Chi Ho

Catherine Leung

Marc Leclerc

Alan M Moreau

Andrew L Shipley

Sarah E Miller

Webb

Ho Chi Leung

Chi

Leclerc

Andrew L ; Miller

Catherine Leclerc

Marc Moreau

Alan M Shipley

Sarah E Webb

Transmembrane H fluxes and the regulation of neural induction in Xenopus laevis

Keywords: H+ fluxes, neural induction, Xenopus laevis, extracellular ion-selective electrode, Zic3 expression H + fluxes, neural induction, Xenopus laevis, extracellular ion-selective electrode, Zic3 expression

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Introduction

In cells, ion channels and transporters located in the plasma and organellar membranes generate ion-specific fluxes (reviewed by [START_REF] Meissner | Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum[END_REF][START_REF] Zhu | TPCs: Endolysosomal channels for Ca 2+ mobilization from acidic organelles triggered by NAADP[END_REF][START_REF] Szabo | Mitochondrial channels: Ion fluxes and more[END_REF][START_REF] Xu | Organellar channels and transporters[END_REF][START_REF] Carraretto | Ion channels in plant bioenergetic organelles, chloroplasts and mitochondria: From molecular identification to function[END_REF]. These generate electrical or chemical signals that play vital roles in specific biological events. The coordinated action of all the channels and transporters located in a membrane within a cell in so-called 'ion channel networks', results in the differential accumulation of ions (and thus electric charge) across this membrane [START_REF] Longden | Measurement of extracellular ion fluxes using the ion-selective self-referencing microelectrode technique[END_REF]. It is these distinct heterogeneities in charge across membranes that govern the overall electrical properties and thus the signaling properties of a particular cell, tissue, organ, or organism [START_REF] Talevi | Electrical characteristics of ascidian egg fragments[END_REF][START_REF] Gusovsky | Formation of second messengers in response to activation of ion channels in excitable cells[END_REF][START_REF] Simone | Non-specific currents at fertilization in sea urchin oocytes[END_REF]Monteiro et al., 2014;Luxardi et al., 2015;[START_REF] Longden | Measurement of extracellular ion fluxes using the ion-selective self-referencing microelectrode technique[END_REF].

It is well established that ion fluxes either into or out of cells, are correlated with fertilization, development, differentiation, growth, regeneration, pattern formation, and homeostatic regulation in a diverse array of biological systems, including animals, fungi, and plants [START_REF] Jaffe | Control of development by ionic currents[END_REF][START_REF] Simone | Non-specific currents at fertilization in sea urchin oocytes[END_REF][START_REF] Tosti | Dynamic roles of ion currents in early development[END_REF][START_REF] Tosti | Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis[END_REF][START_REF] Hunter | Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure[END_REF][START_REF] Tosti | Ion currents in embryo development[END_REF][START_REF] Carvacho | Ion channel function during oocyte maturation and fertilization[END_REF][START_REF] Mclaughlin | Bioelectric signaling in regeneration: Mechanisms of ionic controls of growth and form[END_REF].

In addition to intracellular ion fluxes, developing organs and organisms often also require the activity of ions in the localized extracellular environment [START_REF] Slack | Intracellular and intercellular potentials in the early amphibian embryo[END_REF][START_REF] Jaffe | Control of development by ionic currents[END_REF][START_REF] Jaffe | The role of ionic currents in establishing developmental pattern[END_REF][START_REF] Jaffe | Strong electrical currents leave the primitive streak of chick embryos[END_REF][START_REF] Kline | Ion currents and membrane domains in the cleaving Xenopus egg[END_REF][START_REF] Nuccitelli | The wave of activation current in the egg of the medaka fish[END_REF]Rathore et al., 1988;[START_REF] Altizer | Endogenous electric current is associated with normal development of the vertebrate limb[END_REF]. In plants, for example, one of the earliest reports demonstrated that an endogenous Ca 2+ current was driven through the eggs of the brown fucoid algae Pelvetia fastigiata during zygotic polarization (Robinson and lily (Lilium longiflorum) pollen tubes [START_REF] Weisenseel | The major growth current through lily pollen tubes enters as K + and leaves as H +[END_REF], and H + currents were reported to traverse the growing roots and root hairs of barley (Hordeum vulgare; [START_REF] Weisenseel | Natural H + currents transverse growing roots and root hairs of barley (Hordeum vulgare L.)[END_REF]. These early papers were quickly followed by a plethora of reports of ion fluxes entering and leaving a variety of tip-growing and polarizing plant structures from a wide range of different species [START_REF] Miller | ATPase proton pumping activity is required for adult zebrafish appendage regeneration[END_REF][START_REF] Miller | Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotiana tabacum: spatial relationship proposed between resulting electrical fields and phtophthoran zoospore infection[END_REF][START_REF] Miller | Correlation between profile of ion-current circulation and root development[END_REF][START_REF] Jones | Role of calcium and other ions in directing root hair tip growth in Limnobium stoloniferum: I. Inhibition of tip growth by aluminum[END_REF][START_REF] Kühtreiber | Detection of extracellular calcium gradients with a calcium-specific vibrating electrode[END_REF][START_REF] Feijó | The cytosolic Ca 2+ concentration gradient of Synapis alba root hairs as revealed by Ca 2+ -selective microelectrode tests and fura-dextran ratio imaging[END_REF][START_REF] Messerli | Pulsatile influxes of H + , K + and Ca 2+ lag growth pulses of Lilium longiflorum pollen tubes[END_REF][START_REF] Zonia | Oscillatory chloride efflux at the pollen tube apex has a role in growth and cell volume regulation and is targeted by inositol 3,4,5,6-tetrakisphosphate[END_REF][START_REF] Xu | Application of non-invasive microsensing system to simultaneously measure both H + and O 2 fluxes around the pollen tube[END_REF]. With regards to fungi, early reports described ion currents traversing the growing hyphae of the water mold Achlya bisexualis [START_REF] Kropf | Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry[END_REF] as well as the bread mold Neurospora crassa [START_REF] Mcgillviray | The transhyphal electrical current of Neurospora crassa is carried principally by protons[END_REF]. More recently, transmembrane H + and Ca 2+ fluxes have also been reported during apical growth and gravi-reception in stage 1 sporangiophores of the fungi Phycomyces blakesleeanus (Živanović, 2012).

In animals, Na + currents have been demonstrated in the near vicinity of regenerating limbs of the red-spotted newt Notophthalmus viridescens [START_REF] Borgens | Bioelectricity and regeneration: Large currents leave the stumps of regenerating newt limbs[END_REF], as well as the regenerating tails of Xenopus laevis tadpoles (Tseng and Levin, 2008). Furthermore, transmembrane fluxes of H + and K + have been reported to play a role in the regulation of head and organ size during regeneration of the planaria Schmidtea mediterranea [START_REF] Beane | Bioelectric signaling regulates head and organ size during planarian regeneration[END_REF]; and an efflux of H + has been reported to occur during regeneration of the caudal fin in zebrafish (Danio rerio; Monteiro et al., 2014). In addition, during embryogenesis, an early report demonstrated that the membrane of early Xenopus embryos is highly permeable to K + [START_REF] Slack | Intracellular and intercellular potentials in the early amphibian embryo[END_REF].

Furthermore, effluxes of H + , K + , and HCO 3 -and influxes of K + and Ca 2+ have been reported [START_REF] Moreau | Free calcium changes associated with hormone action in amphibian oocytes[END_REF][START_REF] Faszewski | Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology[END_REF]. Ion fluxes have also been reported around the follicles and eggs of Drosophila melanogaster [START_REF] Overall | Patterns of ionic current through Drosophila follicles and eggs[END_REF], an efflux of H + is reported to initiate development of sea urchin (Strongylocentrotus purpuratus) eggs just after fertilization (Johnson et al., 1976), and more recently Ca 2+ fluxes were found in the cleavage furrow of dividing zebrafish embryos [START_REF] Chan | Inhibition of SOCE disrupts cytokinesis in zebrafish embryos via inhibition of cleavage furrow deepening[END_REF]. An efflux of H + coupled to an influx of Ca 2+ has also been reported to create favorable alkaline internal conditions for calcification events during shell formation of the freshwater common pond snail Lymnaea stagnalis [START_REF] Ebanks | Characterization of the mechanisms for Ca 2+ and HCO 3 -/CO 3 2acquisition for shell formation in embryos of the freshwater common pond snail Lymnaea stagnalis[END_REF].

Ion currents are also associated with key physiological processes, such as those required for homeostatic regulation. For example, transepithelial ion fluxes have been recorded from H + -pump rich cells located in zebrafish skin that help regulate systemic acid-base homeostasis [START_REF] Guh | Oestrogen-related receptor α is required for transepithelial H + secretion in zebrafish[END_REF], and Ca 2+ fluxes have been reported exiting and entering the scales of zebrafish [START_REF] Hung | Assessing the ability of zebrafish scales to contribute to the shortterm homeostatic regulation of [Ca 2+ ] in the extracellular fluid during calcemic challenges[END_REF] and sea trout (Salmo trutta; [START_REF] Jamieson | Short-term homeostatic regulation of blood/interstitial fluid Ca 2+ concentration by the scales of anadromous Johnson JD, Epel D and Paul M[END_REF] as a short-term mechanism that helps in the regulation of the blood/interstitial fluid Ca 2+ concentration during environmental and behavioral-induced calcemic challenges. In addition, ion fluxes have been reported to traverse a wide variety of neurons from a diverse range of species. For example, Ca 2+ fluxes cross the cell membrane of the abdominal ganglion of the sea slug Aplysia californica when it is under oxidative stress [START_REF] Duthie | Use of a vibrating electrode to measure changes in calcium fluxes across the cell membranes of oxidatively challenged Aplysia nerve cells[END_REF]. It has also been proposed that endogenous electrical currents might guide the rostral migration of neuroblasts in the brain of the neonatal mouse (Mus musculus; [START_REF] Cao | Endogenous electric currents might guide rostral migration of neuroblasts[END_REF]. using a scanning ion-selective electrode technique (SIET). This is an ultra-sensitive technique for measuring extracellular ion fluxes in a non-invasive manner via the use of a single ion-sensitive microelectrode (ISM), which is moved repeatedly between two measuring points located in close vicinity to the sample [START_REF] Kühtreiber | Detection of extracellular calcium gradients with a calcium-specific vibrating electrode[END_REF][START_REF] Kochian | Use of an extracellular, ion-selective, vibrating microelectrode system for the quantification of 610[END_REF][START_REF] Smith | Non-invasive ion probes -tools for measuring transmembrane ion flux[END_REF]Felle and Hepler, 1997;[START_REF] Hung | Assessing the ability of zebrafish scales to contribute to the shortterm homeostatic regulation of [Ca 2+ ] in the extracellular fluid during calcemic challenges[END_REF]. Indeed, when the appropriate ion-selective ionophore sensor is loaded into the ISM tip, the detection of various ions at concentrations as low as at the picomolar level is possible with the SIET. For example, in recent years this technique has been used to detect small Ca 2+ fluxes at the surface of excised metatarsal bones in the mouse [START_REF] Marenzana | Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not due to resorption[END_REF][START_REF] Dedic | Calcium fluxes at the bone/plasma interface: Acute effects of parathyroid hormone (PTH) and targeted deletion of PTH/PTH-related peptide (PTHrP) receptor in the osteocytes[END_REF], and around the scales of adult zebrafish [START_REF] Hung | Assessing the ability of zebrafish scales to contribute to the shortterm homeostatic regulation of [Ca 2+ ] in the extracellular fluid during calcemic challenges[END_REF] and sea trout [START_REF] Jamieson | Short-term homeostatic regulation of blood/interstitial fluid Ca 2+ concentration by the scales of anadromous Johnson JD, Epel D and Paul M[END_REF]. It has also been used to measure effluxes of Na + , Cl -, K + , Ca 2+ and H + in wounds made in the skin of C57BL/6 mice [START_REF] Sun | Ionic components of wound current at mouse skin incisional wounds[END_REF], and an efflux of H + in the excretory pore of Caenorhabditis elegans [START_REF] Adlimoghaddam | Localization of K + , H + , Na + and Ca 2+ fluxes to the excretory pore in Caenorhabditis elegans: Application of scanning ion-selective microelectrodes[END_REF]. Prior to the development of the SIET, ion fluxes could still be identified using a simple voltage sensitive vibrating probe [START_REF] Jaffe | An ultrasensitive vibrating probe for measuring steady extracellular currents[END_REF] in combination with an ion-substitution methodology [START_REF] Reid | Non-invasive measurements of bioelectric currents with a vibrating probe[END_REF], where ions were selectively removed from the measuring medium and the effect on the current density recorded.

Alternatively, intracellular Na + activity and pH and could be measured continuously for several hours with sharp recessed-tip microelectrodes (prepared from Na + -or pHsensitive glass, respectively), which were inserted into cells [START_REF] Thomas | Intracellular sodium activity and the sodium pump in snail neurons[END_REF][START_REF] Thomas | Intracellular pH of snail neurones measured with a new pH-sensitive glass microelectrode[END_REF].

Here, we used the SIET to measure H + fluxes around X. laevis embryos between stage 9 to stage 12 (i.e., from ~7 hpf -13.25 hpf), for the duration of gastrulation (Harland, 2000). Neural induction is initiated during blastula stage 8 and continues throughout the gastrula period [START_REF] Baker | Wnt signaling in Xenopus embryos inhibits Bmp4 expression and activates neural development[END_REF][START_REF] Kuroda | Neural induction in Xenopus: Requirement for ectodermal and endomesodermal signals via chordin, noggin, βcatenin, and Cerberus[END_REF], during which the ectodermal cells adopt either a neural or epidermal fate depending on their location with regards to the dorsal/ventral axis. The dorsal ectoderm is triggered to become neuroectoderm and at the same time the anterior-posterior axis is also initiated [START_REF] Doniach | Planar induction of anteroposterior pattern in the developing central nervous system of Xenopus laevis[END_REF][START_REF] Lamb | Fibroblast growth factor is a direct neural inducer, which combined with noggin generates anterior-posterior neural pattern[END_REF] reviewed by [START_REF] Weinstein | Neural induction[END_REF]Hemmati-Brivanlou, 1999, Harland, 2000). It has been reported that in ex vivo planar explants of X. laevis, the intracellular pH (pH i ) increases (indicating a decrease in intracellular [H + ]) specifically in the dorsal ectoderm cells from stage 10.5 to 11.5 (i.e., 11-12.5 hpf) and it was suggested that this change in pH i plays a role in neural induction [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. These experiments were conducted using the intracellular fluorescent pH indicator bis-carboxyethylcarboxyfluorescein-dextran (BCECF-dextran), which was microinjected into 32-cell stage embryos, in conjunction with monitoring via emission ratio microfluorimetry. More recently, the SIET was used to measure distinct H + effluxes in X. laevis embryos from the two-cell stage to stage 6 (i.e., ~1.5 hpf to 3 hpf), and these were reported to play a role in left-right patterning [START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF]. However, these studies did not extend as far as neural induction. Thus, it was of interest to confirm if distinct localized H + fluxes could be measured around X. laevis embryos at the onset of neural induction using this non-invasive method. In addition, vacuolar H + -ATPases (V-ATPases) have been demonstrated to control the intracellular pH in many systems [START_REF] Harvey | Physiology of V-ATPases[END_REF], and in X.

F o r P e e r R e v i e w 7
laevis embryos, V-ATPase subunits have been shown to be localized asymmetrically as so-called 'fingers' extending from the vegetal cytoplasm into the animal hemisphere during the first few hours of development [START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF]. Thus, we also 8 investigated the activity of this ATP-driven H + pump on neural induction by treating embryos from stage 9 to 12 with the V-ATPase inhibitor bafilomycin A1 [START_REF] Bowman | Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells[END_REF]. The embryos were then fixed at stage 22 (i.e., 24 hpf) and the expression of the early neural gene, Zic3 [START_REF] Nakata | Xenopus Zic3, a primary regulator both in neural and neural crest development[END_REF], was determined by in situ hybridization.

The possible role of H + fluxes and the V-ATPase on neural induction in X. laevis is discussed.

F

Embryo production

The procedure used to harvest the embryos was as described previously [START_REF] Kay | Xenopus laevis: Practical Uses in Cell and Molecular Biology[END_REF]. Eggs were obtained from female X. laevis that had been primed between ~3 [START_REF] Nieuwkoop | Normal Table of Xenopus laevis (Daudin): A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis[END_REF].

Scanning Ion-selective Electrode Technique (SIET)

Non-invasive measurements of real-time H + fluxes (pmol cm -2 s -1 ) in the extracellular medium perpendicular to the surface of X. laevis embryos from stages 9 to 12 (i.e., from 7 hpf to 13.25 hpf) were accomplished using a SIET system (custom-designed and built by Applicable Electronics, LLC, New Haven, CT, USA) via an H + ion sensitive microelectrode (ISM). Details about the technique are described in detail in Hung et al.

(2019) where we used a Ca 2+ ISM, with the following modifications for measuring H + fluxes.

For the H + flux measurements, silanized glass microelectrodes were back-filled with pH-buffered electrolyte (100 mM KCl and 50 mM HEPES, pH 7) to a column length of ~1 cm, and then front-filled with Hydrogen Ionophore I Cocktail B (Sigma-Aldrich Corp.)

to a column length of 25 µm. These glass microelectrodes were assembled and then connected to the microelectrode holder to form the ISM of the SIET system. ISMs were calibrated before every experiment to ensure their reliable performance during data collection (Fig. 1a). To achieve this, a three-point calibration was performed using three ion standard solutions. For H + flux measurements, 0.05 M Tris buffers at pH 6, pH 7 and pH 8 were used. A voltage value was taken for each ion standard solution and the (ASET) software (Science Wares Inc., Falmouth, MA). The Nernstian slope was accepted at a theoretical value ± 4 (i.e., for H + : 54.76 -62.76), otherwise, the ISM was recalibrated or replaced [START_REF] Kühtreiber | Detection of extracellular calcium gradients with a calcium-specific vibrating electrode[END_REF]. After calibration, the ISM was also checked before each experiment to ensure that the background system noise was close to 0 pmol/cm 2 /s (Fig. 1b). This background recording was performed using the experimental bathing medium (i.e., 0.1X MMR) in the scanning chamber minus an embryo.

Measurement of extracellular endogenous ionic currents

Before scanning via the SIET began, a dejellied stage 9 embryo was placed in the centre of the scanning chamber mounted in a groove made between two strips of silicon elastomer (Fig. 1ci). The scanning positions of the ISM were then set at a circumferential plane ~200 µm above the equator of the embryo using the computerized motion control system (Fig. 1cii-iv). In this way, the dorsal animal hemisphere could be scanned with the direction of ISM excursion being perpendicular to the surface of the embryo. An initial recording was acquired at a reference position ~5 mm away from the measurement positions. This allowed any background noise to be subtracted from the 'real' signals generated at the embryo surface. In addition to stage 9, scans were also performed around the embryos at stages 10, 11 and 12. The direction of excursion was different at each measurement point since it had to be perpendicular to the surface of the embryo (Fig. 1cii-civ). The calibration and background values (Fig. 1d,e) were checked once again after each experiment to verify that the ISM had functioned in a Nernstian manner throughout the data collection period. After scanning, the embryos

F o r P e e r R e v i e w
12 were raised until they were at 3-4 days post-fertilization (dpf) to ensure that they developed normally (Fig. 1f). No measurements were made after stage 12 because during the neural stages (stages 13 -21), embryos undergo highly dynamic movements [START_REF] Vandenberg | V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis[END_REF], due in part to the dramatic extension of the dorsal marginal zone [START_REF] Keller | The cellular basis of gastrulation in Xenopus laevis: Active, postinvolution convergence and extension by mediolateral interdigitation[END_REF]. This prohibited close surface scanning due to potential damage to the delicate glass ISM.

Pharmacological treatment and in situ hybridization

The possible relationship between V-ATPase function and the development of neural structures was investigated by pharmacological means, using bafilomycin A1 (a V-ATPase antagonist; [START_REF] Bowman | Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells[END_REF]. In situ hybridization was then conducted to detect the presence of the early neural gene, Zic3. The in-situ hybridization method used, was modified from [START_REF] Hemmati-Brivanlou | Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization[END_REF]. Linearized DNA fragments of pBluescript II KS-Zic3 were prepared by digestion with the restriction enzyme, BamH1, for 2-4 hr at 37 o C. To produce a Zic3 DIG-labeled RNA probe, in vitro transcription was then performed using the linearized pBluescript II KS-Zic3, DIG RNA labeling mix and T3

RNA polymerase (11277073910 and 11031163001; Roche Diagnostics) following the manufacturer's instructions. The mRNA pellets were then purified using phenolchloroform extraction, as described in the manufacturer's instructions of the mMESSAGE mMACHINE® transcription kit, and then dissolved in 20 µL RNase-free water.

The final concentration of each mRNA synthesized was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) prior to being microinjected into embryos to induce protein overexpression. 13 Embryos were incubated with 0.1X MMR containing either 0.5 µM bafilomycin A1 and 0.4% DMSO (EMD Millipore; stock solution: 160 µM in DMSO) or 0.4% DMSO alone (control) from ~7 hpf to 13.25 hpf. At 13.25 hpf, the embryos were washed with 0.1X MMR for 3 x 5 min and then incubated in this same medium until they reached 24 hpf (i.e., equivalent to stage 22 in untreated, normally developing embryos). The bafilomycin A1 + DMSO-or DMSO-treated embryos were fixed with MEMFA solution (0.1 M MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO 4 and 3.7% formaldehyde) at ~22 o C for 1 h, and then washed with 2 mM MgCl 2 in PBS for 3 x 20 min. They were then dehydrated by sequential 5-min incubations through PBS containing increasing concentrations (i.e., 25%, 50%, 75%, and 100%) of methanol. The dehydrated embryos were stored for at least a week at -20 o C, after which they were rehydrated by sequential 5-min incubations in PBS containing 0.1% Tween 20 (PBSTw) and decreasing concentrations (i.e., 100%, 75%, 50% and 25%) of methanol. The embryos were then permeabilized with proteinase K solution (10 µg proteinase K, 20 mM Tris (pH 7.6), 2 mM CaCl 2 ) for 7 min.

Permeabilized embryos were washed with 0.1 M triethanolamine, pH7.8 for 2 x 5 min, and then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 5 min.

After subsequent rinsing with PBSTw, the embryos were fixed in PBSTw containing 3.7% formaldehyde for 20 min. They were then washed with PBSTw for 5 x 5 min, and then pre-hybridized with hybridization buffer (Hyb: 50% deionised formamide, 5X saline sodium citrate (SSC), 8.7 UI heparin, 1 mg Torula RNA, 1X Denhardt's, 0.1% Tween 20, 0.1% CHAPS, 50 mM EDTA) at 65 o C for 4 h. The Zic3 DIG-labeled RNA probe (at 1 µg/mL in Hyb) was denatured at 80 o C for 2 min before hybridization. Embryos were incubated with the denatured probe at 65 o C overnight to allow time for it to hybridize with the SSC for 3 x 20 min at 65 o C. Non-hybridized RNA was then digested with 2 µg/mL RNase A in 2X SSC at 37 o C for 30 min, after which embryos were washed with 2X SSC for 10 min at room temperature and 0.2X SSC at 65 o C for 2 x 30 min. The embryos were then blocked with Blocking Reagent (2 g; 11096176001; Roche Diagnostics) in 1X maleic acid buffer (MAB) at room temperature for 1 h, after which they were incubated at room temperature with Blocking Reagent containing alkaline phosphatase-tagged antidigoxigenin (11093274910; Roche Diagnostics), at a dilution of 1:4,000 for 4 h. Embryos were then washed with 1X MAB overnight, after which they were stained with BM-Purple (11442074001; Roche Diagnostics) for ~4 h until purple staining was observed.

The embryos were then fixed in Fixative B (1X SSC containing 10% acetic acid and 9.25% formaldehyde) for 1 h and depigmented in bleach solution (1X SSC containing 5% formamide and 10% H 2 O 2 ) for 2 h. After rinsing with 1X SSC, images were acquired using a Nikon AZ100 Multizoom microscope system.

Top-illuminated stereomicroscopy

To study the morphology of embryos and the localization of mRNA after in situ hybridization, top-illuminated microscopic images were obtained using an AZ100

Multizoom microscope system with a DS-5Mc Color Digital Camera Head in conjunction with the Digital Sight DS-U2 microscope camera controller and ACT-1 Version 2.63 software (Nikon Instruments, Inc., Tokyo, Japan). Top illumination was provided by a Schott KL 1500 electronic light source (Schott AG, Mainz, Germany). For live imaging, embryos were anesthetized with 0.1X MMR containing 0.2 g/L MS-222 just prior to

Results

Identification of endogenous H + currents using the SIET

We conducted extracellular non-invasive SIET measurements around embryos between stage 9 to 12 (i.e., from ~7-13.25 hpf) using an H + -specific ISM (Fig. 1). At each developmental stage, the H + flux measurement made at a reference position ~5 mm from the embryo surface was ~0 pmol cm -2 s -1 . In contrast, the minimum H + efflux measured around the embryos was ~0.1 pmol cm -2 s -1 (Fig. 2). At stage 9 (Fig. 2a), a near-constant H + efflux of ~0.1 pmol cm -2 s -1 was maintained at all the measuring locations around the embryos. At stage 10 (Fig. 2b), the H + effluxes from the left (L), left dorsal (LD), dorsal (D) and right dorsal (RD) locations appeared to be slightly higher than from the other regions, but no significant differences were found between all the measurement locations around the embryos. At stage 11, the H + efflux from the dorsal region (especially in positions LD and D) was elevated, showing values of ~0.2 pmol cm - 2 s -1 , which were significantly higher than the effluxes of ~0.1 pmol cm -2 s -1 detected on the left ventral (LV) side (Fig. 2c). At stage 12, a distinct H + efflux of ~0.55 pmol cm -2 s -1 was detected on the dorsal (D) side of embryos (Fig. 2d). This was significantly higher than the effluxes detected at all the other measurement positions. These same data are also presented on a radial column chart (Fig. 3), which clearly shows that there was an increase in H + efflux from stage 9 to stage 12 in all the measurement locations, with a more prominent H + efflux generated on the dorsal side of embryos. In addition, at stage 12 (i.e., the end of gastrulation), the H + effluxes in the dorsal (D) and right dorsal (RD) locations were significantly greater than those measured in the same locations between stages 9-11. Following the discovery of a distinct H + efflux at the dorsal animal hemisphere of embryos between stages 11 and 12, we investigated whether H + signaling might play a role in neural induction. We treated embryos with bafilomycin A1 to block the activity of this ATP-driven H + pump from stages 9 to 12, and then investigated the effect of this inhibition on the gross morphology of embryos, as well as the expression of the early neural gene Zic3 at 24 hpf. As the stock solution of bafilomycin A1 was prepared in DMSO, some embryos were treated with the same concentration of DMSO alone as controls. In the DMSO-treated control embryos, unfused and fused regions of the neural fold, as well as the cement gland and eye anlagen were all clearly visible at 24 hpf (i.e., stage 22; Fig. 4ai,4aii, see white, blue, yellow, and black arrowheads, respectively), thus suggesting normal development. In contrast, embryos treated with bafilomycin A1 (Fig. 4bi, 4bii), did not complete gastrulation, as shown by the obvious yolk plug at the vegetal pole (pink arrowheads). The DMSO-and bafilomycin A1-treated embryos were fixed at 24 hpf and then in situ hybridization was conducted to visualize the expression of Zic3.

In the DMSO-treated embryos, Zic3 was expressed in the telencephalon, diencephalon, mesencephalon and rhombencephalon (Fig. 4c), as first described by [START_REF] Nakata | Xenopus Zic3, a primary regulator both in neural and neural crest development[END_REF].

In contrast, no Zic3 expression was detected in the bafilomycin A1-treated embryos (Fig. 4d).

Discussion

Transmembrane H + fluxes are known to play a role in regulating the cytoplasmic pH of cells during fertilization and early animal development (Johnson et al., 1976;[START_REF] Gillespie | Changes in intracellular pH and pH regulating mechanisms in somitic cells of the early chick embryo: A study using fluorescent pHsensitive dye[END_REF][START_REF] Baltz | A novel H + permeability dominating intracellular pH in the early mouse embryo[END_REF][START_REF] Faszewski | Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology[END_REF][START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF][START_REF] Ebanks | Characterization of the mechanisms for Ca 2+ and HCO 3 -/CO 3 2acquisition for shell formation in embryos of the freshwater common pond snail Lymnaea stagnalis[END_REF]. Here, we applied the non-invasive extracellular SIET in an H +sensitive configuration and demonstrated the presence of a significantly higher H + efflux on the dorsal side of intact X. laevis embryos at stage 12 when compared with the H + effluxes at the other measuring positions around the embryo (Figs 2 and3). This is the first direct, truly non-invasive measurement of such a phenomenon during the late gastrula stage of X. laevis development. Our new data support and confirm those described previously, where it was reported that X. laevis embryos exhibit an intracellular alkalinization in the dorsal ectoderm cells during neural induction [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. In this earlier report, X. laevis planar explants loaded with the fluorescent pH indicator BCECF-dextran, were monitored by emission ratio microfluorimetry. As the increase in pH i was not detected in ectodermal cells in planar explants dissected from the ventral marginal zone or if the dorsal ectoderm was isolated from the inductive activity of the mesodermal cells, the authors suggested that it might be one of the factors required for determining the anterior neural fate of the ectoderm [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. We suggest that this neuralizing intracellular alkalinization might at least in part contribute to the localized H + efflux across the plasma membranes of the dorsal epithelial cells that we recorded.

We considered it important to confirm and extend the original data reported by [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. For example, some problems have been reported when using BCECF- [START_REF] Weiner | Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule[END_REF][START_REF] Han | Fluorescent indicators for intracellular pH[END_REF]. However, [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF] adopted protocols to minimize possible photobleaching, such as utilizing both continuous and intermittent imaging strategies, as well as incorporating neutral density filters to reduce the intensity of the required excitation light. Another factor to consider is that the early BCECF-dextran imaging experiments were conducted ex vivo with planar embryonic explants [START_REF] Keller | Regional expression, pattern and timing of convergence and extension during gastrulation of Xenopus laevis[END_REF] rather than in intact embryos. The experimental protocol involved microinjecting the reporter dye into the dorsal blastomeres of 32-cell stage embryos, then planar explants were dissected from the dorsal or ventral marginal zones at the start of gastrulation. Furthermore, the external epithelium was removed from the planar explant prior to imaging to improve the optical conditions for microfluorimetry in the underlying layers.

Any of these procedures, therefore, might have induced a pH i -related artefact. Thus, we considered it important to conduct our experiments in intact, normally developing embryos. However, despite the differences in protocol, the similarities between the BCECF-dextran data reported by [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF] and our new SIET-derived data with respect to the spatial nature of the embryonic domain generating the efflux of H + (and the developmental time window), suggest that the localized H + flux is a reproducible phenomenon associated with neural induction in X. laevis. In addition, it suggests that the precautions taken by [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF] to limit the deleterious effects of photobleaching (and thus dye reliability) were largely successful. Furthermore, the ability to recapitulate a developmental event from an intact embryo in an embryonic tissue explant illustrates the robust nature of X. laevis embryos as a developmental et al., 2005). It also demonstrates that tissue explants are effective ex vivo models for deciphering the complex three-dimensional signaling events that take place between different tissue layers [START_REF] Keller | Regional expression, pattern and timing of convergence and extension during gastrulation of Xenopus laevis[END_REF].

Proton pumping vacuolar-ATPases (V-ATPases) have diverse functions from acidifying the lumen of a variety of intracellular organelles [START_REF] Futai | Vacuolar-type ATPase: A proton pump to lysosomal trafficking[END_REF][START_REF] Banerjee | Regulation of V-ATPase activity and organelle pH by phosphatidylinositol phosphate lipids[END_REF] to generating H + effluxes across the plasma membranes of different cell types [START_REF] Nanda | Activation of proton pumping in human neutrophils occurs 15970[END_REF][START_REF] Wagner | Renal vacuolar H + -ATPase[END_REF][START_REF] Jouhou | Depolarization of isolated horizontal cells of fish acidifies their immediate surrounding by activating V-ATPase[END_REF]. As such, they play a key role in the regulation of intra-and intercellular signaling pathways [START_REF] Pamarthy | The curious case of vacuolar ATPase: regulation of signaling pathways[END_REF]. For example, an active V-ATPase was reported to be expressed in the plasma membrane of X. laevis embryos as early as the 4-cell stage, where it was shown to play a key role in the left-right patterning of the embryo [START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF]. It has been suggested that the function of such a V-ATPase-dependent H + efflux is to hyperpolarize the plasma membrane and increase the pH of the cytoplasm [START_REF] Swallow | A vacuolar type H + -ATPase regulates cytoplasmic pH in murine macrophages[END_REF][START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF][START_REF] Vandenberg | V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis[END_REF]. Considering these reports, we treated X.

laevis embryos with the specific V-ATPase inhibitor, bafilomycin A1 [START_REF] Yoshimori | Bafilomycin A1, a specific inhibitor of vacuolar-type H + -ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells[END_REF], from stage 9 to 12, and thus began to explore the possible function(s) of the late gastrula period H + efflux we detected. The inhibition of V-ATPase activity during this key developmental period led to a variable level of inhibition of blastopore closure and the subsequent failure to form early neural structures (Fig. 4b). Clearly, treatment with bafilomycin A1 will inhibit all the cellular V-ATPases located in the plasma membrane as well as those of the intracellular organelles, and thus may result in numerous secondary developmental and physiological effects. For example, they are known to attenuate two-pore channel-mediated Ca 2+ release from lysosomes [START_REF] Patel | Two-pore channels: Regulation by NAADP and customized roles in triggering calcium signals[END_REF]. This might, neurogenesis [START_REF] Guo | TPC2-mediated Ca 2+ signaling is required for axon extension in caudal primary motor neurons in zebrafish embryos[END_REF]. A similar finding has previously been described when X. laevis embryos were treated with bafilomycin for 24 hours starting at the 2-cell stage;

this led to an inhibition gastrulation and convergent extension movements [START_REF] Coombs | WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification[END_REF]. We showed that bafilomycin A1 treatment also led to the downregulation of Zic3 expression (Fig. 4d). This suggests that V-ATPase activity and pH i might also play an important signaling role during the gastrula period with regards to neural gene expression. It is not possible at this time to determine if bafilomycin A1 had a direct effect on the signal transduction pathway leading to the expression of Zic3, or if the effect was secondary; for example, resulting from a cessation of the morphogenic movements that contribute to gastrulation. However, it is becoming clear that neural induction is regulated via a complex interplay between cell-to-cell signaling, inductive interactions, morphogenetic movements, and localized gene expression [START_REF] Keller | Planar induction of convergence and extension of the neural plate by the organizer of Xenopus[END_REF][START_REF] Nakata | Xenopus Zic3, a primary regulator both in neural and neural crest development[END_REF][START_REF] Muñoz-Sanjuán | Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by posttranscriptional mechanisms and TAB3, a novel TAK1-binding protein[END_REF][START_REF] Leclerc | The calcium: an early signal that initiates the formation of the nervous system during embryogenesis[END_REF][START_REF] Stern | Neural induction: 10 years on since the "default model[END_REF][START_REF] Néant | TRPC1 as the missing link between BMP and Ca 2+ signaling pathways during neural specification in amphibians[END_REF]. It will, therefore, require careful and extended experimentation to decipher exactly where and when the H + fluxes occur (perhaps at various times and in different intracellular and/or embryonic locations) during the extended process of neural induction and subsequent neurogenesis. For example, it has been shown via the use of voltage and pH dyes, that a wave of bioelectrical activity travels across the ectoderm of intact X. laevis embryos during neurulation, and that inhibiting V-ATPases leads to abnormalities in craniofacial morphogenesis [START_REF] Vandenberg | V-ATPase-dependent ectodermal voltage and pH regionalization are required for craniofacial morphogenesis[END_REF].

Furthermore, it has been demonstrated that a V-ATPase is expressed strongly in the neural tissues and head of X. laevis during the later tail bud stages (Rutenberg et We did not investigate the effect of bafilomycin A1 on the dorsal H + fluxes.

However, it has previously been reported that H + fluxes measured using a SIET similar to ours across the pseudostratified epithelium in the proximal region of the vas deferens in rat [START_REF] Breton | Regulation of epithelial function, differentiation, and remodeling in the epididymis[END_REF], were drastically reduced by treatment with bafilomycin A1 [START_REF] Smith | Noninvasive measurement of hydrogen and potassium ion flux from single cell and epithelial structures[END_REF]. Bafilomycin A1 has also been shown to reduce [H + ] gradients (again measured using a similar SIET to ours), in the anterior midgut of mosquito (Aedes sp) larvae [START_REF] Boudko | In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pHsensitive microelectrodes[END_REF]. Moreover, in X. laevis embryos, concanamycin (another specific H + -V-ATPase inhibitor; [START_REF] Whyteside | Concanamycin and indolyl pentadieneamide inhibitors of the vacuolar H + -ATPase bind with high affinity to the purified proteolipid subunit of the membrane domain[END_REF] was shown to reduce the efflux of H + (and block the formation of left-right asymmetry) during early development [START_REF] Adams | Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates[END_REF]. This suggests that the H + fluxes we recorded would likely also be inhibited by an H + -V-ATPase inhibitor such as bafilomycin A1 (or concanamycin).

V-ATPase activity has also been shown to be required for (and in some situations it might directly regulate) Wnt signaling [START_REF] Cruciat | Requirement of prorenin receptor and vacuolar H + -ATPasemediated acidification for Wnt signaling[END_REF][START_REF] Wada | Role of vacuolar-type proton ATPase in signal transduction[END_REF][START_REF] Oginuma | Intracellular pH controls WNT downstream of glycolysis in amniote embryos[END_REF]. In addition, a Wnt signal at the cleavage stage in X. laevis embryos has been reported to play a role in activating subsequent neural development [START_REF] Baker | Wnt signaling in Xenopus embryos inhibits Bmp4 expression and activates neural development[END_REF]. It has also been proposed that binding of the Wnt ligand to the Wnt receptor complex requires a V-ATPase-mediated H + efflux [START_REF] Wada | Role of vacuolar-type proton ATPase in signal transduction[END_REF] and that bafilomycin A1 inhibits Wnt signaling [START_REF] Cruciat | Requirement of prorenin receptor and vacuolar H + -ATPasemediated acidification for Wnt signaling[END_REF]. Thus, the precise relationship between V-ATPase activity, H + efflux, and Wnt signaling is still far from clear. Our new data, however, add to the growing evidence first proposed by [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF], for an increase in pH i via a V-ATPase-mediated H + efflux playing a role in regulating neural

F o r P e e r R e v i e w
23 induction in X. laevis embryos. It has, however, also been reported in tail bud cells from chick embryos that increased pH i promotes acetylation of non-enzymatic β-catenin downstream of Wnt signaling [START_REF] Oginuma | Intracellular pH controls WNT downstream of glycolysis in amniote embryos[END_REF], and that acetylated β-catenin promotes mesodermal rather than neural fate [START_REF] Hoffmeyer | Trimethylation and acetylation of B-catenin at lysine 49 represents key elements in ESC pluripotency[END_REF]. There is, therefore, still much to understand with regards to the relationship between pH i dynamics and Wnt signaling during the different phases of neurogenesis in different embryonic domains. In addition to accumulating evidence suggesting the function of a V-ATPase-driven H + extrusion across the plasma membrane, alternate complementary mechanisms might also play a role in the alkalinization of dorsal ectoderm cell cytoplasm during neural induction. These include Na + /H + exchange and Na + -dependent Cl -/HCO 3 - exchange. While the former has been reported during fertilization in sea urchin eggs and in preimplantation mouse embryos [START_REF] Epel | Ionic triggers in the fertilization of sea urchin eggs[END_REF][START_REF] Siyanov | NHE1 is the sodium-hydrogen exchanger active in acute intracellular pH regulation in preimplantation mouse embryos[END_REF], it does not appear to play a role in the early development of X. laevis embryos [START_REF] Webb | Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage[END_REF][START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. For example, when dorsal marginal zone explants were treated with the protein kinase C and Na + /H + exchange agonist TPA (12-O-tetradecanoyl phorbol-13-acetate), the cells experienced an immediate decrease (rather than the expected increase) in pH i and the explants dissociated rapidly [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF]. There is, however. evidence for a role for Na + -dependent Cl -/HCO 3 -exchange in the alkalinization that occurs during neural induction in X. laevis embryos. For example, when explants at early stage 10.5 were treated with the anion transport inhibitor 4, 4'dihydrodiisothiocyanatostilbene-2, 2'-disulfonate, or when the [Na + ] and [Cl -] in the bathing solution was decreased and increased, respectively, then the normal increase in pH i and tissue-specific expression of the neural gene engrailed-2 were both blocked 24 [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF].

It is still unknown if the V-ATPase-generated H + fluxes mediate their effect via hyperpolarization of the plasma membrane or due to an alkalization of the cytoplasm of dorsal ectoderm cells, or indeed via a combination of both processes. Although it has been reported that H + pump-dependent changes in membrane voltage are an early and necessary mechanism to induce X. laevis tail regeneration [START_REF] Adams | H + pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration[END_REF], we suggest that the relatively long lasting slow-changing H + fluxes we recorded are likely to be involved in the intracellular alkalinization that has been reported to be important for a number of early development processes in several species [START_REF] Winkler | Dual ionic controls for the activation of protein synthesis at fertilization[END_REF][START_REF] Baltz | A novel H + permeability dominating intracellular pH in the early mouse embryo[END_REF][START_REF] Phillips | Intracellular pH regulation in Rathore KS, Hodges TK and Robinson KR[END_REF]. It has previously been reported from X. laevis embryos that localized, intracellular Ca 2+ transients triggered by planar signals can induce the expression of the Zic3 gene in the dorsal ectoderm during neural induction [START_REF] Leclerc | Calcium transients triggered by planar signals induce the expression of ZIC3 gene during neural induction in Xenopus[END_REF][START_REF] Batut | The Ca 2+ -induced methyltransferase xPRMT1b controls neural fate in amphibian embryo[END_REF][START_REF] Néant | TRPC1 as the missing link between BMP and Ca 2+ signaling pathways during neural specification in amphibians[END_REF]. We propose that an increase in pH i might be associated in some required/synergistic way with these Ca 2+ transients to induce the expression of an important early primary genes known to regulate the subsequent expression of other essential neural genes [START_REF] Nakata | Xenopus Zic3, a primary regulator both in neural and neural crest development[END_REF][START_REF] Winata | Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish[END_REF]. Such ionic and multistep regulation of gene expression is well recognized [START_REF] Broeck | Electrical-ionic control of gene expression[END_REF][START_REF] Calkhoven | Multiple steps in the regulation of transcriptionfactor level and activity[END_REF]. Indeed, it has previously been reported that when X. laevis dorsal marginal zone planar explants were treated with the anion transport inhibitor 4, 4'-dihydrodiisothiocyanatostilbene-2, 2'-disulfonate to block the normal intracellular alkalinization, then the expression of neural-specific genes (i.e., NCAM and otx2) were either reduced or completely missing [START_REF] Akif Uzman | The role of intracellular alkalinization in the establishment of anterior neural fate in Xenopus[END_REF]. In addition, when were all expressed. Furthermore, alkalinization of the ectoderm at stage 10.5 elicited a rapid increase (i.e., within ~15 min) in the expression of otx2 (Akif [START_REF] Akif Uzman | The role of intracellular alkalinization in the establishment of anterior neural fate in Xenopus[END_REF].

It is also known that gap junction conductance is sensitive to pH i [START_REF] Spray | Gap junctional conductance is a simple and sensitive function of intracellular pH[END_REF][START_REF] Peracchia | Chemical gating of gap junction channels; roles of calcium, pH and calmodulin[END_REF], and it has been suggested that localized Ca 2+ transients generated in the anterior dorsal ectoderm, which are required for neural induction, might pass from cell-to-cell through gap junctions [START_REF] Leclerc | Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos[END_REF][START_REF] Živanović | Surface tip-to-base Ca 2+ and H + ionic fluxes are involved in apical growth and graviperception of the Phycomyces stage I sporangiophore[END_REF][START_REF] Belousov | Neuronal gap junctions: making and breaking connections during development and injury[END_REF]. We suggest that this might, therefore, provide a link between Ca 2+

and H + being involved in the synergistic regulation of Zic3 expression and subsequent neural induction in the dorsal ectoderm of X. laevis embryos. This adds another layer of complexity to the interaction between the multiple ectodermal and endomesodermal signals (including chordin, noggin, BMP4, Wnts, ß-catenin, Cerebus, various Ca 2+ channels, and H + ) and gene expression, which combine to regulate the complex process of neural induction in X. laevis [START_REF] Sater | An increase in intracellular pH during neural induction in Xenopus[END_REF][START_REF] Baker | Wnt signaling in Xenopus embryos inhibits Bmp4 expression and activates neural development[END_REF][START_REF] Kuroda | Neural induction in Xenopus: Requirement for ectodermal and endomesodermal signals via chordin, noggin, βcatenin, and Cerberus[END_REF][START_REF] Stern | Neural induction: 10 years on since the "default model[END_REF][START_REF] Moreau | Calcium signaling during neural induction in Xenopus laevis embryos[END_REF][START_REF] Moreau | Calcium signaling and cell fate determination during neural induction in amphibian embryos[END_REF]2020;[START_REF] Cho | Calcineurin signaling regulates neural induction through antagonizing the BMP pathway[END_REF][START_REF] Néant | TRPC1 as the missing link between BMP and Ca 2+ signaling pathways during neural specification in amphibians[END_REF]. Our new data therefore add to the accumulating evidence, which suggests that intracellular alkalinization might contribute to establishing anterior neural fate in X. laevis embryos.

F

  of the African clawed frog (Xenopus laevis;

  calculated using the Automated Scanning Electrode Technique

  Zic3 mRNA. Embryos were then washed with Hyb for 10 min and 2X

  23 (IBM, Armonk, NY, USA), Microsoft Office Professional Plus Excel 2013 (Microsoft Corp., Redmond, WA, USA), and CorelDRAW X8 (Corel Corp., Ottawa, ON, USA) were used for statistical analysis, graph plotting and figure preparation, respectively. Two-way ANOVA and post hoc Tukey's Honestly Significant Difference tests were performed to analyze the significance of the data.

  pH i due to prolonged photobleaching of the dye, which can result in erroneous measurements

  in various developmental defects including those that arise during

  ectoderm was precociously alkalinized by treatment with methylamine or NH 4 Cl, then NCAM, otx2 and noggin (an anterior neural inducer gene)

Fig. 1 .

 1 Fig. 1. Schematic to show the scanning ion-selective electrode technique (SIET)

Fig. 2 .Fig. 3 .

 23 Fig. 2. SIET measurements showing the H + fluxes recorded around the equatorial

Fig. 4 .detect

 4 Fig. 4. The effect of the V-ATPase blocker, bafilomycin A1 on the gross morphology and

Fig. 1 .

 1 Fig. 1. Schematic to show the scanning ion-selective electrode technique (SIET) procedure. (a) An initial 3point calibration was performed using three "ion standard solutions", with a known concentration of H+. In this case, calibration was performed at pH 6, 7 and 8. (b) An initial background recording was performed in the centre of a scanning chamber in the absence of an experimental embryo. (ci) Experiments were performed in a scanning chamber made of a 35 mm glass bottom microwell dish with pieces of silicon elastomer in the centre. (cii) Typically, the plane of scan was set at ~200 µm above the equator of the embryo. (ciii,civ) Images to show a representative sample scan around the embryo. Visualization at the (ciii) top and (civ) bottom of the embryo allows for precise positioning of the ion-selective microelectrode (ISM) close to the embryo surface. The ↔ indicate the 8 measuring positions around the circumference of embryos. (d) Recalibration and (e) background scans were also performed at the end of each experiment. (f) Scanned embryos were photographed at 3-4 dpf to ensure that they had developed normally. AP, VP, D, V, Ant., and Pos. are animal pole, vegetal pole, dorsal, ventral, anterior, and posterior, respectively. Scale bars are 1 mm (ci and f), and 500 µm (cii).

Fig. 2 .Fig. 3 .Fig. 4 .

 234 Fig. 2. SIET measurements showing the H+ fluxes recorded around the equatorial circumference of X. laevis embryos from stage 9 to 12 (i.e., ~7-13.25 hpf). H+ fluxes were measured in 8 positions around embryos, as shown in the schematic on the upper right corner of panel (a). Fluxes were measured at (a) stage 9; (b) stage 10; (c) stage 11; and (d) stage 12 at an elevation of ~200 µm above the embryonic equator. The data represent the mean ± SEM of n=5 embryos for each stage measured. The reference point (Ref) was measured at a distance of ~5 mm away from the embryo. In (d), the asterisks indicate that H+ efflux data acquired around the embryo were significantly lower (P < 0.05) than those acquired on the dorsal side. Statistical significance was tested by two-way ANOVA and Tukey's honestly significant difference test. 117x123mm (300 x 300 DPI)

Materials and Methods General X. laevis husbandry

	Wild-type X. laevis were obtained from Nasco Agricultural Sciences (Fort Atkinson, WI,
	USA), and maintained in a custom-made recirculating system in the Animal and Plant
	Care Facility at the HKUST. The system had a 5 µm water filter and UV lights for
	disinfection. X. laevis were kept in 17 x 11.5 x 5.5-inch clear plastic tanks at a density of
	either 2-3 females or 4-5 males per tank in 12 L of frog water, which consisted of
	dechlorinated tap water containing 0.06 g/L Instant Ocean synthetic sea salt (Aquarium
	o r Systems, Inc., Mentor, OH, USA). They were maintained on a 12-h light/12-h dark cycle
	P at ~18 o C and with 70% humidity, and they were fed twice a week with ~1 g of Purina
	LabDiet® frog brittle (5LP3; Purina Mills, LLC, Gray Summit, MO, USA) per animal and e e r once a week with blood worms (Hikari, Japan). All the procedures used in this study
	were performed in accordance with the guidelines and regulations set out by the Animal R e v Ethics Committee of the HKUST and by the Department of Health, Hong Kong.
	i e w

Cambridge University PressZygote

Acknowledgments

We thank Mr Chris Shipley of Applicable Electronics, LLC (New Haven, CT, USA) and Mr Eric Karplus of Science Wares Inc., (Falmouth, MA, USA) for donating equipment or software, and technical support. We also thank Prof. H. Benjamin Peng and Ms Frances SL Chan (Division of Life Science, HKUST) for teaching H.C.L. the Xenopus laevis handling techniques described, and Dr Isabelle Néant (CNRS, Université Paul Sabatier) for providing advice about the in situ hybridization protocol.

Financial support

This work was supported by the Hong Kong Research Grants Council General Research Fund (A.L.M., grant numbers 16100115, 16100719). We also acknowledge funding from the Hong Kong Innovation and Technology Commission (ITCPD/17-9).

Conflict of interest

The authors declare none.

Ethical approval

All the procedures used in this study were performed in accordance with the guidelines and regulations set out by the Animal Ethics Committee of the HKUST and by the Department of Health, Hong Kong.


Daniel S Öderstr Öm

Student Member, IEEE Lucas Matana Luza

Student Member, IEEE Heikki Kettunen

Member, IEEE Arto Javanainen

Member, IEEE Wilfrid Farabolini

Antonino Gilardi

Andrea Coronetti

Member, IEEE Christian Poivey

Luigi Dilillo

Electron-Induced Upsets and Stuck Bits in SDRAMs in the Jovian environment

Keywords:

This study investigates the response of SDRAMs to electron irradiation. Stuck bits, SEUs and memory cell degradation is presented in this paper, in a memory that will be part of the ESA JUICE mission.

I. INTRODUCTION

T HERE has been a rise of interest regarding the radiation effects of electrons on components. Much of this interest has been directed towards and motivated by space missions in the Jovian environment such as the European Space Agencys (ESA) JUICE (JUpiter ICy moons Explorer) mission.

This study is on the radiation effects of electron irradiation in a synchronous dynamic random access memory (SDRAM) that will be used aboard JUICE.

One of the main points of interest of this work was to investigate the possibility of stuck bits being induced by electrons. This has, to the best of the authors knowledge, not previously been observed or reported. Previous studies of single event effects (SEE) caused by electron irradiation have been reported in [START_REF] King | Electron-induced single-event upsets in static random access memory[END_REF]- [START_REF] Tali | Mechanisms of electron-induced single-event latchup[END_REF], where single event upsets (SEU) and single event latch-up (SEL) were investigated.

Stuck bits are in this experimental work defined as bits with reoccurring errors, so that the memory cell is returning the same data when it is read ('0' or '1'), independent of the value which was written to the cell. This behaviour is different from other observed SEU, which manifested themselves as a bit error only present in a single reading of the bit in question, but not in any consecutive reads of the cell after it had been rewritten.

In this paper, experimental results of SEE from electrons resulting in stuck bits and bit-flips are presented. The degradation of the cells following these events are studied, and implications of the usage of the studied SDRAM in the JUICE mission are discussed.

II. COMPONENTS AND EXPERIMENTAL SETUP

The tested component that will fly on JUICE is the ISSI 512 Mb SDRAM IS42S86400B, but two other newer ISSI SDRAM memory models were also tested as presented in Table I. The newer models with smaller node sizes (72 nm and 63 nm instead of 110 nm) were only tested at VESPER with 200 MeV electrons. They did however not show any response in terms of SEUs during the irradiation, which is why they were not subjected to irradiation tests with lower energy electrons.

The tested memory devices were all ISSI 512 Mb Single Data Rate (SDR) SDRAMs, with 536,870,912 bits separated on four banks. The IS42S86400B memory's banks are divided in 8192 rows by 2048 columns by 8 bits, while the IS42S16320D/F memories have 8192 rows by 1024 columns by 16 bits in their banks. They all have an operating frequency up to 143 MHz, with a 3.3 V bias and were packaged in 54-pin TSOP-II packages.

A Terasic DE0-CV FPGA development board was used as a memory control board during the experiments, and the samples were mounted on daughter boards connected to the control board through a GPIO pin interface.

The components were tested through dynamic tests, with read and write operations carried out during the irradiation. The memories were continuously running a March C-test which was slightly modified so that it would not have any stand-alone write or read elements [START_REF] Dilillo | Soft errors in commercial off-the-shelf static random access memories[END_REF]. The IS42S86400B memories were operated at 100 MHz, while the IS42S16320D/F memories were operated at 75 MHz, because the D/F memories could not operate without issues at higher frequencies with the cabling and set-up used in the experiment. During all runs the memories were operated with their nominal refresh frequency 128 kHz.

III. TEST FACILITIES

The tests with high-energy electrons (60 -200 MeV) were performed at VESPER (The Very energetic Electron facility for Space Planetary Exploration missions in harsh Radiative environments) at CERN (The European organization for nuclear research). Lower energy electron exposures (20 MeV) was performed at RADEF (RADiation Effects Facility) at the University of Jyv äskyl ä, Finland, with the Clinac (Clinical LINear ACcelerator) at RADEF.

A. VESPER

High-energy electrons between 60 and 200 MeV are provided at VESPER. The VESPER beam is pulsed, and the electrons arrive in short bunches. Trains of bunches were arriving at the device under test (DUT) at a frequency of 3 GHz, and the bunch trains were separated in pulses with a 10 Hz frequency. 100 bunches per pulse were used in these experiments.

A Beam Current Transformer (BCT) was used to monitor the beam current during the runs, and a YAG (Yttrium Aluminium Garnet) screen and cameras were used to monitor the beam spot shape. A camera was filming the scintillating YAG-screen to monitor the beam profile during the run. The beam spot was approximated to be of Gaussian shape. The shape parameters (µ x,y and σ x,y ) was logged by the facility, and provided in the beam log along with the pulse charge monitored by the BCT.

This system for beam monitoring worked rather well with a high intensity beam. As the beam intensity gets lower, and as the beam energy gets lower, the beam profile and charge is monitored less efficiently. This is much due to that the beam spot on the scintillating screen gets diffuse in these cases. The beam fluence uncertainty for this beam has been estimated as 20 %.

B. RADEF

Electrons with energies of 6, 9, 12, 16 and 20 MeV are available at the Clinac at RADEF. This electron beam is also pulsed, and consists of series of 5 µs long pulses with a 5 ms period.

The beam was monitored with in-beam ionization chambers, which were calibrated against an IBA PPC40 dosimeter at maximum dose depth in water. The radiation field has been measured to be uniform within a few percent over the beam area, and this was not monitored actively during the runs. The uncertainty of the reported dose, and electron fluence calculated from this dose, is estimated to be less than 10 %.

IV. RESULTS AND DISCUSSION

A. Observed errors

During the irradiation tests two types of errors ascribed to SEE were observed in the IS42S86400B memory, while none were observed in the IS42S16320D/F memories. One type manifested themselves as bit-flips, which resulted in a single erroneous read of a bit in a word. The other type was stuck bits. These were bits which during consecutive write and read operations were stuck to the same value (either '0' or '1'). They were however generally stuck only for a few write/read cycles. On average, between 5 and 10 errors were detected in total per stuck bit for the IS42S86400B memories. The pattern of errors in the stuck bits can be seen in Fig. 1. In the figure, the red dots represent initial errors, with the previous write and read of the same value in the bit not resulting in an error. The blue dots represent the read of the memory where the previous write and read of the same value in the bit did give an error.

Many of these stuck bits in Fig. 1 were Intermittently Stuck Bits (ISB), i.e. bits that switch between appearing as stuck, and being normally operational. This effect has been discussed e.g. in [START_REF] Chugg | The random telegraph signal behavior of intermittently stuck bits in SDRAMs[END_REF]- [START_REF] Goiffon | Radiation induced variable retention time in dynamic random access memories[END_REF] with stuck bits originating from irradiation with other particle types. ISBs discussed in these publications have, like the ones found here, long periods of time where they are not stuck and causing errors.

The number of errors detected during the tests present a sudden increase at a certain level of fluence. This is shown in Fig. 2 for the SDF3 memory under 123 MeV electron irradiation after 18:30. The same effect was seen in all memories of this model (IS42S86400B), and the point of onset of this error increase is displayed in Table II. The errors up to this point were occurring at random points in time and fluence, and are assumed to originate from SEE induced by the impinging electrons. The rapid increase eventually occurring in all samples of the IS42S86400B memories is in contrast expected to be a cumulative radiation effect. The bits are numbered as 1 -18, with each row displaying the behaviour of one of the observed stuck bits. This error rate increase, and the consecutive annealing of these bits shown later in the paper, present competing effects which might lead to the memory suffering from a similar break down as is seen here, or not, depending on factors such as the electron flux, and how the memory is used. The flux dependence, and behaviour under different test modes, will be studied further during the spring of 2020 in planned experiments at VESPER and RADEF. These results will be included in the final version of the paper.

B. Stuck bit and SEU cross section

The calculated cross sections for the errors obtained before the increase in error rate are presented in Fig. 3. The error bars in the figure represent a 95 % confidence interval with a 20 % beam fluence uncertainty. A similar number of stuck bits and single bit-flips were observed at each of the tested energies, thus the risk of getting a single bit-flip in the component is similar to the risk of getting a stuck bit. Cross section (cm 2 bit -1 )

Cross sections of single bit-flips and stuck bits for electrons Weibull fit average cross section Stuck bits Single bit-flips Fig. 3. Stuck bits and single bit-flip cross sections to electron irradiation under dynamic testing. The Weibull fit of the average cross section is also presented, which will be used later for a discussion of the memory behaviour in the Jovian environment.

C. Retention time distributions

The stuck bit has been damaged by the incident radiation, so that the storage capacitor in the memory cell is not anymore able to hold its charge between data refresh events in the memory. For the single bit-flip, the memory cell storage capacitor is discharged, but after rewriting the memory it is not so damaged that it is not capable of holding the charge between the refresh periods. During post-irradiation analysis, a trend was found that the bits that got stuck during irradiation are more damaged than the ones that suffered a bit flip. This trend is shown in Fig. 4, where the bits that were stuck show errors at lower refresh frequencies, and have a generally larger portion of the population failing at a given refresh frequency.

The trends of the bits that were stuck and had bit-flips during irradiation fall rather close to each other in Fig. 4, and with similar trends. This can indicate that the mechanisms behind the different failure modes are similar to each other, but differ mainly in the severity of the event. There are many studies that suggests displacement damage caused by single particles creating leakage paths from the storage capacitor as the cause for stuck bits in DRAMs, e.g. [START_REF] Chugg | Probing the nature of intermittently stuck bits in dynamic RAM cells[END_REF], [START_REF] Shindou | Bulk damage caused by single protons in SDRAMs[END_REF], [START_REF] Edmonds | Physical mechanisms of ion-induced stuck bits in the hyundai 16M x 4 SDRAM[END_REF]. Since the bits that only had a single bit-flip display similar behaviour in Fig. 4 to the bits that were stuck, it is possible that the cause for the single bit-flips found in this study is the same as for the stuck bits. Looking at the behaviour in Fig. 1, some bits are stuck only twice or a few times, while others are stuck many more times. It is therefore reasonable that the same kind of failure would result in an error at only one occasion.

These distributions were produced by changing the refresh frequency F R of the memories (the abscissas in the figures are the inverse of this, 1/F R ), then writing the memory with all '0' pattern, waiting 60 s, then reading back the memory. Thereafter the procedure was repeated for an all '1' pattern. The sum of the words with errors in these tests is presented on the vertical axis.

The retention time distribution in SDF5 during irradiation with 20 MeV electrons can be seen in Fig. 5. This figure shows the number of words with bits that are stuck at different refresh frequencies at points with increasing dose. The nominal refresh frequency of the memories, 128 kHz, is marked in the figure with a dotted black line.

The retention time distribution in SDF5 post irradiation with 20 MeV electrons can be seen in Fig. 6, where the annealing of the memory is shown. The memories were kept in room temperature, and without bias when not characterized. A complete reversal to the pristine case is not seen in the annealing at this point. Instead the bits failing at a high F R are annealed to a large extent, but the total distribution is approaching a level shifted up to the left of the pristine one in Fig. 6. This shift of the memory cells data retention time to shorter times would indicate a permanent or long lasting damage to them. In the final version of the paper will also be included a high-temperature annealing study, where it will be investigated whether the retention time distribution of the cells will approach the pristine one.

V. IN-FLIGHT PREDICTIONS

Based on the cross sections presented in Fig. 3, and the expected electron environment during the JUICE mission [START_REF] Team | JUICE environment specification[END_REF] which is depicted in Fig. 7 along with the cross section Weibull fits, an estimation of the number of upsets in 0.00 Time between refresh (s)

SDF5 Flipped Stuck All
Normalized population of words with failing bits Fig. 4. The amount of words with bits that fail at certain refresh frequency of the memory (abscissa is 1/F R ). The populations that are shown are the bits which had only one bit-flip during irradiation, bits that were stuck, and all bits in the memory. Data taken post irradiation for the four SDF# memories.

10 -5 10 -4 10 -3 10 -2

Time between data refresh (s) Time between data refresh (s) Fluence (e/cm 2 /sr/MeV) the IS42S86400B memory during the JUICE mission is calculated. The mission duration is in total planned to be 11.1 years, with different phases of the mission receiving different fluxes of particles [START_REF] Team | JUICE environment specification[END_REF]. As a first estimation, the presented differential fluence is multiplied with 4π to assume an isotropic fluence, and also as an approximation an isotropic sensitivity of the component. So far the component has only been tested with electrons at a normal incidence angle. The cross section was then multiplied with the electron fluence, and then integrated, to obtain a first estimation of the number of upsets. The result was 2.44 upsets, which means about 5 events counting both stuck bits and bit-flips.

The number of upsets vary however greatly with variations in the cut-off energy of the Weibull fit, and thus the behaviour of the memory under lower-energy electron irradiation is of relevance. With a lower cut-off energy at 1 MeV, instead of 7 MeV as was used here, the number of errors per upset mode increases to 20. Irradiation with lower energy electrons (down to 6 MeV) is planned to be performed in the RADEF facility to investigate this. This prediction will be further expanded upon, by transporting the electron fluence trough the space-craft wall, and working with the sensitive volumes of the cells. Other important considerations to further expand upon of the behaviour of the memory in space is the effect of acquired dose versus the annealing of the bits. The implications of the dose rate behaviour and points of error rate increase will also be discussed and related to the different phases of the JUICE mission.

Fig. 1 .

 1 Fig. 1. Time structure of the errors from stuck bits in memory SDF3 under 123 MeV electron irradiation during modified March C-test loops.The bits are numbered as 1 -18, with each row displaying the behaviour of one of the observed stuck bits.

Fig. 5 .

 5 Fig. 5. Retention time distribution of SDF5 during 20 MeV electron irradiation at 5 different refresh frequencies. The doses in the figure are doses in water at maximum dose depth.

Fig. 7 .

 7 Fig.7. Total differential electron fluence of the JUICE mission from[START_REF] Team | JUICE environment specification[END_REF], along with the Weibull fits of the electron cross sections.

ID Electron energy (MeV) Bit- flips Stuck bits Fluence at onset of error rate increase (e/cm 2 )

			TABLE II
		SUMMARY OF IRRADIATION RESULTS.
	SDF1 SDF3 SDF4 SDF5 SDB1	200 123 61 20 200	11 12 13 6 0	14 18 13 6 0	1.02 • 10 12 1.41 • 10 12 2.73 • 10 12 3.12 • 10 12 -
	SDE1	200	0	0	-
			TABLE I
	SUMMARY OF SPECIMEN USED IN THE EXPERIMENT.
	ID	Memory	Electron energy (MeV)	Memory size	Node size (nm)
	SDF1	IS42S86400B	200		512 Mb	110
	SDF3	IS42S86400B	123		512 Mb	110
	SDF4	IS42S86400B	61		512 Mb	110
	SDF5	IS42S86400B	20		512 Mb	110
	SDB1 IS42S16320D	200		512 Mb	72
	SDE1	IS42S16320F	200		512 Mb	63

  Errors during the runs in SDF3 with 123 MeV electrons at VESPER. The total errors graph also counts the repeating errors from stuck bits, while the stuck bits graph only shows the onset of new stuck bits.

		[e/cm 2 ]	4	#10 9			Memory SDF3
			Fluence	0 2	15:30:00	16:00:00	16:30:00	17:00:00	17:30:00	18:00:00	18:30:00	19:00:00
	errors	5000
	Total			0
					15:30:00	16:00:00	16:30:00	17:00:00	17:30:00	18:00:00	18:30:00	19:00:00
	bits	1000
	Stuck		500
				0
					15:30:00	16:00:00	16:30:00	17:00:00	17:30:00	18:00:00	18:30:00	19:00:00
	Single bit-flips	0 200 400
					15:30:00	16:00:00	16:30:00	17:00:00	17:30:00	18:00:00	18:30:00	19:00:00
								Time [HH:MM:SS]
	Fig. 2.

  Fig. 6. Retention time distribution of SDF5 after 20 MeV electron irradiation after different annealing times.

		10 6	Retention time distribution SDF5 vs anneal time
			Anneal time
			0 h
		10 5	20 h 40 h
			63 h
	Words with errors	10 2 10 3 10 4	400 h Pristine Fr = 128 kHz
		10 1
		10 0
		10 16
		10 14
		10 12
		10 10
		10 8
		10 6
			10 -1	10 0	10 1 Energy (MeV)	10 2	10 3

VI. CONCLUSION

In this paper is shown that high-energy electron irradiation can cause stuck bits and bit-flips in SDRAMs as SEEs, and consequences of this for the JUICE mission is discussed. The mechanism behind the two failure modes are suggested to be one and the same, based on the similarity of the cell degradation and the similarity of the reaction cross sections.

The electron irradiation is found to degrade the memory cells on a long term time scale, but annealing of the most damaged cells during testing was observed. Further annealing studies at high temperatures will be conducted.

The effects that are found are related to the JUICE environment, and further predictions and studies of the behaviour of the memory in the Jovian environment will be conducted.

The results presented here are part of a project (RADSAGA) that has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 721624. This work was also supported by the European Space Agency ESA/ESTEC under Contract 4000124504/18/NL/KML/zk.


Junhao Wen

Elina Thibeau-Sutre

Jorge Samper-Gonzalez

Alexandre M Routier

Simona Bottani

Didier Dormont

Stanley Durrleman

Olivier Colliot

Ninon Burgos

How serious is data leakage in deep learning studies on Alzheimer's disease classification?

Data Leakage

3 categories identified: 3 possible labels

• Clear when data leakage is explicitly witnessed • Unclear when no sufficient explanation is offered • None detected otherwise 1. Biased split Data extracted from the same individual is distributed in both the train and the test sets.

Late split

Test / train split is performed after another procedure (feature selection, pretraining…).

No independent test set

The performance is evaluated on the train and / or validation sets.

Evaluation of the impact of biased split: • Methods

• Results

• Conclusion • Downloads @AramisLabParis

Search engines:

Literature Search

All the papers that were analyzed in the literature search may be found at www.zotero.org/groups/2337160/ad-dl Data leakage is a common problem in the literature (42% of surveyed papers).

Moreover, it has a serious impact on performance evaluation, as demonstrated by the strong differences in accuracies in both the literature and our experiments.

Thus the current literature of the domain may overestimate the performance of deep learning systems for automatic diagnosis of Alzheimer's disease.

In recent years, there has been a strong interest in the use of deep learning (DL) for assisting diagnosis of brain diseases from neuroimaging data. Unbiased evaluation of their performances is critical to assess their potential clinical value.

A major source of bias is data leakage, that can be difficult to detect for nonspecialists.

In this study, focusing on the case of Alzheimer's disease (AD) diagnosis from T1 MRI using convolutional neural networks (CNN), we performed a rigorous literature search, assessed the prevalence of data leakage and analysed its causes. Additionally, we demonstrated the phenomenon of data leakage in a controlled setting by focusing on the impact of the data split strategy.


CNN architecture and training

Mask optimization

Visualization approach to assess the robustness of neural networks for medical image classification Elina Thibeau--Sutre 1 , Olivier Colliot 1 , Didier Dormont 1,2 , Ninon Burgos 1 1 ARAMIS Lab, ICM, Inserm U1127, CNRS UMR 7225, Sorbonne University, Inria, Paris, France 2 AP-HP, Department of Neuroradiology, Pitié-Salpêtrière Hospital, Paris, France elina.thibeausutre@icm-institute.org

• Methods

•

Results

• Conclusion

• Bibliography @AramisLabParis

Materials

Deep learning methods have shown a high performance potential for medical image analysis. However, explaining their decisions is not trivial and could be helpful to achieve better results and know how far they can be trusted.

Many methods have been developed in order to explain the decisions of classifiers, but their outputs are not always robust or meaningful [1] and they remain difficult to interpret.

In this study, we adapted to 3D medical images the method of [2] which relies on two visualization methods extensively used: occlusion and saliency maps. A mask of input size is applied to increase values voxel-wise. The image 𝑋 . / masked by 𝑚 at voxel 𝑢 is defined as:

𝑋 . / u = m u X u + (1 -m u )
The optimal mask covers a minimal amount of voxels in connected parts of the image and transform a set of patients in controls for the CNN.

𝑚 * = argmin . 𝑓 # * 𝑋 . / + 𝜆 : 1 -𝑚 ; < ; < + 𝜆 = 𝛻𝑚 ; ?

; ?

Metrics of evaluation:

• prob CNN : output probability of the CNN for the true class for an input masked by two masks optimized in two different contexts. 0=similar / 1=dissimilar

• ROI-based: cosine similarity between the vector of the densities of the masks in each ROI of AAL2. 0=dissimilar / 1=similar We demonstrated the robustness of our visualization method by showing the small impact of hyperparameters choice on the resulting mask.

Then we could apply this visualization method to assess the robustness of CNN training and found out that the patterns identified are not robust, though the set of most highlighted ROIs is coherent with previous knowledge on AD.

  with Random Search [4] on training + validation. Performance (balanced accuracy) • Training / Validation (ADNI): 0.89 • Test (ADNI): 0.88 • Test (AIBL): 0.90 CNN robustness Mask robustness à No overfitting detected Objective: During training, the weights 𝑤 were optimized to maximize the score function 𝑓 # on a set of images 𝑋 as follows 𝑤 * = argmax # 𝑓 # (𝑋).

Figure 1 .Figure 2 .

 12 Figure 1. Grid search on 𝜆 : and 𝜆 = hyperparameters

Table 1 .

 1 ROI-based similarity across different values of 𝜆 :

  Coherent with prior knowledge on AD à Stability across different sets of hyperparameters

	3D Convolution + BatchNorm + LeakyReLU
	Max pooling	Dropout (0.79)	Fully-connected
		Top 5 of more masked ROIs across the 5 folds
		hippocampus, parahippocampal gyri, fusiform gyri, amygdalae,
		putamen, pallidum, temporal gyri, thalamus

[1] Adebayo et al, 2018, 'Sanity checks for saliency maps' [2] Fong and Vedaldi, 2017 'Interpretable Explanations of Black Boxes by Meaningful Perturbation' [3] Routier et al, 2018 'Clinica: an open source software platform for reproducible clinical neuroscience studies' [4] Begstra and Bengio, 2012 'Random Search for Hyper-Parameter Optimization' à CNN training is not robust towards the regions identified à


Elina Thibeau-Sutre
email: elina.thibeausutre@icm-institute.org

Olivier Colliot

Didier Dormont

Ninon Burgos

CNN training Interpretability Identification of unlabeled latent subtypes with saliency maps

Synthetic data

Deep learning methods have shown a high performance potential for medical image analysis. However, explaining their decisions is not trivial and could be helpful to discover new associations and know how far they can be trusted.

The reliability of interpretability methods is difficult to evaluate, and saliency metrics may not be reliable themselves [1]. Moreover, it is often unclear what the method is proving, and what are its properties or drawbacks [2].

In this study, we used saliency maps on convolutional neural networks, and focused on their ability to accurately highlight patterns of latent subtypes grouped under the same labels in a simulation framework.

Architecture Task classification controls vs atrophied

• Two overlapping classes: controls / atrophied • Composite atrophied label: typical /atypical • Regional atrophy: top / bottom

Benchmark Baseline results

Method Evaluation criteria

The saliency maps better represent the typical subtype than the atypical one for a small number of samples whereas both subtypes are better represented when the dataset size is large.

Separability and specificity of saliency maps can be improved using data augmentation or by changing the optimization criterion, though these techniques cannot be coupled easily.

[ 1 ]

 1 Tomsett et al., 2020, 'Sanity Checks for Saliency Metrics' [2] Lipton, 2018, 'The mythos of model interpretability' [3] Simonyan et al., 2013, 'Deep Inside Convolutional Networks: Visualising Image Classification Models and Saliency Maps' [3] corresponds to the mean gradients of the controls node with respect to the image of an atrophied group. Then intensities are related to the changes needed to transform this image into a sample of the control group. 3 criteria were designed to evaluate the separability of typical and atypical subtypes based on CNN feature maps or saliency maps of the typical or atypical group: a. CNN feature maps subtyping: mean adjusted rand index between 10 K-means clustering and the true labels. b. Saliency maps separability: (invert for atypical) -𝑦 < if 𝑥 -𝑦 < 1 else 𝑥 -𝑦 -0.5 |𝑠𝑜𝑓𝑡𝑚𝑎𝑥(𝑥) -𝑦| Criteria are evaluated on test dataset only.When a criterion is applied to an atypical saliency map, the symbol † is used.Values associated to randomness: a=0.00 ; b=b †=1.00 ; c=c †=1.00Default training (no data augmentation + CE)