent excitatory synaptic transmission by dopaminergic axons, as well as modulatory effects of GABAB receptors, respectively. Under our recording conditions, optogenetic stimulation of VTA axons with brief (1 ms) flashes of blue light reliably evoked inward IPSCs in direct- and indirect-pathway SPNs that were blocked by the GABAA receptor antagonists picrotoxin  presynaptic synthetic enzymes, (2) presynaptic vesicular transporters, (3) postsynaptic receptors, and (4) a biochemical mechanism for inactivation. In the case of GABAergic signaling at dopaminergic synapses, conditions 2 and 3 are respectively satisfied by the presence of VMAT2 in DA neurons and GABAhe brain . Its genhe brain . Importamuscimol . Thus, wA receptors. By contrast, mGAT1 antagonism significantly affected decay kinetics: whereas oIPSC decay time constants remained stable in control recordings , they were prolonged by a factor of 3 in the presence of SKF 89976A  and Gad1 or Gad2 . We focused our analyses to regions highlighted in Vmat2 and Gad1, as only 4 out of 958 Vmat2+ neurons (0.4%) showed weak signal for Gad1 in SNc and lateral VTA (Gad2 mRNA in these regions (Vmat2+ cells (0.6%) were weakly Gad2+, despite the presence of numerous brightly-labeled GABAergic neurons in neighboring substantia nigra pars reticulata (SNr) and within the SNc and lateral VTA. We confirmed our ability to detect co-labeling by examining the histaminergic tuberomamillary nucleus (TMN) and dopaminergic A13 cell group (Previous reports indicate that up to 10% of midbrain DA neurons in rat contain detectable levels of mRNA for the 65 kDa isoform of glutamic acid decarboxylase (GAD65) ; 2004. Teral VTA . To our  regions : only 7 ll group , both ofll group .10.7554/Gad1 or Gad2  or Gad2 (0 out of 526 TH+ neurons) promoters  to label catecholaminergic neurons. However, the high density of GABAergic axons converging into the substantia nigra, combined with the low concentration of GADs in cell bodies compared to axon terminals prevented us from clearly identifying DA neurons that expressed GADs (not shown). We instead imaged coronal brain sections from knock-in mice  expressi or Gad2 . Cre exp or Gad2  and TH eromoters . CollectVmat2 and Abat (the gene encoding GABA transaminase) revealed strong co-labeling in 701 out of 738 DA neurons (95%) within the SNc and lateral VTA  are the two major plasma membrane GABA transporters expressed in the mouse midbrain (Vmat2 and mGat1 or mGat4 to establish whether either isoform is present in DA neurons (mGat1 in 667 out of 748 Vmat2+ neurons (89%), particularly within the SNc. mGat4 localized predominantly to glial cells, consistent with previous reports (Vmat2+ neurons).Our data indicate that inhibitory synaptic transmission from DA neurons does not depend on synthesis of GABA by either GADs or GABA transaminase. In the central nervous system, several synapses rely on neurotransmitter reuptake across the plasma membrane as opposed to de novo synthesis to sustain synaptic transmission . It is tmidbrain . We ther neurons . Surpris reports , althougThe expression of mRNA for mGAT1 and mGAT4 in DA neurons raises the possibility that DA neurons acquire GABA not through de novo synthesis, but rather through plasma membrane uptake. Being almost exclusively composed of GABAergic neurons, the striatum represents a rich source of extracellular GABA for dopaminergic terminals . InhibitA receptor-mediated current in SPNs  serves as a substrate for VGAT  functions as a GABAfor VGAT , and (3)for VGAT , glycineobserved . In addiobserved , and appobserved . Moreoveobserved , perigloobserved , as wellobserved , suggestSynaptic transmission requires constant refilling of new and recycled vesicles, which is contingent upon the availability of transmitter in the cytosol. Most GABAergic neurons sustain inhibitory transmission by maintaining high cytosolic concentrations of GABA using de novo synthesis from glutamate by GAD67 and to a lesser extent GAD65 . Given tMost classical transmitters are transported back into the presynaptic terminal after vesicular release using plasma membrane transporters. This process is important not only for limiting the duration of synaptic currents and preventing extrasynaptic spillover, but also for replenishing presynaptic transmitter levels . In factA receptors. Phasic activation of DA neurons may thereby dampen ongoing cortical and thalamic drive onto SPNs to limit DA receptor-mediated plastic changes to synaptic inputs most strongly activated by salient or rewarding stimuli. In addition, the mechanism adopted by DA neurons to obtain GABA may confer these cells the flexibility to dynamically and locally control GABAergic transmission across their extensive axonal arbors. For instance, VTA neurons projecting to NAc and medial prefrontal cortex may only co-release GABA in the former, where extracellular GABA levels are high compared to cortex  or Drd1a-tdTomato  BAC transgenic mice to permit distinction between direct- and indirect-pathway SPNs  were used. For stereotaxic viral injections, postnatal day 18–25 mice were anesthetized with isoflurane, placed in a small animal stereotaxic frame  and injected with 1 μl of an adeno-associated virus  encoding Cre-dependent ChR2 (AAV2/8.EF1α.DIO.hChR2(H134R)-mCherry or AAV2/8.EF1α.DIO.hChR2(H134R)-EYFP), as described previously   . The knoad1-Egfp  and Gad2Cre mice . The lat(+ cells . Mice weGad1 (RP_040324_01_F01), Gad2 (RP_071018_02_B07), Vmat2 (RP_071218_02_B08), mouse Gat1 (RP_071204_04_H01), mouse Gat4 (RP_050428_04_D04), and Abat (RP_050301_02_C02). For in vitro transcription, each cDNA was cloned out from a mouse brain cDNA library using the primer sets suggested in the in situ hybridization data portal from the Allen Institute for Brain Science. Two fluorescein- or digoxigenin-labeled riboprobes generated by an in vitro transcription method  were hybridized simultaneously and stained by fluorescein or Cy3 chromogens, respectively. After staining, sections were mounted with Prolong Gold antifade reagent with DAPI nuclear stain .Mice were anesthetized with isoflurane and transcardially perfused with phosphate buffered saline followed by 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer. 50-micrometer coronal brain sections were subject to immunohistochemical staining for tyrosine hydroxylase , as described previously . Endogen3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4 and 11 glucose (295 mOsm·kg−1). Parasagittal slices of striatum (275-μm thick) were subsequently obtained in cold choline-based cutting solution . Following 15 min recovery in ACSF at 34°C, slices were kept at room temperature (20–22°C) until use. All solutions were constantly bubbled with 95% O2/5% CO2. Whole-cell voltage-clamp recordings were established from direct- and indirect-pathway SPNs in dorsal or ventral striatum resting 18–75 μm below the slice surface (median: 40 μm) in ACSF warmed to 32–34°C. Direct-pathway SPNs were identified as tdTomato+ cells in Drd1a-tdTomato mice or EGFP–cells in Drd2-EGFP mice, and indirect-pathway SPNs as tdTomato−or EGFP+ cells in Drd1a-tdTomato and Drd2-EGFP mice, respectively. Patch pipettes (2–4 MΩ) were filled with (in mM) 125 CsCl, 10 TEA-Cl, 10 HEPES, 0.1 Cs-EGTA, 3.3 QX-314 , 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-Phosphocreatine . The recording perfusate always contained NBQX (10 μM) and R-CPP (10 μM) to block AMPA and NMDA receptor-mediated inward currents, respectively, as well as CGP55845 (2–5 μM) to prevent GABAB receptor-evoked pre- and post-synaptic modulation. For some experiments . To activate ChR2-expressing fibers, light from a 473-nm laser  was focused on the back aperture of the microscope objective to produce wide-field illumination of the recorded cell. Brief pulses of light  were delivered at the recording site at 30 s intervals under control of the acquisition software. Epifluorescence illumination was used sparingly to minimize ChR2 activation prior to recording. eIPSCs were evoked using constant-current pulses  delivered every 15 s through a bipolar tungsten stimulating electrodes positioned within striatum, 100–200 μm away from the recorded cell. Constant-potential amperometry was performed as before  written in MATLAB (Mathworks). Electrophysiology data were analyzed offline using Igor Pro . In figures, voltage-clamp traces represent the averaged waveform of 3–5 consecutive acquisitions. Averaged waveforms were used to obtain current latency, peak amplitude, 10–90% rise time and decay time constant. The latter was estimated by measuring the time elapsed from the peak of the IPSC to 1/e (36.8%) of the peak amplitude. Detection threshold for sIPSCs was set at 40 pA to facilitate event detection in the presence of large and noisy tonic GABA currents. For pharmacological analyses in Brain sections processed for in situ hybridization or immunofluorescence were imaged with an Olympus VS110 slide-scanning microscope. High-resolution images of regions of interest were subsequently acquired with a Zeiss LSM 510 META confocal microscope (Harvard NeuroDiscovery Center). Individual imaging planes were overlaid and quantified for colocalization in ImageJ (NIH) and thresholded for display in Photoshop (Adobe). Confocal images in figures represent maximum intensity projections of 3-µm confocal stacks. Membrane currents were amplified and low-pass filtered at 3 kHz using a Multiclamp 700B amplifier , digitized at 10 kHz and acquired using National Instruments acquisition boards and a custom version of ScanImage ( review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  These data make several specific, testable predictions that would enhance the strength of the main interpretation. For example, increasing extracellular GABA should increase the amplitude of DA-IPSCs through an mGAT-dependent mechanism. Does bath application of GABA, or perhaps extracellular GABA uncaging, increase DA-IPSCs? And is this effect blocked by the acute presence of mGAT blockers during said GABA application? 2) A second prediction is that rundown of transmission induced by SKF should be activity dependent. I found it confusing at first that 3) The authors deal with a number of potential confounding factors such as the increased tonic GABA currents produced by GABA transporter inhibition, showing that this does not affect the kinetics of quantal GABA events from sources other than dopamine neurons. On the other hand, it is remarkable that the inhibitor does not affect the kinetics of these other events – is it so selective for GABA release from dopamine neurons because the transporter is only expressed on those terminals? It would be helpful if the authors could address this surprising result – previous work has indicated expression of GAT-1 by SPNs and an effect of a different inhibitor (NO-711) on amplitude and decay time of events evoked by local depolarization .4) Treatment with GAT-1 inhibitor for longer times effectively eliminates the GABA release by dopamine neurons, as anticipated if it depends on the GABA taken up from other cells. However, it is also possible that dopamine neurons make GABA themselves, and the main argument used here against this is the apparent lack of expression by a variety of criteria. Although these seem reasonable, other work cited by the authors in a different context  along wi5) The authors also suggest that GABA corelease involves both mesostriatal and mesolimbic projections, in contrast to the apparent corelease of glutamate by a subset of VTA neurons. However,  We are thankful for the reviewers’ prompt evaluation of our manuscript and for their valuable comments and suggestions. We are delighted that our work was reviewed favorably and described as “straightforward”, “carefully conducted” and “well-presented”. We agree with the reviewers that the results described in our study are an important and necessary first step to elucidate the mysterious role of fast-acting neurotransmission from midbrain dopamine neurons. However, we take exception with the notion that the principal findings of this study were in any way predictable from prior work. Although our initial study  pointed Please find below our response to each of the reviewer’s comments. We hope the reviewers will find our revised manuscript improved and suitable for publication.1) These data make several specific, testable predictions that would enhance the strength of the main interpretation. For example, increasing extracellular GABA should increase the amplitude of DA-IPSCs through an mGAT-dependent mechanism. Does bath application of GABA, or perhaps extracellular GABA uncaging, increase DA-IPSCs? And is this effect blocked by the acute presence of mGAT blockers during said GABA application?(cited) showed that GAT activity can rescue transmission in conventional GABAergic synapses when GABA is bath applied in the presence of gabazine. This experiment would reveal the timecourse over which DA neurons take up GABA from the extracellular space and package it into synaptic vesicles, and it would be important to contrast the timecourse of the authors' novel mechanism with that of enzymatic synthesis at more traditional GABA synapses. This experiment would enhance also the claim that GAT supplies the GABA used for synaptic vesicles.We performed the experiment suggested by the reviewers, in which we show that bath application of GABA increases the amplitude of IPSCs evoked by dopaminergic neuron stimulation in a GAT-dependent manner. These data are consistent with the claim that GABA is the transmitter released, and that it is supplied by GATs for vesicular release. These results are now shown in Technical limitations unfortunately prevented us from performing the second series of experiments, which would have allowed us to contrast the time course of GABA uptake and vesicular filling in dopamine neurons with that of ‘classical’ GABAergic synapses that depend on GABA synthesis. The studies by 2) A second prediction is that rundown of transmission induced by SKF should be activity dependent. I found it confusing at first thatshows little effect of SKF on IPSC amplitude andshows a large effect, and I am sure readers will also be caught off guard. The difference is that with time the synapses in SKF are depleted of GABA. Therefore if the synapses were stimulated one might expect to accelerate the process, confirming that it reflects depletion of vesicular GABA rather than a side effect of the drug.We apologize for the confusion and thank the reviewers for pointing out the need to clarify the description of our experimental manipulations. As the reviewers indicate, the major difference between Following the reviewers’ suggestion, we now provide evidence in 3) The authors deal with a number of potential confounding factors such as the increased tonic GABA currents produced by GABA transporter inhibition, showing that this does not affect the kinetics of quantal GABA events from sources other than dopamine neurons. On the other hand, it is remarkable that the inhibitor does not affect the kinetics of these other events – is it so selective for GABA release from dopamine neurons because the transporter is only expressed on those terminals? It would be helpful if the authors could address this surprising result – previous work has indicated expression of GAT-1 by SPNs and an effect of a different inhibitor (NO-711) on amplitude and decay time of events evoked by local depolarization .The different sensitivity of IPSCs originating from dopamine neurons vs. ‘classical’ GABAergic neurons to SKF89976A is indeed remarkable, pointing to fundamental structural and/or biochemical differences between both synapses. It is unlikely that selective expression of mGAT1 in dopamine neurons fully accounts for this difference, as mGAT1 is expressed widely throughout the brain, including in the striatum. However, it is possible that dopaminergic synapses rely exclusively on mGAT1 for membrane GABA transport, whereas classical GABAergic synapses may utilize other transporters in the presence of SKF89976A, such as mGAT4. Indeed, the tonic GABA current evoked by acute bath application of SKF89976A and SNAP-5114 (a mGAT4 antagonist) is more than twice that of SKF89976A alone . Additional factors may contribute to differences in the sensitivity of IPSC kinetics to GAT blockers, including GABAA receptor subunit composition , GABAA rThe reviewers correctly note that another group  previous4) Treatment with GAT-1 inhibitor for longer times effectively eliminates the GABA release by dopamine neurons, as anticipated if it depends on the GABA taken up from other cells. However, it is also possible that dopamine neurons make GABA themselves, and the main argument used here against this is the apparent lack of expression by a variety of criteria. Although these seem reasonable, other work cited by the authors in a different context  along with other work they do cite suggests expression of GAD by a subset of dopamine neurons. Rather than rely on the anatomy, it would help greatly to demonstrate that inactivation of GAD specifically in dopamine neurons does not eliminate the dopaminergic IPSCs. This might require a substantial amount of work that would lie outside the scope of this manuscript , but it would certainly be helpful. However, showing that expression of a GAD isoform in dopamine neurons makes the dopaminergic IPSCs resistant to inhibition of GAT-1 would be considerably simpler.+ and VMAT2+ terminals in the striatum of mice and rats, but did not examine the distribution of GADs. We only know of two studies having directly investigated whether SNc and VTA dopamine neurons express GADs , but we currently do not have access to them. Carrying out the suggested GAD knockout experiment would require a substantial amount of work  that is not motivated by prior or current evidence for GAD expression in midbrain dopamine neurons. We therefore agree with the reviewers that this experiment is beyond the scope of this manuscript.Global and conditional knockout mice for GAD65 and GAD67 exist, respectively  that are currently not at our disposal and would take several months to generate and characterize. We agree that it would nicely demonstrate that the inhibition of oIPSCs by GAT antagonists does not stem from unintended effects of these drugs on synaptic vesicle exocytosis or GABA synthesis by GADs. However, this experiment would, by itself, offer little additional insight as to the molecular mechanism normally responsible for providing GABA to midbrain dopamine neurons. Control experiments are now provided in 5) The authors also suggest that GABA corelease involves both mesostriatal and mesolimbic projections, in contrast to the apparent corelease of glutamate by a subset of VTA neurons. However,also showed that only ∼10 % of striatal terminals apparently contain GABA, raising the question whether the dopaminergic IPSCs derive from a subset of both populations, or all of them. It would be great if the authors could address this point, and one way might be to determine whether GAT-1 inhibition influences dopamine release measured by voltammetry-GABA corelease by many terminals should have some effect on total dopamine release, but involvement of only a subset would produce minimal if any effect. This would also help to address one important functional outcome of corelease that has not yet been examined.+ synaptic vesicles, and 3) that the concentration of GABA is large enough to be detected in a sizeable fraction of dopaminergic terminals.The study by Nevertheless, the question raised by the reviewers is important, as establishing whether GABA co-release is common to all midbrain dopamine neurons or a unique feature of a subset of cells is essential to determine its functional relevance. It should be noted that regardless of the molecular mechanisms engaged or the fraction of midbrain dopamine neurons implicated, GABA co-transmission may be a general feature of monoaminergic neurons, as opposed to an exception, suggesting an important role for dopamine-GABA co-transmission : light-evoked dopamine release is unaffected by inhibition of GABAA receptors  or by acThe suggestion that GABA co-release in the striatum may apply to all midbrain dopamine neurons (as opposed to a subset of SNc and VTA neurons) is based on several observations. First, oIPSCs are large and present in every SPN studied to date, as opposed to glutamatergic events, which are significantly smaller in amplitude and only observed in a fraction SPNs . Second,


Evidence shows that there are reductions in gray matter volume (GMV) and changes in long association white matter fibres within the left insula-temporoparietal junction (TPJ) during the early stages of psychotic disorders but less is known about short association fibres (sAFs). In this study we sought to characterise the changes in sAFs and associated volumetric changes of the left insula-TPJ during the early stages of psychosis. Magnetic resonance imaging was obtained from a sample of young people with psychosis (n = 42) and healthy controls (n = 45), and cortical parcellations of the left insula-TPJ were used as seeding masks to reconstruct 13 sAFs. Compared to healthy counterparts, the psychosis group showed significant reductions in fractional anisotropy (FA) in the sAFs connecting the superior (STG) and middle temporal gyri (MTG) and as well as reduced GMV within the inferior temporal gyrus and increased white matter volume (WMV) within Heschl's gyrus (HG). Furthermore, adolescent-onset psychosis subjects (onset 18 year or earlier) showed FA reductions in the STG-HG sAF when compared to adult-onset subjects, but this was not associated with changes in GMV nor WMV of the STG or HG. These findings suggest that during the early stages of psychosis, changes in sAFs and associated cortical GMV and WMV appear to occur independently, however age of onset of a psychotic syndrome/disorder influences the pattern of neuroanatomical abnormalities. Short association fibres (sAFs) comprise either local association fibres (U-fibres of Mynert) Superficial cerebral WM is comprised of striatal, thalamic, pontine and commissural fibres as well as long and short association fibres The principle aim of this study was to delineate sAFs in young healthy individuals and compare these with sAFs in young people at the early stages of psychosis. Based on previous research into superficial WM changes in older psychosis subjects Building on our recent investigation into long association fibres in early psychosis A psychiatrist assessed all patients to determine the nature and history of any mental health problems. By consensus of the senior investigators (IBH and ES), subjects were categorized according to DSM-IV-TR criteria A psychiatrist or trained research psychologist conducted the clinical assessment (in a semi-structured interview format) to determine the nature and history of any mental health problems using the Brain and Mind Research Institute Structured Interview for Neurobiological Studies 2) were acquired prior to the acquisition of 69 images (each containing 55 slices) with uniform gradient loading (b = 1159 s/mm2).Subjects underwent MRI scanning using a 3-Tesla GE MR750 Discovery scanner  at the Brain and Mind Research Institute, Camperdown, NSW Australia. To enable neuroanatomical analysis at high resolution (0.9 mm isotropic resolution), we acquired a customized MP-RAGE 3D T1-weighted sequence: repetition time (TR) = 7264 msec; echo time (TE)  = 2784 msec; flip angle  = 15; coronal orientation; field of view (FOV)  = 230 mm; matrix of 256×256; total slices  = 196. Next, whole brain diffusion-weighted images were acquired using an echo planar imaging sequence: TR  = 7000 ms; TE  = 68 ms; slice thickness  = 2.0 mm; FOV = 230×230 mm; acquisition matrix  = 256×256; axial orientation; 69 gradient directions. Eight images without gradient loading  Human Brain Atlas (version 3) http://surfer.nmr.mgh.harvard.edu/) as previously described To create seeding targets for reconstructing sAF, cortical gyri demarcated by the Desikan–Killiany atlas Using DTI-TK, the FA scalar information from the IIT Atlas was extracted and a binary WM map was built with a lower threshold of 0.1 to capture the detail of these finer tracts. Nine cortical parcellations from the Desikan–Killiany atlas were used as inclusion targets to delineate the sAF within the left insula-TPJ, namely the superior temporal gyrus (STG), transverse temporal gyrus , supramarginal gyrus (SMG), inferior parietal lobe (IPL), fusiform (FUS), inferior temporal gyrus (ITG), middle temporal gyrus (MTG), the banks of the superior temporal sulcus (BANKS) and the insula (INSULA). Tracking was performed using two gyri as inclusion regions of interest (ROIs) with the remaining seven gyri used as exclusion ROIs. To further remove extraneous superficial fibres  from sAFs, exclusion ROIs of the lateral occipital and midline were also applied and stray fibres were manually removed using ITK-SNAP. Pairing of some ROIs resulted in less than 50 streamlines per connection and were subsequently excluded from the study.Whereas long association fibre templates are widely available, the present study required the creation of sAFs templates –13. ThisTo determine the GMV and WMV of each ROI, FreeSurfer software package version 5.1 t-tests. Volumetric differences were evaluated using an MANCOVA analysing intracranial-corrected ROI volumes between groups, controlling for age and gender. Significance was set at p<0.05 (two-tailed).Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 22.0 for Windows). Between groups demographic analyses were conducted using chi-square tests for categorical data. One-way analysis of variance (ANOVA) with posthoc Bonferroni pairwise comparisons was used to assess differences in age, predicted IQ, HDRS, K10 and DASS. The age of onset of psychosis, duration of illness and BPRS scores was assessed using independent-samples p<0.05 which converted to a t-statistic threshold of 1.66.Within the DTI-TK program we used the tract-specific analysis generalised linear model with 10,000 permutations to determine group differences in FA, AD and RD measurements. Significance was set at p = .018) and the adolescent-onset group was significantly younger age  than the adolescent-onset group  or control group . The psychosis subjects had significantly worse HDRS, K10 and DASS clinical scores than controls.There were no significant group differences in handedness or predicted IQ between controls, adolescent-onset psychosis and adult-onset psychosis subjects . Collectp<.001) and duration of illness .The mean age of onset of psychosis was 17.6 years (SD 4.2) with a mean duration of illness of 5.2 years (SD 4.0). The adolescent-onset and adult-onset psychosis subjects did not differ in HDRS, K10, DASS or BPRS scores but did differ in age of onset  that exhibited reduced FA  of reduced AD in psychosis subjects compared to controls, but none reached levels of significance (t>1.66) but none reached levels of significance  as well as showing a trend of reduced FA within the STG-MTG . Within the STG-HG and STG-MTG there were several small clusters (i.e. t>1.66) of reduced AD in adolescent-onset psychosis subjects compared to controls, but none of these reached levels of significance, with no RD clusters found. There were no statistically significant differences in the 13 sAF reconstructions between adult-onset psychosis subjects and healthy counterparts.Compared to controls, the adolescent-onset psychosis subjects showed reduced FA within the STG-HG  but subsequent pairwise comparisons of simple effects showed that left ITG mean GMV was reduced in the psychosis group relative to the control group , but subsequent pairwise comparisons of simple effects showed that the mean left HG WMW was increased in the psychosis group relative to the control group  and the adolescent-onset psychosis group showed significantly reduced MTG WMV compared with the adult-onset psychosis group .Comparing WMVs between groups, the overall MANCOVA did not show a main effect , WM disorganisation  or WM reorganisation  Previous investigations have identified abnormalities in superficial WM within the left temporoparietal and frontal regions per se, and follow-up replications of these techniques in different population samples would help clarify the nature of these changes in illness progression.There are several limitations to the present study that warrant discussion. It is important to note that the continuous medial representation framework that was used to model the sAFs optimally represents ribbon-like structures such as u-fibres. However, the FUS-ITG sAF had a more complex structure that could not be optimally fitted by the model (Dice similarity coefficient 0.75) and thus these findings need to be interpreted with this constraint in mind. Next, to create sAFs that only included streamlines connecting the seed regions without extraneous inclusions, we set strict exclusion seeds that may have excluded genuine streamlines – it is our belief that this would affect only a small number of streamlines per sAF and is preferential to including misleading reconstructions. Additionally, we chose a shape-based technique to find a balance between deterministic tractography that does not accommodate crossing fibres, and probabilistic tractography that will overestimate tracts – future investigations should model at least two directions (preferably three directions) within each voxel to reconstruct estimated pathways in regions of crossing fibres. As a cross-sectional study with 42 psychosis subjects, the study is underpowered to examine the influence of medication. Finally, variations in AD and RD may be a consequence of partial volume effects or underlying tissue structure Our findings indicate that in psychosis there is a significant GMV reduction within the left ITG that is accompanied by reduced FA in the sAFs connecting the MTG and STG. Furthermore, in adolescent-onset psychosis there is reduced FA in the sAFs connecting HG and STG independent of volumetric changes in these two structures, and future investigations should elucidate the interface between sAFs and connecting gyri and how these changes relate to disease progression over time.Figure S1Short association fibres connecting the superior and middle temporal gyri.(TIFF)Click here for additional data file.Figure S2Short association fibres connecting the fusiform to the inferior temporal gyrus.(TIFF)Click here for additional data file.Figure S3Short association fibres connecting the insula cortex to Heschl's gyrus.(TIFF)Click here for additional data file.Figure S4Short association fibres connecting the banks of the superior temporl sulcus to the middle temporal gyrus.(TIFF)Click here for additional data file.Figure S5Short association fibres connecting the insula cortex to the supramarginal gyrus.(TIFF)Click here for additional data file.Figure S6Short association fibres connecting the insula cortex to the supramarginal gyrus.(TIFF)Click here for additional data file.Figure S7Short association fibres connecting the supramarginal gyrus to the inferior parietal region.(TIFF)Click here for additional data file.Figure S8Short association fibres connecting the banks of the superior temporal sulcus to the superior temporal gyrus.(TIFF)Click here for additional data file.Figure S9Short association fibres connecting the fusiform to the superior temporal gyrus.(TIFF)Click here for additional data file.Figure S10Short association fibres connecting the Heschls' gyrus to the superior temporal gyrus.(TIFF)Click here for additional data file.Figure S11Short association fibres connecting the insula cortex to the superior temporal gyrus.(TIFF)Click here for additional data file.Figure S12Short association fibres connecting the inferior parietal cortex to the superior temporal gyrus.(TIFF)Click here for additional data file.Figure S13Short association fibres connecting the supramarginal gyrus to the superior temporal gyrus.(TIFF)Click here for additional data file.


Arabidopsis can recognize and respond differentially to insect species at the transcriptional level using a genome wide microarray. Transcriptional reprogramming was characterized using co-expression analysis in damaged and undamaged leaves at two times in response to mechanical wounding and four insect species. In all, 2778 (10.6%) of annotated genes on the array were differentially expressed in at least one treatment. Responses differed mainly between aphid and caterpillar and sampling times. Responses to aphids and caterpillars shared only 10% of up-regulated and 8% of down-regulated genes. Responses to two caterpillars shared 21 and 12% of up- and down-regulated genes, whereas responses to the two aphids shared only 7 and 4% of up-regulated and down-regulated genes. Overlap in genes expressed between 6 and 24 h was 3–15%, and depended on the insect species. Responses in attacked and unattacked leaves differed at 6 h but converged by 24 h. Genes responding to the insects are also responsive to many stressors and included primary metabolism. Aphids down-regulated amino acid catabolism; caterpillars stimulated production of amino acids involved in glucosinolate synthesis. Co-expression analysis revealed 17 response networks. Transcription factors were a major portion of differentially expressed genes throughout and responsive genes shared most of the known or postulated binding sites. However, cis-element composition of genes down regulated by the aphid M. persicae was unique, as were those of genes down-regulated by caterpillars. As many as 20 cis-elements were over-represented in one or more treatments, including some from well-characterized classes and others as yet uncharacterized. We suggest that transcriptional changes elicited by wounding and insects are heavily influenced by transcription factors and involve both enrichment of a common set of cis-elements and a unique enrichment of a few cis-elements in responding genes.We tested the hypothesis that  Approximately a quarter of all described eukaryotic species are insects that feed on plants, and as a group they are thought to exert strong selection on plants to detect and repel them  to wounding, to chewing insects (caterpillars) and to stylet feeders (aphids). We used a genome-wide Arabidopsis microarray to examine all transcriptional changes at two time intervals after mechanical wounding or being attacked by four insect species. The insect species were two aphids (phloem feeders) and two caterpillars (leaf chewers). Both feeding types included one with an extremely broad diet  and another with a narrow diet  focused on Brassicaceae, the plant family to which A. thaliana belongs. This design enabled testing the hypothesis that Arabidopsis can recognize and respond individually to these insects at the transcriptional level. Common patterns in fundamental reprogramming of plant metabolism were identified using gene enrichment tests. Candidate genes that may determine species-specific responses were identified with coexpression network analysis. Frequencies determined for coregulated gene promoters linked to genes differentially expressed suggest that reprogramming of expression patterns elicited by wounding and different insect species involve both a common set and a unique, small set of cis-elements.To identify HAMPs that address these experimental issues, we report here a fully replicated study of genome-wide transcriptional responses by A. thaliana ecotype Columbia Wild-Type (Col WT) to attack by larvae of two leaf chewing caterpillar species and by adults and nymphs of two phloem feeding aphid species (Table Spodoptera exigua (Hübner) (Noctuidae), which feeds on as many as 20 plant families including Brassicaceae  (Pieridae), which feeds exclusively on plants in the family Brassicaceae, to which Arabidopsis belongs , a broad generalist feeding on species in many plant families including Brassicaceae, and Brevicoryne brassicae (L.), whose feeding is limited to the Brassicaceae . Eggs of S. exigua were obtained from Benzon Research and larvae were reared on artificial diet (Bio-Serv). Pieris rapae was maintained as a culture in our lab on pak-choi and originated from the Carolina Biological Supply Company. Both caterpillar species were transferred to Col WT plants one day before the experiments for acclimation to the new host. Plant seeds were vernalized in 2% agar and sown into 6 × 5 cm pots containing sterile Metromix 200 soil (Sun Gro Horticulture). Plants were chamber grown at 22 ± 1°C, 65 ± 5% relative humidity, and 200 μmol m−2s−1 light intensity on a 8:16 (L:D) photoperiod. Plants were watered as needed and fertilized every other watering with 21-7-7 Miracle-Gro (Scotts Company).We assessed the transcriptional responses in rosette leaves of 4-week-old es Table . The catN = 6–10) caterpillars of both species were allowed to feed for 2–4 h to generate 6 leaves of similar age per plant with ~20% leaf area removed. Caterpillars were wrangled as needed with a size 0 camel's hair brush to concentrate their feeding on the 6 leaves sampled. Caterpillars were removed when sufficient damage was achieved and the plants were returned to the growth chamber. The mechanical wounding treatment was designed to approximate insect damage to tissues by running a sterile pattern wheel across both sides of the midrib of 6 leaves of similar age on each plant, once at the beginning of the caterpillar treatment and again half way through the caterpillar wrangling period. Control plants were jiggled with the brush to simulate leaf movement caused by caterpillar wrangling or mechanical wounding. Damaged leaves  were harvested for gene expression 6 and 24 h after the start of caterpillar damage or wounding. Unwounded leaves (“systemic”) were harvested separately from size-matched damaged or wounded leaves. Leaves from 3 to 4 plants per treatment  were pooled for each of the four biological replicates.The treatments are summarized in Table N = 20) were placed on plants whose rosettes were caged at the soil line by transparent mylar cylinders  with tops of fine mesh gauze (<0.01 mm mesh wide) to maintain air exchange. Controls were caged plants without aphids and all plants were returned to the growth chamber. After 1 week, all cages and aphids were removed and control plants were jiggled with a camel's hair brush to simulate leaf movement caused by aphid removal. Plants were returned to the growth chamber and whole plants were harvested per treatment (aphid and control) for gene expression 6 and 24 h after aphid removal.Treatment of plants with aphids was different from that for caterpillars because aphids have effects on plants that are much weaker and slower to develop than those of caterpillars  by vortexing, and incubated at 65°C for 5 min with regular mixing. Cell debris was pelleted by centrifugation  and the supernatant was extracted twice with 3 ml chloroform with the aqueous phase recovered each time after centrifugation . RNA was precipitated from this phase at room temperature for 5 min with 0.5 volumes each of 0.8 M sodium citrate and isopropanol. The RNA pellet obtained after centrifugation  was washed with 70% ethanol, recovered again by centrifugation, air dried for 5 min and resuspended in 200 μl nuclease free water. The RNA was further purified by standard ethanolic sodium acetate precipitation at −20°C overnight. Following a wash with 70% ethanol the pelleted RNA  was air dried and resuspended in nuclease free water to an approximate concentration of 5 μg/μl whose actual concentration was determined spectrophotometrically. Quality of randomly selected samples of RNA was determined using a 2100 Bioanalyzer (Agilent Technologies).Arabidopsis oligonucleotide targets (Qiagen Operon), 12 housekeeping gene oligos (Qiagen Operon) as positive controls and 16 oligos with no similarity to any Arabidopsis gene (4 synthesized human genes and 12 others) as negative controls and internal, spike controls was previously described .Design and production of microarrays with 26,090 Total RNA isolated as previously described  between treatment and corresponding control, as previously described  and an estimate of the false discovery rate (FDR) based on the distribution of parametric p-values  and also displayed a more than two-fold change between treatment and control . Lower N = 17) in the caterpillar treatments at 6 and 24 h in both local and systemic tissue because of their species-specific expression pattern,  that used the centroid method and the maximum number of possible clusters set to 16, or the number of treatments. The percentage overlap in gene expression between treatments was calculated as the (# genes in common/(sum # genes elicited by both treatments–# genes in common).p-value (EASE score) and clusters with a Group Enrichment Score > 1 were examined further. A Benjamini-Hochberg p < 0.05 correcting for multiple comparisons was considered to be statistically enriched; values significant at the uncorrected p-value were also indicated in some comparisons. Similar analyses were run using MAPMAN and PAGEMAN but no significant enrichments were detected with a Benjamin-Hochberg correction for multiple comparisons. Thus, we concluded that the large number of genes of unknown function in this study made DAVID a more useful tool because it incorporates a larger number of databases.To identify functional patterns in the large number of genes differentially expressed in response to insects, we used the DAVID gene enrichment analysis tool  were converted to Affymetrix GeneChip identities using the “at to AGI converter” tool   as described previously  Sequence Database for all genes whose expression was significantly affected by insect feeding or mechanical wounding. First, the database of motifs found in plant cis-acting regulatory DNA elements  and cluster analysis was performed with the tallied data to identify specific cis-element fingerprints possibly unique to insect treatments. Treatments were clustered on the basis of similarities of their cis-element distributions using the SAS VARCLUS procedure (SAS Institute).Several on-line databases and bioinformatics tools were used to conduct three separate analyses to identify potential Arabidopsis transcription factor binding sites and 30,067 predicted promoters, was used to search the downloaded sequences for enriched, known transcription factor binding sites and cis-elements in gene promoters. The ATHENA algorithm conducts a student's t-test to determine whether motifs in a given sample set are significantly different from a random distribution in the genome. Motif occurrences with a p < 0.00001 were designated as “enriched.”Second, the ATHENA search tool  than at 24 h   than at 24 h  Transcriptional responses to the two aphids across both times only shared a 4–8% overlap in differentially expressed genes. Both caterpillars elicited similar numbers of differentially expressed genes at 6 h and 24 h  Figure . More geArabidopsis to herbivores were related to the insects' dietary breadth. The two dietary generalists upregulated more genes than did their respective dietary specialists  between them for downregulated genes.Transcriptional responses of The DAVID Functional Clustering Tool was used to identify biological functions significantly enriched among differentially expressed genes. About two thirds of the genes differentially expressed in response to our treatments have some functional or structural information in TAIR. DAVID used TAIR and added information from other species for genes with little annotation in TAIR. Although most of these ontological data were collected in contexts other than responses to insects, we can use them to infer some functions of proteins encoded by genes influenced by our treatments.Pieris rapae elicited transcriptional changes in 8 and S. exigua in 10 stimulus pathways linked to our treatments by DAVID at least once; each species elicited only two changes by the 6 h sampling. The two aphid species elicited fewer stress-related responses than did the caterpillars. Brevicoryne brassicae elicited changes in 4 stress-related pathways while M. persicae altered expression in 6 of the 14 relevant pathways. The transcriptional gene set most often altered by all the insects was response to hexose stimulus (6 of 16 possible interactions), but no single response gene set in this analysis characterized responses to insect elicitation as different from wounding or other stressors.Transcriptional responses to herbivory overlapped broadly with responses to 14 other biotic and abiotic stressors and stimuli, including responses to wounding, pathogens, cold, starvation, nutrient supply, metal ions, osmotic stress, reactive oxygen species, radiation, and light quantity and quality Table . CaterpiP. rapae elicited changes in 11 of the 12; no other insect treatment elicited changes in more than five. Both caterpillars and aphids elicited changes in expression of genes associated with cell wall metabolism, although their impacts were different , two of which are known to influence levels of trehalose-6-phosphate in vivo; when reduced in TPS mutants, starch accumulation is inhibited and sugar phosphates accumulate  which makes asparagine by amidation of aspartate using glutamate or ammonium as the amide donor , a key enzyme in the assimilation of inorganic nitrogen and reassimilation of ammonia released by photorespiration, and the translational inhibitor GCN2, whose phosphorylation of eIF2-alpha reduces overall protein synthesis  in all leaf types Figure . There wCaterpillar treatments (but not wounding or aphids) elicited expression of genes associated with responses to abscisic acid (ABA) in all leaf types at both times Figure . All of S. exigua and responses of genes associated with lectins were upregulated only at 24 h in leaves damaged by P. rapae. Ethylene signaling pathway genes were upregulated in both local and systemic leaves only at 24 h by S. exigua. Responses of genes involved in sulfur metabolism were upregulated at both time points in leaves damaged by S. exigua. Both species elicited responses in genes involved in auxin signaling at 6 h, but in different leaf types, systemic for P. rapae and local for S. exigua. Most of the auxin signaling-related genes in the enriched functional category were downregulated by insects  by caterpillars and age-matched undamaged (“systemic”) leaves on the same plants. DAVID identified 8 functional categories that were significantly enriched in one or more combinations of leaf type, caterpillar and time Table . Two of P. rapae compared to only in systemic leaves at both times for S. exigua. Wound-typical, other than JA responses, continued into the 24 h sampling of systemic leaves in P. rapae treatments. JA- and wound-related responses comprised upregulation except for two genes, which were downregulated by both caterpillar species in all leaves at 24 h , ORA47 (AT1G74930), WRKY40 (AT1G80840), AP2C1 (AT2G28900), CCR-4-NOT (AT3G44260), SYP122 (AT3G52400), and STZ (AT1G27730)  was associated with mechanical wounding . For aphids, the “yellow,” “salmon” and “brown” modules were associated with responses to B. brassicae, whereas the “black,” “dark turquoise,” “midnight blue,” “brown” and “dark green” modules were associated with responses to M. persicae.The weighted gene coexpression analysis identified a non-random network architecture of 18 modules of coexpressed genes , caterpillars (“red”), and aphids (“midnightblue”)  were statistically associated with responses to M. persicae at 6 h  in genes co-expressed in response to individual treatments  which require that expression changes of individual genes be confirmed. The larger number of genes upregulated by the two dietary generalists is consistent with the commonly held hypothesis that dietary specialists have evolved a stealthier way to exploit their host plants than dietary generalists  and glucose oxidase (GOX) are in the regurgitant of S. exigua  by the kinase GENERAL CONTROL NON-REPRESSIBLE 2  by RUBPCAse activase mediates the change in primary metabolism which in turn attenuates JA-induced defense responses following chewing insect attack . Caterpillars uniquely elicited expression of genes associated with ABA signaling, which in N. attenuata is due in part to the inhibitory effect of an elicitor in caterpillar oral secretions on ABA catabolism , and SnRK1 and TOR kinases is likely to provide additional insight (Robaglia et al., In summary, our results provide a comprehensive overview of cis-element analysis, edited manuscript. Trupti Joshi—Provided advice on cis-element analysis, edited manuscript. Dong Xu —Provided advice on cis-element analysis, edited manuscript. Joerg Bohlmann—Helped design microarray experiments, edited manuscript. Jack Schultz—Helped design microarray and insect experiments, assisted with the analysis and interpretation of data, conducted hierarchical clustering, edited manuscript, authored funding for project.Heidi M. Appel—Helped design array and insect experiments, conducted insect experiments, prepared manuscript, analyzed and interpreted data. Howard Fescemyer—Helped design microarray experiments, prepared RNA for microarray analysis, edited manuscript. Juergen Ehlting—Helped design and conduct microarray experiments. David Weston—Conducted network cluster analysis. Erin Rehrig—Conducted qRT-PCR validation of microarray and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gasterosteus aculeatus) exposed to predation risk produce offspring with altered behavior, metabolism and stress physiology. Here, we investigate the effect of maternal exposure to predation risk on the embryonic transcriptome in sticklebacks. Using RNA-sequencing we compared genome-wide transcription in three day post-fertilization embryos of predator-exposed and control mothers. There were hundreds of differentially expressed transcripts between embryos of predator-exposed mothers and embryos of control mothers including several non-coding RNAs. Gene Ontology analysis revealed biological pathways involved in metabolism, epigenetic inheritance, and neural proliferation and differentiation that differed between treatments. Interestingly, predation risk is associated with an accelerated life history in many vertebrates, and several of the genes and biological pathways that were identified in this study suggest that maternal exposure to predation risk accelerates the timing of embryonic development. Consistent with this hypothesis, embryos of predator-exposed mothers were larger than embryos of control mothers. These findings point to some of the molecular mechanisms that might underlie maternal effects.There is growing evidence for nongenetic effects of maternal experience on offspring. For example, previous studies have shown that female threespined stickleback fish ( Non-genetic maternal effects, or the influence of a mother's phenotype on her offsprings' phenotype, are widespread across taxa Studies on diverse organisms have shown that maternal stress can have long-lasting effects on offspring traits including survival Predators are one of the most important naturally-occurring stressors for animals in natural populations Despite growing appreciation of maternal effects occur across taxa and traits, we are just beginning to understand the molecular mechanisms by which maternal experience influences offspring. Therefore, we used RNA-sequencing technology to compare the stickleback embryonic transcriptomes of predator-exposed mothers and unexposed (control) mothers. We measured embryos at three days post-fertilization (dpf) because in zebrafish it is the earliest period when maternally-derived RNAs are thought to be fully degraded We made the following predictions about the types of genes and pathways likely to be influenced by maternal exposure to predation risk. First, because embryos from maternally-stressed stickleback mothers had higher initial respiration of oxygen Second, stress influences brain gene expression e.g.  thereforThird, previous studies have shown that exposing fish embryos to elevated cortisol alters the expression of genes encoding insulin-like growth factor, growth hormone, thyroid hormone receptors, and growth hormone receptors Fourth, predation pressure is associated with a relatively ‘fast’ life history  in many taxa .ad libitum once daily frozen bloodworms, brine shrimp, mysis shrimp, and cyclopeez.Juvenile threespine sticklebacks were caught by baited trap from the Navarro River, CA in July, 2011 and transported to the University of Illinois . Fish were housed until adulthood (approximately 1 year of age) in 37L tanks (53L x 33W x 24H cm) with gravel-bottom, artificial plant refugia, a flow-through system with UV, charcoal, particulate and biological filters that remove olfactory cues and a photoperiod that mimics seasonal changes at a density of 10 fish/tank. Fish were fed Cottus spp, SL: 7 cm) to simulate predation risk. Sculpin of this size are a known predator of stickleback eggs and juveniles in the Navarro River . Females were chased 29.4±7.3 days (mean ± standard error); chased females were exposed to simulated predation risk prior to and during gravidity. Males and control females were not chased. Females were wild caught therefore it is possible that they experienced predation risk prior to the experiment, but they were randomly assigned to either the predator-exposed or control treatment. A promising future direction is to evaluate for how many generations in sticklebacks the effects of maternal exposure to predation risk persist. Further, it is possible that some of the genes differentially expressed in embryos as a result of maternal exposure to predation risk are not specific to predation risk, but instead are common to all stressors or to maternal effects in general. Future studies that expose mothers to multiple and various treatments could potentially distinguish those genes specific to predator-induced maternal effects.In August, 2012 when sticklebacks showed reproductive morphology  they were separated by sex. Males were housed individually in 9L tanks, females were housed in groups of four in 26.5L tanks (36L x 33W x 24H cm) with opaque external shading. Tanks of females were randomly assigned as control or predator exposed. The females in predator exposed tanks were chased daily for 30 seconds at a randomly chosen time with a rubber model of a sculpin  of MS-222 anesthetic. Testes were dissected from individual males, macerated, and the suspension was used to fertilize eggs in a glass petri dish. Egg fertilization was confirmed by observation under a light microscope. Clutches were obtained from n = 8 control and n = 8 predator-exposed females. Eggs were transferred to mesh-bottom cups in 9 L tanks with air bubblers directly beneath developing clutches. All embryo tanks were treated with 100 uL of methylene blue solution to prevent fungal infection. Unfertilized and infected eggs were removed daily. We sampled whole embryos at 72.040±0.012 (mean ± standard error) hours post-fertilization (3 dpf). By 3 dpf our embryos had pigmented eyes, melanopores, and intermittent tail spasms In our study gene expression was measured in whole embryos. As a result, many tissue-specific expression changes would not have been detected, particularly for genes that exhibit low levels of expression or are expressed in minute tissues. Measuring sex- and tissue-specific embryonic expression at multiple time-points is an obvious task for future study. Also, measuring cortisol in mothers, eggs, and offspring would be useful toward understanding possible mechanisms for the observed effects on gene expression, and is an obvious future direction. In this experiment it was our priority to obtain sufficient RNA from embryos for sequencing. Since the sacrifice of adult fish or viable eggs is necessary for the measurement of cortisol in sticklebacks it was therefore not possible to set aside a sufficient quantity of mothers, eggs, or offspring for steroid measurement.To obtain sufficient quantity of RNA for quantitation, quality control, and template for cDNA library creation, we haphazardly selected and pooled ten embryos from each mother . Pools represent independent mothers and so are treated as biological replicates. Embryo pools were homogenized and total RNA extracted from the homogenate using the AllPrep DNA/RNA Micro Kit  according to the manufacturer's instructions as in . BrieflyFor all samples cDNA libraries were constructed from 1 ug total RNA using the TruSeq Stranded RNAseq Sample Prep kit . These were quantitated on a Qubit fluorometer , run on an Agilent bioanalyzer chip, diluted to 10 nM, and quantitated again with qPCR. The library construction process selectively purified for mRNA using oligodT primers. Each of the 16 samples was individually barcoded. All samples were combined into a single pool which was divided in half and sequenced on two single-end lanes on an Illumina HiSeq 2000 (TruSeq SBS sequencing kit version 3) at the Roy J. Carver Biotechnology Center . RNA-sequencing of embryo samples produced over 356 million reads each 100 nt in length with average quality scores over 30; there were 22.28±0.48 million reads (mean ± standard error) per sample.exactTest function in EdgeR . Second, we used Cufflinks/Cuffdiff (version 2.1.1). Cuffdiff employs an algorithm that probabilistically divides ambiguous fragments among genomic loci and then counts expression data at the transcript level, resulting in counts of Fragments Per Kilobase of exon per Million mapped reads  using TopHat 2 (version 2.0.8). Trimmed reads expressed at very low level were filtered out (described below). Across all samples 88.95±0.18 percent (mean ± stderr) of trimmed reads mapped to the genome, and 51.87±0.79 percent (mean ± stderr) that mapped to the genome mapped uniquely within known or predicted genes, consistent with other RNA-seq studies that used genomes with comparable assembly and annotation quality ds FPKM, . EdgeR uds FPKM, . The lisds FPKM, . The majhttp://www.genenames.org); 101 gene symbols for the remaining Ensembl IDs were obtained manually from the Ensembl database  based on 1-to-1 orthology to other vertebrate species, for a total of 393 genes (∼86% of the dataset) with gene symbols. To visualize the general relationships between sample genome-wide expression levels we used the plotPCA command in R to visualize the clustering of samples on the first and second principle components.Stickleback Ensembl gene IDs were downloaded from BioMart (toplevel.fa version 70 ensemble.org) for all 455 differentially expressed genes along with 292 of the corresponding gene symbols The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus H. sapiens. A total of n = 317 of the genes with gene symbols (∼81%) were successfully recognized within DAVID. GO annotations representing biological processes (GOTERM_BP_FAT), cellular components (GOTERM_CC_FAT), molecular functions (GOTERM_MF_FAT), and KEGG pathways were considered significant with a FDR of 0.05. The DAVID analysis was carried out a second time using Human Ensembl IDs corresponding to 15504 of the 17440 detected stickleback genes (∼89%) to test the reliability of using genome as background. The majority of the 317 genes with gene symbols (>80%) were not represented in the output when Human Ensembl IDs converted from the list of detected stickleback genes were used as background, likely due to incomplete annotation of genes with official gene symbols within DAVID. Using the 17440 detected (i.e. expressed) genes as background instead of the whole genome reduced the total number of terms that met the FDR threshold but did not change the nature of the terms obtained (results not shown). Due to the fact that some gene symbols might not match between stickleback and humans and to test the effect of species-specific quality of annotation within DAVID we carried out additional analyses. The first additional analysis was run using mouse annotations and mouse genome background: 294 of 455 genes from the list of differentially expressed genes (64.6%) had corresponding gene symbols within DAVID. The second additional analysis was run using zebrafish annotations and zebrafish genome as background: 165 of the 455 differentially-expressed genes (36.3%) had corresponding gene symbols within DAVID. Finally to evaluate whether some of the identified GO terms might reflect spurious associations, a random list of n = 455 genes were selected from the total 17440 expressed genes in stickleback embryos and run through DAVID using human annotations and genome as background. This procedure was repeated three times. In all 71–75% of genes in the lists were mapped within DAVID, and all three trials produced no significant GO_BP, GO_CC, GO_MF, or KEGG pathways below the FDR = 0.05 cutoff.To identify enriched gene ontology (GO) categories the list of 393 genes with gene symbols was analyzed with the DAVID Functional Annotation Tool v6.7;  using thA separate functional classification of the genes differentially expressed in stickleback embryos was performed using Ingenuity Pathways Analysis (IPA)  using Human Ensembl Gene IDs converted from the 455 Stickleback Ensembl Gene IDs in BioMart. There were corresponding Human Ensembl Gene IDs for n = 302 of the 455 stickleback genes in the dataset (∼66%). However because the human genome has multiple orthologs per gene relative to the stickleback genome, the number of Human Ensembl Gene IDs (n = 596) we obtained was greater than the number of genes in the original dataset. Gene identifiers, their fold changes, and FDR values for the 596 human genes were uploaded to IPA and queried against the Ingenuity database to identify biological functions enriched in the dataset, at default stringency. A total of n = 334 of the 596 Human Ensembl Gene IDs (∼56%) were successfully recognized by IPA.To understand whether changes in gene expression relate to morphological changes we measured total length and eye diameter on a subset of embryos. Clutches were thawed, decanted to glass petri dishes and photographed at 15× magnification under an Olympus SZX12 stereo light microscope with a DF Plapo 1x-PF objective and an Olympus DP26 camera. The anterior/posterior axis of each embryo  , includiPathway analysis in IPA revealed 13 clusters that represent cell proliferation, hematopoeisis, sensory organ development , adhesion of immune cells, kidney development, and metabolism of amino acids .Several genes involved in oxygen transport, ATP synthesis, and other metabolic processes were significantly upregulated in maternally-stressed embryos including embryonic hemoglobin alpha (hbae1) and beta (hbbe1.1), all of the major cytochrome c oxidase subunits , cytochrome b (CYTB), and lactate dehydrogenase D (LDHD). The gene encoding thyroid hormone receptor interactor 13 (TRIP13) was downregulated.Numerous genes involved in the differentiation of neurons and the formation and survival of neurites were differentially expressed between maternal treatments . These iIn three day post-fertilization sticklebacks the HPI axis is not yet active . Therefore we did not expect to observe the differential expression of genes related to cortisol synthesis in this study. As predicted, several genes central to HPI axis function were not expressed at all , including corticotropin-releasing hormone (CRH), the CRH receptor 1 (CRHR1), one gene encoding proopiomelanocortin (POMC), the adrenocorticotropic hormone receptor (MC2R), and one of the genes encoding 3-β-hydroxysteroid dehydrogenase (HSD3B2), which is involved in the de novo synthesis of cortisol. Interestingly, however, some genes related to HPI axis functioning were expressed at low levels but were not significantly different between maternal treatments, including the second gene encoding POMC, and the genes encoding proprotein convertase 1 (PCSK1) and steroidogenic acute regulatory protein (star). Several genes involved in the production of cortisol precursors, and the reception and metabolism of cortisol were expressed at detectable levels but were not differentially expressed between maternal treatments, including one of the genes encoding 3-β-hydroxysteroid dehydrogenase (HSD3B7), the genes encoding 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) and 3 (11-beta-hsd3), and the two genes that code for glucocorticoid receptor (NR3C1). There was one gene, the FK506 binding protein 4 (FKBP4), that was differentially expressed . Also differentially expressed in maternally-stressed embryos were genes that are known to have altered expression in tissues treated with the synthetic glucocorticoid dexamethasone, including downregulation of C3 and retinoic acid receptor gamma (RARG), and upregulation of SOD1, CISH, NFKBIA, and IGFBP2.Glucocorticoids can affect immunity We detected the differential expression of several genes involved in normal development, including genes responsible for the genome-wide epigenetic control of gene expression. Several genes involved in the maintenance of epigenetic modifications  were difInterestingly, genes involved in sexual differentiation were also differentially expressed between treatments. Enzymes responsible for the metabolism of sex steroids were upregulated including steroid sulfatase (STS) and 17β hydroxysteroid dehydrogenase 10 (HSD17B10). Gonadotropin-releasing hormone receptor 1 (GNRHR1), responsible for the reception of GNRH and subsequent release of LH and FSH during sexual maturation, was downregulated. Also upregulated were the male sex-determining protein (DMRT1Y) and one gene encoding sex determining region Y-box 4 (SOX4).Several non-coding RNAs were differentially expressed in embryos as a result of maternal exposure to predation risk including upregulation of many small nucleolar RNAs , upregulation of two novel microRNAs , and downregulation of the microRNA miR-9a.1,14.4 = 5.241, p = 0.038; 1,16.1 = 0.629, p = 0.439).Embryos of mothers exposed to predation risk were longer along the anterior-posterior axis  than embryos of control mothers . Also, we detected a significant effect of maternal stress on the expression of genes shown to be similarly influenced in humans and other mammal cells by exposure to the synthetic glucocorticoid dexamethasone. It is therefore possible that some of the genes differentially expressed in stickleback embryos in response to maternal stress were activated or repressed through the action of maternal cortisol, though cortisol was not measured in this experiment. Further study is needed to understand the magnitude and tissue specificity by which maternal glucocorticoids might influence the embryonic transcriptome.Consistent with the known effects of glucocorticoids on immune function, we detected the differential regulation of several immune genes, including genes involved in the innate and adaptive immune responses. Previous studies in humans Sexual differentiation in sticklebacks does not begin until the primordial germ cells (PGCs) within the gonads of females begin to proliferate at 11 days post-fertilization Several factors involved in the epigenetic silencing of gene transcription, including multiple DNA-methyltransferases and two histone proteins, were differentially expressed in maternally-stressed embryos. DNMT3 is responsible for de novo methylation, specifically methylation at CpG sites, during development The unbiased whole transcriptome approach we used allowed us to identify the differential expression of several small RNAs that are not picked up in traditional microarray studies. We identified several microRNAs differentially expressed between maternally stressed and control embryos including several small nucleolar RNAs . Some snoRNAs are thought to regulate specific alternatively spliced transcripts We also detected several novel differentially expressed miRNAs including one (ENSGACG00000022837) that was expressed more than 193-fold higher in maternally-stressed embryos than control embryos (FDR  = 0.003). It is possible that some of these known or novel miRNAs are involved in the epigenetic transfer of maternal stress to offspring. For example a miRNA in zebrafish (miR-430) accelerates the deadenylation of maternal mRNAs during embryonic development, promoting their clearance from embryos These results suggest that early stickleback embryos respond to maternal exposure to predation risk via changes in gene expression. Embryos of predator-exposed mothers had greater expression of genes involved in neurite formation, the development of the visual system and aerobic and anaerobic metabolism. Embryos of predator-exposed mothers were also larger. Together these results suggest that offspring of mothers exposed to predation risk undergo a general acceleration of the developmental program. Given that a faster life history is associated with high predation environments Figure S1The intersection of upregulated, downregulated, and total differentially expressed genes as identified in EdgeR and Cuffdiff from the same alignment of reads to the stickleback reference genome .(DOC)Click here for additional data file.Figure S2comparison of fold change data of n = 108 genes identified via both Bowtie-edgeR and Cufflinks-Cuffdiff analyses as differentially expressed in stickleback embryos exposed to maternal stress. While Cuffdiff generally called a higher fold change than edgeR (slope  = 0.88), there was nevertheless a tight correlation between the two (R-square  = 0.855). All genes were found to be upregulated (quadrant I) or downregulated (quadrant III) in both analyses.(DOC)Click here for additional data file.Figure S3A Principle Components Analysis of general gene expression patterns of each sample as determined by RNA-seq and edgeR differential expression analysis. Blue squares represent embryos of mothers exposed to predation risk and red circles represent embryos of control mothers. Each data point represents total RNA from 10 pooled embryos from a single mother.(DOC)Click here for additional data file.Figure S4A diagram illustrating the measurements taken of stickleback embryo length (A) and eye diameter (B). Morphological data were then compared between embryos of mothers exposed to a predator and embryos of control mothers.(DOC)Click here for additional data file.Table S1The stickleback genes found to be differentially expressed in 3dpf embryos of mothers exposed to a predator compared to embryos of control mothers. Direction: “up”  =  higher expression in embryos of mothers exposed to a predator compared to embryos of control mothers, “down”  =  lower expression in embryos of mothers exposed to a predator compared to embryos of control mothers. FDR  =  false discovery rate , logCPM  =  log(adjusted counts per million reads) from edgeR analysis, FPKM  =  fragments per kilobase of exon per million mapped reads, the count method used in Cufflinks analysis, “shared?”  =  whether a gene was identified as differentially expressed using both EdgeR and Cuffdiff analyses (yes) or in one analysis but not the other (blank).(XLS)Click here for additional data file.Table S2Mus musculus gene annotations and genome background within the DAVID Functional Annotation Tool (v6.7). Only terms with FDR <0.05 and ≥2 genes were included.The most overrepresented biological annotations from the list of genes differentially expressed between 3dpf stickleback embryos exposed to maternal stress and embryos of control mothers as measured via RNA-seq. Genes were mapped to their associated biological process (BP_FAT), cellular component (CC_FAT), molecular function (MF_FAT), and KEGG pathways using (XLS)Click here for additional data file.Table S3Danio rerio gene annotations and genome background within the DAVID Functional Annotation Tool (v6.7). Only terms with FDR <0.05 and ≥2 genes were included.GO terms and KEGG pathways obtained using (XLSX)Click here for additional data file.


Crash mice, which possess a dominant mutation in Celsr1, an orthologue of the core planar cell polarity (PCP) Drosophila protein Flamingo . We observe a failure of ventral enclosure in Crash mutants suggesting that defective epidermal spreading might underlie some ventral wall birth defects.Epiboly is a morphogenetic process that is employed in the surface ectoderm of anamniotes during gastrulation to cover the entire embryo. We propose here that mammals also utilise this process to expand the epidermis and enclose the body cavity and spinal cord with a protective surface covering. Our data supports a model whereby epidermal spreading is driven by the primary establishment of the epidermal basal progenitor monolayer through radial cell intercalation of a multi-layered epithelium towards the basal lamina. By using a suspension organotypic culture strategy, we find that this process is fibronectin-dependent and autonomous to the skin. The radial cell rearrangements that drive epidermal spreading also require ROCK activity but are driven by cell protrusions and not myosin II contractility. Epidermal progenitor monolayer formation and epidermal spreading are delayed in  Summary: The nascent mammalian epidermis spreads to enclose the embryo trunk through a process akin to epiboly, which has important implications for human birth defects of the abdominal wall. Epiboly is one of a series of complex tissue movements that shape the basic body plan of species of amphibian and fish embryos . In amphIn mammals, the surface ectoderm of the developing embryo transforms, through a process of tissue stratification, into the skin (epidermis), which protects our bodies from mechanical, chemical and microbial insult. Stratification of the epidermis can be visualised in the mouse embryo from around embryonic day (E)13.5 (depending on mouse strain) and is marked by the appearance of the suprabasal layer . DelaminXenopus, we find that epidermal thinning and spreading is dependent on fibronectin but also requires ROCK activity and actin-rich lamellipodial protrusions, but not myosin II contractility. Epidermal spreading is disrupted in the Celsr1 mouse mutant Crsh and correlates with defects in ventral closure of the embryonic body in this mutant. These findings argue that the mammalian embryonic skin encloses the embryo through a morphogenetic strategy utilised by anamniotes to spread their surface ectoderm and provide new insights into the underlying basis of abdominal wall defects.Although we understand the embryonic origins of the skin , very liFig. S1A–C). The latter also revealed that flank ectoderm did not fully enclose the embryo body even by E14 . Our findings therefore reveal a hitherto unrecognised morphogenetic process in the mid-gestation mammalian embryo: the dorsal and ventral enclosure of the embryonic body surface. This is an important period of development and correlates with an increase in the circumference of the embryonic trunk but not its anterior-posterior (forelimb-to-hindlimb) length .To investigate early epidermal development we analysed wild-type mouse embryos staged between E13.25–E13.75 when the epidermis had not yet stratified. We examined hematoxylin and eosin (H&E)-stained wax-embedded transverse sections and observed several intriguing features along the dorso-ventral extent of the E13.25 embryonic body surface that had disappeared by E13.75. H&E staining was most intense in the embryo flank . Quantification of cell packing index (i.e. the number of cells per unit area) revealed a significant increase in basal layer cell packing  between E13.25–E13.5 and disappearance of the middle layer of cells by E13.5 . Suprabasal cells appeared during the squamous phase at around E13.75 and were marked by cytokeratin-1 . We consistently distinguished a period of 9–12 h between the onset of surface ectoderm spreading at E13.25 and the appearance of epidermal suprabasal cells around E13.75 (n>8 litters). Taken together, therefore, our data suggest that epidermal progenitor layer formation is coupled to surface ectoderm spreading and dorsal and ventral enclosure of the embryonic body.Subsequent staining with markers of basal progenitors confirmed the emergence of superficial periderm from E13.25 (Xenopus (ex vivo) culture. We made explants from wild-type flank and cultured them for 15 h, at which point the epidermis had begun stratification and expressed cytokeratin-1 . We first tested variations on the well-established Trowell culture , albeit columnar and not squamous . Lumox culture therefore recapitulated the in vivo situation , and we considered it a suitable organotypic culture method to study formation of the epidermal basal monolayer.Our data revealed that formation of the skin progenitor monolayer correlated with the conversion of a disordered multi-cell layered surface epithelium (hereafter immature epidermis) into an orderly basal monolayer with overlying squamous periderm. This transformation correlated with spreading of the embryo surface and was strikingly reminiscent of epiboly in Xenopus . To test culture ; Fig. 2An=4 timecourse experiments) but then recovered (4–6 h) and eventually spread beyond its original size after 8–10 h in culture , and a high cell-death index in the superficial periderm . Taken together, these data suggest that the increases in epidermal thickness and middle layer and superficial layer (periderm) packing resulted from cell rearrangement although cell proliferation must also play a role. Importantly, basal cell packing hardly changed, indicating that planar cell shape changes did not contribute to surface area reduction. It seems most likely therefore that both basal cells and middle cells were displaced superficially, thus thickening the middle and superficial periderm layers during this period. Three-dimensional reconstruction of live cells from explants in which we had induced a mosaic expression of membrane GFP (mGFP), further supported this hypothesis  and correlated with the formation of a columnar basal monolayer  with no overlying superficial periderm cells, as observed in vivo , supporting the hypothesis that Lumox culture recapitulates progenitor monolayer formation in the embryo.A full recovery and an increase in surface area beyond their original size was consistently observed in explants by 8 h  and, importantly, very few basal layer telophase divisions were horizontal  revealed that middle layer cells moved basally and basal layer cells became columnar (Movies 1–3) consistent with our model of radial intercalation. Taken together, the data argue against surface shedding of middle cells as explants recover surface area. Superficial or periderm cells remained at the tissue surface, arguing against any significant contribution to the basal layer . We predicted that the absence of a middle layer would prevent spreading. We found that these later explants behaved as the earlier stage explants  except that they recovered little of their surface area and did not expand beyond their original size , when the middle layer had mostly disappeared  cell intercalation mechanism in which middle layer and basal layer cells of the disordered multi-cell layered surface epithelium generate the basal monolayer. A mechanism of radial intercalation explains how the surface area of the embryonic surface ectoderm expands during this first phase of spreading when basal cells become more orderly and increasingly well packed L. Our exryo body L. It is Our explant assays support an epiboly-type mechanism for the epidermal spreading process associated with dorsal and ventral body enclosure. Molecular studies in teleosts (zebrafish) have implicated the Celsr family of adhesion G-protein-coupled receptors in epiboly . Celsr1 Celsr1, named Crash . Timing of mammary gland formation was similar between Crsh mutants and wild-type littermates (data not shown) ruling out any developmental delay. To test whether the failure of ventral enclosure correlated with defective epidermal spreading, we performed a timecourse of Crsh flank skin explant spreading in Lumox culture. We found that despite a predominance of horizontal cell divisions in the basal layer , Crsh homozygote explants were significantly less able to recover their surface area from 4 h to 10 h in culture compared to wild-type littermates  argue that the thickened surface epithelium apparent in the mutant at E13.75 in vivo , a key component of the core planar cell polarity (PCP) pathway . We also observed that the timing of basal monolayer formation was similar in fz6-knockout embryos and wild-type littermates  despite clear later stage planar polarisation phenotypes in the skin of the homozygote mutant . This regional pattern explained the variability in basal layer cell packing from E13.25 to E13.5 in vivo  whereas laminin was uniformly enriched at the basal lamina (data not shown). Conversely, where we found basal monolayer formation in Crsh homozygote epidermis . We also observed abnormal fibronectin deposition in the surface epithelium of E12.5–E13 Crsh homozygotes compared to wild-type, with the homozygote mutants exhibiting fibronectin deposition at both superficial and basal interfaces . Taken together, we conclude that deposition of fibronectin within the immature epidermis was defective in Crsh homozygotes.In wild-type, we were also struck by the increasing enrichment of fibronectin at the boundary between epidermis and underlying dermis  H,I. Vincpidermis M–P′, fibpidermis H and vinXenopus is also demonstrably fibronectin-dependent  peptides, which mask integrin-binding sites on fibronectin, thereby inhibiting integrin–fibronectin interactions and adhesion , whereas explants from littermates incubated with the non-inhibitory RGE peptide  as observed in wild-type  basal lamina and move basal-ward, increasing the occupancy of the basal layer to drive epidermal spreading.Epiboly in ependent . The abeadhesion . We testadhesion A. Staini peptide  formed aild-type M. This iCrsh homozygotes, which also exhibit neural tube defects (Celsr1 and Rho kinase (ROCK) activity. Equally compelling were reports that mouse mutants in ROCK1 and ROCK2 display abdominal wall defects  when compared to DMSO-treated controls . We next asked whether ROCK activity was linked to actomyosin contractility, as reported in the avian neural tube  despite a significant reduction in cell division when used at higher concentrations  but their surface area did not reduce significantly  at both cell vertices and cell interfaces H. Actin-ficantly A. Taken ficantly . Arp2/3 ficantly . Inhibitficantly A,F–G″. Nficantly H. Cell pficantly J but so ficantly I. Importficantly H, suggesProgenitor epithelia are essential building blocks for tissue and organ construction. The findings of this study argue that the progenitor monolayer epithelium  of the mammalian skin is established through a morphogenetic process akin to epiboly in anamniotes that concomitantly spreads the surface ectoderm of the mammalian embryo to enclose the body. In anamniotes (frogs and fish), epiboly expands the ectoderm to cover the entire embryo at gastrulation stages. Epiboly-like cell intercalation of definitive endodermal cells has also been reported during mouse gastrulation . We propin vivo and ex vivo we find that basal monolayer formation correlates with the disappearance of a middle cell layer from the multi-cell layered epithelium of the surface ectoderm. Our explant experiments support a model for radial intercalation of middle cells towards the epidermal basal lamina as the cellular basis for basal monolayer formation. The importance of epidermal spreading for establishing an orderly basal monolayer is further supported by the failure of both these processes in the Celsr1 mouse mutant, Crash. Importantly, radial intercalation of middle cells into the basal layer would contribute new basal cells during an important period of circumferential growth of the embryo body  and thus ensure expansion of the surface ectoderm at this time. Radial intercalation offers the embryo a rapid route to increasing basal layer occupancy. Indeed, it is unlikely that horizontal cell divisions could alone achieve the required increase in basal cell numbers between E13.25 and E13.5. Furthermore, our ex-vivo data suggest that cell division maintains a middle layer of cells between E13.25–E13.5. We do expect, however, that horizontal cell divisions within the established progenitor monolayer directly contribute to tissue spreading between E13.5 and E13.75.Both Crsh, revealed defects in ventral enclosure which might correlate with abdominal wall defects observed in this mutant  and the fz6-knockout mouse (C57BL6) have been described previously  of each z-stack were generated for each image. All telophase divisions and their underlying basal lamina were individually cropped and the resulting images rotated (3D-opacity mode) until the segregation of each chromatid pair could be measured relative to the basal lamina (marked by staining for fibronectin). One snapshot was taken from different sides of any given division. Angles of cell division relative to the basal lamina were measured using the ruler tool in Adobe Photoshop and the mean angle for each division calculated. In each case, a line was first drawn along the basal lamina and then through the centre of each chromatid, which appeared in side-view . view see F. Polar For frozen sections, epidermis was assessed on both sides of four longitudinal sections and scored for cell depth along their anterior–posterior extent. Individual basal cells sitting on the basal lamina were scored. Local cell thickness was quantified by counting cells overlying each cell sitting on the basal lamina. For whole-mounted epidermis and explants, multiple images of whole-mount immunostained tissue were analysed using Volocity. Different cell groups from different areas of the image were individually cropped and an image snapshot exported into Adobe-Photoshop. The depth of the epidermis was measured using the ruler tool and converted with reference to the scale bar from Volocity.Multiple images of whole-mount immunostained tissue were analysed using Volocity. Cell packing data were collected by counting nuclei along 100-μm sample lengths in multiple cropped images taken from different areas of the image and at different orientations. For any one image, the number of nuclei in each layer along every 100-μm sample length was scored.Contour length of embryonic epidermis from transverse paraffin sections and from images of whole embryos taken on a Leica dissecting microscope was measured in ImageJ.For whole-mount immunostained tissue, the mitotic index was scored using immunostaining for phospho-histone H3 (explants). Cell death index was scored by immunostaining for active caspase-3. The mitotic index and cell death index was calculated for each 1000 basal cells contained within every image analysed. At least three explants from two different litters were analysed or three embryo whole-mounts in each case.Embryos were dissected from the uterus in HBSS (Sigma) and flank skin dissected away from both sides of the embryo using Lumsden scissors as follows: back skin was first cut along the lateral edge of the spinal cord from the forelimb to the hindlimb. Just inside of the forelimb a 90° turn was executed and the epidermis was cut to just beyond the level of the forelimb. Epidermis was similarly cut from the spinal cord to the just beyond the level of the hindlimb and a rectangular piece of flank skin removed. Tissue intrinsic morphological criteria described above were used to determine the stage of epidermal morphogenesis for dissection purposes.−4 M adenine (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 5 µg/ml insulin (Sigma), 10−10 M cholera enterotoxin (Sigma), 10 ng/ml EGF (Peprotech)] and cultured at 37°C under 5% CO2 (A significant layer of mesoderm was left attached to the epidermis. Explants were immediately placed onto a solid support [PET-track-etched-membrane (Becton-Dickinson) or nucleopore-track-etched-membrane (Whatman)]. Explants cultured on PET-membranes were mounted on a sterile-grid support suspended on a Center-well organ culture dish , overlain with a small amount of epidermal medium [DMEM (Invitrogen) supplemented with fetal bovine serum (10%), 1.8×10r 5% CO2 . NucleopEpidermis and dermis were peeled away from the underlying mesoderm. ‘Peeled’ explants were placed immediately into Lumox dishes (Starstedt) containing the epidermal medium described above. Such explants were free to curl up in culture bringing together the edges, which eventually sealed after 15 h. As a result, the dermis was internalised and covered. Explants were cultured for up to 24 h and fixed in 4% formaldehyde in PBS for either 1 h (whole-mount immunohistochemistry) or 24 h (paraffin sectioning).n=4) and each explant individually subjected to whole-mount immunostaining to label cell membranes (E-cadherin), mitotic index (phospho-histone H3) and cell death (activated caspase-3).Epidermis was dissected as described for Trowell culture, immersed into a droplet of HBSS, epidermal side up and captured under a glass coverslip secured by two spots of vacuum grease. Slight pressure was exerted to flatten the explant and a bright-field image was taken at low magnification using a Leica dissecting microscope. The glass coverslip was then carefully lifted and the explant transferred onto a membrane support (Trowell culture) or the epidermis was peeled away from the mesoderm and transferred into suspension culture. After the appropriate culture time, Trowell culture explants were lifted into a droplet of HBSS and again captured under a glass coverslip secured by two spots of vacuum grease. Slight pressure was exerted to flatten the explant and a bright-field image was taken at the same magnification as pre-explant. Sharp forceps were used to uncurl Lumox explants (4 h to 10 h) or break into the sealed Lumox explant and open up the tissue (15–24 h). In the latter, the explant was then cut at four equidistant sites from the edge to just inside the centre so that the explant could be opened out flat like a flower with four petals. All explants were lifted into a droplet of HBSS and captured under a glass coverslip secured by two spots of vacuum grease. Slight pressure was exerted to flatten the explant and a bright-field image was taken at the same magnification as pre-explant. For all explants, surface area was measured by measuring the cumulative width and lengths of rectangular sections  of each explant image both prior and subsequent to culture. For the timecourse experiment, at each time point the change in surface area of several explants was measured (Stock solutions of Jasplakinolide (Sigma), cytochalasinB (Sigma), Y27632 (Stemgent), Blebbistatin (Sigma) and CK666 (Tocris) were made in DMSO. RGD and RGE peptides  were resuspended in PBS and added to the culture media to the final concentrations described.z-stack image was taken at 25× magnification (water dipping objective) using a Leica SP5 laser scanning confocal microscope. The explant was gently moved away from under the coverslip and allowed to develop further without constraint. This process was repeated at a second time point. To register the same group of cells over time a defined area of the explant was marked with DiI on the explant surface. Successful z-stack images of these cells were cropped in the xy and z planes to register the superficial cells, which remained at the tissue surface, and superimposed using Imaris8.0 3D imaging software . Individual cell shapes in the merged image were generated manually using each optical section of the z-stack and the surfaces function in Surpass view (Imaris). The centre of the nuclear mass for each fluorescent cell was concurrently measured by identifying the z-position of the top and bottom of the nuclear space and calculating the central point. The z-position was measured from the top of the superimposed z-stack image. Statistical analysis was the paired t-test.Mosaic mGFP was induced by injecting pregnant females with 1.7 mg Tamoxifen with progesterone 2 days prior to explanting. Explants were dissected as described above and allowed to develop in Lumox culture for 3–7 h. At the appropriate time, individual explants were gently pushed under a glass coverslip secured with vacuum grease and immobilised by a gentle pressure on the coverslip which slightly flattened the explant. A