K Smaali

David Guérin

V Passi

L Ordronneau

A Carella

Thierry Melin

Emmanuel Dubois

Dominique Vuillaume

J P Simonato

Stéphane Lenfant

T Mélin

Physical study by surface characterizations of sarin sensor on the basis of chemically functionalized silicon nanoribbon field effect transistor

INTRODUCTION

The func[onaliza[on of semiconductor surfaces with organic molecules has become an increasingly popular topic because of its poten[al in many applica[ons, especially gas sensors. To improve the sensi[vity of these sensors based on func[onalized semiconductors, a preferred approach is to increase the surface to volume ra[o by using nanostructures like nanowires 1;2;3 or nano--ribbons. This allows to maximize the amount of molecules or biomolecules immobilized by volume for a given planar surface of semiconductor [START_REF] Hu | Chemistry and Physics in One Dimension: Synthesis and Proper[es of Nanowires and Nanotubes[END_REF] . Kelvin Probe Force Microscopy (KPFM) is of a great interest to understand quan[ta[vely the electronic proper[es of these nano--structures with a high precision [START_REF] Melitz | Kelvin probe force microscopy and its applica[on[END_REF] .

KPFM is capable to map the contact poten[al difference (CPD) at the nanometer scale, it gives access to quan[ta[ve measurements of the electronic proper[es of a func[onalized surface by Self Assembled Monolayers (SAM) 6;7;8 , DNA [START_REF] Richards | Kelvin Probe Force Microscopy Analysis of the Covalent Func[onaliza[on and DNA Modifica[on of Gallium Phosphide Nanorods[END_REF] or organic nanostructures 10;11 . These electronic proper[es include the work func[on [START_REF] Delalande | Chemical func[onaliza[on of electrodes for detec[on of gaseous nerve agents with carbon nanotube field--effect transistors[END_REF] , i.e. the energy gap between the Fermi level and the vacuum level, and dipole moments [START_REF] Celle | Interface dipole: Effects on threshold voltage and mobility for both amorphous and poly--crystalline organic field effect transistors[END_REF] . KPFM has also been used frequently on single nanowire to characterize Ohmic and Schoiky contacts [START_REF] Bercu | Characteriza[ons of Ohmic and Schoiky--behaving contacts of a single ZnO nanowire[END_REF] , localized trapped charges 15;16;17;18;19 , dopant distribu[ons 20;21 or Schoiky junc[on deple[on region 22 . KPFM presents several advantages such as (i) a quan [ NanoRibbon Field Effect Transistor (SiNR--FET) 24;25 . Beier sensi[vity was obtained on these sensors by reducing wire width from a few µm to 25 nm 26;27 . Thanks to these size modifica[ons which increased the surface to volume ra[o of the conduc[ve channel, the current gain of the SiNR--FET was improved by 4 decades. In the present paper, we report on a direct measurement of the CPD at the nanometer scale by performing KPFM study on SiNR--FET biased at the opera[ng voltages. These results are compared with Kelvin Probe measurements at the macroscopic scale. The SiNR surface was modified by a SAM selec[vely sensi[ve to organophosphorus gases. The diphenyl chlorophosphate DPCP was used as a simulant of OP nerve agents. The drain--source current (IDS) ploied versus the gate voltage (VG) shows the ambipolar behavior of the FET. The exposure of func[onalized SiNR--FET to vapors of DPCP induced a strong modifica[on of the IDS--VG curve [START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF] . In the following, a more detailed characteriza[on of the SiNR--FET is presented in order to explain the observed electrical proper[es.

The main aims of this work are to associate different surface characteriza[on techniques as KPFM, Kelvin Probe and ToF--SIMS and electrical measurements in order to (i) understand the physical and chemical effects occurring on the sensor surface; (ii) op[mizing the sensor and (iii) demonstrate the poten[al of the KPFM technique for electrical device studies. Our study focuses on three aspects: (i) an electrosta[c aspect for the different chemical steps ("naked" SiNR, aeer surface func[onaliza[on by the SAM, and aeer DPCP exposure) by comparing the CPD mapping measured by KPFM on the biased SiNR--FET with Kelvin Probe measurement realized on large surfaces with the same chemical steps; (ii) a chemical specia[on using ToF--SIMS 2D element mapping experiments to localize precisely the reac[on of molecules on the surface of the Si--NR FET for each chemical step; (iii) an applica[ve aspect with the demonstra[on of a prototype evaluated in real condi[ons with sarin, a wellknown chemical warfare agent.

MATERIALS AND METHODS

Device fabrica4on. Silicon Nano--Ribbons (Si--NR) were formed from silicon on insulator (SoI) wafers using e--beam lithography and dry reac[ve--ion etching steps, following the procedure described elsewhere [START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF] (see also suppor[ng informa [on). The structure of the SiNR--FET (figure 1a) is based on a Si--NR (70 nm thick, (100)--oriented, p--doped with Boron at 10 15 atom.cm --3 ) with different lengths and widths (4 x 4 µm; 4 x 1 µm) connected by source and drain Ti/Au (10/100nm) contacts paierned by e--beam lithography and a lie--off process. The thickness of the buried oxide (BOX) of the SOI wafer is 140 nm, and serves as the gate dielectric layer combined to a back--gate command through the silicon wafer handler. The SiNRs were func[onalized by covalent graeing through thermal hydrosilyla[on of 1 onto HF--pretreated substrate (more details in the suppor[ng informa[on). Compound 1 named 3--(4--ethynylbenzyl)--1,5,7--trimethyl--3--Azabicyclo[3.3.1]nonane--7--methanol is hereaeer referred to as TABINOL. Diphenylchlorophosphate (or DPCP) was used as a simulant of nerve agents (sarin) because of its similar structure and chemical reac[vity, but much lower toxicity. TABINOL reacts with DPCP and forms azaadamantane quaternary ammonium salt following the reac[on presented in figure 1b (more informa[on for the chemistry synthesis and reac[on are detailed elsewhere [START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF] ). For a more detailed surface characteriza[on, large pieces (1cm x 1cm) of silicon wafers were also func[onalized with TABINOL and exposed to DPCP using the same protocols.

Contact angle measurements. We measured the water contact angle (on the 1 cm² sample) with a

remote--computer controlled goniometer system (DIGIDROP by GBX, France). We deposited a drop (10--30 µL) of deionized water (18 MΩ.cm) on the surface and the projected image was acquired and stored by the computer. Contact angles were extracted by a contrast contour image analysis soeware.

These angles were determined a few seconds aeer applica[on of the drop. These measurements were carried out in a clean room (ISO 6) where the rela[ve humidity (50%) and temperature (22°C) are controlled. The precision of these measurements is ± 2°.

Thickness measurements.

We recorded spectroscopic ellipsometry data (on the 1 cm² sample) in the visible range using an UVISEL (Horiba Jobin Yvon) Spectroscopic Ellipsometer equipped with DeltaPsi 2 data analysis soeware. The system acquired a spectrum ranging from 2 to 4.5 eV (corresponding to 300 to 750 nm) with intervals of 0.1 eV (or 15 nm). Data were taken at an angle of incidence of 70°, and the compensator was set at 45.0°. We fiied the data by a regression analysis to a film--on-substrate model as described by their thickness and their complex refrac[ve indexes. We es[mated the accuracy of the SAM thickness measurements to ± 1 Å.

ToF--SIMS. ToF--SIMS spectra measurements and images were carried out in posi[ve and nega[ve modes using a ToF--SIMS V instrument (ION--TOF GmbH Germany) on the Si--NRFET. This instrument is equipped with a Bi liquid metal ion gun (LMIG). Pulsed Bi3 + primary ions have been used for analysis (25 keV) in both bunch and burst alignment modes. Spectra and images were taken from an area of 35µm x 35µm. In bunch mode, mass resolu[on was > 8500 at m/z= 196, 249, 294, 312 and 314, for C12H22NO + , C12H10PO4 --, C21H28N + , C21H30NO + , C21H32NO + , respec[vely.

KP

KPFM measurements. On the Si--NR FET, we measured, locally, CPD on the silicon channel by Kelvin

Probe Force Microscopy (KPFM). KPFM measurements were carried out at room temperature with a Dimension 3100 from Veeco Inc., purged with a flow of dry nitrogen atmosphere and controlled by the Nanoscope v5.30R2 soeware. Images were processed using WSxM 5.0 soeware from Nanotec Electrónica [START_REF] Horcas | WSXM: A soeware for scanning probe microscopy and a tool for nanotechnology[END_REF] . We used Pt/Ir (0.95/0. by sweeping the gate voltage from --10 V to 10 V and then from 10 V to --10 V with a 1 V step at a fixed VDS. Each value of VG corresponds approximately to 10 lines in the KPFM image. Values on Si--NR presented at a fixed VG were an average of about 23 pixels corresponding to the length of the Si--NR, and an average of these pixels on the 10 lines obtained at the same VG.

We also recorded I--V and KPFM sequen[ally. In this case, I--V characteriza[ons were first recorded in a glove box filled with clean nitrogen (O2 < 1 ppm, H2O < 1 ppm) and then transferred to the Dimension 3100 for KPFM characteriza[on with no applied bias on the SiNR--FET. These measurements were done on the same device before and aeer each chemical steps, i.e., we measured: i) the "naked" Si--NR aeer HF passiva[on, ii) the Si--NR func[onalized with TABINOL before DPCP exposure; and iii) aeer DPCP exposure.

SURFACE CHARACTERIZATIONS

Water contact angle and thickness

Water contact angle and thickness measurements are quick methods to assess the effec[veness of the chemical reac[ons on the Si surface. [START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF] ). Since the naked Si--H surface (aeer cleaning) is highly hydrophobic (water contact angle ~90° [START_REF] Williams | WETTING OF THIN--LAYERS OF SIO2 BY WATER[END_REF] ), the decrease of the water contact angle to 66° is consistent with the presence of a more hydrophilic group at the outer surface, mainly the primary alcohol group. Upon reac[on with DPCP, the thickness is slightly increased (depending on the dura[on of DPCP exposure) and the water contact angle decreases slightly. These results are consistent with previous experiments. [START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF]

KP and KPFM measurements on large samples

Since the top surface of the nanoribbons is (100) and sidewalls are (110) orientated, and to compare with localized KPFM on the Si--NR FETs (see next sec[on), we measured the CPD on large samples for these two orienta[ons, and also on (111) surfaces for the sake of completeness. Figure 3 Firstly, we note that there is a difference in the CPD values when similarly treated samples are compared for different orienta[ons. This varia[on is aiributed to the fact that the surface termina[on of silicon for the different orienta[ons is not the same, i.e., forma[on of di--hydrides, mono--hydrides and a combina[on of mono-- and tri--hydrides on (100), ( 110) and ( 111) orienta[ons, respec[vely. [START_REF] Chabal | Infrared spectroscopy of Si(111) and Si(100) surfaces aeer HF treatment: hydrogen termina[on and surface morphology[END_REF] . For the naked Si surfaces, these different chemical environments lead to varia[ons in the Si work func[on 34; 35 . Aeer graeing the TABINOL, the CPD values are increased, again with rela[ve amplitude which is orienta[on dependent. Graeing molecules on Si surfaces is well known to induce interface dipoles which modulate the work--func[on. 36;37;38 This work--func[on varia[on depends on the density of molecules and on the surface orienta[on, and it is also known that the density of graeed molecules depends on the Si surface orienta[on. 39;40;41;42 However, for the two Si orienta[ons of interest in this work, i.e. ( 100) and ( 110), the increase of the CPD aeer the graeing of TABINOL is almost the same (Figure 3). Aeer exposure to DPCP, the varia[ons of CPD are weak.

In order to compare the KP and KPFM measurements, large surfaces (1 cm x 1 cm) of silicon substrate

were measured before and aeer TABINOL graeing by KP and KPFM (Table 2). The graeing of TABINOL

ToF--SIMS

Since the surface characteriza[on techniques (contact angle, ellipsometry) are not applicable to the and from fragmenta[on of the organophosphorus compounds (PO3 --, PO2 --). Indeed, as specified in Table S1 (suppor[ng informa[on), characteris[c TABINOL fragment ions were observed before and aeer DPCP whereas PO3 --and PO2 --ions were only observed aeer exposure to organophosphorus molecules. Moreover, molecular imaging (Fig. 4) shows that these laier fragment ions are selec[vely localized on the Si--NR (4×4 µm) confirming the effec[veness of the DPCP reac[on on NR--Si FET func[onalized with TABINOL. Note that some of these fragments were also observed on the neighboring gold contact electrodes since amine and phosphorus deriva[ves are also known to readily chemisorb on this metal. However, the most intense signal comes clearly from the Si--NR region (yellow spots in Fig. 4).

KPFM AND I--V RESULTS ON SI--NR FET AND DISCUSSION

Sequen4al approach

In a first step, I--V and KPFM measurements were done sequen[ally as described above. These measurements were done to establish the first trends of the effects of the chemical treatments on the electrical behaviors of the devices. We present in the following the IV and KPFM results obtained on Si--NR with the larger surfaces (4 x 4 µm²).

Transfer curves of the ambipolar SiNR--FET are highly dependent of the surface func[onaliza[on of the Si--NR (figure 5). Aeer TABINOL graeing, the main effect is a shie towards more nega[ve voltages.

Here These CPD drops with TABINOL graeing are comparable: (i) same sign: the graeing of TABINOL is associated to an increase of the CPD, corresponding to a decrease of the work func[on (Eq. 3); (ii) a weak varia[on (around 14%) on could be explained by the fact than the density of graeed TABINOL molecules is not exactly the same for large surface and Si--NR. This good consistency between measurements on microstructures and on large surfaces validates the side capacitance correc[on (sec[on SI--4) with the use of Eq. 2.

To establish a correla[on between the IV curves and the KPFM experiments, the effect of the chemical steps (TABINOL graeing and then DPCP exposure) was determined using a couple of parameters: ΔVm (shie of the current minimum picked from the IDS--VG curve) and Φ (change in the CPD). Since we have measured the back and forth IV curves, we considered two voltage shies: ΔVmfor and ΔVmrev for the forward and reverse IV traces, respec[vely. The graeing of a TABINOL monolayer induces a drop of the CPD * in the ~ + 302 mV range and a voltage shie of the IV curves in the 4.2--4.4 V range (Fig. 5). Note that the IV hysteresis (in Fig. 5 the difference between ΔVmfor and ΔVmrev)

remains the same as for the "naked" Si--NR FETs, i.e. of the order of 3.6--3.8 V. On the contrary, DPCP expose induces a weak CPD * change, = --61 mV, but a significant larger IV hysteresis (ca. 4.7 V) (Fig. 5). Since the TABINOL graeing does not increase the IV hysteresis, we can discard the trapping of

TAB Φ TAB Φ TAB Φ TAB Φ DPCP Φ
charges as the origin of the electrical modifica[ons of the Si--NR FETs. Thus, we assume that the decrease of CPD is mainly due to the dipole moment of the TABINOL graeed on the silicon surface.

We can es[mate this dipole using the Helmholtz equa[on: (Eq. 4)

where N is the surface density of molecules in the SAM, es[mated around 2.5 x 10 14 cm --2 from previous work [START_REF] Celle | Self--assembled monolayers for electrode fabrica[on and efficient threshold voltage control of organic transistors with amorphous semiconductor layer[END_REF] , Pz is the dipole moment of the molecule perpendicular to the surface, ε0 is the vacuum dielectric permi'vity, εSAM is the rela[ve permi'vity of the SAM (here 2.5). From Eq.

Direct measurement by KPFM of polarized Si--NR FET

Effect of V G on the SP of Si--NR at V DS = 0 V

To analyze more precisely these charge effects, KPFM were performed on the Si--NR FETs biased by different back gate (VG) and source--drain (VDS) poten[als. In the case of "naked" Si--NR before any chemical step, the effect of the back gate (VG) on the SP measured by KPFM was studied for a source-drain poten[al VDS = 0 V (figure 7). For VG = 0 V, the CPD profile is similar to the previous one presented in figure 6 with a CPD difference between the SI--NR "naked" and gold comprised between 1 on the CPD values in Fig. 7--b, the corrected CPD * obtained is not significant, in view of the low values of CPD measured in absence of VDS. In that later case, we do not have any VG dependence of the CPD. It means that surface poten[al of the Si--NR is not modulated by the applied back--gate. A possible explana[on would be that (i) the KPFM characteriza[on system is not sufficiently sensible to detect varia[on of the CPD with the back gate poten[al (VG) on Si--NR, or (ii) that the real effect is masked by the side capacitance effects. At the silicon/gold interface, we observe a clear voltage drop that reveals a Schoiky contact formed by the gold electrode with the Si--NR [START_REF] Bercu | Characteriza[ons of Ohmic and Schoiky--behaving contacts of a single ZnO nanowire[END_REF] . Moreover, peaks are present mainly at the right interface, and the peak height increase with the bias (peak height up to 350mV for VG = 10V). A possible origin of these peaks is due to the presence of localized charges at the Si--NR/electrode interface, caused by a mismatch between metallic layers and consequently by the presence of oxidized Ti locally.

A KPFM scan on a same segment of the "naked" Si--NR (inset in figure 8) versus VG and at VDS = 0 V (i.e. no charge transport through the Si--NR) shows the reversible effect of the back gate poten[al on the Si--NR effec[ve CPD (figure 8). Star[ng on top of the image at VG = --10 V, the back gate voltage increases to VG = 10 V in the middle of the image, and finally decreases to VG = --10 V at the boiom of the image (step of 1V every 10 lines approximately). The two CPD profiles at VG = --10V (lines at the top and boiom of the image) are similar showing the reversible effect of VG. We can no[ce here the absence of hysteresis between the forward bias (from --10V to 10V) compared to the reverse bias (from +10V to --10V) (the two half images are symmetric).

Effect of V G on the SP of Si--NR at V DS = --2 V

KPFM characteriza[ons were subsequently performed by applying a source--drain poten[al (VDS = --2 V) on a "naked" Si--NR (figure 9--b). The main differences on CPD profiles between VDS = 0 V and VDS = --2 V are: (i) the shie of the right electrode CPD from 0 V (figure 8) to --2 V (figure 9--b) due to the polariza[on of this electrode; to measure exactly this difference of --2 V on CPD between source and drain electrodes, it is necessary to take the value measured at a distance of X = 0 µm and X = 110 µm of the Si--NR, due to the presence of the transi[on region (side capacitance effect as discussed above); (ii) the important varia[on of the CPD value for VDS = --2 V: for VG = +10 V (ON state of the Si--NR FET with an electron conduc[on), the maximum value in the middle of the NR is equal to CPDSi--NR = 5.97 V, for VDS = --2 V and for VG = --10 V (ON state of the Si--NR FET with a hole conduc[on), the minimum value is equal to CPDSi--NR = --6.86 V. Corrected from the side effects (Eq. 1), the corrected CPD * of the Si--NR varies between 5.7 V and --7.3 V (varia[ons of the CPD value aeer correc[ons are inferior 6%). This important varia[on of KPFM signal observed here (compared to the absence of VG-dependency at VDS=0 (Fig. 7--b) is explained by the presence of carriers (electrons for posi[ve VG and holes for nega[ve VG) into the Si--NR during the KPFM measurement. Fig. 10 shows the CPD dependence of the Si--NR surface (average value on the Si--NR see methods) with VG, and the corresponding IV data acquired simultaneously during the KPFM measurements (see methods). Here, the CPD * on Si--NR (CPD * Si--NR) presents an important hysteresis. The maximum of the CPD * hysteresis was measured for VG = --3 V with a varia[on ΔCPD * = 2,5V between the two curves. FET is in OFF state for VG ~ --2 to --3 V. In figure 10, the IV curves also display a small hysteresis, with its maximum of ΔVG around 1V for gate voltages at the current minima (Vm ≈ --3 and --2V). This maximum of the IV hysteresis is observed at approximately the same poten[al as for the maximum on the Si--NR CPD hysteresis (at VG = --3 V see above). This hysteresis behavior can be due to trapping and detrapping of charges at the Si--NR surface caused by the presence of traps at the molecule/Si interface. 46;47

Effect of the chemicals func@onaliza@on on SP

The comparison of the Si--NR FET transfer curve and CPD dependence with VG acquired simultaneously on the Si--NR FET func[onalized by TABINOL, before and aeer DPCP exposure shows clearly an effect of the simulant of nerve agents on the measured proper[es (Fig. 11). For the transfer curves, the same behavior as previously described (Fig. 5) is observed with the DPCP exposure: a shie of the curve to posi[ve gate voltage bias (shie ΔVm = 2 V and ΔVm = 3 V for the forward and reverse bias respec[vely). For the CPD dependence with VG, as observed on "naked" Si--NR at VDS = --2 V, the CPD on Si--NR is not perfectly linear with VG, and presents a hystere[c behavior. DPCP increases the CPD hysteresis between the forward and reverse bias measured ΔCPD = 0.47 V and ΔCPD = 1.17 V (at of the hysteresis with DPCP exposure, the IV hysteresis is ≈ 2V aeer TABINOL graeing and ≈ 4V aeer reac[on with DPCP. Note that results presented in figure 11 are not directly comparable with those presented on "naked" Si--NR at figure 10; for TABINOL and DPCP in figure 11 characteriza[ons were done at VDS = --1 V while before chemical modifica[on in figure 10 VDS is --2 V. Nevertheless, the trend is the same as for the sequen[al method (sec[on 4.1): TABINOL graeing mainly modifies the CPD, while the IV hysteresis remains unchanged, the reac[on with DPCP mainly increases hysteresis in both the IV curve and the VG--dependency of CPD.

DETECTION OF SARIN WITH TABINOL--MODIFIED SI--NR FET

Set up

The detailed procedure for the fabrica[on of the Si--NR FET used for sarin test are described in [START_REF] Passi | High Gain and Fast Detec[on of Warfare Agents Using Back--Gated Silicon--Nanowired MOSFETs[END_REF] . The TABINOL modified Si--NR FETs are connected onto a pluggable plas[c card by gold wire bonding to source and drains electrodes and with silver paste deposited onto the back for gate electrode. Three of these sensors were place in a 3--way [ght fluidic cell with BNC connectors and connected to three independent detectors where drain--source and gate--source voltages are applied while the drain source current is measured as a func[on of [me (See Suppor[ng Informa[on). The prototype is mainly divided into three parts: a microcontroller, a func[onalized SiNW--FET as transductor, and digital--analog converter interfacing the FET with a computer. The detectors are able to measure very low current over a range of six decades. The common range of measurement for our devices is 10 --10 A --10 --4 A.

Results

A sampling cycle begins with a calibra[on step which consists in sweeping drain--source current versus gate voltage of the transistor at a constant bias voltage VDS = -1V. Then, the minimum value of the off--current is iden[fied with the corresponding gate voltage (VBG). This value of VBG is named V0, and is then applied to monitor IDS as a func[on of [me. It has been shown previously that reac[on with OPs causes a shie to more posi[ve VBG with an average varia[on ΔVm of (7.3V ± 3.5V). A series of tests with a real chemical warfare nerve agent, the Sarin, have been carried out with TABINOL func[onalized SiNW--FET on the portable detector [START_REF] Clavaguera | Development of an autonomous detector for sensing of nerve agents based on func[onalized silicon nanowire field-effect transistors[END_REF] . Exposure to Sarin vapors induced a modifica[on of the transfer curves as observed with the nerve agent simulants described in this ar[cle. The IDS-VBG curves are shieed to more posi[ve gate--voltage bias by a few volts (∼5V) (Figure 12a). The portable detector sets automa[cally VBG to --7.5 V as opera[ng voltage. Figure 12b shows a stable IDS, at this gate voltage, before exposure to Sarin vapors (40 ppm measured with ppbRAE detector from RAE Systems). Aeer exposure, IDS rise aeer few seconds with a steep current increase. A plateau is reached within few minutes. The prototype is able to accurately measure IDS down to hundreds of pA.

The very stable value of a few pA measured before exposi[on and the IDS value measured just aeer the exposure are lower than the limit of detec[on of the prototype and are inaccurate but this is not detrimental to efficient Sarin detec[on.

Two factors could explain the longer dead [me observed for Sarin compare to DPCP exposure. First, the flow cell used for Sarin exposure was not op[mized and is possibly affected by fluidic problems.

Secondly, Sarin could exhibit a lower reac[vity than DPCP due to the higher bond dissocia[on energy (D 0 at 298 K) of P--F bond (439 kJ.mol --1 , Sarin) when compared to P--Cl bond (289 kJ.mol --1 , DPCP).

However, the detec[on is fast with a high on/off ra[o, which demonstrates the ability of this technique to detect efficiently traces of Sarin.

CONCLUSIONS

Silicon surface orientation

Si TABINOL +DPCP

CPD (mV)

CPD by KPFM (mV) CPD by KP (mV)

V G =-10V ON h + V G =10V ON e - CPD (V) @ V DS =-2V x (µm) V G =10V V G =7,5V V G =5V V G =2,5V V G =0V V G =-2,5V V G =-5V V G =-7,5V V G =-10V

NANO--RIBBON SI FET

Silicon nanoribbons were fabricated using e--beam lithography and reacLve--ion etching (RIE) on SoI (Silicon on Insulator) substrates. The top silicon (p--type) layer, 70 nm thick, was doped with 10 15 cm -3 boron impuriLes. The thickness of the silicon dioxide is 140 nm on a silicon substrate (10 Ω.cm) serving as the boZom gate. A thin layer of 45 nm of negaLve resist (HSQ FOX 12) was deposited by spin--coaLng on clean and deoxidized SoI substrate. The resist was developed by dipping the substrate in tetramethyl ammonium hydroxide (TMAH) 25 % for 1 min, aaer e--beam exposiLon. Combining HSQ resist as mask with RIE with a SF6 15 sccm / N2 10 sccm / O2 10 sccm plasma (10 mT, 50 W, 80 s in Plasmalab system from Oxford Instrument), we obtained nanoribbons with different lengths and widths (4x4 µm, 4x1 µm, 2x1µm, 2x0.2µm) connected to 30 µm x 30 µm silicon squares for the source and drain contacts. Finally, HSQ resist was removed by wet etching (HF 1%, 1 min). This step reduced the thickness of exposed silicon dioxide to around 134 nm as measured by ellipsometry. The large (100 µm x 100 µm) metal contacts on the 30 µm x 30 µm silicon pads were paZerned by e--beam lithography with a double layer resist (copolymer EL 10 % -- MMA 17.5 % and PMMA 3 % 495 K), 610 nm and 60 nm thick, respecLvely, deposited by spin coaLng. Aaer e--beam exposiLon, the resist was developed with a soluLon of 1/3 Methyl isobutyl ketone (MIBK) and 2/3 isopropanol for 60 s. Metal layer (Ltanium 10 nm and gold 100 nm) was deposited using an e--beam evaporator in high vacuum. The lia--off was carried out in acetone bath. S 2

GRAFTING OF TABINOL

The same procedure was followed for the funcLonalizaLon of the SoI chip bearing silicon nanoribbons of various dimensions and for the silicon wafer used for contact angle measurements, ellipsometry and KP measurements. DeoxidaLon of subtrates and hydrosilylaLon reacLons were realized in a nitrogen glovebox (H2O and O2 < 1 ppm).

Schlenk glassware was dried in an oven at 120°C for 2 days. Hydrofluoric acid (1 % HF in water), mesitylene and deionized water were degassed for 15 min by nitrogen bubbling.

Mesitylene was stored over 4 Å molecular sieves for 48h before use. The substrates were sonicated 5 min in acetone then 5 min in isopropanol. They were immersed in the degassed 1 % HF soluLon for 20 seconds and then thoroughly rinsed with degassed deionized water. Aaer drying under nitrogen flow, they were introduced in a schlenk flask containing a 10 --3 M soluLon of TABINOL in anhydrous mesitylene (3 mg in 10 mL).

The schlenk was sealed then transferred in an oil bath at 160 °C for 2 h. The chip and the Si wafer were sonicated 2 min successively in toluene, acetone then isopropanol, and finally they were dried under nitrogen flow.

DPCP EXPOSURE PROTOCOL

The funcLonalized silicon nanoribbons were fully characterized before and aaer 1 h exposure to DPCP vapors. In a typical experiment, two drops of DPCP were deposited in a 50 mL closed Petri dish to generate a stabilized vapor pressure of DPCP, the chip was then introduced carefully so that it was not in direct contact with the liquid Using the experimental KPFM data ploZed as a funcLon of Vds (data not shown here), we obtain a set of two equaLons which enable to derive the fracLon of C'2 and C'3 probing the NR (αNR) and the adjacent Au metallizaLon (αAu), respecLvely. We find αNR ≈35%, αAu≈30%. The fracLon of C'2 and C'3 probing the SiO2 CPD (αox) is αox=1--αNR --αAu ≈35%. A similar work also enables the determinaLon of the fracLon of C'4 probing the SiO2 layer (α'ox≈87%) and the Au metallizaLon (α'Au≈13%).

From these esLmaLons, we obtain the relaLonship between the measured CPD signal in the center of the NR of the FET device, as a funcLon of the CPD of the NR, oxide (i.e. Thus, these features may partly explain the difference of ΦTAB=CPDTAB --CPDref when measured by KP (in vacuum) --about 400 mV, and by KPFM in a N2 purged ambient (likely with a certain amount of residual oxygen).

TOF--SIMS: TABLE

TEST OF SENSORS WITH SARIN

  ta[ve measurements of the CPD value at the nanometer scale with a poten[al sensi[vity around few mV; (ii) it is a nondestruc[ve technique; (iii) direct characteriza[on of polarized device without important perturba[on due to the absence of contact between the [p and the surface 23 ; (iv) applicability on conduc[ng and insula[ng substrates. Recently, we have demonstrated a highly sensi[ve (sub ppm) and selec[ve nerve agent sensor based on electrical transduc[on of a chemical reac[on occurring on the surface of a func[onalized Silicon

  measurements. Contact poten[al difference (CPD) was measured on large pieces (1 cm x 1 cm) of wafers/samples using a macroscopic Kelvin probe (KP) system (from Kelvin Probe Technologies) in ultra--high vacuum ~ 4--5x10 --10 Torr at 24°C. The Kelvin probe technique measures the contact poten[al difference between two surfaces brought in close proximity. For sake of clarity and consistence with respect to Kelvin probe force microscopy experiments described hereinaeer, we define the CPD as: CPD = (W[p --Ws)/e (Eq. 1) with Ws and W[p are the work func[ons of the sample and the [p respec[vely, and e the absolute magnitude of electron charge (see SI for experimental details). In the actual Kelvin probe instrument a metallic [p is vibrated at a frequency of 80 Hz. Using a high--gain, low noise amplifier, the AC(ω) current generated by the oscilla[on is monitored. Gradual ramping of the counter poten[al and finding zero AC current gives CPD.

  shows the results for the naked Si surface (just aeer oxide removal and cleaning), aeer graeing TABINOL and aeer exposure to room temperature DPCP vapor pressure (for 1 h). Kelvin probe measurements can only provide rela[ve values, i.e. difference of work func[on between the [p and surface under study. This varia[on of work func[on is related to the CPD defined in Eq. 1. We defined the rela[ve values, noted Φ (i.e. ΦTAB=CPDTAB -- CPDref and ΦDPCP= CPDDPCP--CPDTAB), with the reference samples being the naked Si surface.

  in both cases, induces an increase of the CPD of ΦTAB = CPDTAB -CPDREF ~ + 350 mV and ~ + 399 mV for KPFM and KP respec[vely, with CPDREF and CPDTAB the measured CPD on naked Si--NR and aeer TABINOL graeing, respec[vely. This varia[on of 49 mV (14% of the CPD value) between the KP and SI part 6 the air exposure effect on the CPD measured by KP). In KPFM (or KP), the posi[ve (or nega[ve) values for the CPD correspond to a lower (or higher) WF for the sample than the [p. In both techniques, the varia[on measured aeer TABINOL graeing, was associated to an increase of the CPD, corresponding to a decrease of the sample work func[on. These varia[ons measured by the two instruments are in good agreement. These results validate the comparison between measurements by KP and KPFM, which are mutually consistent.

  Si--NRFET (too small surfaces), we used 2D mapping ToF--SIMS experiments to check the surface func[onaliza[on of the nanowire transistors (aeer TABINOL graeing) and to assess the reac[on of tabinol with DPCP. Thanks to molecular imaging mass spectrometry (see Fig.4), we could highlight precisely the localized graeing of TABINOL on the silicon nanoribbon and then the reac[on with DPCP by the observa[on of certain fragment ions resul[ng from TABINOL (C12H22N + , C12H22NO + , C21H32NO + )

  the voltage shie is calculated from the voltage posi[on, Vm, of the minimum current in the IV curves. We have ΔVm ~ 4.2 V and 4.4 V for the forward and reverse bias sweeps, respec[vely. The exposi[on to DPCP vapor induced: (i) a shie of the curves to posi[ve gate voltages (shie of ΔVm = 3.3 V and ΔVm = 4.4 V, respec[vely, for the forward and reverse bias sweep as shown in figure 5); (ii) an increase of the hysteresis behavior. The shie of the IV curves may be due to several factors: (i) charge trapping/crea[on in the SAMs or at the SAM/Si--NR interface; (ii) charge transfer between silicon and molecules (induced interface dipole), (iii) modifica[on of molecular dipole. Each of these three factors leads normally to a change in the silicon SP of the Si--NR, and KPFM is used to gain more insights in the physical origins of the IV shie. KPFM measurements, as expected, show significant changes of the CPD of the Si--NR with the chemical func[onaliza[on (figure 6). KPFM images and CPD profiles show a clear homogeneity on the Si--NRs. The experimental CPD (obtained by averaging on the Si--NR in Fig. 6) were corrected using Eq. 2, with values of Vox and VAU extracted from the KPFM image (Fig. 6) on the oxide region and on the gold electrode, respec[vely. We can no[ce than VAU remains stable aeer TABINOL graeing (VAU ~ +36 mV and VAU ~ +40 mV before and aeer TABINOL graeing respec[vely) and weak shies aeer DPCP exposure (VAU ~ +85 mV aeer DPCP exposure). This CPD shie with the DPCP exposure is explained by the chemisorp[on of few DPCP molecules on the gold electrode as observed by ToF--SIMS (see sec[on 3.3). With the CPD correc[on (using Eq. 2), a significant increase = CPD * TAB -- CPD * REF ~ + 302 mV of the CPD * with the TABINOL graeing is observed, and a weak decrease of = CPD * DPCP --TAB Φ DPCP Φ 10 CPD * TAB ~ -- 61 mV is measured aeer exposure to DPCP, with CPD * REF, CPD * TAB and CPD * DPCP being the corrected CPD (with Eq. 2) on naked Si--NR, aeer TABINOL graeing, and aeer DPCP exposure, respec[vely. The same study was systema[cally repeated for various Si--NR FETs with other Si--NR geometries. On these various devices, dispersion was observed on CPD values (varia[ons of 80% in maximum) and on IV characteris[cs. This dispersion may be explained by the uncertainty on the oxida[on of silicon; KPFM measurements are done in N2 purged atmosphere, thus likely with residual oxygen. But in most cases, the same trends on the CPD than those presented before were observed. If we compare these measurements on Si--NRFET with those on large sample by KPFM (sec[on 3.2), we obtained ~ + 302 mV ~ + 350 mV for the Si--NR FET and for large surface, respec[vely.

  4 we es[mate a value of dipole moment perpendicular to the surface Pz ~ 0.81 D. Since, the KPFM measurement shows a decrease of the WF aeer TABINOL graeing, this dipole has its posi[ve side poin[ng out the TABINOL SAM. This is consistent with the presence of ter[ary amines at the end of the TABINOL. The dipole moment for a single TABINOL molecule in vacuum is es[mated ~ 0.44 D with the MOPAC soeware (ChemOffice Soeware). Considering the classical electrosta[cs of the FET device, a poten[al change of Φ at a distance d (through a material with a dielectric constant εSAM) from the Si channel (virtual top gate) is roughly equivalent to a change ΔVG of the back-- gate voltage through a gate oxide of thickness tox (dielectric constant εox) according to ΔVG = (tox/d)(εSAM/εox ) Φ.With tox = 140 nm, eox = 3.9, d= 1.7 nm (from thickness measured by ellipsometer, see sec[on 3.1) a back--gate shie of 4.2--4.4 V roughly corresponds to Φ of 80--84 mV, i.e. of the same order of magnitude as the KPFM measurements.Aeer DPCP exposure, the situa[on is different. We have measured a weak CPD change from KPFM, ΔΦ = --61 mV, but a sensi[vely larger IV hysteresis (ca. 4.7 V). During DPCP exposure, TABINOL reacts with DPCP and produces azaadamantane quaternary ammonium salt and diphenyl phosphate counter ions on the Si--NR FET surface[START_REF] Clavaguera | Sub--ppm Detec[on of Nerve Agents Using Chemically Func[onalized Silicon Nanoribbon Field--Effect Transistors[END_REF] . Posi[ve charges (N + ) created on top of the monolayer aeer treatment with DPCP (see Fig.1) can eventually modify the CPD. However, a nega[vely charged counter--ion is also created and the monolayer is likely to remain neutral with no further contribu[on to the CPD. Diphenyl phosphate counter ions are randomly oriented on the surface, and, in average, Φ TAB = NP z ε 0 ε SAM we no dot expect a contribu[on of its specific dipole to the CPD. These features are consistent with the weak ΦDPC measured by KPFM, as well as by KP and KPFM on large surfaces. "Mobile" phosphonate ions present in the SAM aeer DPCP exposure can cause the more important hysteresis observed in the IV characteris[cs (4.7 V in Figs.5).

  360 --390 mV. At VG ≠ 0, the smooth transi[on region in the CPD profile outside the Si--NR region extending over the gold electrodes corresponds to side capacitances between the device surface and the KPFM [p 29 (see SI). The length of this region (around 50 µm) is very close to the total width of the AFM [p comprised between 45 - 55 µm according to technical data, showing that the capacitance formed by the different surfaces of the device and the sides of KPFM [p is not negligible. KPFM signals have to be corrected to es[mate the real CPD (Eq. 1). 44;45 Fig. 7--b shows a linear varia[on of the uncorrected effec[ve CPD (value measured on the middle of Si--NR channel) with VG. By using Eq.

From

  the higher back gate poten[als (VG = --10 V and 10 V), we can es[mate that VG induces a varia[on on CPD * Si--NR comprised between 33--42 % of the applied VG. So for VDS = --2 V, we clearly observe by KPFM measurement (i) a modifica[on of the Si--NR CPD with the back gate poten[al (VG) around 68 ± 7 % of VG, and (ii) an important hysteresis of the CPD with VG. The transfer IV curve of the Si--NR FET (Fig 10) is qualita[vely similar to that presented in figure 5 for the sequen[al method, with an ambipolar behavior: the Si--NR FET transistor is in ON state for nega[ve VG < --4 V (conduc[on by holes) and for posi[ve VG (conduc[on by electrons), and the Si--NR

  In summary, we have performed surface characteriza[ons of a sarin detector by KPFM, KP and ToF--SIMS. Simultaneous current--voltage characteriza[on and KPFM mapping on nerve agent sensor based on chemical reac[on occurring on SiNR--FET surface at each chemical steps, were achieved. By this approach, we observed clear correla[ons of the hysteresis between the CPD measured on the Si--NR and the transfer curve of the transistor. More important CPD values were obtained under biased source--drain electrodes and can be explained by the presence of charge carriers into the Si--NR. Comparison between KPFM measurements on the Si--NR FET and KP measurements on large Si TABINOL graeing and a weak decrease aeer the reac[on with DPCP. ToF--SIMS analysis showed the presence of OP molecules essen[ally localized on the Si--NR aeer the SiNR--FET exposure, confirming the effec[veness and selec[vity of the sensor. Finally a sensi[ve prototype exposed to real Sarin vapors and based on the Si--NR FET was successfully demonstrated. This work shows that KPFM is a powerful tool in order to understand the electrical behavior of devices based on chemical surface modifica[ons, and paves the way (i) to an analyze in func[on of the molecular surface coverage due to the sensibility of these surface characteriza[ons and (ii) to the development of commercial sensi[ve, compact, portable, low--cost, and low--consump[on sensors based on the func[onalized Si--NR by OP sensors. 47. Douheret, O.; Anand, S.; Glatzel, T.; Maknys, K.; Sadewasser, S., Characteriza[on of quantum wells by cross--sec[onal Kelvin probe force microscopy. Applied Physics LeTers 2004, 85 (22), 5245--5247.

Figure 1 .Figure 2 .

 12 Figure 1. (a) TM--AFM image a SiNR--FET (70 nm thick, 4 µm length and 4 µm width) and its source and

Figure 3 .

 3 Figure 3. CPD values measured for the naked Si surface, and Si surface treated with TABINOL and

Figure 4 .

 4 Figure 4. (a) High resolu[on Tof--SIMS images (in nega[ve mode) of PO2 --ions observed aeer exposure

Figure 5 .

 5 Figure 5. Typical IDS--VG curves at VDS=--4V measured on 4 x 4 µm² Si--NR FET with "naked" Si (-■-); Si--

Figure 6 .Figure 7 . 34 - 10 Figure 8 .Figure 9 .

 67341089 Figure 6. Tapping AFM image, KPFM image and experimental CPD profile of Si--NR FET of 4 x 4 µm²

Figure 10 .

 10 Figure 10. IDS--VG curves at VDS = --2 V measured on the Si--NR FET 4 x 4 µm² before chemical

Figure 11 .Figure 12 .

 1112 Figure 11. IDS--VG curves at VDS = --1 V measured on the Si--NR FET (4 x 4 µm²) with TABINOL

  Figure SI--1. (a) SchemaLcs representaLon of side capacitances between Lp and surface

  back--gate), and Au metallizaLon pads: CPD = α1. VNR + (α2+α3).(αNR . VNR + αox.Vox + αAu.VAU) + α4.(α'ox.Vox+α'Au. VAu) Numerically, one obtains for the AFM Lp at the center of the Si--NR: CPD = 0.57 VNR + 0.27 Vox + 0.16 VAu When used at fixed VAu (i.e. at fixed Vds condiLons, and without comparing different chemical steps for the NR--FET device), the variaLons of the CPD can be thus corrected from the back--gate bias VG (assuming Vox=VG), and from side capacitance effects, using an effecLve CPD value: CPD* = VNR = (CPD--0.27VG -0.16 VAu)/0.57 = 1.75 (CPD -0.27 VG -0.16 VAu)

  Stability of TABINOL monolayer was verified by placing the funcLonalized large Si pieces in ambient air for 30 min over duraLon of day. It was observed that degradaLon of TABINOL occurred on this exposure to air. The measured CPD by KP shows an increase by about 50 mV aaer a day. It is likely that due to the steric hindrance of the TABINOL not all the surface hydride Si--H sites are bonded with the TABINOL. These hydrid bonds are very sensiLve to the ambient air and they can form hydroxyl (OH) groups when kept in ambient air. Thus when the TABINOL graaed samples are kept in ambient air the hybrid groups form hydroxyl groups and slowly lead to the reformaLon of a naLve oxide at the TABINOL/Si interface. Similarly, bare silicon surfaces were measured by KP at various stages, i.e., immediately aaer removal of the naLve oxide by HF--5% treatment, and when exposed to air for intervals of 6, 12, 18, 24 and 48h, respecLvely. We observed a gradual reducLon of the CPD values indicaLng the growth of an oxide layer. For example, the CPD decreases by about 200 mV aaer 12h of exposure and about 300 mV aaer 48h.The values of the CPD stabilize aaer 48h indicaLng the presence of a stable naLve oxide layer. The values of CPD did not show much variaLon aaer exposed to air for 48h.

  Tedlar bags are commonly used gas sampling bags. Tedlar bags of 20 L were filled in advance with Sarin, determined by weight. The concentraLon of Sarin was expressed in S 9 ppm and was calculated from the weight of CWA and the air volume in the bag and measured with ppbRAE detector.

Figure SI-- 2 .

 2 Figure SI--2. Experimental setup picture

3 .

 3 Connect all components to the flow cell.

4 . 10 5.

 410 Open valve to vacuum pump to create vacuum, down to --0.8 bar.S Close valve to vacuum pump. 6. Open valve to Tedlar bag with sarin. Sarin flows into the flow cell.

7 .

 7 Switch on flow to the ppbRAE detector. 8. Keep flow for at least 10 min.

9 .

 9 Close valve to Tedlar bag. 10. Allow fresh air from the room to enter the flow cell. Keep the flow with the ppbRAE detector on. 11. Flush system with air unLl the ppbRAE detector shows background value.

Figure SI-- 3 .

 3 Figure SI--3. Experimental setup descripLon

Table 1

 1

	gives the results obtained aeer TABINOL

Table 1 .

 1 Water contact angles and thickness values as measured on large pieces (1 cm 2 ) of treated Si surfaces.

		Water Contact Angle (°)	Thickness (Å)
	TABINOL	66	16.5
	TABINOL + DPCP	60	17.8 (1h) --19.3 (2h)

Table 2 .

 2 KP and KPFM characteriza[ons on large surfaces aeer and before TABINOL graeing on silicon (same silicon than for Si--NR fabrica[on). Rela[ve varia[ons of the CPD with the TABINOL graeing are also given.

Table SI -

 SI -1. CharacterisLc fragment ions observed by ToF--SIMS. Mass resoluLon was >7000 for all selected fragments (>8500 for m/z = 196, 249, 294, 312 and 314) Experimentally, the Kelvin probe technique measures the contact potenLal difference (CPDmeas) between two surfaces brought in close proximity as: CPDmeas = (Ws --WLp)/e with Ws and WLp the work funcLon of the sample and the Lp, respecLvely, and e the electron charge. But in order to compare directly CPD values with the same definiLon than KPFM (see Eq. 3 in arLcle), the CPD value presented in KP (CPD) is the opposite of

	Tabinol monolayer	A.er reac0on with DPCP
	Mass (m/z) Assignment	Mass (m/z) Assignment
	145.05	C9H9Si +	145.05	C9H9Si +
	196.17	C12H22NO +	196.17	C12H22NO +
	294.22	C21H28N +	294.22	C21H28N +
	312.23	C21H30NO +	312.23	C21H30NO +
	314.22	C21H32NO +	314.22	C21H32NO +
			62.96	PO2 --
			78.96	PO3 --
			154.98	C6H4PO3 --
			170.97	C6H4PO4 --
	CPDmeas:

6. KP MEASUREMENTS ON SAMPLES EXPOSED TO AIR.

ACKNOWLEDGEMENTS

We thank Dr. Nicolas Nuns (Unité de Catalyse et Chimie du Solide - University of Lille CNRS) for his assistance and advice for the ToF--SIMS analyses. Bruno Simonnet (ID3 Technologies) and Dominique Boichon (NBCSys) are acknowledged for their help in the device fabrica[on for experiments on real Sarin gas; Dominique Deresmes (IEMN) for his assistance on KPFM. This work has been financially supported by ANR, grant n°ANR--10--CSOSG--003, project CAMIGAZ and the French RENATECH network.

ASSOCIATED CONTENT

Supporting information. The Supporting Information is available: Fabrication of Nanoribbon Si FET, grafting of TABINOL, DPCP exposure protocol, KPFM side capacitance corrections, ToF-SIMS: table, KP measurements on samples exposed to air, test of sensors with Sarin.

AUTHOR INFORMATION Corresponding Author

* E--mail: stephane.lenfant@iemn.univ--lille1.fr. Phone : +33 3


Fabrice G Petit
email: fabrice.petit@inserm.fr

Soazik P Jamin

Pierre-Yves Kernanec

Emmanuelle Becker

Guillaume Halet

Michael Primig
email: michael.primig@inserm.fr

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition

Keywords: Exosc10, RNA exosome, nucleolus precursor body (NPB), embryogenesis

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10 -/-mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein's role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10 -/-mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

Introduction

Cell division requires a tightly orchestrated balance between synthesis, modification and degradation of RNAs. The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 contributes to this process in cooperation with the nuclear RNA exosome [START_REF] Kilchert | The regulation and functions of the nuclear RNA exosome complex[END_REF], regulatory proteins [START_REF] Eberle | An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster[END_REF][START_REF] Wagschal | Microprocessor, Setx, Xrn2, and Rrp6 cooperate to induce premature termination of transcription by RNAPII[END_REF], and perhaps alone via its ability to bind RNA [START_REF] Wasmuth | The Rrp6 C-terminal domain binds RNA and activates the nuclear RNA exosome[END_REF]. Extensive work using model organisms such as yeast, fly and mouse has revealed numerous EXOSC10/Rrp6 substrates [START_REF] Davidson | Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control[END_REF][START_REF] Gudipati | Extensive degradation of RNA precursors by the exosome in wild-type cells[END_REF][START_REF] Kuai | A nuclear degradation pathway controls the abundance of normal mRNAs in Saccharomyces cerevisiae[END_REF][START_REF] Lardenois | Execution of the meiotic noncoding RNA expression program and the onset of gametogenesis in yeast require the conserved exosome subunit Rrp6[END_REF][START_REF] Neil | Widespread bidirectional promoters are the major source of cryptic transcripts in yeast[END_REF][START_REF] Pefanis | RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity[END_REF][START_REF] Schneider | Transcriptome-wide analysis of exosome targets[END_REF][START_REF] Xu | Bidirectional promoters generate pervasive transcription in yeast[END_REF], roles in rRNA processing [START_REF] Briggs | Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3' end formation[END_REF][START_REF] Davidson | Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control[END_REF], DNA double strand break repair [START_REF] Domingo-Prim | EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks[END_REF][START_REF] J O U R N A L P R E -P R O O F Marin-Vicente | RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination[END_REF], protein network interactors [START_REF] Meldal | The complex portal--an encyclopaedia of macromolecular complexes[END_REF], and the threedimensional structure of the enzyme [START_REF] Makino | Crystal structure of an RNA-bound 11subunit eukaryotic exosome complex[END_REF][START_REF] Midtgaard | Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain[END_REF][START_REF] Wasmuth | Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA[END_REF]. Targeted gene inactivation studies revealed important functions for mouse EXOSC10 in gametogenesis, erythropoiesis, B-cell enhancer function and forebrain development [START_REF] Jamin | EXOSC10/Rrp6 is post-translationally regulated in male germ cells and controls the onset of spermatogenesis[END_REF][START_REF] Mehta | RNAregulatory exosome complex confers cellular survival to promote erythropoiesis[END_REF][START_REF] Pefanis | RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity[END_REF][START_REF] Ulmke | Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling[END_REF][START_REF] Wu | EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes[END_REF]. Human EXOSC10 is important for cell division of cultured cancer cells [START_REF] Blomen | Gene essentiality and synthetic lethality in haploid human cells[END_REF] and was associated with the polymyositis/scleroderma overlap syndrome [START_REF] Bluthner | Cloning and characterization of the cDNA coding for a polymyositis-scleroderma overlap syndrome-related nucleolar 100-kD protein[END_REF][START_REF] Ge | Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen[END_REF][START_REF] Mahler | Novel aspects of autoantibodies to the PM/Scl complex: clinical, genetic and diagnostic insights[END_REF].

Mouse embryogenesis is initiated when the fertilized egg (zygote) progresses through three rounds of mitotic divisions to form an eight-cell embryo, which undergoes polarization and compaction at embryonic (e) day e2.5; reviewed in [START_REF] Clift | Restarting life: fertilization and the transition from meiosis to mitosis[END_REF]. The embryo then develops into the morula, followed by the blastocyst, which contains a cavity (blastocoel) and the inner cells that differentiate into the primitive endoderm and the epiblast, thereby establishing, together with the trophectoderm, the first three cell lineages. These early developmental steps culminate in the implantation of the embryo at e4.5 [START_REF] Chazaud | Lineage specification in the mouse preimplantation embryo[END_REF][START_REF] Zernicka-Goetz | Making a firm decision: multifaceted regulation of cell fate in the early mouse embryo[END_REF]. Initial stages of embryo development depend upon maternal transcripts that are gradually replaced during the maternal-to-zygotic transition (MZT) stage after zygotic genome activation (ZGA) is initiated. During this process, the maternal transcripts are replaced by newly synthesized and processed RNAs encoded by the embryo's genome [START_REF] Jukam | Zygotic Genome Activation in Vertebrates[END_REF].

The nucleolus is the largest sub-nuclear structure of eukaryotic cells where, among other processes, ribosome biogenesis occurs; nucleoli form at so-called nucleolar organizing J o u r n a l P r e -p r o o f regions (NORs) and have protein, DNA and RNA components organized in a biomolecular condensate [START_REF] Lafontaine | The nucleolus as a multiphase liquid condensate[END_REF]. As opposed to somatic cells, mammalian oocytes and pre-blastocyst stage embryos contain compact nucleolus precursor bodies (NPBs) of unknown composition. NPBs have been considered to be important for the formation of active nucleoli as sites of aggregation for nucleolar proteins; however, is has been proposed that NPBs may indeed predominantly (or even exclusively) exert their essential function in embryogenesis by establishing chromatin domains; reviewed in [START_REF] Fulka | Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries[END_REF].

Our objectives were to gain insights into the expression, localization and function of the conserved 3'-5' exoribonuclease EXOSC10/Rrp6 during embryogenesis in a gene inactivation mouse model.

Results and Discussion

Exosc10 -/-mice show a prenatal lethality phenotype. We generated a knockout line using Exosc10 tm1a(KOMP)Wtsi embryonic stem (ES) cells and the "knockout-first" strategy [START_REF] Austin | The knockout mouse project[END_REF][START_REF] Skarnes | A conditional knockout resource for the genomewide study of mouse gene function[END_REF][START_REF] Testa | A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles[END_REF]) (see Materials and Methods for more details; Fig. 1A). Next, we crossed Exosc10 +/-male and female mice showing no discernable phenotype to generate offspring. We genotyped 931 newborn mice (including 230 dead newborn pups) and detected 315 wild type mice (33.83%) and 616 heterozygous mice (66.17%) but no homozygous mutants; we note that the Exosc10 +/+ and Exosc10 +/-alleles show the expected Mendelian distribution (Fig. 1B,C). This result revealed a novel essential role for Exosc10 during embryo development.

Exosc10 is expressed in oocytes and during all stages of early embryogenesis.

Based on the results of our genetic analysis we set out to determine the early embryonic expression profile of Exosc10 and the protein's subcellular localization pattern in oocytes and blastomeres.

Ovarian folliculogenesis is an important process that prepares the mouse oocyte for embryonic development. The oocyte accumulates maternal RNAs and proteins that decline rapidly after fertilization and reach a minimum at the two-cell stage where the major wave of ZGA occurs in the mouse; reviewed in [START_REF] Li | Maternal control of early mouse development[END_REF][START_REF] Schulz | Mechanisms regulating zygotic genome activation[END_REF][START_REF] Zhang | Maternal control of early embryogenesis in mammals[END_REF]. Successive developmental stages mark the beginning of rRNA gene transcription, the transition from nucleolus precursor bodies (NPB) to mature nucleoli, and embryo compaction when morulae and blastocysts form before the implantation step (Fig. 2A) [START_REF] Baran | Nopp 140 involvement in nucleologenesis of mouse preimplantation embryos[END_REF][START_REF] Zatsepina | The step-wise assembly of a functional nucleolus in preimplantation mouse embryos involves the cajal (coiled) body[END_REF]; for review, see (Kresoja-Rakic and Santoro, J o u r n a l P r e -p r o o f 2019). Since pre-implantation embryonic development involves maternal and embryonic mRNAs/proteins we sought to gain insights into the expression profile of Exosc10 during this phase. To this end, we first mated Exosc10 +/+ female mice at the age of two to six months with vasectomized or fertile C57BL/6N males to obtain oocytes and embryos, respectively.

Next, we analyzed the expression of Exosc10 at oocyte and zygote to blastocyst stages by PCR assays using a wild type allele-specific pair of oligonucleotide primers and found that Exosc10 mRNA is expressed at all stages (Fig. 2B andC). To determine if Exosc10 mRNA present in the oocyte is of maternal origin we mated Exosc10 +/-females with vasectomized males, and analysed the oocytes by PCR genotyping for the mutant allele and by RT-PCR for the wild type mRNA. The results show that Exosc10 -metaphase II (MII) oocytes contain wild type Exosc10 mRNA (Fig. 2D, lanes 4 and 5).

EXOSC10 localizes to the nucleolus precursor body (NPB) in early embryonic cells.

In oocytes we observed a diffuse cytoplasmic signal for EXOSC10 (Fig. 2E). In zygotes we detected a diffuse signal in the cytoplasm and the pronuclei (Fig. 2E). From the two-cell stage onward we found that EXOSC10 accumulates in the nucleus (Fig. 2E). In metaphase and anaphase cells, where the nuclear envelope disintegrates and condensed chromosomes are clearly visible, we observe a weak and diffuse cytoplasmic signal (Fig. 3A). However, in newly formed nuclei containing a nuclear envelope and chromatin the protein is concentrated in the nucleus (Fig. 3A, three-cell panels). In four-cell embryos EXOSC10 is also present in foci (Fig. 3B andC).

Eukaryotic cells contain speckles and paraspeckles, which are membraneless structures composed of long non-coding RNAs (lncRNAs), splicing factors and other RNA binding proteins; reviewed in [START_REF] Chen | Genome organization around nuclear speckles[END_REF][START_REF] Pisani | Nuclear paraspeckles function in mediating gene regulatory and apoptotic pathways[END_REF]. We performed a colocalization assay using marker proteins for speckles (SC35) and paraspeckles (PSF) and found no overlap with the EXOSC10 signal in blastomeres (Fig. 3B andC). This confirms earlier results obtained with cultured fibroblasts and a different anti-EXOSC10 antibody; see Fig. S7 in reference [START_REF] Fei | Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution[END_REF]. Finally, we observed that EXOSC10 accumulates discontinuously at the NPB periphery (Fig. 4A, blastomeres 1a-f and 4g-j) or completely surrounds the NPB, where it colocalizes with the nucleolar marker B23, in different blastomeres of a four-or six-cell embryo (Fig. 4A, blastomeres 2k-l, and Fig. 4B).

Our immunofluorescence data reveal that in blastomeres from the two-cell embryo stage onward EXOSC10 localizes to the periphery of NPBs and to the nucleus, while the protein appears to diffuse into the cytoplasm in oocytes and metaphase blastomeres that lack J o u r n a l P r e -p r o o f structured nuclei. Localization to the NPB is in agreement with the protein's well established function in ribosomal RNA processing [START_REF] Davidson | Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control[END_REF][START_REF] Kent | The 5' external transcribed spacer in mouse ribosomal RNA contains two cleavage sites[END_REF] and may point to a potential role in establishing chromatin domains [START_REF] Chun | RNA surveillance controls 3D genome structure via stable cohesin-chromosome interaction[END_REF][START_REF] Fulka | Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries[END_REF]. A critical role for EXOSC10 during cell growth and division via controlling protein translation is also supported by work on the anti-cancer drug 5-FU. This compound inhibits the ribonucleolytic activities of the yeast, fly and human EXOSC10 orthologs, which provides a straightforward explanation for the drug's interference with late rRNA processing (notably of 5.8S rRNA), ribosome biogenesis and translation elongation by inhibiting the ribonucleolytic activity of EXOSC10 [START_REF] Abou Elela | Role of the 5.8S rRNA in ribosome translocation[END_REF][START_REF] Burger | Chemotherapeutic drugs inhibit ribosome biogenesis at various levels[END_REF][START_REF] Fang | 5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs[END_REF][START_REF] Hoskins | Evidence for distinct DNA-and RNA-based mechanisms of 5-fluorouracil cytotoxicity in Saccharomyces cerevisiae[END_REF][START_REF] Kammler | The RNA exosome component hRrp6 is a target for 5-fluorouracil in human cells[END_REF][START_REF] Silverstein | The incorporation of 5fluorouracil into RNA affects the ribonucleolytic activity of the exosome subunit Rrp6[END_REF].

Exosc10 -/-embryos arrest at the eight-cell embryo/morula stage. Next, we asked at which stage homozygous mutant mice undergo a developmental arrest in utero. We first analyzed 30 embryos from six independent litters at day e7.5 and observed 10 wild type (33.3%), 20 heterozygous (66.7%), and no homozygous mutant embryos (Fig. 5A). Further analysis of 49 embryos from 10 litters at day e6.5 revealed eight wild type (16.3%), 41 heterozygous (83.7%) and still no homozygous mutant embryos (Fig. 5A). Finally, we genotyped 32 embryos from seven litters at day e4.5 and found 11 wild type (34.4%), 21 heterozygous (65.6%) but no mutant lacking Exosc10 (Fig. 5A). These results demonstrate a homozygous mutant arrest phenotype during early embryogenesis prior to the embryo implantation step.

Given that maternal wild type Exosc10 mRNA is present in Exosc10 -oocytes, we assumed that these embryos are able to reach the two-cell stage before embryonic genome activation occurs and maternal RNA decays. To determine the point at which homozygous mutants arrest beyond that stage, we cultured two-cell embryos for five days. We first asked if our in vitro culture conditions allowed for embryogenesis to proceed efficiently (Fig. 5B). To this end, we incubated wild type and heterozygous embryos stemming from crosses between eight Exosc10 +/+ females and Exosc10 +/-males, and found that our culture conditions did not significantly affect embryo development, since 96% (48/50 embryos) reached the blastocyst stage, while only 4% (2/50) died at the eight-cell embryo/morula stage (Fig. 5C). Next, we analysed embryos stemming from crosses between 12 Exosc10 +/-females and Exosc10 +/- males and found that 33% (36/109) undergo a developmental arrest at the eight-cell embryo/morula stage (Fig. 5B andC). Bearing in mind that 25% of the embryos from a heterozygous intercross are expected to be homozygous mutants, and that the vast majority of J o u r n a l P r e -p r o o f wild type and heterozygous embryos develop normally, this result suggests that most arrested embryos lack functional EXOSC10.

Finally, we sought to further substantiate our findings by genotyping cultured embryos using wild type and mutant allele-specific primer pairs (Fig. 5D). First, we assayed 37 embryos from control crosses between Exosc10 +/+ females and Exosc10 +/-males and found one wild type (100%) but no heterozygous mutant at the eight-cell embryo/morula stage, while 17 (47.2%) homozygous and 19 (52.8%) heterozygous embryos developed into blastocysts (Fig. 5E). In comparison, we genotyped 53 embryos from Exosc10 +/-intercrosses, and observed two wild types (16.7%), four heterozygotes (33.3%) and six homozygotes (50%) among 12 embryos arrested at the eight-cell embryo/morula stage, and 11 wild types (26.8%), 30 heterozygotes (73.2%) and no homozygotes among 41 embryos at the blastocyst stage (Fig. 5E).

Given the known roles of EXOSC10, a plausible interpretation of our data is that the arrest phenotype is due to disrupted nucleolar rRNA processing, which leads to impaired ribosome biogenesis and translation elongation (Abou Elela and Nazar, 1997; [START_REF] Davidson | Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control[END_REF][START_REF] Knight | Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis[END_REF][START_REF] Kobylecki | Elimination of 01/A'-A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3[END_REF]. We note that UBF -a co-factor of RNA Polymerase I (POLR1B), which is important for rRNA transcription -shows peripheral NPB localization similar to EXOSC10's pattern [START_REF] Kone | Three-Dimensional Distribution of UBF and Nopp140 in Relationship to Ribosomal DNA Transcription During Mouse Preimplantation Development[END_REF] and mutant embryos lacking functional UBF or POLR1B arrest at the morula stage [START_REF] Chen | Early pre-implantation lethality in mice carrying truncated mutation in the RNA polymerase 1-2 gene[END_REF][START_REF] Hamdane | Disruption of the UBF gene induces aberrant somatic nucleolar bodies and disrupts embryo nucleolar precursor bodies[END_REF]; for review, see [START_REF] Kresoja-Rakic | Nucleolus and rRNA Gene Chromatin in Early Embryo Development[END_REF]. Moreover, mice lacking the NPBassociated ribosome biogenesis factor PES1 also show an early phenotype by failing to progress normally through the eight-cell embryo/morula/blastocyst steps [START_REF] Lerch-Gaggl | Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation[END_REF]. In addition to impaired ribosome biogenesis, it is conceivable that altered RNA exosome-dependent and cohesin-mediated genome architecture also contributes to Exosc10's embryonic lethal phenotype [START_REF] Chun | RNA surveillance controls 3D genome structure via stable cohesin-chromosome interaction[END_REF]. This is a tempting idea in the context of earlier findings that implicate NPBs and nucleoli in the spatial organization of the genome; reviewed in [START_REF] Fulka | Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries[END_REF][START_REF] Kresoja-Rakic | Nucleolus and rRNA Gene Chromatin in Early Embryo Development[END_REF].

Conclusion

Collectively, our results suggest that Exosc10 -/-mutants arrest at the eight-cell/morula transition prior to formation of the blastocyst. It will be exciting to learn more about the effects disease-related EXOSC10 alleles might have on cell division, growth and J o u r n a l P r e -p r o o f development; reviewed in [START_REF] Stuparevic | Regulation of the conserved 3'-5' exoribonuclease EXOSC10/Rrp6 during cell division, development and cancer[END_REF], especially since interpreting how mutations might alter protein function is greatly facilitated by the novel AlphaFold protein structure prediction algorithm [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF]. Generation of Exosc10 knockout (KO) mice. We generated the Exosc10 KO mouse strain using Exosc10 tm1a(KOMP)Wtsi embryonic stem (ES) cell clones EPD0567_5_B04 and EPD0567_5_F03, which were provided by the Knockout Mouse Project (KOMP) funded by the NIH and obtained via the KOMP Repository (www.komp.org) funded by the NCRR-NIH [START_REF] Jamin | EXOSC10/Rrp6 is post-translationally regulated in male germ cells and controls the onset of spermatogenesis[END_REF]. Exosc10 knockout first, promoter driven mice were generated based on the "knockout-first" design, where a trapping cassette including the En2 splice acceptor (En2 SA) and SV40 polyadenylation (pA) sequences is inserted within the second intron of Exosc10 such that splicing between the first and second exons and En2 SA creates a knockout Exosc10 allele [START_REF] Skarnes | A conditional knockout resource for the genomewide study of mouse gene function[END_REF][START_REF] Testa | A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles[END_REF]. ES cell clones were used to generate chimeric mice at Institut Clinique de la Souris (ICS, www.ics-mci.fr). After germline transmission, Exosc10 +/-mice were transferred to our local animal facility. Two-cell embryo culture. We mated Exosc10 +/+ or Exosc10 +/-females with stud Exosc10 +/- male mice and recovered two-cell stage (gestational day 2; GD2) embryos by flushing approximately 0.5 ml of EmbryoMax Advanced KSOM medium (Merck-Sigma, MR-101-D) into the oviduct. We placed embryos in a 96-well plate containing 100 l of Advanced KSOM medium, cultured them in a humidified incubator with 5% CO2 at 37°C and J o u r n a l P r e -p r o o f monitored them daily (2-4PM) from day two (two-cell stage, GD2) to day six. Embryos where collected and rinsed three times in H2O before processing them for PCR genotyping.

Materials and methods

Ethics

We documented the process using a CELENA S Digital Imaging System (Logos Biosystems).

Unfertilized oocyte and embryo recovery. We obtained unfertilized oocytes by mating Exosc10 +/+ or Exosc10 +/-female mice with vasectomized males. Mice were sacrificed the day a vaginal plug was observed (GD1) or e0.5). Under a stereo dissecting microscope we cut the ampulla region of the oviduct and collected the cumulus oocyte mass in prewarmed M2 medium (Sigma, France). We removed the cumulus mass from ovulated eggs by incubating them for 5-10 min in M2 medium supplemented with 1 mg/ml hyaluronidase (Sigma) at 37°C. To recover embryos from zygote to morula stages (GD1 to GD3), we inserted a 30gauge needle attached to a 1 ml syringe filled with prewarmed M2 into the oviduct and flushed the embryos out. To collect blastocysts and post implantation embryos (GD4 and GD6 to GD8), we inserted a 27-gauge needle attached to a 1 ml syringe filled with M2 into the uterine horn and flushed the embryos out. Oocytes and embryos were rinsed twice in M2 and three times in H2O before processing for reverse transcription or PCR. France), 0.1 mM mixed primers (Ef2/Er3/L3f/Lxr, 1:1:1:1, Eurogentec, France), 1x Q solution and 0.017 units of HotStar Taq Plus DNA polymerase (Qiagen, France). PCR was at 95°C for 5 min, 35 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min and 72°C for 10 min.

Oligonucleotide primers for mouse and embryo genotyping and the detection of mRNA.

Embryo genotyping. We collected embryos at e4.5, e6.5 and e7.5 and digested them in 6 µl of 50 mM NaOH at 95°C for 10 min before adding 0.5 µl of 1 M Tris-HCl pH 8.0. For genotyping, we set the parameter at 40 cycles. For cultured embryos, we lysed samples in 8 µl of lysis buffer PBND containing 0.1 mg/ml Proteinase K (Roche) [START_REF] Scavizzi | Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach[END_REF] and incubated them at 56°C for 30 min before we inactivated Proteinase K at 95°C for 10 min. PCR was set at 45 cycles using Ef/325as for wild type and Neo-s/Neo-as for mutant alleles.

Reverse transcription and PCR analysis.

We isolated total RNA from oocytes to blastocysts using the Arcturus PicoPure kit (Applied Biosystems). We extracted RNA in 50 µl of Extraction buffer, eluted total RNA in 10 µl of Elution buffer and reverse transcribed RNA in 20 µl using the iScript cDNA synthesis kit (Bio-Rad). We incubated the samples for 5 min at 25°C, 20 min at 46°C and 1 min at 95°C. PCR was performed using 2 µl of RT sample following the mouse genotyping protocol for 42 cycles. We used 390s/503as primers for wild type Exosc10 mRNA and GAPDH-s/GAPDH-as primers as a control.

Detection of the maternal Exosc10 mRNA. We obtained unfertilized MII oocytes from

Exosc10 +/-female mice and rinsed them in H2O before transferring them into a PCR tube in a variable volume of 1-5 µl H2O. We processed samples in a thermal cycler during three cycles at 65°C for 5 min and 4°C for 1 min. Subsequently, the RNA was reverse transcribed (RT) using the iScript cDNA synthesis kit (Bio-Rad) in a final volume of 10 µl. Therefore, the resulting samples contain both genomic DNA and cDNA. We assayed the KO allele and the wt mRNA using Ef/Kr and 390s/503as primer sets, respectively. We performed PCRs at 45 cycles using 10 µl of RT sample in a final volume of 20 µl.

Immunofluorescence (IF) staining.

For IF/confocal analyses 27 Exosc10 +/+ or C57BL/6NRj female mice (Janvier Labs, Le Genest Saint Isle, France) were used to collect 52 oocytes, 35 zygotes, 46 two-cell embryos, 83 four-cell embryos, seven six/eight-cell embryos, eight morulae and one early blastocyst. Confocal images were generated with four oocytes, six zygotes, 12 two-cell embryos, 23 four-cell embryos, three six/eight-cell embryos, four J o u r n a l P r e -p r o o f morulae and one early blastocyst. We collected unfertilized oocytes and embryos in M2 medium and fixed them in 4% paraformaldehyde (Electron Microscopy Sciences) 1x PBS for 30 min at room temperature. We permeabilized the samples in 5% BSA-0.5% Triton X-100 1x PBS for 30 min at room temperature before rinsing them in blocking buffer (5% BSA in 1x PBS). The first antibody diluted in blocking buffer (1:200) was incubated at 4°C overnight. A few samples were incubated in blocking buffer only ("no primary antibody") as a negative control. The next day, we washed the samples three times in 1x PBS before adding the secondary antibody (1:500 in blocking buffer) for 60 min at room temperature. We washed the samples three times in 1x PBS and kept them in a 1% BSA 1x PBS solution until mounting. Antibodies used in this study were against EXOSC10 (Abcam, ab50558), B23 (Sigma, B0556), PSF (Sigma, P2860) and SC-35 (Sigma, S4045). Secondary antibodies were Alexa Fluor 488 (Life Technologies, chicken anti-rabbit antibody A-21441) and Alexa Fluor 555 (Life Technologies, donkey anti-mouse antibody A-31570).

Confocal imaging, image processing and analysis. We kept unfertilized oocytes and embryos (zygotes to blastocysts) in 1% BSA 1x PBS after immunofluorescence staining before we placed them on glass-bottom dishes (MatTek Life Sciences, Ashland, MA, USA) for inspection with a Leica SP5 confocal microscope equipped with a Plan-Apo 63x/1.4 HC oil immersion objective and lasers with excitation lines at 488 nm, 561 nm and 633 nm. We used To-Pro-3 to stain DNA (far red-fluorescent stain, Invitrogen). We acquired image stacks at 1 µm intervals and processed them using the Application Suite Advanced Fluorescence software (Leica). ImageJ (version 1.53j) software available from the NIH (https://imagej.nih.gov/ij/) was used to create composite images by merging the red and green channels and to obtain intensity RGB profile plots.

J o u r n a l P r e -p r o o f

  Statement. Studies involving animals, including housing and care, method of euthanasia and experimental protocols were conducted in accordance with the French regulation for Laboratory Animal Care, issued from the transposition of the European Directive 2010/63/UE. This Directive stipulates in article 1, alinea 2 that euthanizing laboratory animals for the sole purpose of investigating their tissues does not require prior authorization by an ethics committee. Vasectomized mice used in this study were part of a project approved by the French ministry of research (APAFIS#25237-2020042715505401 v2). The animal facility was licensed by the French Ministry of Agriculture (C35-238-19). Animals were sacrificed using CO2, according to a method in Annex IV of the directive. All experiments were supervised by Fabrice G. Petit who is licensed for animal experimentation by the French Ministry of Agriculture (A92-313).

  Ef2 -TCT AAG CCT GAC AGC ATG AAT TGA ACC Er3 -GTG GTG TAG ACT TGT GAT ACT GC L3f -TGC AAG GAG AAA CCC CAG TGT TAC G Lxr -TTA TCA TTA ATT GCG TTG CGC CAT C Ef -GAT GGA GCG AGC AAG CTT CTG Kr -CCA ACA GCT TCC CCA CAA CGG 325as -CTG CGA CAG CCA TGG TAC T 390s -AAT GAC GTG ATA TTG GAG AGA GTG 503as -GGA TAC TAT TGT TTT GGG GAC CT Neo-s -GCC CTG AAT GAA CTG CAG G Neo-as -CAC GGG TAG CCA ACG CTA T GAPDH-s -TGG ATC TGA CAT GCC GCC TG GAPDH-as -ATG TAG GCC ATG AGG TCC AC Mouse genotyping. We digested tail tips collected at weaning and from sacrificed animals in 600 µl of 50 mM NaOH at 95°C for 60 min and before adding 50 µl of 1 M Tris-HCl pH 8.0 and centrifuging samples as 13,400 x g for 5 min. Then we added 1 µl of supernatant to a J o u r n a l P r e -p r o o f final volume of 15 µl containing 1x CoralLoad PCR buffer, 0.1 mM dNTP (Invitrogen,

Figure 1 .

 1 Figure 1. Gene deletion strategy and embryonic lethal phenotype. (A) A schematic shows Exosc10 wild type (wt) and mutant (KO) alleles. Critical exons are numbered. The knockoutfirst allele generated by KOMP allows the inactivation of Exosc10 via splicing between exon 1-2 and the mouse En2 splice acceptor (En2 SA). Annotated black arrows symbolize PCR primers producing positive amplicons for genotyping and expression assays. (B) A representative genotyping PCR assay is given. Lane 1 contains markers (M) and lanes 2-4 contain samples from wild type (+/+) and heterozygous (+/-) or homozygous mutants (-/-). The PCR amplicon is 405 bp for wt and 191 bp for KO. (C) A chart summarizes the genotypes observed for newborns from heterozygous intercrosses. Percentages are shown and the numbers of mice analysed are given in parentheses.

Figure 2 .Figure 3 .

 23 Figure 2. EXOSC10 expression and localization during early embryogenesis. (A) A schematic shows critical phases during folliculogenesis and embryogenesis. Important steps are indicated by blue arrowheads. The timing and levels of maternal and embryonic gene expression patterns, formation of the nucleolus and ribosomal RNA expression are shown. (B) A schematic of the first four Exosc10 exons locates PCR primers used to assay mRNA expression. The output of PCR expression assays with or without the reverse transcription step (-/+RT) is shown. Lane 3 contains the markers (M). An arrow marks the diagnostic bands for which the size is given in bases. (C) The output of PCR expression assays is shown. Lanes 1 and 2 contain a positive control (RT) and markers (M). Lanes 3-14 contain duplicate samples from an unfertilized egg (Oocyte), a fertilized egg (Zygote), two-and eight-cell embryos (2-cell, 8-cell), morula and blastocyst (Blast). GAPDH was used as a control. (D) The output of PCR expression assays is shown. Lanes 1-4 contain controls for RNA (Testicular RNA, RT+/+), genomic DNA (gDNA+/-), RT/gDNA and markers (M). The remaining lanes contain six samples from female gametes as shown. Diagnostic bands for wild type (wt) and deletion (KO) alleles are indicated. (E) Confocal images of an oocyte, a zygote and early embryos are given. The top row shows brightfield images, the middle and bottom rows show immunofluorescence images of cells assayed for the target protein (EXOSC10) in green and DNA (To-Pro3) in magenta, respectively. The zygote's pronuclei (PN) are indicated. An insert in the image showing the zygote delineates the localization of the second PN. Scale bars (including the insert) are 20 m.

Figure 4 .

 4 Figure 4. Localization of EXOSC10 at the periphery of the nucleolar precursor bodies. (A) Z-stack images of co-staining patterns for the target protein (EXOSC10, green) and a nucleolar marker protein (B23, red) in a four-cell embryo (brightfield) are shown. Intervals between images are 1 µm for blastomeres 1 and 4, and 3 µm for blastomere 2. Schematics show the positions of confocal sections within individual blastomeres as indicated. The fluorescence intensity was measured along the yellow lines drawn on the composite images (yellow arrows mark the direction of reading) and the corresponding intensity profiles were displayed underneath. White arrowheads mark EXOSC10 foci and partial or complete peripheral signals. Scale bars are 20 µm and 5 µm (1a-f, 4g-j, 2k-l). (B) Confocal images of a six-cell embryo stained for EXOSC10 (green), B23 (red) and DNA (To-Pro3, magenta) are shown. White arrowheads mark EXOSC10 foci and partial or complete peripheral signals.Scale bars are 20 µm for the six-cell embryo brightfield, 10 µm for the enlargement of a blastomere, and 2.5 µm for the merge insert.

Figure 5 .

 5 Figure 5. Homozygous mutant developmental arrest phenotype during in utero and in vitro embryogenesis. (A) A table summarizes the genotypes of embryos collected at e7.5, e6.5 and e4.5 (columns 1 and 2). The numbers of litters (column 3), embryos (column 4) and observed genotypes (columns 5-7) are shown. The data for wild type mice and hetero/homozygous mutants are color-coded for clarity. Numbers of embryos assayed are indicated and percentages are given in parentheses in columns 5 and 6. Scale bars are 100

Acknowledgments

We thank the Knockout Mouse Project (KOMP), the Institut Clinique de la Souris (ICS) and the UMS 3480 -US 018 Biosit Facility Ibisa Microscopy Rennes Imaging Center (MRic-Photonics) for providing excellent services.

Funding

This project was supported by research grants from La Ligue Contre le Cancer committees CD29 and CD35 awarded to M. Primig. Additional funding was provided by Inserm, EHESP and the University of Rennes 1. The funding sources were not involved in the design of our study.

J o u r n a l P r e -p r o o f


Pierre Dias

Colin Kerouanton

Sandrine

Children's daily

Children's daily mobility changes and outdoor parenting profiles: From travel and place patterns to educational requirements Dias Pierre1 ; Kerouanton Colin 2 ; Depeau Sandrine1

Context

Children's mobility is an important issue to understand brakes and levers of the change of travel behavior inside the family. For the past 30 years, a decline in children's independent and active travels has been observed and explained by many factors (spatial, social, psychological). Among them, we can advance the parenting role.in childrenurban environment relationships. that operates in children's mobility socialization.

Conclusion

By focusing on active travels profiles of children, these first results show the changes in children's mobility between the 5th and the 6th grade. Then, it also raises question about the role of parents' educative profile, which are based on child-parent interactions in the choices of mobility but also on the exchanges with children and trusting relationships with the child from the outside. They make it possible to question the process of socialization to mobility and its role in encouraging active mobility. Future data processing should make it possible to define urban educational cultures by associating travel profiles.

Aims & main questions

1/ Development of children's daily travel behaviors during the transition from primary to secondary school; 2/ Children's mobility profiles for each school level;

3/ How these children's mobility profiles are part of parenting style related to mobility.

Methods and process

Data were collected longitudinally (2018-2019) using an integrated device (MK MOBIBACK) mixing GPS data loggers (tracking over five days) and a complementary survey on tablets allowing : 1/ to enrich GPS data (mainly places and trips), 2/ to question children's socialization, autonomy, free-times and family attitudes about their child's independence.

Field & particpants

Families (n=86) interviewed in Rennes (n= 42) and in south urban periphery (n=44). Children and one of their parents were surveyed in both the 5th (n children= 90) and 6th grades (n children = 59). Between an important transition for families that often involves a change in the mobility habits of children.

27 th IAPS conference -5-8 July 2022, ISCTE

Main results

-Overconfidence or bias of overestimation of their abilities for children in primary school less experienced in moving independently. Those children are then more willing to make unplanned trips to middle school. For unknown routes, no greater ease of movement among middle school students.

-Children's ability to imagine certain modes of transport to compensate for the effects of increased distance. I.e Use/knowledge of transport networks are used as cognitive support.

Flexible parents are also those who may experience anxiety, prescriptive recommendations, and car coaching. Does not seem to favor the development of a strong autonomy, this is observed especially for girls.

Analysis: MCA and a HAC with selected variables in primary and secondary school.

Observation of 4 groups of evolutions according to selected variables Contact: sandrine.depeau@univ-rennes2.fr