Beilei Du
email: beilei.du@ep5.rub.de

Sebastian Mohr

Dirk Luggenhölscher

Uwe Czarnetzki

L A Kennedy

An atmospheric pressure self-pulsing micro thin cathode discharge

Keywords: numbers: 52.25.Os, 52.50.Dg, 52.50.Nr, 52.70.Ds, 52.70.Kz, 52.80.Mg References

A micro thin cathode discharge (µTCD) is a modified micro hollow cathode discharge (MHCD) where the anode is much thicker than the one in a MHCD, which allows a stable operation at atmospheric pressure. The µTCD was operated at different pressures in argon by a DC voltage. At atmospheric pressure, the µTCD operates in a self-pulsing mode with current peaks with a duration of a few nanoseconds and a current density up to 10 5 A/cm 2 , supplied by the charge stored in the capacitance of the discharge device. The plasma emission was observed by an ICCD camera. Both, the voltage -current characteristic and the ICCD image indicate that this self-pulsing is due to a periodic ignition of a spark discharge. The electron density was measured by the Stark broadening of the H β -line with an admixture of 1% hydrogen. In the periodic spark mode the electron density is of the order of 10 16 cm -3 . The direct measurement of the discharge current in these kind of discharges is not possible, since it is driven by the inherent capacitance of the discharge device. Therefore, an electrical model including stray capacitances is set up. The modelled results agree very well with the measured currents and voltages.

Introduction

As a kind of microcavity discharge, micro hollow cathode discharges have simple electrode geometries [1] and have found various applications in lighting as UV radiation sources [2 -6], as plasma cathodes to generate large volumes of atmospheric pressure plasmas [7] as well as for analytical applications [8]. Basically, a MHCD consist of two electrode layers (anode and cathode) separated by a dielectric layer. Each layer is several 100 µm thick. The discharge is developed in a hole (several 100 µm in diameter) which penetrates the cathode and the dielectric. The anode layer can have various shapes [9]. In the current research, the common anode shape is identical to the cathode [8, 10 -12].

Under certain conditions, the MHCD shows self-pulsing with oscillations of the discharge voltage and current [9 -12]. At medium pressure, several tens of hectopascal, the MHCD can be operated in a selfpulsing mode with a current peak of several milliamperes and with a duration of several microseconds. Rousseau and coworkers [12] showed that the self-pulsing is related to a periodic transition between two modes: the plasma is confined inside the hole and the plasma expands outside the hole onto the cathode backside. On the other hand, the MHCD can be operated in another self-pulsing mode with a current peak which is much higher than several milliamperes and with a duration of only several nanoseconds. This self-pulsing mode was reported by Schoenbach [9] firstly in a MHCD with a closed anode at over-atmospheric pressure. In our study, we chose an anode with the same hole diameter as the one of the cathode, but which is much thicker (1 mm) than the cathode and the dielectric, since the thick anode provides the mechanical stability for the whole discharge setup.

The time resolved discharge voltage and current measurement showed that our discharge operates in both self-pulsing modes. In our study, we focus on the self-pulsing with high current peaks with nanosecond a duration at atmospheric pressure. The plasma current density and the ICCD image of the plasma volume indicate that this self-pulsing mode is related to a periodically repeated ignition of a spark discharge. The thinness of the cathode (in our case 100 µm) leads to enable the cathode to heat very quickly and to emit electrons by thermionic emission with a high current density. Although we have no detailed measurements on the hypothesis of a hot cathode spot, reversal of the polarity of the applied voltage inhibits the development into the self-pulsing regime. Thus, we prefer to call this device micro thin cathode discharge (µTCD) instead of micro hollow cathode discharge. The electron density in argon was measured by the Stark broadening of the H β -line with an admixture of 1% hydrogen. The time averaged electron density can reach up to 10 16 cm -3 . In the voltage and current measurement, a peak current as high as several amperes was measured. Due to a 500 kΩ series resistor, the peak current measured on the anode side should be in the milliampere range. An electrical model was built and carefully analyzed. The existence of a stray capacitance is the reason for the measured high peal current. Very good agreement between the modelling results and the measurement was obtained.

The construction of the µTCD and the experimental setup are introduced in the next chapter, followed by the measurement results including the analyses in chapter 3. Finally, conclusions are drawn.

Experimental setup

Because of the expected high gas temperature of about 1000 K [13], molybdenum and alumina were used as electrodes and dielectric layer respectively. Different from the usual MHCD with symmetric electrodes, the electrodes of the µTCD are asymmetric (Figure 1a). One electrode, which was used as anode, is much larger and thicker than the other one. The dielectric layer has the same thickness as the cathode and separates the electrodes. To avoid an unwanted electrode edge ignition, the dielectric is a little larger than the anode. The plasma is developed in a mechanically drilled hole with a diameter of 200 µm penetrating the sandwich structure. The three layers are pressed together by a PVC holder fixed by screws (Figure 1b). The thick and large anode provides mechanical stability for the fragile alumina layer. The holder has an axial opening with 5 mm diameter in order to avoid obstructing the plasma development on the electrode surface. The capacitance of the entire discharge setup including the PVC holder is 120 pF, measured by a LCR meter. The discharge is generated by a DC voltage supply. To avoid an overheating of the electrodes, the applied voltage was pulsed with 10 Hz and with 1% duty cycle. A 500 kΩ series resistor R 1 and a 100 Ω measurement resistor R 2 were connected in series at the cathode side and the anode side respectively (Figure 2). High voltage probes (Lecroy PPE20kV) were used to measure the discharge voltage and the current via the measurement resistor R 2 . A fast oscilloscope (Lecroy Wavepro 7100A) was used to read out the temporally resolved potentials, V 1 and V 2 . The discharge voltage U d and the current I R via R 2 , are deduced from the measured potentials:

2 1 V V U d - = , 2 2 R V I R = .
In a DC discharge, I R equals the plasma current. When the discharge operates in the self-pulsing mode, according to the experimental setup, the AC part of the plasma current I pl cannot be directly measured on R 2 but can be deduced from the change of the discharge voltage in case that the external current measured on R 2 is much smaller than the discharge current inside the capacitor: The emission spectrum of the µTCD was recorded by a 2m-spectrograph with 0.01 nm spectral resolution equipped with an intensified diode array detector. The gate-width is 33 ms which is longer than the duration of the applied voltage. Therefore, the emission is time integrated. An optical fibre mounted at the cathode side guides the emission light to the spectrograph, i.e. the emission spectrum is spatially integrated, too. In order to obtain the emission image of the plasma volume, a microscope objective with 17 mm working distance (Nikon CFI L Plan Epi SLWD 50X) was mounted in front of the camera objective (Figure 3). In this way, the image is magnified. The magnification depends on the focal length of both objectives. The focal lengths of the microscope objective f m and the camera objective f c are 4 mm and 50 mm respectively. The magnification is then:

5 . 12 = = m c f f M .
Because of the limitation of the vacuum chamber, the µTCD cannot be placed in the working distance of the microscope objective. An achromatic lens with 50 mm focal length was used to image the plasma volume outside the vacuum chamber in a ratio of 1:1. This image is magnified by the objective system and recorded.

Results and discussion

Two self-pulsing modes

Self-pulsing at atmospheric pressure

At atmospheric pressure temporally resolved voltage and current measurements showed that the µTCD operates in a self-pulsing mode with a very stable frequency (Figure 4a). Zooming in Figure 4a, the voltage drops very fast to zero (Figure 4b). When the voltage drops, the plasma current I pl reaches a peak value as high as 30 A, and lasts only several nanoseconds. According to the discharge cross section, the current density is as high as 10 5 A/cm 2 . The current I R via the resistor R 2 is only 10% of the plasma current, but much higher than the limit due to the series resistor. The current I R will be discussed in section 3.3 along with an electrical model. Figure 4c

Self-pulsing at medium pressure

At medium pressure the µTCD shows a self-pulsing similar to the self-pulsing in a MHCD. The voltage oscillates but does not drop to zero. The peak current is in the range of milliamperes with a duration of a few microseconds (Figure 5a), which is obviously longer than the current peak width in the self-pulsing at atmospheric pressure showed in Figure 4b. This self-pulsing is explained by Rousseau and coworkers [11,12]. The oscillation of the discharge voltage and current is related to a periodic transition between two modes: the plasma is confined inside the hole and the plasma expands outside the hole onto the cathode backside. The time integrated ICCD image of the µTCD shows that the hot electrons are located in the centre of the hole (Figure 5b). Although the emission during the expansion onto the cathode backside surface is much weaker than the emission from the plasma confined inside the hole, one can still recognize this mode in Figure 5b.

The exposure times for the images in Figure 4c and Figure 5c are both 1 ms which is the duration of the applied voltage. The self-pulsing frequencies for both cases are about 25 kHz which means that each exposure integrates over about 25 pulses. However, the duration of each single pulse in the case of the spark mode is much shorter (several nanoseconds) than in the other case. Therefore, the integrated maximum intensity for the spark mode is only one order of magnitude higher. The discharge is always constricted at the edge when it goes into the spark mode. At pressures close to the threshold, the transition to this mode can be stochastic. Although the location at the edge of the discharge in the spark mode can vary, it is usually fixed over a large number of pulses.

The µTCD shows two self-pulsing modes in different pressure regions. At medium pressure the selfpulsing is due to the plasma expansion which is the same as in a MHCD, although the µTCD anode is much thicker than the one of the MHCD. At atmospheric pressure the self-pulsing discharge produces strongly localized sparks. In this case, the thinness of the cathode is more important than its hollow structure.

Electron density

The electron density was measured by the Stark broadening of the H β -line.

⋅ = ∆ , ( 1
)
where λ is the wavelength, T is the gas temperature and m is the atomic weight of the hydrogen atom.

The dipole interaction of an excited atom with the induced dipole of a ground state atom results in the van der Waals broadening. The FWHM of the van der Waals broadening profile in approximated by the following expression which is strictly applicable to hydrogen and similar atomic structures [19]:

] [ ] [ ] [ ] [ 10 3 3 10 3 5 2 6 2 2 18 - -         ⋅ ≈ ∆ cm N u K T C nm µ λ λ , ( 2
)
where λ is the wavelength, T is the gas temperature, µ is the reduced mass of the atom-perturber (argon) and N is the perturber density. C 6 is the interaction constant and is calculated according to [17]. In case of this experiment, the interaction constant is 3•10 -31 .

The gas temperature is estimated to be 500 K at medium pressure and 1000 K at atmospheric pressure [8]. With these gas temperatures, the FWHMs of both Doppler broadening and van der Waals broadening contribute less than 10% to the line profile. Therefore, the contribution of Doppler broadening and van der Waals broadening was neglected in our measurement.

The interaction of the emitter atoms with charged particles results in the Stark broadening. The FWHM of the Stark broadening profile is approximated by following relation [20]:

68116 . 0 3 17 ] [ 10 8 . 4 ] [         ⋅ = ∆ - cm n nm e λ .
(3)

Due to the low H β emission intensity, the entrance slit of the spectrograph is widely opened. This leads to the instrumental broadening with a Gaussian profile with a FWHM of 0.12 nm, which is not negligible. A Voigt function is fitted to the measurement profile (Figure 6). The electron density is obtained from the remaining Lorentzian part of the Voigt profile. This does not include uncertainties resulting from spatial integration in the measurement and the applied line broadening model in the analysis. Both uncertainties are difficult to quantify. Figure 7 shows the time and spatially integrated electron density and the plasma current at different pressures with 1.2 kV applied voltage. Regarding the dependency between the electron density and the gas pressure, Figure 7 can be divided into two regions. Region 1: the electron density increases linearly with the pressure. In this region, the temporally resolved voltage and current show neither oscillation, as showed in Figure 5a, nor the periodic sparks. The discharge operates in DC mode with plasma current of several milliamperes;

Region 2: the electron density increases to the order of 10 16 cm -3 and stays constant. In this region, the µTCD shows the self-pulsing due to the periodic ignition of the spark discharge, where the peak currents are several tens of amperes. It should be mentioned that the plasma currents are measured in pure Ar discharges, where the discharge operates in the periodic spark mode above 600 hPa. The pressure where the transition between region 1 and region 2 occurs depends on the gas composition and the applied voltage, and varies between 300 hPa and 700 hPa.

Discussion of the measured current

The µTCD self-pulsing mode (the periodic spark mode) in the pure argon discharge appears at pressures from several 300 hPa up to atmospheric pressure with frequencies ranging from several kilohertz up to several tens of kilohertz. With the same applied voltage, the self-pulsing frequency decreases with the increasing of the pressure, while the breakdown voltage increase (Figure 8a).

Further, Figure 8b shows that the self-pulsing frequency at different pressures depends on the ratio of the applied voltage U 0 to the self-pulsing breakdown voltage U b as indicated in Figure 4a. In order to explain this behaviour, an equivalent circuit is made (Figure 9) with a plasma resistance R pl parallel to the µTCD setup capacitance C d . This model is also used by Rousseau and coworkers [12] to describe the self-pulsing at medium pressure. Graves et al [21] used a more elaborate model that includes also the differential resistance which is approximated by a third order polynomial. However, in their case the discharge oscillates between the Townsend, normal, and abnormal glow regimes. In our case of the self-pulsing at high pressure, transition is made to the high current spark regime, characterized by a negative differential resistance. The corresponding instability is terminated by the finite charge in the capacitor that limits the discharge time to only a few nanoseconds. As a first and simple approach here only a fixed plasma resistance is assumed. This allows already description of some of the major characteristics. More elaborate models are desirable including also thermionic emission from the surface. However, this would go beyond the scope of this work.

Following the equivalent circuit in Figure 9, the discharge voltage U d can be deduced as follows:

( )

d C R t d e U U ⋅ - - = 1 1 0 . ( 4
)
At t = t b , the discharge voltage U d reaches the breakdown voltage U b , then Equation 4 can be written in following expression with f = 1/t b :

1 0 1 1 ln -                 - ⋅ - = U U C R f b d .
(5)

Adjusting the R 1 C d parameter in Equation 5 until the calculated curve fits the measured profile (Figure 8), the µTCD setup capacitance C d is determined. The capacitance C d is about 130 pF, 10 pF above the value measured by the LCR meter. This 10 pF can be contributed by the stray capacitances originating from coaxial cables in the experimental setup. With this assumption, the equivalent circuit was modified by a stray capacitance C s parallel to the µTCD capacitance C d (Figure 10). The existence of the stray capacitance is also discussed about in [12]. During the charging process, the discharge voltage increases, the current I R is the charging current of the µTCD capacitance (Figure 10a). Once the discharge voltage reaches the breakdown voltage, the discharging process begins (Figure 10b). The discharge voltage drops to zero. The discharging current of the stray capacitance contributes to the current I R . Therefore, the measured value of I R is much higher than it should be due to the limit by the series resistor R 1 . Following the modified equivalent circuit (Figure 10), the charging and the discharging processes are described by two differential equations.

Charging process:

0 1 1 1 1 2 1 2 1 2 = ⋅ +         + + + total s d total s s d total I C R C R I C R C R C R I & & & ; ( 6
)
Discharging process:

( )

d pl s pl pl d pl s pl s d d pl pl C R C R R U I R R R R C R C R I C R R R C R C R I ⋅ ⋅ =         + + ⋅ +         + + + + 2 1 0 1 1 2 2 2 1 2 2 1 1 1 1 1 1 & & & . (7)
The differential equation for the charging process (Equation 6) is easily solved with initial conditions ( )

1 0 0 R U t I total = =
pl d pl pl R I C R I I & + = , pl pl d I R U ⋅ = .
The quantities of the parameters in the upper differential equations except the plasma resistance R pl are given in Table 1. Adjusting the value of the plasma resistance R pl , one can obtain the best agreement between the measured and simulated currents when R pl is 14.5 Ω. The simulation results are shown in Figure 11.

Once the discharge voltage reaches the breakdown voltage, the discharge is ignited (Figure 11a): The discharge voltage drops to zero immediately. At the same time, the plasma current I pl reaches 30 A, and the current I R is 1 A. Quantitatively, the measured (Figure 4a) and the simulated peak currents agree with each other excellently. The good agreement between the measurement results and the simulation results confirms the existence of the stray capacitance.

The measured temporal development of the discharge voltage and current I R is of the order of the temporal resolution of the high voltage probe. Therefore, it is possible that the development of the discharge voltage and current I R is probably faster. However, with the assumption of the plasma resistance R pl , one can obtain a good agreement about the current durations between the simulation (Figure 11b) and the measurement (Figure 4b). Both, the measurement and the simulation show that the plasma current duration is shorter than the current I R , which is basically the discharging current of the stray capacitance.

On the other hand, the plasma resistance R pl is connected to the plasma conductivity σ:

pl pl pl A l R ⋅ = σ , ( 8
)
where A pl and l pl are the cross section area and the length of the plasma volume. A pl is the full cross section of the hole; l pl is assumed to be 200 µm. , where e is the elementary charge, m e is the mass of the electron, ν = 4•10 12 s -1 is the collision frequency [22]. Thus, the electron density can be deduced with assumptions of the plasma volume and the plasma resistance, which is obtained from the fitting procedure described before. The deduced electron density is 6.3•10 16 cm -3 , three times more than the measured density. One should notice that the electron density was not measured in pure argon, but with an admixture of 1% hydrogen. Due to this amount of hydrogen, the electron density is lower than the density in the pure argon discharge. Therefore, the discrepancy of factor 3 is reasonable. However, it should be pointed out that the determination of electron density from the fitted plasma parameter is certainly not a very precise method since it is based on a series of simplifying assumptions.

Nevertheless, the rather good agreement is a strong indication that the order of magnitude is correct and that the rapid discharging of the capacitor is due to the formation of a rather dense plasma on a nanosecond-scale.

Conclusions

The micro thin cathode discharge shows two self-pulsing modes: 1) at medium pressure, similar to a micro hollow cathode discharge, related to the periodic transition between plasma expansion on the cathode backside and collapse back inside the hole; 2) at higher pressure up to atmospheric pressure, thermionic electron emission occurs on the thin cathode hole edge. The µTCD repeats charging and igniting. A spark discharge is characterized by the discharge voltage and current oscillation as well as the ICCD image. In the periodic spark mode, the electron density in argon was measured to be as high

Figure 1 .

 1 Figure 1. Cross section of the µTCD electrode geometry (a) and with the holder (b).

  is the capacitance of µTCD device.

Figure 2 .

 2 Figure 2. Experimental setup.

Figure 3 .

 3 Figure 3. Concept for imaging the plasma volume.

Figure 5

 5 Figure 4. a) The temporally resolved discharge voltage and the currents at 950 hPa with 1000 V applied voltage; b) Zoom of one single current peak; c) ICCD image of µTCD at 1000 hPa with 900 V applied voltage. The black circle in the projection indicates the orifice of the discharge hole.

  Figure 6 shows the time averaged recorded H β -line at 900 hPa with 1.2 kV applied voltage. Besides the Stark broadening and the instrumental broadening, the Doppler broadening, the natural broadening, the resonance broadening and the van der Waals broadening also contribute to the line profile. Considering the experimental conditions, the natural broadening and the resonance broadening are negligible compared to the other broadening effects. The FWHM of the Doppler broadening profile is determined by the following expression [18]:

Figure 6 .

 6 Figure 6. Solid line: the recorded H β -line at 900 hPa with 1.2 kV applied voltage; dashed line: Voigt fit with consideration of the instrumental broadening.

Figure 7 .

 7 Figure 7. Average electron density and the plasma current in the µTCD with 1.2 kV applied voltage at various pressures. The plasma currents are measured in the pure Ar discharges. The measurement uncertainty of the electron density is about the size of the symbols.

Figure 8 .

 8 Figure 8. a) The self-pulsing frequency and the breakdown voltage in the µTCD with 600 V applied voltage at various pressures. b) The self-pulsing frequency of the µTCD depends on the ratio of the applied voltage U 0 to the breakdown voltage U b . The breakdown voltage depends on the gas pressure and the dimension of the plasma volume.

Figure 9 .Figure 10 .

 910 Figure 9. The equivalent circuit. The measurement resistance R 2 is neglected.

  C s = 10 pF, as obtained above. The current I R and the discharge voltage U d are deduced: process the plasma current is zero. When the discharge voltage U d reaches the breakdown voltage U b , the discharging process begins. With initial conditions current I pl is obtained by solving Equation 7. The current I R and the discharge voltage U d are deduced as follows:

  Further, the plasma conductivity is proportional to the electron density n e :

Figure 11 .

 11 Figure 11. Simulation results (950 hPa, 1000 V): a) The temporally resolved discharge voltage and currents; b) Zoom of one single current peak.

  as 10 16 cm -3 by the Stark broadening of the H β -line. Both the measurement and the analysis of an equivalent circuit proved the existence of stray capacitances in the experimental setup, which results in the measured high current peak. The modelled temporal development of discharge voltage and current agrees very well with the measurement result.

Table 1 .

 1 Parameters used in Equation 6 and 7.

	Applied voltage	U 0	-1000 V
	Breakdown voltage	U c	-440 V
	Series resistance	R 1	500 kΩ
	Measurement resistance	R 2	100 Ω
	µTCD capacitance	C d	120 pF
	Stray capacitance	C s	10 pF

Acknowledgments

This work was supported by the "German Research Foundation" (DFG) in the frame of the "Reaserch Unit FOR 1123 -Physics of Microplasmas" and the Research School of the Ruhr-University Bochum.

The authors would also like to thank Bernd Becker, Frank Kremer and Thomas Zierow for the technical support.


Fanette Lasoudris

Céline Cousin

Armelle Prevost-Blondel

Nadine Martin-Garcia

Issam Abd-Alsamad

Nicolas Ortonne

Jean-Pierre Farcet

Flavia Castellano

Valérie Molinier-Frenkel

IL4I1: an inhibitor of the CD8(+) antitumor T-cell response in vivo

teaching and research institutions in France or abroad, or from public or private research centers.

  Figure 1


Elise C Lelièvre

Jérome E Roger

José- Alain Sahel

Reiner A Veitia

Olivier Goureau

X Guillonneau
email: xavier.guillonneau@inserm.fr

A Regulatory Domain Is Required for Foxn4 Activity During Retinogenesis

Keywords: Foxn4, Foxn1, Forkhead, retinogenesis, activation domain

Foxn4, a member of the N-family forkhead transcription factors, controls fate-decision in mouse retina and spinal cord as well as in zebrafish heart. Analysis of Foxn4 amino acid sequence revealed the presence of a region homologous to the activation domain of its close relative Foxn1 in between Cterminal amino acids 402 and 455 of Foxn4 protein. The requirement of Foxn4 putative activation domain remains to be elucidated. Using a gain-of function approach in the rat and chick retinal explants, we report that deletion of Foxn4 putative activation domain results in a complete loss of its activity during retinogenesis. Target promoter transcription assay indicates that this domain is critical for Foxn4 transcriptional regulatory properties in vitro. Accordingly, in chick retinal explants, this domain is required for proper regulation of target retinogenenic factors expression by Foxn4. Thus our study demonstrates that the domain between amino acids 402 and 455 is necessary for Foxn4 transcriptional activity both in vitro and in the retina.

[2]

INTRODUCTION

Forkhead (Fkh) transcription factors form an evolutionarily conserved family of proteins involved in a variety of biological processes [START_REF] Kaestner | Unified nomenclature for the winged helix/forkhead transcription factors[END_REF]. These transcription factors act either as transactivators or as transrepressors of target gene expression. Their transcriptional properties rely notably on the presence of two specific domains, a transcriptional regulatory domain (either an activation or a repression domain) and a DNA-binding domain (DBD) and are regulated by posttranslational modifications [START_REF] Wijchers | In control of biology: of mice, men and Foxes[END_REF].

Foxn1 and Foxn4 are two closely related forkhead transcription factors belonging to the Nfamily [START_REF] Hannenhalli | The evolution of Fox genes and their role in development and disease[END_REF] involved in cell differentiation during development. Foxn1 drives differentiation in hair follicles and in the thymus (for review [START_REF] Wijchers | In control of biology: of mice, men and Foxes[END_REF]). Foxn4 is required for the specification of amacrine and horizontal cells during mouse retinogenesis [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF], the specification of ventral interneurons in mouse spinal cord [START_REF] Li | Foxn4 acts synergistically with Mash1 to specify subtype identity of V2 interneurons in the spinal cord[END_REF] and was more recently shown to be essential for atrio-ventricular canal formation in zebrafish heart [START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF]. Foxn1 protein contains a regulatory domain that acts as an activator of transcription [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF], deleted in almost all reported mutations of Foxn1 leading to T-cell immunodeficiency and congenital alopecia (reviewed in [START_REF] Boehm | Genetic dissection of thymus development in mouse and zebrafish[END_REF]). With respect to the similarity of sequence with Foxn1, a putative activation domain (AD) was described in the C-terminal end of Foxn4 protein (Fig. 1A) [START_REF] Gouge | Foxn4--a new member of the forkhead gene family is expressed in the retina[END_REF]. However, the requirement of this domain and its function as a transcriptional activator or repressor hasn't been studied so far. To assess the functional relevance of Foxn4 putative AD, we designed a variant of Foxn4 in which the putative AD domain was deleted (Foxn4AD). We analyzed its activity in vivo, using gain-of-function approaches in the embryonic retina, and in vitro and demonstrated that Foxn4 AD is required for Foxn4 activity.

MATERIALS AND METHODS

Animals and Ethics Statement

Gallus gallus white leghorn embryos were obtained from Haas (Kaltenhouse, France). Sprague-Dawley OFA rats were purchased from Charles River. Animals were sacrificed following approval by the French Minister of Agriculture (authorization 75-865-RENEWAL delivered on April 2010) according to the recommendations of our local ethical committee (CNREEA5 Charles Darwin (France)).

Plasmids

The well-characterized mouse Foxn4 cDNA was cloned using specific primers into pDONR221 (pDONR) vector (Invitrogen). Mutation/deletion of activation domain (aa 402 to 455) were performed by PCR-based mutagenesis using specific primers available upon request. pDONR plasmids were recombined with pCIG expression vector [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF].

Electroporation, retinal explant culture and dissociation Rat retinal explants or whole chick retinas were electroporated with pCIG vectors (1 g/l) as described previously [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. Rat retinal explants were cultured on polycarbonate filter discs as previously detailled [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. Whole chick retinas were cultured as floating explants in DMEM, 10% FBS, 1% Penicillin/Streptomycin. For cell counting experiments, after the indicated days of in vitro (DIV) culture, retinal cells were dissociated with trypsin, plated on poly-Lysine coated glasses and fixed for 10 min in 4% PFA before immunostaining.

[3] For cell sorting experiments, whole chick retinas were cultured for 36 h, then dissociated and a total of 10 5 GFP-positive cells [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF] were collected for RNA extraction using Vantage Sorter (BD Biosciences).

Immunostaining

The following antibodies were used: anti-rhodopsin (kind gift of R.Molday, Vancouver), anti-syntaxin (Sigma-Aldrich), anti-VC1.1 (Sigma), anti-recoverin (Millipore), anti-visinin (Developmental Studies Hybridoma Bank) antibodies. Secondary Alexa Fluor antibodies were obtained from Molecular Probes (Invitrogen). Cells were immunostained as previously described [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF]. Fluorescent staining signals were captured with a DM 5500 microscope (Leica) and analyzed with MetaMorph software (Molecular Devices).

Reporter gene assays A 1 kb genomic DNA fragment upstream of Tbx2 ATG containing Foxn4 responsive sites was cloned into pGL3-basic vector (Promega). Renilla luciferase reporter driven by the herpes simplex virus thymidine kinase promoter (pRL-TK) was used as an internal control for transfection efficiency. 611W cells [START_REF] Tan | Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice[END_REF] were cultured in DMEM medium, 10% FBS and 1% Gentamicin, seeded in 24-wells plate and transfected with pCIG expression vectors and the pTbx2-pGL3 reporter plasmid using the calcium-phosphate method [START_REF] Sambrook | Molecular Cloning : A laboratory manual[END_REF]. Luciferase activity quantification was performed using Dual-Luciferase ® Reporter Assay System (Promega) and a Tristar LB940 luminometer (Berthold technologies). Relative luciferase activity is the ratio of Firefly over Renilla luciferase activities.

RNA isolation and quantification by real-time PCR Total RNAs were extracted with Nucleospin RNAII Kit (Macherey Nagels). Real-time PCR was performed using 7300 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Reactions were performed in a 20 µl final volume with Power SYBR® Green PCR Master Mix (Applied Biosystems). Primers are available upon request. For quantification on GFPpositive sorted cells, all samples (pCIG n=5, pCIG-Foxn4 n=5, pCIG-Foxn4-AD n=3) were subjected to two independent retrotranscriptions. For each retrotranscription and each sample, qPCR were run in triplicate and quantification was repeated twice. Data represent the mean ± SEM of all values of each experimental group.

Western blotting and nuclear extract preparation Western blotting was conducted as described previously [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF] using the following primary antibodies : mouse anti-HA (MMS 101R, Covance), goat anti-laminB (sc-6216, SantaCruz), goat anti-GAPDH and mouse anti-actin (Sigma). For nuclear extract preparation, total protein extracts were collected in hypotonic buffer (20mM HEPES pH7.9, 1mM Na 3 VO 4 , 1mM Na Glycerophoshate, 5mM EDTA pH7.5, 1mM EGTA pH7.5, 1mM DTT, Protease inhibitors (IP) (Calbiochem, Merck4Biosciences)) plus 0.2% NP40, incubated on ice 15 minutes and centrifugated 20 secondes at 16000 g. After three washes in hypotonic buffer, pellets were suspended in saline buffer containing IP (120 mM NaCl, 20% glycerol in hypotonic buffer) incubated 30 minutes at 4°C and centrifugated 20 minutes at 16000 g to collect the supernatant (Nuclear Extract, NE).

Statistical analyses [4]

Statistical analyses were conducted using one way analysis of variance followed by Tukey's multiple comparison tests (Prism 5.0, Graphpad software). (*) p<0.05 (**) p<0.01, (***) p<0.001. If not indicated differently, statistical significances for values compared to pCIG are reported.

RESULTS and DISCUSSION

Foxn4 activity during retinogenesis.

The vertebrate retina is composed of six types of neurons. They all derive from multipotent progenitor cells that differentiate in an evolutionarily conserved chronological order [START_REF] Young | Cell differentiation in the retina of the mouse[END_REF][START_REF] Livesey | Vertebrate neural cell-fate determination: lessons from the retina[END_REF]. Ganglion cells, amacrine cells (AC), horizontal cells (HC), cone photoreceptors are generated first while rod photoreceptors and bipolar cells are generated last. In mice retina, Foxn4 is expressed from embryonic day 11.5 (E11.5) to postnatal day 6 (P6) in the zone of progenitor cells [START_REF] Gouge | Foxn4--a new member of the forkhead gene family is expressed in the retina[END_REF][START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. Foxn4 overexpression during development increases the number of syntaxinpositive HC/AC and decreases the number of recoverin-positive photoreceptors [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. Here, we show through quantitative PCR analysis that Foxn4 was highly expressed during rat retinal development and became undetectable by P7 (Fig. 1B). Foxn4 was not detected in other adult rat tissues tested except in the thymus (Fig. 1C). The similar temporal expression pattern in mouse and rat retina [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF] suggests that Foxn4 might have a similar function among rodents. To assay its role on the specification of early and late born cells during rat retinogenesis, Foxn4 was overexpressed in rat retinal explants at E16 and P0 that is just prior the early and late waves of cell differentiation, respectively [START_REF] Rapaport | Timing and topography of cell genesis in the rat retina[END_REF]. For that purpose we cloned Foxn4 cDNA in the bicistronic pCIG-eGFP (Green Fluorescent Protein) expression vector [START_REF] Roger | Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells[END_REF] (Fig. 2A) and control pCIG-eGFP and pCIG-Foxn4-eGFP vectors were electroporated in E16 or P0 rat retinal explants. After 5 or 7 days of in vitro culture (DIV) following E16 electroporation, we stained cell-dissociated explants with anti-GFP and anti-syntaxin to identify AC/HC or anti-recoverin antibodies that label early born cone photoreceptors at these stages (Fig. 2B). After 3 or 6 DIV following P0 electroporation, dissociated cells were stained with anti-GFP and anti-rhodospsin for late born rod photoreceptors. Misexpression of Foxn4 in rat retina at E16 resulted in a 1.9-and 2.3-fold increase in the number of syntaxin-GFP double-positive cells at 5 and 7 DIV, respectively (Fig. 2C), and a 4.9-fold reduction in the number of recoverin-GFP double-positive cone photoreceptors at these two time points (Fig. 2D). Moreover, misexpression at P0 of Foxn4 strongly decreased the number of rhodopsin-GFP double-positive late born rod photoreceptors after 3 or 6 DIV (Fig. 2E). Together these data confirm the phenotype previously observed in the mouse after misexpression of Foxn4 by an in vivo retroviral approach [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF]. Hereafter, we chose Foxn4-induced changes in retinal cell type representation as an appropriate marker of wild type Foxn4 activity.

Activity of Foxn4AD variant in the retina.

Transcription activity of the Fox family member is thought to be dependent of the presence of a regulatory domain in addition to their DBD. Foxn1 is the only member of the N-family in which the activity of an trans-Activation Domain (AD) has been demonstrated. In mouse, it is located between amino acid (aa) 509 and 562 [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF]. Gouge et al. have initially described a putative activation domain located in the C-terminal part of the mouse Foxn4 protein [START_REF] Gouge | Foxn4--a new member of the forkhead gene family is expressed in the retina[END_REF]. Since that, C-terminal putative activation domains with various boundaries have been hypothesized in chick, xenopus, human and zebrafish Foxn4 proteins [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF]Schlake, 2000;[START_REF] Gouge | Foxn4--a new member of the forkhead gene family is expressed in the retina[END_REF][START_REF] Boije | Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina[END_REF][START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF]. Here we have tested the requirement of the region between aa402 and aa455 for Foxn4 activity (reference sequence NP_683737) (Fig. 1A). This region is rich in acidic amino acids and has been delimited to encompass the highly conserved amino acids of previously described [5] activation domains [START_REF] Boije | Temporal and spatial expression of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina[END_REF][START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF]. A Foxn4AD cDNA missing aa402 to aa455 was generated and recombined within the pCIG vector (Fig. 2A) to allow for misexpression in the retina and, concomitantly, recombined in frame with the HA-tag of pRCAS plasmid to ensure the proper synthesis and the stability of the Foxn4ΔAD tagged-protein in chick fibroblast cells (DF1). Foxn4AD protein was expressed to a nearly equal level to the wild type Foxn4 protein (Fig. 2A, WCE). Moreover, purification of nuclear extracts indicated that Foxn4AD was still present in the nucleus (Fig. 2A,NE). Overexpression at E16 of Foxn4AD failed to increase the percentage of syntaxin-GFP double-positive early-born AC/HC cells and to decrease the percentage of recoverin-GFP double-positive early born cone cells (Fig. 2C,D). Furthermore, as opposed to Foxn4, Foxn4AD did not modify the number of rhodopsin-GFP double-positive late born rod photoreceptors (Fig. 2E) when misexpressed at P0. Thus these results indicate that Foxn4 AD is required for Foxn4 effects on retinal cell type representation in vivo. A dominant negative effect of the AD defective form of Foxn4 could have been expected. However none of the characteristics of Foxn4 knock-out mouse phenotype [START_REF] Li | Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors[END_REF], that is a decrease of syntaxin-positive amacrine/horizontal cells and an increase of recoverin-positive photoreceptor cells, was observed following forced expression of Foxn4AD.

Transcriptional properties of Foxn4 variants in vitro.

Loss of Foxn4AD activity during retinal development strongly suggests that this putative AD domain is necessary for the transcriptional regulatory properties of Foxn4. Thus we tested the transcriptional activity of Foxn4 and this variant with an in vitro reporter assay using a Tbx2 promoter, recently described as a direct target of Foxn4 in the zebrafish heart, driving the expression of the luciferase gene [START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF]. For this purpose, we co-transfected 661W mouse retinal cells with Foxn4 or Foxn4AD and the pTbx2-pGL3 reporter plasmid. Foxn4 transfection reduced by 2.7-fold the basal level of Tbx2 promoter transcription (Fig. 3A). On the contrary, deletion of Foxn4 AD increased by 1.9-fold Tbx2 transcription level when compared to pCIG vector (Fig. 3A) showing that this domain might be indeed a trans-regulatory domain of Foxn4 transcriptional activity. Transactivation properties of Foxn1 AD rely mostly on the presence of four aspartic acid residues [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF]. Three of them are conserved within the Foxn4 AD and thus might be involved in Foxn4 AD regulatory activity (Fig. 3B). Mutation of these aspartic acid residues, separately, did not significantly change the reporter transcription level when compared to Foxn4 suggesting that none of these residues are involved in Foxn4 trans-repression ability (Fig. 3A). The effect of the combination of the three mutations should be further studied. However, in genetic screen tests of affected individuals, it is unlikely that all three mutations would be identified in a single patient.

Foxn4 AD regulatory properties in vivo.

We next monitored Foxn4 AD functional relevance in the regulation of Foxn4 candidate target genes involved in retinogenesis using the chick retina that offers a reliable model to study transcriptional regulation following the overexpression of a transcription factor [START_REF] Tetreault | The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 transactivation[END_REF].

To validate this model, we first analyzed whether Foxn4 misexpression in the cone-dominated chick retina biased cell specification. Whole E5 chick retinas were electroporated with pCIG, pCIG-Foxn4 or pCIG-Foxn4AD GFP-encoding plasmids and collected after 3 DIV corresponding approximately to E8 in vivo. At this stage most amacrine/horizontal cells and photoreceptors were differentiated in the chick retina [START_REF] Prada | Spatial and Temporal Patterns of Neurogenesis in the Chick Retina[END_REF]. Dissociated cells were stained with anti-GFP and antisyntaxin or anti-VC1.1 antibodies to identify AC/HC [START_REF] Alexiades | Subsets of retinal progenitors display temporally regulated and distinct biases in the fates of their progeny[END_REF]. Anti-visinin antibody was used to label all photoreceptors (cones and rods) as both types of photoreceptors are born concomitantly in chick retina [START_REF] Prada | Spatial and Temporal Patterns of Neurogenesis in the Chick Retina[END_REF]. Misexpression of Foxn4 in chick retina increased by 2- [6] fold the number of syntaxin-GFP double-positive cells (Fig. 4A) and VC1.1-GFP double-positive cells (Fig. 4B) at 3 DIV. On the contrary, no significant increase of syntaxin-GFP or VC1.1-GFP cells was observed following pCIG-Foxn4AD electroporation (Fig. 4A-B). As for photoreceptors, the number of visinin-GFP double-positive cells was decreased by three fold in Foxn4 misexpressing retinas and this effect was abolished following deletion of Foxn4 AD (Fig. 4C). Together results in mouse, chick and rat retinas suggest that the function of Foxn4 may be conserved among vertebrates at least concerning amacrine/horizontal and photoreceptor cell specification.

We next assessed gene regulation in pCIG, pCIG-Foxn4 or pCIG-Foxn4AD transfected E5 chick retinas. To study early changes in candidate gene expression, electroporated cells were harvested as shortly as 36 h after electroporation. This condition, soon after the onset of GFP expression, was chosen as the earliest time point where a sufficient number of GFP-expressing cells can be collected. We then isolated GFP-positive electroporated cells by Fluorescence Activated Cell Sorting and quantified by quantitative Polymerase Chain reaction (qPCR) the expression level of candidate genes selected according to their known function during retinogenesis (Fig. 4D). We first analyzed genes that are representative of the AC or/and HC pathways. NeuroAB is expressed in GABAergic AC during chick retinogenesis [START_REF] Ohkawara | A novel basic helix-loop-helix (bHLH) transcriptional repressor, NeuroAB, expressed in bipolar and amacrine cells in the chick retina[END_REF] and Prox1 and Lim1 are HC specific genes [START_REF] Dyer | Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina[END_REF][START_REF] Poche | Lim1 is essential for the correct laminar positioning of retinal horizontal cells[END_REF][START_REF] Poche | Retinal horizontal cells: challenging paradigms of neural development and cancer biology[END_REF][START_REF] Suga | LIM family transcription factors regulate the subtypespecific morphogenesis of retinal horizontal cells at post-migratory stages[END_REF]. Loss of Foxn4 AD abolished the Foxn4mediated NeuroAB upregulation. Foxn4 misexpression induced a 3.5-and 2.5-fold increase in Prox1 and Lim1 mRNA expression levels, respectively. However, Lim1 and Prox1 were not upregulated when Foxn4AD was overexpressed (Fig. 4D). Ptf1a is a bHLH transcription factor required for AC and HC specification that acts as a downstream target of Foxn4 in the mouse retina [START_REF] Fujitani | Ptf1a determines horizontal and amacrine cell fates during mouse retinal development[END_REF]. Deletion of Foxn4 AD abolished the Ptf1a upregulation observed following Foxn4 misexpression. We next studied genes involved in specifying photoreceptors and their subtypes. The homeodomain transcription factors Otx2 and RaxL are essential for photoreceptor cell development in the retina [START_REF] Chen | The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation[END_REF][START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development[END_REF] and are expressed in photoreceptor cells in chick retina [START_REF] Bovolenta | Implication of OTX2 in pigment epithelium determination and neural retina differentiation[END_REF][START_REF] Chen | The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation[END_REF][START_REF] Plouhinec | Comparative analysis of gnathostome Otx gene expression patterns in the developing eye: implications for the functional evolution of the multigene family[END_REF]. Nr2e3 is detected in the photoreceptor cell layer in chick retina [START_REF] Kobayashi | Expression and functional analysis of Nr2e3, a photoreceptor-specific nuclear receptor, suggest common mechanisms in retinal development between avians and mammals[END_REF] and is critical for rod specification [START_REF] Cheng | Photoreceptorspecific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors[END_REF]. Foxn4 strongly downregulated Otx2, Nr2e3 and RaxL in chick electroporated retinal cells (Fig. 4D). Loss of its AD decreased the repressive activity of Foxn4 on Otx2 albeit not significantly and abolished Foxn4 repression of Nr2e3 (Fig. 4D). Foxn4AD repression of RaxL was significantly lower compared to wild type Foxn4 (Fig. 4D). Furthermore, we found that Atoh7 involved in ganglion [START_REF] Liu | The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development[END_REF] and photoreceptor cell [START_REF] Ma | A role of ath5 in inducing neuroD and the photoreceptor pathway[END_REF] genesis in chick was inhibited following Foxn4 overexpression while deletion of Foxn4 AD decreased significantly this repression (Fig. 4D). Therefore, in vivo Foxn4 transcriptional regulation of its target genes during retinogenesis is dependent on its putative AD. Loss of this AD only affects Foxn4 regulation of its target genes since AD deletion did not change the expression level of Hes1, a marker of proliferating cells which is not modified following Foxn4 misexpression (Fig. 4D).

Together our findings support that Foxn4 Activation Domain is indeed involved in Foxn4 transcriptional regulation properties both in vivo and in vitro.

We here show that Foxn4AD was detected in the nuclear cell extracts as reported for the AD-deleted form of Foxn1 StL (Schorpp, 2000). Thus, the loss of trans-activation and/or transrepression activity of Foxn4AD could possibly result from an alteration of its DNA binding property or by a loss of interaction with its cofactors, as previously stated for Foxn family members [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF]Schorpp, 2000;[START_REF] Gouge | Foxn4--a new member of the forkhead gene family is expressed in the retina[END_REF]. We show that the AD-deleted form of Foxn4 was able to activate the Tbx2 promoter. The deleted domain is enriched in negatively charged acidic amino acids, a feature of transcription domains, that favor the interaction with the general transcription machinery [START_REF] Mitchell | Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins[END_REF]. It is therefore likely that this domain is involved in the regulation of [7] transcription initiation by recruiting co-factors. It is also possible that Foxn4 AD is required for the binding of Foxn4 to DNA. However, other Fkh members such as Foxn1, Foxa1 (HFN3 Foxa2 (HFN3were still capable of binding DNA regardless the lengh of deletions of their C terminal and/or N-terminal domains if their DBD sequences are preserved [START_REF] Lai | HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally[END_REF][START_REF] Lai | Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head[END_REF][START_REF] Pani | Hepatocyte nuclear factor 3 beta contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein[END_REF][START_REF] Qian | Analysis of hepatocyte nuclear factor-3 beta protein domains required for transcriptional activation and nuclear targeting[END_REF]Schlake, 2000). Using various experimental conditions, we were not able to detect a specific binding of mouse Foxn4 proteins or their mutant forms to zebrafish Tbx2b or mouse Tbx2 promoter specific probes by electromobility shift assay (EMSA). The identification of a consensus sequence of mouse Foxn4 binding sites will be required to perform EMSA experiments to determine if Foxn4 AD is involved in Foxn4 binding to DNA. Selected genes in the AC/HC pathway were systematically upregulated by Foxn4. Whether this transcriptional regulation direct or not remains to be elucidated. Foxn4 repressed Tbx2 in the 661W cell line suggesting that Foxn4 is a transcriptional repressor contrary to Foxn1, and acts indirectly on the transcription AC/HC genes in vivo. However, its transcriptional regulatory properties might be cellular context-and gene-dependent. Notably, Foxn4 downregulated Tbx2 in 611W cell line while it positively regulates it in the zebrafish heart but has no effect on it in zebrafish retina [START_REF] Chi | Foxn4 directly regulates tbx2b expression and atrioventricular canal formation[END_REF]. As already described for other Fkh transcription factors [START_REF] Wijchers | In control of biology: of mice, men and Foxes[END_REF], Foxn4 might therefore act either as a repressor or as an activator depending on the recruitment of co-repressors or co-activators by its AD. In light of our study, the putative trans-activation domain of Foxn4 should thus be referred to as a transcriptional "trans-regulatory domain".

So far most of the mutations of the Fox family associated with pathologies have been found in the DBD [START_REF] Lehmann | Fox's in development and disease[END_REF]. However, the deletion of the trans-activation domain of Foxn1 in the nude mice has been proposed as a molecular explanation for the majority of known loss-of-function alleles of whn genes [START_REF] Schuddekopf | The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain[END_REF][START_REF] Boehm | Genetic dissection of thymus development in mouse and zebrafish[END_REF]. Taken together our results and others strongly suggest that the regulatory domain is critical for Foxn protein family activity and may be an additional target of mutation resulting in a loss-of-function phenotype. Identification of cofactors interacting with Foxn4 regulatory domain within Foxn4-expressing structures in vivo are now required to fully understand tissue-dependent Foxn4 transcriptional regulatory properties and may also help to decipher the genetic of human retinal, heart or spinal cord defects.

ACKNOWLEDGEMENTS

We thank J. Dreux for critical reading of the manuscript. This work was financed by INSERM, Retina France, EU (LSHG-CT-2005-512036, ERC-StG-210345), French ANR (ANR-Geno-031-03, ANR-08-MNPS-003).

[8]

[11] [14]


Thomas Antoni
email: thomas.antoni@spectro.jussieu.fr

Aurélien G Kuhn

Tristan Briant

Pierre-François Cohadon

Antoine Heidmann

Rémy Braive

Alexios Beveratos

Izo Abram

Luc Le Gratiet

Isabelle Sagnes

Isabelle Robert-Philip

Deformable two-dimensional photonic crystal slab for cavity optomechanics

We have designed photonic crystal suspended membranes with optimized optical and mechanical properties for cavity optomechanics. Such resonators sustain vibration modes in the megahertz range with quality factors of a few thousand. Thanks to a two-dimensional square lattice of holes, their reflectivity at normal incidence at 1064 nm reaches values as high as 95 %. These two features, combined with the very low mass of the membrane, open the way to the use of such periodic structures as deformable end-mirrors in Fabry-Perot cavities for the investigation of cavity optomechanical effects.

Cavity optomechanics based on mechanically deformable optical microcavities has attracted a lot of attention over the past few years in view of applications in sensing and monitoring of mechanical motion. A fundamental motivation behind such work resides in the possibility of optically cooling the mechanical motion, in view of reaching the quantum ground state of the mechanical system [1,2]. Downsizing the mechanical resonator allows one to enhance the optomechanical coupling by reducing the effective mass of the vibration modes. Among the various nanomechanical oscillators presently investigated, photonic crystal slabs may be good candidates, in particular due to the versatility of their optical properties combined with their very low mass. For instance, a "defect" in a planar one-or two-dimensional periodic dielectric structure can offer a strong confinement for both photons and phonons simultaneously inside a very small volume, thus exhibiting a strong optomechanical coupling [3,4]. The optomechanical response of two coupled membranes, each pierced as a perfect two-dimensional periodic dielectric structure sustaining band-edge Bloch modes, has also been investigated [5].

In this paper, we present a new optomechanical device based on a suspended photonic crystal membrane that can be used as a deformable end-mirror in a Fabry-Perot cavity optomechanical system. We investigate both its mechanical response (frequencies and quality factors of the vibration modes) and its optical response (reflectivity at normal incidence).

The photonic crystal consists of an InP slab pierced with a square lattice of holes. The whole structure is grown by metal-organic vapor phase epitaxy on an InP substrate, starting with a 1 µm InGaAs sacrificial layer and then the InP slab layer. Inductively coupled plasma etching of the photonic crystal is performed on the top InP layer using a SiN hard mask defined by electronbeam lithography and dry etching. To suspend the InP membrane, the sacrificial layer under the membrane is removed by selective wet etching [6].

We were able to suspend membranes as large as 30 × 30 µm 2 , using several clamping solutions as described in the following. We have optimized the photonic crystal structure in order to maximize its optical reflectivity at normal incidence: transmission through the slab and inplane losses can be canceled out by coupling the incident radiation to Bloch modes with a flat dispersion curve at the Γ point of the Brillouin zone. Moreover, it is desirable to design a mirror with broadband reflectivity so as to overcome fluctuations of the spectral position of the high-reflectivity zone, which may be due to defects and inhomogeneities introduced during the slab processing. As suggested in [7], this can be obtained by coupling two non-degenerate but overlapping slow Bloch modes close to the Γ point with the radiated modes in the vertical direction. Finite Difference Time Domain MEEP code [8] and eigenmode solver [9] MPB have been used to determine adequate parameters for the photonic crystal slabs in order to reach a near unity reflectivity over more than 50 nm around 1064 nm. The simulations are performed for an infinite structure, taking into account the evanescent coupling and cavity effects with the underlying substrate.

Fully clamped 30 × 30 µm 2 samples with a thickness of 260 nm have been processed to measure the slab's reflectivity as a function of the optical wavelength by use of a Fourier transform infrared spectroscopy setup. The corresponding spectral behavior is shown in Figure 2 for a photonic crystal structure corresponding to a square array of holes, with a period of 725 nm and different diameters (see Figure 1). The measured band edges well coincide with reflectivity calculations, in particular the cut-off wavelengths of the large plateau around 1064 nm, and the resonance about 1010 nm. The spectral displacement of this resonance with increasing hole diameters is also well reproduced. The dip in the middle of the large plateau, mostly visible in the calculated and measured spectra but with a depth that depends on the hole diameter, is a consequence of the splitting between the two underlying Bloch modes. Note that due to finite size effects and optical diffraction, as well as to alignment and focusing difficulties, the presented results are arbitrary normalized so that absolute values of the curves are irrelevant. The reduced visibility of the experimental reflectivity may be due as well to finite size effects, as the beam waist is of the order of the photonic crystal area, or to non-uniform hole diameters over the membrane (resulting from proximity effects during the electron beam lithography of such large photonic crystal surfaces [10]), or to the roughness of the substrate behind the membrane, hence distorting the photonic crystal resonances. Cavity operation of such membranes requires a very small beam waist to minimize clipping loss. As a consequence the reflectivity may be affected by the resulting beam divergence, about 6 • for a waist of 3 µm. To investigate this effect, and also to measure an absolute value of the reflectivity, we have used a small 10 × 20 µm 2 fully clamped membrane and focused a laser beam to a waist as small as 2.5 µm with a microscope objective (0.65 numerical aperture). In addition, we have measured the dependence of the reflectivity on the hole diameter. Experimental results obtained by comparing the reflected power to the one reflected by a 99.94% commercial mirror are shown in Figure 1. They are in good agreement with FDTD simulations, demonstrating that divergence effects do not limit the reflectivity. The absolute reflectivity reaches values as high as 95% ± 8%.

For the investigation of the mechanical response of the membrane, we have built a Michelson-like interferometer in which the membrane is the end-mirror in one of the arms. The light from a 1064 nm Nd:YAG laser source is focused on the membrane with the microscope objective, down to a beam waist of 2.5 µm. The membrane position is adjusted thanks to a motorized 3-axis translation stage. The setup is inserted into a vacuum chamber pumped down to 10 -4 mbar.

The interference between the two arms is monitored by a photodiode and sent to a network analyzer which is also used to activate the resonator via a piezoelectric stack. On the aforementioned membranes, typical results show mechanical modes with resonance frequencies in the megahertz range and with quality factors about 1000, due to clamping losses. We consequently have tried different geometries and best results have been obtained with 10×20 µm 2 suspended membranes, with a thickness of 210 nm. The membrane is suspended by four bridges with optimized position, length and width (see inset in Fig. 3) [11]: they are symmetrically located 5.9 µm from the center of the membrane, at the node of the fundamental longitudinal mode (0,0), as computed with Comsol Multiphysics. We have tested samples with auxiliary bridges length ranging from 2 µm to 12 µm, and with a width down to 0.5 µm.

The spectrum of the mechanical response obtained with a membrane suspended by 8 µm-long auxiliary bridges is shown in Figure 3. It displays six mechanical modes whose frequencies Ω m /2π are in the megahertz range, as expected from finite-element simulations, with the mode of interest about 2.5 MHz. The estimated effective mass of this mode is of the order of 150 pg. The quality factors Q m of the mechanical modes are in-ferred via a ring-down technique, by measuring the decay time of the vibration mode after switching off the membrane actuation at the resonance frequency. Quality factors ranging from 2 000 to 10 000 are obtained, without relevant dependence on the observed mode. We have investigated various dissipation mechanisms that may limit these quality factors. For instance, we have checked that Q m does not depend on pressure below 10 -1 mbar, ruling out air viscous damping and the well known squeezedfilm damping [12]. Using Zener model [13], we have estimated that thermoelastic damping is negligible as well. Another mechanism that can be eliminated is the clamping losses corresponding to dissipation into the substrate, since Q m has no clear dependence on the length or the position of the four auxiliary bridges.

Two dominant dissipation channels can consequently be foreseen in our system. The first one corresponds to surface effects [14,15], whose impact goes up for increasing surface-to-volume ratio, estimated to 10 µm -1 (or 1 over 500 atoms) with our structures. Another loss mechanism is related to crystal defects of the membrane, either induced in the bulk material during processing, or due to a non-intentional residual doping, estimated at about 10 17 cm -3 [16,17].

Note that the bending of the membrane distinguishable in Figure 3 is due to residual stress and yields to a radius of curvature of about 100 µm. This is not an issue for the design of a cavity since coupling mirrors with smaller radius of curvature are achievable [18].

Large optomechanical effects are expected by combin-ing such mechanical properties to high optical reflectivities, making these photonic-crystal slabs promising candidates for cavity optomechanics experiments. As an example, the shot-noise limited displacement sensitivity with a single-ended Fabry-Perot cavity would already be large enough to observe the Brownian and quantum motions of the membrane. It is given by :

δx shot = λ 16F I in T + L T , (1)
where F is the cavity finesse, T the transmission of the coupling mirror and L the cavity losses (including the membrane reflectivity). Based on our experimental results, we find δx shot ≃ 2 × 10 -17 m/ √ Hz for realistic parameters: incident power P in = (hc/λ) I in = 5 mW, F ≃ 100, L = 5%, and T ≃ 1%. This value is four orders of magnitude smaller than the Brownian motion amplitude of the fundamental mode at 300 K (δx T = 2Q m k B T /(M Ω 3 m ) ≃ 2 × 10 -13 m/ √ Hz), and of the same order of magnitude as its zero-point motion (δx T ≃ 5 × 10 -17 m/ √ Hz at T 150 µK). Further improvement of the mechanical quality factors may allow the experimental demonstration of the ground state of these resonators, by performing cryogenic and laser coolings of the membrane either used as an endmirror in a Fabry-Perot cavity, or as a high-reflectivity membrane in "membrane-in-the-middle" setups [19].

This research has been partially funded by the FP7 Specific Targeted Research Projects QNems, and by the C'Nano Ile-de-France project Naomi.

FIG. 1 .

 1 FIG. 1. Left: Scanning electron microscope close-up view of the photonic crystal structure. Right: measured (red dots) and simulated (black curve) reflectivity at 1064 nm of a 10 × 20 × 0.26 µm 3 fully clamped photonic crystal.

FIG. 2 .

 2 FIG. 2. Normalized experimental (red solid line) and simulated (black dashed line) reflectivities as a function of the wavelength, for a 30 × 30 × 0.26 µm 3 fully clamped photonic crystal membrane with holes diameters of 372 nm (left) and 392 nm (right).

FIG. 3 .

 3 FIG. 3. Displacement spectrum of a 10 × 20 × 0.21 µm 3 photonic crystal membrane, clamped by four 8 µm-long and 1 µmwide auxiliary bridges, as shown in the scanning electron microscope image. The simulated vibration profile is shown for each mechanical mode.