Martin Robert-De-Saint-Vincent

Jean-Philippe Brantut

Christian J Bordé

Alain Aspect

Thomas Bourdel

Philippe Bouyer

J.-P Brantut

A quantum trampoline for ultra-cold atoms

Keywords: 03.75.Dg -Atom and neutron interferometry 06.30.Gv -Velocity, acceleration, and rotation 05.60.Gg -Quantum transport

published or not. The documents may come

Introduction. -Atoms in free fall are remarkable test masses for measuring gravity, with a host of applications from underground survey to tests of the equivalence principle [1]. Because of the quantized character of atom-light interaction, the acceleration of free falling atoms can be precisely measured with lasers, for instance by comparing the velocity change of atoms by absorption or emission of a single photon to the gravity induced velocity change in a precisely determined time [2]. Furthermore, atom interferometry exploits the quantum nature of matter-waves [3][START_REF]Atom Interferometry[END_REF][START_REF] Peters | [END_REF][6]. In both cases, an accurate measurement of gravity demands a long time of free fall, but it is a priori limited by the size of the vacuum chamber in which the measurement takes place [7][8][9]. It is possible to overcome this limitation by bouncing many times the atoms on an atomic mirror [10], realizing a trampoline for atoms [11,12]. This scheme can be used to fold the trajectories within a standard light-pulse atom gravimeter [12].

Here we show how to operate a quantum trampoline based on a periodically applied imperfect Bragg mirror, which not only reflects upwards the falling atoms, but also acts as a beam splitter that separates and recombines the atomic wave packets. This results in multiple-wave [13][14][15][16] atom interference, evidenced by an efficient suspension of the atoms even though successive leaks at each imperfect reflection would classically lead to a complete loss of the atoms. This suspension is obtained at a precise tuning of the trampoline period, whose value yields directly the local value of gravity g. Our scheme can be generalized to other interferometer geometries, such as in [17,18] replacing perfect Bragg reflections with imperfect ones.

A classical trampoline for atoms can be experimentally realized by periodically bouncing them with perfect Bragg mirrors. These mirrors are based on atom diffraction by a periodic optical potential [19], i.e. a vertical standing wave of period λ/2, where λ is the laser wavelength. The interaction between the atoms and the optical potential leads to vertical velocity changes quantized in units of 2V R , where V R = h/λm is the one photon recoil velocity of an atom of mass m (h is the Planck constant). For long interaction pulses, the applied potential can be considered as time independent, and the atom kinetic energy has to be conserved. This requirement is fulfilled by changing the vertical velocity component from -V R to +V R , and vice-versa. We call this process a resonant velocity transfer. Perfect Bragg reflection, yielding only resonant velocity transfer, is obtained by choosing appropriate duration and intensity of the pulse [12]. When a perfect Bragg reflection is applied on atoms freely falling with a velocity -V R , they bounce upward with a velocity +V R . After a time T 0 = 2V R /g (≈ 1.2 ms for 87 Rb), the reflected atoms have again a velocity -V R because of the downwards acceleration of gravity g. Repeating this sequence with a period T 0 allows to suspend the atoms at an almost constant altitude (thick line in Fig. 1). This is a classical trampoline [11,12].

To operate the trampoline in the quantum regime, we use imperfect Bragg reflections, associated with short laser Fig. 1: Atom trajectories in the quantum trampoline. a, atoms, diffracted by periodically applied imperfect Bragg mirror, explore various paths which eventually recombine. The probability of a trajectory is represented by the line thickness. The thick line corresponds to a classical trampoline associated with perfect Bragg reflections. The arrows mark the loss channels at -3V R . b, an imperfect Bragg reflection : an incoming matter-wave with vertical velocity -V R is mainly Bragg reflected to +V R (thick line). A small fraction is also diffracted to higher velocities (+3V R and -3V R ), and a smaller one transmitted without deviation. c, elementary interferometer : from a zero-order trajectory (thick line), an atom can be diffracted to +3V R , at point A in the example shown. It is then Bragg reflected from +V R to -V R at point B1 one period later, and finally recombines at point C with the zero-order trajectory, thanks to diffraction from -3V R to +V R .

pulses, for which the kinetic energy conservation requirement is relaxed: Heisenberg time-energy relation permits energy to change by about h/τ for a pulse of duration τ . Choosing τ ≈ h/4mV 2 R allows us to obtain additional velocity changes from -V R to secondary diffracted components with velocities ±3V R (Fig. 1b), hereafter referred as non-resonant velocity transfers. The matter-wave packet is thus split into various components that eventually recombine, resulting in a richer situation where atomic interference plays a dramatic role (Fig. 1a). For our experimental conditions (τ ≈ 35 µs), transition from -V R to +V R occurs with a probability of 0.93, while the amplitudes ǫ of the components diffracted to ±3V R correspond to a probability |ǫ| 2 ≈ 0.03 (|ǫ| ≈ 0.17). The amplitudes of higher velocity components are negligible, and the probability to remain at -V R is 0.01. A similar situation occurs for an atom with initial velocity +V R : transition to -V R happens with probability 0.93 and to ±3V R with probability |ǫ| 2 ≈ 0.03.

We operate our quantum trampoline as follows. An all-optically produced ultra-cold sample of 1.5 × 10 5 87 Rb atoms in the F = 1 hyperfine level [20] is released from the trap with a rms vertical velocity spread of 0.1 V R for the Bose-Einstein condensate and 0.6 V R for the thermal cloud. In this work, the condensate fraction is limited to 0.2. After 600 µs (≈ T 0 /2) of free fall, such that the mean atomic velocity reaches -V R because of gravity, we start to apply imperfect Bragg reflections with a period T , close to the classical suspension period T 0 . More precisely, a retroreflected circularly-polarized beam of intensity 4 mW, 6.3 GHz red-detuned with respect to the nearest available atomic transition from F = 1 is then periodically applied for a pulse duration τ ≈ 35 µs. The successive diffraction events result in several atomic trajectories that coherently recombine in each output channel, as presented in Fig. 1a.

After N pulses, we wait for a 2 ms time of flight and detect the atoms through absorption imaging with resonant light. We observe distinct wave-packets (Fig. 2). The atoms situated in the circle at the top have been suspended against gravity, while the distinct packets below correspond to falling atoms. These atoms have acquired a velocity -3V R after one of the laser pulses, and then continue to fall, unaffected by the subsequent pulses. The difference between Fig. 2a and2b shows that a small change in the pulse period T results in a dramatic change in the number of suspended atoms. When suspension is maximum (Fig. 2b), the losses are strongly suppressed except for the two lowest spots, which correspond to atoms that have been lost at points A and B of Fig. 1. This behavior is due to quantum interferences between the vari-ous trajectories as presented in Fig. 1a : for an adequate pulse period T , the interferences are constructive in the suspended trajectories and destructive in the falling ones, except at points A and B where no interference can happen. This blocking of the 'leaking channels' is analogous to the suppression of light transmission through a multilayer dielectric mirror.

Our quantum trampoline is a multiple-wave interferometer, where the fraction of atoms in each output port is equal to the square modulus of the sum of the amplitudes associated with all trajectories that coherently recombine at the end. We classify the contributing trajectories with respect to the number of non-resonant velocity transfers. The zero-order path is the one which is reflected from -V R to +V R at each pulse (trajectory ABCD...O in Fig. 1a and Fig. 1c). This is the path associated with the largest output amplitude (of square modulus 0.93 N ). The firstorder paths are the ones which are once deviated upwards from the zero-order path, and recombine with it after twice the period T (for example, the trajectories AB 1 CD...O or ABC 1 D...O in Fig. 1a). All these paths have the same accumulated interferometric phase that depends on the pulse period T . Their amplitude, proportional to |ǫ| 2 , is small but the number of such paths increases as the number of pulses N . Their total contribution to the probability amplitude at O scales as N |ǫ| 2 . Higher order paths, with more than 2 non-resonant transfers, are less probable individually, but their number increases faster with the number of pulses. As a consequence, they can have a major contribution to the final probability amplitude. More precisely, multiple-wave interference plays an important role when N |ǫ| 2 becomes of the order of 1.

Figure 3 shows the fraction of suspended atoms for a 10pulse quantum trampoline where the interference between the zero-and first-order paths dominates since N |ǫ| 2 ≈ 0.3 is small compared to 1. When the pulse period T is changed, we observe interference fringes with characteristic spacing ∆T = 16.6(2) µs, in agreement with the calculation for the elementary interferometer (Fig. 1c) [START_REF]φ0 -4πgT 2 /λ, where φ0 is a constant resulting from the sum of the phase shifts acquired during the diffraction events[END_REF]: ∆T = λ/4gT . We also observe an additional modulation with a fringe spacing of ∆T ′ = 33 µs, about twice . It can be understood by considering the interferometers from A to O 1 and from A to O 2 , such as AB

1 C 1 D 1 ...O 1 and ABC 1 D 1 ...O 1 . The corresponding fringe spacing ∆T ′ = λ/4|V R -gT | is equal to 2∆T for T = T 0 . The output ports O 1 , O 2 of
these additional interferometers are 14 µm above or below O. In our absorption images, we do not distinguish the various ports and the observed signal is thus the sum of the intensities of the two fringe patterns. In addition, the total interference pattern is included in a broad envelop due to the mirror velocity selectivity as predicted for a classical trampoline in [11] and first observed in [12].

We model our quantum trampoline in a semi-classical approximation [START_REF]spread ∆p and position spread ∆x such that (i) (∆p/m)N T < ∆x , (ii) ∆p/m ≪ V R , and (iii) ∆x < 14 µm[END_REF]. It makes use of complex amplitudes calculated along the classical trajectories plotted in Fig. 1a. During each free-fall, the accumulated phase is given by the action along the trajectory and, for each The overall envelope is due to the velocity selectivity [11] while the modulation is due to quantum interference. The solid line corresponds to our model as presented in the text. b, position of three consecutive fringe maxima around the highest maximum, as a function of the pulse duration, showing the influence of the phase shift φ0 imprinted by the diffraction pulses : Dots are experimental points, with error bars reflecting the experimental uncertainties. Solid lines come from the theoretical model using g=9.809 m.s -2 . diffraction pulse, the matrix of transfer amplitudes between the various inputs and outputs is calculated by solving the Schrödinger equation in momentum space. At each output O of the interferometer, we sum the amplitudes of all possible trajectories from the input A to that output and take the square modulus to get its probability. For comparison with our observations, the fraction of suspended atoms is taken as the sum of the probabilities at all outputs O i . Finally, we take into account the finite temperature of the initial atomic sample by summing the results over the distribution of initial velocities. This model reproduces accurately the whole interference pattern of Fig. 3a.

When we increase the number of pulses so that N |ǫ| 2 ≈ 1, the contribution from higher order paths is not negligible and we enter a regime of multiple-wave interference. Fig. 4a shows a comparison of the fringes for the cases of 10 and 20 consecutive pulses. After 20 pulses, we observe a clear deviation from a sinusoidal pattern, the fringe width decreases and the contrast increases to almost 1. As plotted in Fig. 4b, the fringe half-width at half-maximum decreases from 4.1 µs after 10 pulses, where N |ǫ| 2 ≈ 0.3, to 2.1 µs after 30 pulses, where N |ǫ| 2 ≈ 0.3. The relative contributions to the output amplitude at O of zero-, first-, and second-order paths increase from 1, 0.26, and 0.01 respectively in the case of 10 pulses to 1, 0.9, 0.32 in the case of 30 pulses. The finesse of our interferometer, i.e. the ratio of the full-width at half-maximum of the resonances to Fig. 4: Multiple-wave quantum trampoline. a, suspended fraction as a function of the pulse period, for 10 (diamonds) and 20 (triangles) pulses. The contrast evolves from 0.6 to almost 1 and the fringe width is significantly reduced as highlighted by the vertical dashed lines at half-maximum. Lines : calculated suspended fraction, with g = 9.809 m.s -2 selected as fitting parameter. b, fringe half-width at half-maximum as a function of the number of pulses N . The narrowing of the fringes with increasing N is an evidence of the stronger contribution of higher order paths to the interference pattern.

the fringe spacing, is 4 after 30 pulses. This increase of the finesse is an evidence of the stronger contribution of the higher order paths when the number N of pulses increases, as expected for a multiple-wave interferometer [13][14][15].

Our quantum trampoline is sensitive to gravity. From the position of the broad envelop associated with the classical trampoline, we can deduce g=9.8 (1). The same value, with a similar accuracy can also be inferred from the fringe spacing. However, a measure of the absolute fringe position allows us to reach a better accuracy. For this, we need to take into account an additional phase [START_REF]φ0 -4πgT 2 /λ, where φ0 is a constant resulting from the sum of the phase shifts acquired during the diffraction events[END_REF] φ 0 resulting from the diffraction events, which varies with the pulse duration (Fig. 3b). We calculate precisely this phase with our model, and use it to fit the data on Fig. 4a with g as the only fitting parameter. We find g = 9.809(4) m.s -2 , in agreement with the known value of g in Palaiseau (9.8095 m.s -2 from WGS84). The uncertainty is due to our signal-to-noise ratio, and to standing-wave power fluctuations which affect the complex diffraction amplitudes. There are several possibilities to improve our setup. First, a higher number of bounces is achievable, for example starting from a condensate in a trap with weaker confinement, for which the velocity spread after release is reduced [12]. Second, an adequate shaping of the pulses temporal envelope [START_REF] Keller | [END_REF] could favor well chosen diffracted orders, and increase the number of contributing trajectories, resulting into a higher finesse of the fringe pattern. Third, using a standing wave with a smaller wavelength or atoms with a reduced mass (such as helium or lithium), the time between bounces would increase and the precision on g could be improved by several orders of magnitudes.

Conclusion. -We have presented a quantum trampoline and used it as a proof-of-principle simple and compact gravimeter, where atoms are held in a volume of few cubic micrometers. Further investigations are needed to study the systematic effects and limitations of our interferometric scheme and to compare it with other compact sensors [2,12,27]. Our scheme, where the atomic wavefunction is repeatedly split and recombined, is likely to be weakly sensitive to atom interaction or to laser phase-noise thanks to averaging over many diffraction events. Beyond the prospect of miniaturized gravito-inertial sensors, our setup has potential applications for measuring fundamental forces at small distances [25,26]. It also opens perspectives for new types of interferometers and new sensor geometries. Suspended atoms could be used for atomic clock applications [16] or to build additional interferometers in the horizontal plane. The interrogation time would then not be limited by the size of the experimental chamber. The realization of a multidimensional interferometer measuring simultaneously the acceleration in three dimensions seems possible [24]. Our quantum trampoline differs dramatically from its classical analogue, where the random velocity transfers would result in atom losses. It provides another clear demonstration of the dichotomy between classical and quantum dynamics [28][29][30].

Fig. 2 :

 2 Fig. 2: Outputs and losses in a 15-pulse quantum trampoline. a and b, absorption images taken 2 ms after the last pulse (O in Fig. 1), in the case of destructive (a) and constructive (b) interference in the suspended channel. The pulse periods are T =1.206 ms and T =1.198 ms respectively. The image extends over 3 mm vertically. The atoms suspended against gravity lie in the circle at the top of the images. The spots below correspond to loss channels and are strongly suppressed in b compared to a due to the interference blocking the loss channels. The two lowest spots correspond to losses at points A and B of Fig. 1a, which cannot be suppressed by interference.

Fig. 3 :

 3 Fig.3: Interference fringes for a 10-pulse quantum trampoline. a, fraction of suspended atom as a function of the pulse period. The overall envelope is due to the velocity selectivity[11] while the modulation is due to quantum interference. The solid line corresponds to our model as presented in the text. b, position of three consecutive fringe maxima around the highest maximum, as a function of the pulse duration, showing the influence of the phase shift φ0 imprinted by the diffraction pulses : Dots are experimental points, with error bars reflecting the experimental uncertainties. Solid lines come from the theoretical model using g=9.809 m.s -2 .

We acknowledge F. Moron and A. Villing for technical assistance, R. A. Nyman, J.-F. Clément and B. Allard for their work on the apparatus, F. Impens for helpful discussions. This research was supported by CNRS, CNES as part of the ICE project, Direction Général de l'Armement, the project "blanc" MélaBoFérIA from ANR, IFRAF ; by the STREP program FINAQS of the European Union and by the MAP program SAI of the European Space Agency (ESA).


Linda Kessler

Rui Gao

Nalan Tetik-Elsherbiny

Olga Lityagina

Azhar Zhailauova

Yonggang Ren

Felix A Trogisch

Julio Cordero

Yanliang Dou

Yinuo Wang

Evgeny Chichelnitskiy

Joscha Alexander Kraske

Patricia Laura Schäfer

Chi-Chung Wu

Guillermo Barreto

Michael Potente

Thomas Wieland

Roxana Ola

Joerg Heineke

Prof. Dr Gergana Dobreva
email: gergana.dobreva@medma.uni-heidelberg.de

Rnf20 shapes the endothelial control of heart morphogenesis and function

Keywords: endothelial-cardiomyocyte crosstalk, heart development, congenital heart disease, endothelial-to-mesenchymal transition (EndMT), Rnf20, H2B ubiquitination (H2Bub), cardiac progenitor cells, cardiomyocyte binucleation, premature cardiomyocyte maturation

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The heart is a complex organ composed of distinct cell types, which shape its structural, mechanical and electrical properties [START_REF] Meilhac | The deployment of cell lineages that form the mammalian heart[END_REF]. In particular, the crosstalk between endothelial cells and cardiomyocytes plays a crucial role both for normal cardiac morphogenesis and in cardiac disease. However little is known about the molecular players safeguarding physiological signaling between these cell types. During embryogenesis, endocardial endothelial cells (ECs) and myocardial precursors arise bilaterally within the mesoderm and through migration and embryo folding coalesce at the ventral midline to form a primitive heart tube consisting of an inner endocardial layer and an outer myocardial layer [START_REF] Feulner | Endocardial Regulation of Cardiac Development[END_REF][START_REF] Tian | Importance of myocyte-nonmyocyte interactions in cardiac development and disease[END_REF]. The two layers are separated by a dense layer of extracellular matrix (ECM), termed cardiac jelly, but communicate through paracrine signals.

After formation of the initial heart tube, the heart grows by the addition of second heart field (SHF) progenitor cells to its anterior and venous poles [START_REF] Cai | Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart[END_REF][START_REF] Gao | Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate[END_REF][START_REF] Meilhac | The deployment of cell lineages that form the mammalian heart[END_REF][START_REF] Vincent | How to make a heart: the origin and regulation of cardiac progenitor cells[END_REF][START_REF] Witzel | The LIM protein Ajuba restricts the second heart field progenitor pool by regulating Isl1 activity[END_REF]. These SHF precursors, marked by the expression of the transcription factor (TF) Isl1, are multipotent and give rise to the distinct lineages in the heart [START_REF] Caputo | The Isl1/Ldb1 Complex Orchestrates Genome-wide Chromatin Organization to Instruct Differentiation of Multipotent Cardiac Progenitors[END_REF][START_REF] Moretti | Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification[END_REF][START_REF] Zhuang | Expression of Isl1 during mouse development[END_REF], including myocardial and endocardial cells. The endocardium has recently gained attention due to its remarkable cellular plasticity, as well as its important signaling function during heart development, disease, and regeneration [START_REF] Zhang | Endocardial Cell Plasticity in Cardiac Development, Diseases and Regeneration[END_REF].

On one hand, it forms a vascular endothelial layer contiguous with the rest of the vasculature, which provides nutrients and oxygen to the heart. On the other hand, it receives and secretes signals that instruct heart morphogenesis and function, e.g. trabeculation, cardiac valve and septum formation, and myocardial compaction, thereby functioning as a crucial signaling interface [START_REF] Feulner | Endocardial Regulation of Cardiac Development[END_REF][START_REF] Kim | Endothelial-Myocardial Angiocrine Signaling in Heart Development[END_REF][START_REF] Tian | Importance of myocyte-nonmyocyte interactions in cardiac development and disease[END_REF]. With the formation of the compact myocardium intramyocardial vessels develop, which also signal to the subjacent cardiomyocytes (CMs) to regulate cardiac growth, contractility and rhythmicity. The importance of ECs for heart development has been demonstrated by endothelial-specific gene inactivation of different components of the Notch, vascular endothelial growth factor (VEGF), bone morphogenic protein (BMP), transforming growth factor (TGF), fibroblast growth factor (FGF), Neuregulin-1 (Nrg1)/Erbb2/b4 and angiopoietin-1 signaling pathways [START_REF] Feulner | Endocardial Regulation of Cardiac Development[END_REF][START_REF] Kim | Endothelial-Myocardial Angiocrine Signaling in Heart Development[END_REF][START_REF] Tian | Importance of myocyte-nonmyocyte interactions in cardiac development and disease[END_REF]. However, how signal-dependent transcription is coordinated during heart development is largely unknown.

At the molecular level, these dynamic cell-cell interactions induce signaling cascades, which converge in the nucleus to induce transcriptional and epigenetic programming that directs cell fate and function [START_REF] Elsherbiny | Epigenetic memory of cell fate commitment[END_REF]), while at the same time allowing a certain level of cell plasticity that is critical for proper cardiac morphogenesis and tissue healing following environmental stress. The classical model of inducible gene expression is based on signal-dependent RNA polymerase II (Pol II) recruitment and transcription initiation by inducible binding of transcription factors and epigenetic modifiers to promoters and enhancers. However, more recent studies have shown that inducible transcription is often regulated after transcription initiation, by pausing and release of promoter-proximal Pol II [START_REF] Hargreaves | Control of inducible gene expression by signal-dependent transcriptional elongation[END_REF][START_REF] Levine | Paused RNA polymerase II as a developmental checkpoint[END_REF][START_REF] Liu | Ready, pause, go: regulation of RNA polymerase II pausing and release by cellular signaling pathways[END_REF]. Many factors have been identified to regulate Pol II pausing and elongation, including Rnf20, the major E3 ubiquitin ligase responsible for monoubiquitination of histone H2B at lysine 120 (H2BK120ub, H2Bub11) [START_REF] Kim | RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells[END_REF][START_REF] Kim | The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions[END_REF][START_REF] Pavri | Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II[END_REF]. Mechanistically, Rnf20 plays a key role in transcriptional control, by altering nucleosome stability and acting as a signaling hub for downstream processes associated with transcript pausing and elongation by Pol II, such as cotranscriptional mRNA processing [START_REF] Chen | PAF1, a Molecular Regulator of Promoter-Proximal Pausing by RNA Polymerase II[END_REF][START_REF] Fuchs | Writing and reading H2B monoubiquitylation[END_REF][START_REF] Pavri | Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II[END_REF][START_REF] Shema | RNF20 inhibits TFIIS-facilitated transcriptional elongation to suppress pro-oncogenic gene expression[END_REF]. The important function of Rnf20 for proper heart development was revealed by exome sequencing data, which identified a de novo mutation in RNF20 to be associated with congenital heart defects in human patients [START_REF] Zaidi | De novo mutations in histonemodifying genes in congenital heart disease[END_REF]. Further work in Xenopus found that Rnf20 depletion results in defects in left-right (LR) asymmetry likely due to impaired cilia motility [START_REF] Robson | Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility[END_REF], while a recent study revealed a critical role of Rnf20 in CMs for CM maturation in the early postnatal window [START_REF] Vandusen | Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation[END_REF]. Despite 6 these interesting findings, the role of Rnf20 in the different cardiac cell types for ensuring proper cardiogenesis is largely unexplored.

In this study, we have addressed the role of Rnf20 in heart development. We find that Rnf20 is highly expressed in SHF progenitors and orchestrate SHF morphogenesis. Rnf20 ablation in Isl1 positive (Isl1+) cardiovascular progenitors and their progeny results in severe cardiac and vascular abnormalities, including shorter outflow tract (OFT), hypoplastic ventricular wall, abnormal trabeculation and disorganized endocardium. Functional and molecular analyses revealed that Rnf20 plays a central role in cardiac ECs for proper cardiac morphogenesis and function by inhibiting endothelial-to-mesenchymal transition (EndMT) and aberrant angiocrine signaling that induce CM cell cycle withdrawal and arrhythmic beating behavior.

Results

Rnf20 is crucial for SHF development

The transcription factor Isl1 orchestrates a complex gene regulatory network driving SHF development and Isl1 ablation results in loss of SHF derivatives, including the right ventricle (RV), OFT and large portions of the atria [START_REF] Cai | Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart[END_REF][START_REF] Caputo | The Isl1/Ldb1 Complex Orchestrates Genome-wide Chromatin Organization to Instruct Differentiation of Multipotent Cardiac Progenitors[END_REF][START_REF] Gao | Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate[END_REF]. To further unravel the molecular determinants of Isl1-mediated SHF development, we performed a protein array screen for protein interaction partners of Isl1 and identified Rnf20, an E3 H2Bub1 ligase, which tightly associates with Pol II. Co-immunoprecipitation experiments in cardiac progenitors confirmed the interaction, while GST pulldown assays further validated that the binding of Isl1 and Rnf20 is direct, suggesting that Isl1 might regulate transcription through its direct interaction with Rnf20 (Figure 1A). Analysis of single cell RNA-Sequencing (RNA-Seq) datasets of E8.25 embryos [START_REF] Xiong | Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intraorgan Crosstalk During Cardiogenesis[END_REF] showed higher expression of Rnf20 in SHF progenitors, further supporting a role of Rnf20 in Isl1-driven SHF development (Figure 1B). To understand the function of Rnf20 in the developing heart, we first conditionally ablated Rnf20 in mice using Isl1-Cre, which deletes in Isl1+ cardiovascular progenitors and their downstream progeny, i.e. the myocardium and the endocardium. Rnf20-deficient embryos died by E14.5 with severe cardiac and vascular abnormalities, including shorter OFT, hypoplastic ventricular wall, abnormal trabeculation and disorganized endocardium (Figure 1C-G). Coimmunostaining for the proliferation marker EdU together with isolectin B4 (IB4), an endothelial marker, and myosin heavy chain (MF20), a CM marker, revealed significantly decreased CM proliferation in the right and the left ventricle (LV) of E12.5 Rnf20-deficient hearts (Figure 1H, 1I). Immunostaining for H2Bub1, a histone modification catalyzed by Rnf20, revealed complete loss of H2Bub1 in the RV, whereas CMs in the LV were positive for H2Bub1 upon Rnf20depletion using the Isl1-Cre deleter (Figure 1J, 1K). Interestingly, the endocardial cells in the RV, as well as most of those in the LV, were negative for H2Bub1 (Figure 1K), suggesting that Rnf20 might play a role in endocardial cells, which is important for CM proliferation.

Rnf20 controls extracellular matrix dynamics and cell signaling essential for SHF development

To elucidate Rnf20's downstream effectors governing cardiogenesis, we performed RNA-Seq of OFT and RV dissected from E9.5 and E10.5 control and Isl1 cre/+ Rnf20 -/fl embryos (Figure 2A-C, Figure S1A, Table S1). Gene Ontology (GO) pathway analysis of E9.5 hearts revealed an over-representation for genes linked to cell migration and adhesion, ECM organization, angiogenesis as well as transcriptional regulation, including TFs involved in EndMT such as Snai1, Snai2 and Msx2. In contrast, genes downregulated upon Rnf20 loss of function (LOF) were linked to cell cycle, Notch signalling and muscle contraction (Figure 2C). However, at E10.5, genes involved in blood vessel and heart development as well as cell-cell signalling were significantly downregulated upon Rnf20 LOF, whereas upregulated genes upon Rnf20 loss were mainly involved in sarcomere organization, regulation of muscle contraction and similarly cell-cell signalling (Figure 2D, Figure S1B), suggesting a dynamic function of Rnf20 during cardiogenesis. In line with the known function of Rnf20 in transcriptional elongation, differentially expressed genes upon Rnf20 loss have a low pausing index (Figure S1D-F).

Since Rnf20 interacts with Isl1, which induces chromatin opening of key SHF genes [START_REF] Gao | Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate[END_REF], we next analysed chromatin accessibility at Rnf20 target genes. We found a major decrease of chromatin accessibility at Rnf20 targets in Isl1-/-mice (Figure S1C), supporting the notion that Isl1 and Rnf20 work together to instruct SHF morphogenesis. Furthermore, we observed a significant overlap between Rnf20 targets and genes deregulated in dissected OFT and RV of E10.5 Isl1 haploinsufficient mice [START_REF] Gao | Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate[END_REF], particularly for ECM genes and a number of proteases of the Adamts family (A disintegrin and metallopeptidase with thrombospondin motif), i.e. Adamts2/3/10/20 and matrix metalloproteinases (Mmps) (Figure 2E).

Since genes involved in cell-cell signalling were deregulated upon Rnf20 depletion, we next performed ligand-receptor analysis (Figure 2F, Table S2). Among the downregulated ligands we found Angpt1 and Bmp10, which play key roles in chamber morphogenesis, trabeculation and myocardial compaction, while among the downregulated receptors we uncovered Tie1, Pdgfra, Kdr, Notch3 and Notch4 (Figure 2F). Importantly, genes involved in endocardialmyocardial interactions, e.g. Vegfr2 (Kdr), Notch1/2/3 and the Notch target genes Hey1, Hes1, Erbb2 and Ccnd1 were significantly downregulated in endocardial cells isolated from Rnf20 deficient E10.5 hearts (Figure 2G). Rnf20 has been shown to be stimulate Notch reporter gene expression and Rnf20 loss of function resulted in Notch signaling defects in Drosophila [START_REF] Bray | Bre1 is required for Notch signaling and histone modification[END_REF]. Therefore, we next studied whether Notch1 and Rnf20 interact. Indeed, coimmunoprecipitation analysis revealed that the active Notch1 intracellular domain (N1ICD) interacts with Rnf20 (Figure 2H). Further, by intersecting transcriptomics datasets of hearts mutant for the principal transcriptional mediator of Notch signalling, Rbpj [START_REF] Klaus | Wnt/betacatenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells[END_REF][START_REF] Macgrogan | Notch and interacting signalling pathways in cardiac development, disease, and regeneration[END_REF], and for Rnf20 we observed a significant overlap (Figure 2I), suggesting that Rnf20 works together with Notch1 to regulate cardiac morphogenesis. Notch1 controls cardiac jelly dynamics through regulating Adamts1 and Mmp2 levels (Del [START_REF] Monte-Nieto | Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation[END_REF]. Mmp2, Mmp14 and a number of proteases of Adam (A disintegrin and metalloproteinases) and Adamts (A disintegrin and metallopeptidase with thrombospondin motif) families were also significantly downregulated upon Rnf20 LOF (Figure 2J). Consistent with the reduced expression of Mmps and Adam-and Adamts-family members, alcian blue staining revealed excessive accumulation of cardiac jelly upon Rnf20 LOF, particularly around the ventricular trabeculae and the endocardial cushions (Figure 2K).

Rnf20 plays a key role in cardiac ECs for heart development and function

Rnf20 has been shown to regulate CM maturation after birth [START_REF] Vandusen | Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation[END_REF], however its function in cardiac ECs is largely unknown. Interestingly, H2Bub1 was depleted in the RV and the majority of the LV endocardium, but was absent only in right ventricular CMs upon Rnf20 ablation in SHF progenitors using the Isl1-Cre line, while CM proliferation was significantly decreased in both ventricles. Moreover, we identified many critical pathways involved in EC-CM communications to be significantly deregulated upon Rnf20 LOF. These observations suggest that Rnf20 might play an important role in ECs for cardiac 10 morphogenesis, which prompted us to further examine Rnf20 function in cardiac ECs. To this end, we inactivated Rnf20 using a Tie2-Cre deleter, which recombines in ECs and some hematopoietic cells [START_REF] Koni | Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow[END_REF]. Tie2-Cre-mediated Rnf20 deletion resulted in early embryonic lethality around E10.5 (Figure 3A). Tie2 Cre Rnf20 fl/fl mutant embryos displayed shortened OFT and small RV -structures generated by Isl1+ progenitor cells (Figure 3B), supporting the notion that endothelial Rnf20 is important for SHF development.

To be able to study the role of endothelial Rnf20 at different embryonic stages we next used the tamoxifen-inducible Cdh5-CreERT2 and Pdgfb-CreERT2 endothelial-targeting lines [START_REF] Claxton | Efficient, inducible Cre-recombinase activation in vascular endothelium[END_REF][START_REF] Wang | Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis[END_REF] to induce Rnf20 ablation, which resulted in similar phenotypes (Figure 3C-K, Figure S2, data not shown). Endothelial deletion of Rnf20 in ECs following administration of tamoxifen from E8.5 resulted in thinner compact layer, fewer or lessdeveloped trabeculae and a disorganized interventricular septum (IVS) at E12.5 (Figure 3C, Figure S2A) and lethality by E14.5. Interestingly, in contrast to wild-type embryos, in which endocardial cells typically show elongated nuclei and thin cell bodies lining the myocardium, Rnf20-depleted endocardial cells appeared disorganized and did not line the trabeculae (Figure 3C). Tamoxifen administration from E9.5 onwards resulted in similar cardiovascular abnormalities, i.e. reduced trabecular myocardium, thinner compact myocardium and reduced IVS wall thickness together with ventricular septal defects (VSD) (Figure 3D-F, Figure S2B, S2C). We next studied whether the observed alterations are the result of altered CM proliferation. Indeed, immunostaining for the mitotic marker Ser10 phosphorylated-histone H3 (pH3) in combination with the CM marker MF20 revealed a significant decrease in CM proliferation (Figure 3G,3H). Furthermore, stainings with Vybrant DyeCycle DNA dye followed by FACS analysis, which can be used to distinguish between mononucleated and binucleated CMs [START_REF] Windmueller | Direct Comparison of Mononucleated and Binucleated Cardiomyocytes Reveals Molecular Mechanisms Underlying Distinct Proliferative Competencies[END_REF], found significant increase in binucleated CMs in Rnf20 iEC-KO hearts (Figure 3I). Further, echocardiography measurements revealed irregular contractility of the RV as well as reduced ejection fraction, fractional shortening and cardiac output in Rnf20 iEC-KO embryos, indicating cardiac dysfunction (Figure 3J, 3K, Figure S2D).

Direct co-differentiation of endocardial cells and CMs from pluripotent stem cells reveals perfusion independent effects of endothelial Rnf20 deletion on CMs

To study the role of Rnf20 in ECs in controlling CM behavior without the potential confounding side effects of altered tissue perfusion in malformed hearts, we established protocols for directed differentiation of murine ESCs (mESCs) in Nkx2.5+/CD31+ endocardial cells (based on [START_REF] Mikryukov | BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors[END_REF]), as well as protocols for co-differentiation of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs from common cardiovascular progenitors (Figure 4A-F). Both co-differentiation and co-culture of CMs with ESC-derived endocardial ECs resulted in increased CM contractility and differentiation, as well as the expression of genes involved in EC-CM crosstalk (Figure 4D-H), suggesting that co-differentiation is a suitable system to study the CM-EC crosstalk during cardiogenesis. In contrast, co-culture of CMs with hemogenic ECs (Nkx2.5-/CD31+) did not significantly alter CM contractility. We then established induced pluripotent stem cells (iPSCs) from control (Cdh5 CreERT2 -negative-Rnf20 fl/fl ) and Rnf20 iEC-KO (Cdh5 CreERT2 -positive-Rnf20 fl/fl ) fibroblasts, which allow us to ablate Rnf20 specifically in ECs at distinct steps during cardiovascular differentiation using 4hydroxytamoxifen (4-OHT) (Figure 4I). Adding 4-OHT to the differentiation medium at day 5 (cardiovascular progenitors) to induce Rnf20 ablation resulted in decreased CM proliferation and increased number of binucleated CMs, while proliferation of ECs was also reduced (Figure 4J-L). Analysis of movies of spontaneously beating CMs using MYOCYTER [START_REF] Grune | The "MYOCYTER" -Convert cellular and cardiac contractions into numbers with ImageJ[END_REF] revealed arrhythmicity (Figure 4M), similar to the phenotype observed in vivo. These findings suggest that the observed in vivo phenotypes are a direct consequence of Rnf20 ablation.

Rnf20 inhibits endothelial-to-mesenchymal transition (EndMT)

To further understand the mechanisms underlying the impact of endothelial Rnf20 on CMs, we performed single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO hearts as well as bulk RNA-Seq of isolated cardiac ECs and CMs (Figure 5A, Figure S3A-C). Similar to previous studies, 12 we detected several distinct cell types with transcriptional signatures of different CM subtypes, endocardial cells, cardiac cushions, valves, vascular ECs, hemogenic endocardium, smooth muscle cells and others [START_REF] Xiao | Hippo Signaling Plays an Essential Role in Cell State Transitions during Cardiac Fibroblast Development[END_REF] (Figure 5B). Consistent with our histological studies, single-cell RNA-Seq analysis revealed major changes in ventricular CM number, cell cycle activity determined by the expression of Mki67 (Marker of Proliferation Ki-67), and maturation as defined by the expression of mitochondrial marker genes such as mt-Co2, mt-Nd2, mt-Nd1 and mt-Atp6 (Figure 5B,5C). We next re-clustered the EC populations to investigate alterations in Rnf20-deficient ECs in greater detail. This approach identified seven clusters, including three distinct endocardial cell populations, a proliferative endocardial population, one coronary EC population, as well as endocardial valve and AV cushion cells (Figure 5D). The proliferative endocardial cluster, marked by the Mki67 (Marker of proliferation Ki-67), was strongly reduced in Rnf20-deficient hearts (Figure 5D,5E), consistent with the reduced endocardial cell proliferation in our in vitro co-differentiation system (Figure 4J, 4K). Interestingly, we observed many genes involved in ECM organization to be upregulated particularly in endocardial clusters, EndoC2+3 and endocardial valve cells (Figure 5E, 5F). Similarly, bulk RNA-Seq analysis revealed that genes upregulated in Rnf20deficient ECs show over-representation of GO terms linked to connective tissue development and cell-cell signaling by Wnts and Bmps, while downregulated genes were related to chromatin modification, transcriptional activation, cell proliferation and communication (Figure 5G, Figure S3D). Gene set enrichment analysis further supported an important role of Rnf20 in the regulation of genes involved in cell-cell interactions (Figure S3E). Interestingly many typical markers of EndMT, e.g. fibronectin (Fn1), vimentin (Vim), Icam1, Mmp2, a large set of collagens as well as TFs driving EndMT in the heart such as Hand2 and Foxc1, were highly upregulated upon Rnf20 loss suggesting that Rnf20 safeguards EC identity by preventing EndMT (Figure 5G, Figure S3D,S3F). Immunostaining confirmed the high expression of Vim in Rnf20 iEC-KO endocardium (Figure 5H). Similarly, depletion of Rnf20 in HUVECs resulted in upregulation of genes involved in ECM organization and downregulation of genes regulating cell proliferation (Figure S3G). Further, Rnf20-deficient HUVECs showed increased migratory behavior (Figure S3H-J), supporting a function of Rnf20 in inhibiting EndMT.

Rnf20 controls cell-cell signaling-dependent transcription

Interestingly, we detected significant transcriptional changes in CMs upon Rnf20 deletion in ECs (Figure 5I, Figure S3D). Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos. Similar to ECs, genes downregulated in CMs were linked to chromatin modification, transcriptional activation and cell proliferation (Figure 5I, Figure S3D). Among the genes downregulated upon Rnf20 depletion was the RhoGEF gene Ect2 (Figure S3D), which regulates cytokinesis. Loss of Ect2 function has been shown to induce CM binucleation and cell-cycle withdrawal, and is sufficient to suppress the CM proliferation during cardiac regeneration [START_REF] Liu | Control of cytokinesis by beta-adrenergic receptors indicates an approach for regulating cardiomyocyte endowment[END_REF][START_REF] Windmueller | Direct Comparison of Mononucleated and Binucleated Cardiomyocytes Reveals Molecular Mechanisms Underlying Distinct Proliferative Competencies[END_REF]. Interestingly, we detected significant changes in Rnf20 levels in CMs upon Rnf20 deletion in ECs. Similarly, Rnf20 levels were significantly upregulated in CMs upon co-culture with ECs (Figure S3K).

To identify upstream regulatory factors driving cardiac dysfunction in Rnf20 iEC-KO embryos, we performed assay for transposase-accessible chromatin (ATAC) sequencing (ATAC-Seq) [START_REF] Buenrostro | Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position[END_REF] of ECs and CMs isolated from control and Rnf20 iEC-KO hearts.

Endothelial depletion of Rnf20 resulted in major changes in chromatin accessibility in both ECs and CMs (Figure 6A, 6B, Figure S4A,4B). Similar to the transcriptomics data, genes that gained chromatin accessibility in ECs were highly enriched in GO terms linked to cell-cell signaling and vasculature development, while genes that gained open chromatin in CMs were linked to heart development, Ca 2+ homeostasis and cell-cell signaling (Figure 6B). Genes that lost chromatin accessibility in ECs were similarly linked to cell-cell signaling and specifically to Wnt signaling as well as embryonic development. In CMs, genes linked to cell differentiation, cardiac septum development and Wnt signaling showed reduced open chromatin (Figure 6B).

Through integrative analysis, we identified genes whose transcriptional level positively correlate with chromatin accessibility at their respective genomic loci, indicating binding or loss of binding of transcriptional activator (Figure 6C, 6D, 6F, 6G, Figure S4C, S4D). We also observed genes whose transcriptional level inversely correlate with their chromatin accessibility, indicative of transcriptional repressor binding. We next performed Transcription factor Occupancy prediction By Investigation of ATAC-Seq Signal (TOBIAS) footprinting analysis (Bentsen et al., 2020a) to pinpoint transcriptional activators and repressors of Rnf20dependent transcription. We identified multiple TFs, including Stat5, Pparg, Pax3 and Nkx2.5 etc. as potential transcriptional activators that are highly bound in Rnf20 iEC-KO ECs compared to control ECs, and Hes6, Msx1 and Bcl6 as potential repressors of Rnf20-dependent transcription (Figure S4E). Analogous analysis of Rnf20 iEC-KO CMs pointed to Pparg, Hand1, Tp53 as transcriptional activators engaged in CMs upon Rnf20 LOF in ECs, while Tbx20, Irf8/9 as potential transcriptional repressors (Figure S4F). TFs binding to regulatory regions are highly dynamic; hence, differential TF binding does not necessarily result in altered chromatin accessibility. Therefore, we next examined the differential binding of TFs at all ATAC peaks in control and Rnf20 iEC-KO associated only to deregulated genes (Figure 6E, 6H). We found Foxc1, Hif1a, Smad4, Tbx2 and Pparg to bind highly at Rnf20-targets in Rnf20 iEC-KO ECs, while key EC identity TFs (Etv2, Fli1, Erg) and TFs involved in Notch signaling showed decreased binding (Figure 6E). Particularly, TFs involved in EndMT (Sox9, Hif1a, Tbx2/3, Smads) were highly bound at genes upregulated upon Rnf20 LOF (Figure 6I). In CMs, tumor suppressors restricting cell proliferation (Tp53 and Tp73) were highly enriched at Rnf20 target genes, while E2 factor (E2F) family members showed lower binding, consistent with the decreased CM proliferation in Rnf20 iEC-KO hearts (Figure 6H). SOX9 overexpression is sufficient to activate mesenchymal genes in HUVECs and induce EndMT [START_REF] Fuglerud | SOX9 reprograms endothelial cells by altering the chromatin landscape[END_REF], thus we next compared gene expression profiles of ECs overexpressing Sox9 and lacking Rnf20. We found a high degree of overlap (31%), particularly in genes upregulated in both cases (Figure 6J).

Genes significantly upregulated upon Sox9 overexpression and Rnf20 LOF belonged to GO terms including cell adhesion, ECM organization as well as genes involved in regulation of blood pressure (Figure 6K). Given the high degree of overlap, we next tested whether Sox9 upregulation could induce CM binucleation and thus cell cycle withdrawal. We observed significantly increased number of binucleated CMs upon co-culture with ECs overexpressing Sox9, suggesting that Sox9 might regulate an EndMT transcriptional program resulting in premature CM maturation upon Rnf20 LOF (Figure 6L,6M).

Rnf20 inhibits aberrant signaling promoting CM binucleation, cell cycle withdrawal and irregular contractility

Since we observed a dramatic impact of Rnf20 LOF in ECs on CMs and genes involved in cellcell signaling and ECM organization were highly deregulated in Rnf20 iEC-KO , we next performed ligand-receptor analysis to pinpoint the angiocrine and juxtacrine signals from Rnf20-deficient ECs and receptors on ECs or CMs that may result in cardiac dysfunction (Figure 7A,7B).

Interestingly, we found a major increase in EC-CM signaling upon Rnf20 LOF (Figure 7A).

Among others, we identified interactions between collagens, integrins and signaling receptors together with Transforming Growth Factor Beta Induced (Tgfbi) and the tissue inhibitor of metalloproteinases-3 (Timp3), which have been shown to inhibit CM proliferation [START_REF] Rhee | Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular noncompaction[END_REF], to be highly upregulated in Rnf20-deficient ECs. On the other hand, Fgf signals were decreased upon Rnf20 EC LOF (Figure 7B). Similarly, we observed increased signaling derived from CMs upon Rnf20 LOF in ECs. We next selected highly altered EC receptors, angiocrine factors and EC-derived ECM proteins to assess their role on CM proliferation and binucleation (Figure 7B). To this end, we infected HUVECs with lentiviruses carrying overexpression constructs of altered EC-derived factors as well as signaling receptors on ECs, and co-cultured them with neonatal CMs 2 days after infection (Figure 7C). Indeed, many of the selected factors blocked proliferation and/or induced CM binucleation, e.g. PCOLCE2, COL23A1, COL5A1, ICAM1, TGFBI, TIMP3 as well as TGFBR3 and PIEZO1 (Figure 7D, 7E, Figure S5A-C). In addition, we observed changes in CM beating rate and / or abnormal contractility upon overexpression of PCOLCE2, COL23A1, TIMP3, MMP14, TGFBR3 and PIEZO1 in ECs (Figure 7F, 7G, Figure S5D). Together, these data support a key function of 16 Rnf20 in inhibiting aberrant angiocrine signaling promoting CM cell cycle withdrawal and abnormal contractility.

Discussion

In this study, we show that Rnf20 is a central determinant of heart development that controls EC-CM crosstalk during cardiac morphogenesis at different levels. First, Rnf20 controls extracellular matrix dynamics and cell signaling essential for second heart field development.

Second, it safeguards EC identity and function by preventing EndMT. Third, it inhibits vicious angiocrine signaling and ECM deposition that cause precocious CM binucleation, cell cycle withdrawal and irregular contractility. We further identify specific factors derived from Rnf20deficient ECs, e.g. collagens, Pcolce2, Icam1, Mmp14, Tgfbi, Tgfb3, Piezo1, etc. that modulate CM function (Figure 7H).

Rnf20 controls extracellular matrix dynamics and cell signaling essential for second heart field development

In a protein array screen using recombinant Isl1 protein we identified Rnf20 as a putative direct interacting partner of Isl1. Deletion of Rnf20 in Isl1+ SHF progenitors resulted in major cardiovascular defects and deregulation of a multitude of common targets, supporting a key role of the Isl1-Rnf20 complex in SHF development. Interestingly, a large number of Isl1-Rnf20 target genes were linked to ECM organization and cell signaling, including genes involved in Notch signaling. Notch signaling, which plays a central role in EC function, regulates trabeculation, OFT and valve morphogenesis, and alterations in endocardial Notch signaling result in cardiac abnormalities [START_REF] Monte-Nieto | Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation[END_REF][START_REF] Grego-Bessa | Notch signaling is essential for ventricular chamber development[END_REF]. The Drosophila homologues of Rnf20, dBre1, was shown to be required for the expression of Notch target genes by coupling H2Bub1 to H3K4me3 and transcriptional activation in Drosophila development [START_REF] Bray | Bre1 is required for Notch signaling and histone modification[END_REF]. In line with this study, we found a significant overlap between target genes of Rnf20 and Rbpj, the transcription factor mediating canonical Notch signaling.

Among the common targets, particularly interesting were genes involved in ECM organization, consistent with the important role of Notch1 in the regulation of cardiac jelly dynamics (Del [START_REF] Monte-Nieto | Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation[END_REF]. Cardiac jelly deposition and degradation is crucial for cardiac morphogenesis. While depletion of key components of the cardiac jelly, such as versican or hyaluronan synthase-2, results in loss of trabeculation and severe cardiac chamber defects, proper degradation of the cardiac jelly by Adam and Adamts family members is also required for myocardial remodeling and valve formation [START_REF] Nandadasa | The multiple, complex roles of versican and its proteolytic turnover by ADAMTS proteases during embryogenesis[END_REF]. Cardiac jelly degradation was compromised upon Rnf20 depletion, suggesting that the observed defects in myocardial growth and maturation as well as EC-CM signaling, could be due to the inability of Rnf20-deficient cells to degrade the cardiac jelly. In addition, we observed a significant decrease in Angpt1-Tie1/Tek signaling that was also shown to regulate cardiac jelly degradation [START_REF] Kim | Myocardial Angiopoietin-1 Controls Atrial Chamber Morphogenesis by Spatiotemporal Degradation of Cardiac Jelly[END_REF], which could contribute to the observed excessive cardiac jelly in Rnf20 knockout hearts. Together, our data suggest an important role of the Isl1-Rnf20 complex in controlling the ECM dynamics and SHF development.

Rnf20 maintains physiological angiocrine signaling levels that shape the structural and functional properties of the heart

Our results point to a crucial function of Rnf20 in ECs for maintaining physiological angiocrine signaling. Chromatin accessibility analysis identified genes involved in cell-cell signaling as main Rnf20 targets upregulated upon Rnf20 LOF. In the last decade it became clear that inducible transcription is mainly regulated by Pol II pausing and its release into active elongation [START_REF] Hargreaves | Control of inducible gene expression by signal-dependent transcriptional elongation[END_REF][START_REF] Levine | Paused RNA polymerase II as a developmental checkpoint[END_REF][START_REF] Liu | Ready, pause, go: regulation of RNA polymerase II pausing and release by cellular signaling pathways[END_REF]. Rnf20 and Rnf20-mediated H2Bub1 work in concert with the FACT complex to regulate elongation by Pol II [START_REF] Pavri | Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II[END_REF]), but at the same time can induce transcriptional pausing by inhibiting the recruitment of TFIIS, a factor involved in the release of stalled Pol II [START_REF] Shema | RNF20 inhibits TFIIS-facilitated transcriptional elongation to suppress pro-oncogenic gene expression[END_REF]. In addition, the Pol II-associated Factor 1 (PAF1), which mediates the interaction of Rnf20 with Pol II, was shown to function as a key regulator of Pol II pausing [START_REF] Shema | RNF20 inhibits TFIIS-facilitated transcriptional elongation to suppress pro-oncogenic gene expression[END_REF]. Thus, Rnf20 could induce transcriptional pausing in the developing heart, thereby keeping signal-inducible transcription in check. In such a scenario, loss of Rnf20 would result in paused Pol II release and higher expression of genes involved in cell-cell communication. In line with this notion, our results revealed that Rnf20 is essential in maintaining physiological angiocrine signaling, which shapes the structural and functional properties of the heart. Indeed, inducible ablation of Rnf20 in cardiac ECs resulted in upregulation of multitude of angiocrine factors and ECM proteins coupled to major defects in CM proliferation, myocardial growth and cardiac contractility. CM dysfunction could result from altered tissue perfusion in malformed hearts. However, by using a novel cell-culture based system for co-differentiation of cardiac endothelial cells and cardiomyocytes from pluripotent stem cells, we confirmed the critical role of Rnf20 in ECs for CM proliferation and contractility. A large number of upregulated genes upon Rnf20 LOF are ECM proteins, which alter tissue stiffness and biomechanical signaling in the heart. Cardiac stiffness gradually increases during embryonic development and after birth, and maintenance of physiological levels of cardiac stiffness is detrimental for proper CM proliferation, maturation and contractility throughout life [START_REF] Munch | Sensing and Responding of Cardiomyocytes to Changes of Tissue Stiffness in the Diseased Heart[END_REF]. Systematic overexpression of selected factors using genome-wide libraries in ECs and co-culture with CMs revealed many novel molecules that regulate CM binucleation, proliferation and contractility, e.g. Pcolce2, Col5a1, Col23a1, Icam1, Mmp14, Tgfb3, Piezo1, etc., together with already known factors such as Tgfbi and Timp3 [START_REF] Rhee | Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular noncompaction[END_REF]. Collagens are crosslinked through lysyl oxidases (Lox, Loxl), which increases their stiffness. Together with many collagens, Loxl2 was significantly upregulated upon Rnf20 LOF, which might increase cardiac stiffness above the physiological level and thereby induce premature CM differentiation, binucleation and cell cycle withdrawal. LOXL2 is upregulated in diseased human hearts and LOXL2 inhibition protects against stress-induced cardiac dysfunction [START_REF] Yang | Targeting LOXL2 for cardiac interstitial fibrosis and heart failure treatment[END_REF], suggesting that Loxl2 inhibition might be beneficial in CHD caused by Rnf20 LOF.

Rnf20 safeguards EC identity by inhibiting EndMT

Our data revealed that Rnf20 safeguards EC identity. Rnf20-deficient EC show increased migratory capacity and upregulation of many typical markers of EndMT [START_REF] Dejana | The molecular basis of endothelial cell plasticity[END_REF], e.g. Fn1, Vim, Icam1, Mmp2, multiple collagens, as well as TFs driving EndMT in the heart such as Hand2, Foxc1 and Notch, suggesting that Rnf20 safeguards EC identity by preventing EndMT. TF footprint analysis at accessible chromatin regions predicted significantly increased 20 binding of Sox9 and decreased binding of master TFs controlling EC fate, including Etv2 [START_REF] Gong | ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage[END_REF], Erg and Fli1 as well as downsteam effectors of the Notch pathway such as Hes1, required for SHF development [START_REF] Rochais | Hes1 is expressed in the second heart field and is required for outflow tract development[END_REF]. Sox9 regulates ECM production and organization and plays an important role in EndMT in the developing heart [START_REF] Akiyama | Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa[END_REF][START_REF] Hanley | Ectopic SOX9 mediates extracellular matrix deposition characteristic of organ fibrosis[END_REF][START_REF] Lincoln | Sox9 is required for precursor cell expansion and extracellular matrix organization during mouse heart valve development[END_REF][START_REF] Scharf | Inactivation of Sox9 in fibroblasts reduces cardiac fibrosis and inflammation[END_REF]. Sox9 overexpression is sufficient to activate mesenchymal genes in ECs and induce EndMT [START_REF] Fuglerud | SOX9 reprograms endothelial cells by altering the chromatin landscape[END_REF], while silencing of Erg and Fli similarly triggers EndMT [START_REF] Nagai | Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition[END_REF]. In line with increased Sox9 binding in Rnf20 iEC-KO ECs, we found a large overlap of genes involved in cell adhesion, collagen and elastic fibril organization as well as genes involved in regulation of blood pressure upon Sox9 overexpression and Rnf20 LOF. Furthermore, Sox9 overexpression in ECs resulted in increased binucleation, pointing to Sox9 as a critical mediator of Rnf20 function in the heart. Interestingly, a recent study revealed that the relative levels of Notch signaling and Sox9 control EC cell fate and plasticity in a skin wound healing experimental model [START_REF] Zhao | Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors[END_REF]. We found that Rnf20 LOF reduces Notch signaling, while enhancing Sox9 binding to chromatin, suggesting that Rnf20 might be a critical regulator of this fine balance. Importantly, many Rnf20-Sox9 targets are upregulated in heart disease, where transient mesenchymal activation promotes EC migration and proliferation to regenerate the vascular network, but in the long-run might be deleterious. Thus, unraveling the mechanisms that control Rnf20 expression and function to enhance Notch and repress Sox9 activity might provide novel therapeutic avenues for cardiovascular diseases. (A) Co-immunoprecipitation showing interaction between Isl1 and Rnf20 (top). GST pull down assays with recombinant GST protein or GST-Rnf20 fusion protein and recombinant Isl1 protein, showing that the interaction between those two proteins is direct (bottom).

(B) Violin plot of Rnf20 expression levels in Isl1+ and NKX2.5+ single cells of E8.25 embryos [START_REF] Xiong | Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intraorgan Crosstalk During Cardiogenesis[END_REF]. Each black dot represents a single cell. FHF, First heart field. SHF, Second heart field.

(C) Gross appearance of control (Isl1 +/+ Rnf20 +/fl ) and Isl1 cre/+ Rnf20 -/fl embryos at E9.5 and E11.5. Scale bars, 2 mm (left), 1 mm (right). (D) Gene ontology pathway enrichment analysis and representative up-(red) and downregulated (blue) genes in E10.5 Isl1 cre/+ Rnf20 -/fl compared to control hearts.

(E) Heatmap representation of genes involved in extracellular matrix organization and cell differentiation in dissected OFT and RV of Rnf20-deficient and Isl1 haploinsufficient hearts [START_REF] Gao | Pioneering function of Isl1 in the epigenetic control of cardiomyocyte cell fate[END_REF].

(F) Circos plot of the significantly changed ligand-receptor interactions at E10.5 upon Rnf20 LOF in Isl1+ cardiovascular precursors. Red arrows represent upregulated ligands, blue arrows represent downregulated ligands and arrow thickness indicates the probability for interaction, i.e. interaction score [START_REF] Skelly | Single-Cell Transcriptional Profiling Reveals Cellular Diversity and Intercommunication in the Mouse Heart[END_REF].

(G) Relative mRNA expression levels of genes involved in endothelial-myocardial interactions in ECs isolated from control and Rnf20 KO E10.5 RV and OFT (n= 3). (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from [START_REF] Palpant | Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells[END_REF].

(G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER.

Isolation and culture of neonatal rat cardiomyocytes (rCM)

Hearts were collected from 1-3 days old rats, the atria removed and ventricles minced into small pieces on ice with 1x ADS buffer (pH 7.35). 50 hearts per falcon were digested with Collagenase Type II (Worthington: LS004176) and Pancreatin (Sigma P3292) and loaded to a Percoll gradient with different densities to separate into CMs, ECs and FBs. CMs (2.5×10 5 /well) were collected and seeded on plates coated with 0.5% gelatine and used for indirect co-culture or directly co-cultured with HUVECs.

Establishment of stable HUVEC cell lines by lentiviral infection and co-culture with rCM

HEK293T at 60-80% confluence were transfected with plasmids amplified from an overexpressing lentiviral library together with 0.975µg CMVΔR8.74 and 0.525µg VGV.G packaging plasmids using X-tremeGENE™ HP DNA Transfection Reagent (Roche, 6366236001) according to the manufacturer instructions. 48 hours after the transfection the virus-containing supernatants were used to infect HUVECs at a 30-60% confluence. Polybrene (8 mg/ml) was added 1:1000 to the infection medium. For Sox9 overexpression, HUVECs at 85-90% confluence were infected with approx. 10 6 IFU/mL of adSox9 or adβGal particles in EC full medium for 48 hours. Afterwards, wild type rat cardiomyocytes were plated on the confluent monolayers of HUVECs at a density of 300,000 cells per well of a 6 well-plate in EC media. Contractility, binucleation and proliferation were examined at 2 or 4 days of co-culture.

Contraction analysis

Movies of spontaneous beating were acquired for at least 20 seconds with a Leica DMI8 microscope and a 10x or 20x objective. Contractility parameters such as beating rate and the amplitude were obtained by analysis with the ImageJ macro MYOCYTER [START_REF] Grune | The "MYOCYTER" -Convert cellular and cardiac contractions into numbers with ImageJ[END_REF]. Amplitude curves were visualized by OriginPro 2020b.

51

Histology

Embryos or dissected hearts were fixed with 4% paraformaldehyde, dehydrated by increasing percentage of ethanol and stored at -20°C until paraffin embedding. Paraffin embedded samples were cut into 7 µm sections. Hematoxylin and Eosin Y staining (Sigma-Aldrich, GHS116, HT110216) as well as Alcian Blue and Nuclear Fast Red (Sigma-Aldrich, 101647, 100121) were performed according to the manufacturer's instruction. Sections were mounted with ROTI®Histokitt (Carl Roth, 6638.1).

Immunofluorescence staining and imaging

For immunofluorescence staining on paraffin heart sections slides were first dewaxed and heat-induced epitope retrieval was performed by boiling for 10 minutes at 400 Watt in a pressure cooker filled with freshly prepared 10 mM sodium citrate solution with a pH of 6.0.

After cooling down, the slides were blocked in a humidified chamber for 1 hour in blocking buffer consisting of 0.5% Triton X-100 and 10% FBS in PBS. The following primary antibodies were used in blocking buffer and incubated overnight at 4°C in a humidified chamber: antimyosin heavy chain (MF20) (1:5, Dshb), anti-phospho-Histone H3 (Ser10) (1:300, Millipore, 06-570), anti-Ubiquityl-Histone H2B (Lys120) (1:100, Cell Signaling, 5546) and anti-Vimentin (1:50, Cell Signaling, 5741). After three washing steps with PBS, combinations of the corresponding secondary antibodies, conjugated to Alexa Fluor 555 or Alexa Fluor 488 (1:400, Thermo Fisher Scientific) with the conjugated primary antibody Isolectin B4 (IB4) Alexa Fluor 647 (1:40, Thermo Fisher Scientific, I32450) were added in blocking buffer and incubated for 1 hour at room temperature. The slides were then washed 3 times with PBS and stained with DAPI in blocking buffer (1:1000) for 5 minutes at room temperature, followed by 3 PBS washing steps. Sections were mounted with Mowiol 4-88 (Millipore, 475904) mounting medium.

For immunofluorescence staining on rCM cells were permeabilized for 10 minutes with 0.5% Triton X-100 in PBS. After blocking in 3% BSA in PBS for 30 min, rCM were incubated with α-Actinin (Sigma-Aldrich, A7811) antibody in 0.5% Triton X-100/ 1% BSA/ 1xPBS overnight in a 52 humidified chamber at 4°C. After three consecutive 5 minutes washes in PBS, cells were incubated for a further 1 hour with a corresponding secondary antibody followed by DAPI staining.

Immunostaining as well as HE and Alcian Blue/Nuclear Fast Red images were acquired using Zeiss Axio Scan Z1 Digital Slide Scanner with a 10x or 20x objective and analyzed using Zen 3.5 blue software or using Leica DMi8 microscope and analyzed using Leica Application Suite X software.

Echocardiographic assessment of cardiac dimensions and function

Fetal echocardiography was performed with Vevo 3100 imaging system (FUJIFILM VisualSonics, Toronto, Canada) via trans-abdominal ultrasound of pregnant mice. Twodimensional B-mode and one-dimensional M-mode tracings of E14.5 mouse embryonic hearts were recorded in a four-chambered view with a 50MHz MX700 transducer. Left-and rightventricular diameter at end-diastole and end-systole, cardiac output, ventricular ejection fraction (EF) and fractional shortening (FS) were calculated with the integrated cardiac measurement package, where at least three consecutive cardiac cycles were used for analysis.

Embryonic cardiomyocyte and endothelial cell isolation

Hearts from E14.5 embryos were isolated and incubated 30 minutes at 37°C on a rotating shaker with 200 µl of digestion solution per embryo consisting out of 0.05 mg/ml DNase I (Worthington, 2139) and 1 mg/ml Collagenase I (Worthington, 4197) diluted in HBSS (Thermo Fisher Scientific, 14175053) until complete digestion. Reaction was stopped by adding DMEM medium containing 10% FBS or FACS buffer (10% FBS in PBS). For FACS sorting of samples for ATAC-Seq and RNA-Seq applications, cells were centrifuged at 400xg and 4°C for 10 minutes and resupended in 1 ml of ESC DMEM medium and incubated with 3 µl CD31 coupled Dynabeads per embryo rotating for 30 minutes at room temperature. After incubation of the cells with the Dynabeads, CD31 positive cells (Endothelial cells) were captured by a magnet.

53

The supernatant was used for the CM fraction and the beads were washed one more time with ESC DMEM medium and twice with PBS before proceeding with ATAC-Seq and RNA isolation.

Flow cytometry

Cultured cells were dissociated with StemPro Accutase for 20 minutes at 37°C. Cells from E14.5 whole hearts were isolated by digestion with DNase I and Collagenase I as described before. Cells were transferred to a U-bottom-96-well plate (100 000 -200 000 cells/well) and first stained with 50 µl/well of Fixable Viability Dye eFluor 450 (1:800, Thermo Fisher Scientific, 65-0863-14). All used antibodies were titrated with their corresponding isotypes. The following antibodies and dyes were used for extracellular staining with a FACS buffer containing 10% scRNA-Sequencing analysis 10x Genomics scRNA-Sequencing from control and Cdh5 CreERT2 -Rnf20 fl/fl KO hearts was analyzed using cellranger count function and refdata-gex-mm10-2020-A genome (Zheng et al., 2017). Merging of control and KO matrices to analyze expression differences in the subpopulations was done by cellranger aggr function. Resulting clusters were identified with the help of published datasets [START_REF] Hill | A cellular atlas of Pitx2-dependent cardiac development[END_REF][START_REF] Rhee | Endocardial/endothelial angiocrines regulate cardiomyocyte development and maturation and induce features of ventricular noncompaction[END_REF]. Clusters identified as ECs or CMs were reclustered by the use of 10x Genomics Loupe Browser to obtain a more detailed characterization these population upon Rnf20 LOF. The same procedure was done for CMs.

Volcano plots of the reclustered EC populations were extracted from the Loupe Browser.

Public available scRNA-Seq [START_REF] Xiong | Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intraorgan Crosstalk During Cardiogenesis[END_REF] gene expression lists from E8.25 embryos were downloaded from NCBI (GSE108963) and violin plots of Rnf20 expression in NKX2.5 and Isl1 positive cells were prepared using the R package Seurat [START_REF] Hao | Integrated analysis of multimodal single-cell data[END_REF].

RNA isolation, cDNA synthesis and qPCR

RNA for RNA-Seq was isolated using the RNeasy Plus Universal Mini Kit (QIAGEN,73404) according to the manufacturer's instructions. For real-time PCR analysis cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368813) and real-time qPCR was performed using either the PowerUp SYBR Green Master Mix (Applied Biosystems, A25742) or the qPCRBIO SyGreen Blue Mix Hi-ROX (PCR 55 Biosystems, PB20.16-51). Cycle numbers were normalized to these of α-Tubulin (Tuba1a) and/or ribosomal 18S.

RNA-Sequencing

Right ventricle and outflow tract were dissected from E9.5 and E10.5 control and Isl1 cre/+ -Rnf20 - /fl embryos for RNA isolation and RNA-Seq. Control and Rnf20 iEC-KO ECs and CMs for sequencing applications were isolated from E14.5 embryonic hearts as described above. RNA was isolated using the RNeasy Plus Universal Mini kit (Qiagen, 73404). Quality control and library preparation were done by BGI and sequencing was performed on a BGISEQ-500 platform.

RNA-Sequencing data analysis

After read quality control with the MultiQC tool [START_REF] Ewels | MultiQC: summarize analysis results for multiple tools and samples in a single report[END_REF] RNA-Seq 50 bp single-end sequencing reads were trimmed of TruSeq3-SE adapters using Trimmomatic-0.39 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. Trimmed reads were mapped to the mm10 reference genome using STAR (-alignIntronMin 20 -alignIntronMax 500000) [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF] and counted using the analyzeRepeats.pl function (rna mm10 -strand both -count genes -noadj) from HOMER [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF] after creating the tag directories with makeTagDirectory. Differential expression was quantified and normalized by edgeR [START_REF] Robinson | edgeR: a Bioconductor package for differential expression analysis of digital gene expression data[END_REF] and annotated by biomaRt [START_REF] Durinck | Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt[END_REF] using a custom script. Differentially accessible peaks were filtered by R for a p-value of 0.05 and a Log2(FoldChange) of ≥ 0.58 and ≤ -0.58. Gene ontology pathway analysis was performed using either WEB-based GEne SeT AnaLysis Toolkit [START_REF] Liao | WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs[END_REF] or DAVID Bioinformatics Resources 6.8 [START_REF] Huang Da | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF]. The PCA plots were obtained using prcomp function of R package ggfortify (Yuan Tang, 2016) and visualization by ggplot2 [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF]. Volcano plots were designed using EnhancedVolcano R package [START_REF] Blighe | EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling[END_REF]. Gene Set Enrichment Analysis (GSEA) was performed by the use of the R packages clusterProfiler [START_REF] Wu | clusterProfiler 4.0: A universal enrichment tool for interpreting omics data[END_REF], enrichplot [START_REF] Yu | enrichplot: Visualization of Functional Enrichment Result[END_REF] and ggplot2. All heatmaps were prepared by the R packages pheatmap [START_REF] Kolde | pheatmap: Pretty Heatmaps[END_REF] and RColorBrewer (Neuwirth, 56 2014) and represent the row-based Z-scores calculated from normalized counts. Ligand-Receptor analysis was based on the database from [START_REF] Skelly | Single-Cell Transcriptional Profiling Reveals Cellular Diversity and Intercommunication in the Mouse Heart[END_REF]. In detail, differentially regulated ligands upon Rnf20 depletion in the different cell types were paired with expressed receptors. Further, an interaction score based on the Log2FC of the differentially expressed ligand and on the expression levels of both the ligand and receptor was calculated to sort the ligand-receptor pairs by relevance. Interactions of ligand-receptor pairs were visualized with the help of the R package circlize [START_REF] Gu | circlize Implements and enhances circular visualization in R[END_REF]. All data were analyzed with the same parameters.

ATAC-Sequencing and RNA harvesting for RNA-Sequencing of E14.5 Rnf20 iEC-KO cells ECs and CMs from Cdh5 CreERT2 -Rnf20 fl/fl E14.5 embryo hearts were processed using a modified protocol from [START_REF] Corces | An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues[END_REF]. In brief, 30 000 cells of CMs and 10 000 cells of ECs were centrifuged at 500xg for 5 minutes at 4°C and washed with PBS. The cell pellet was resuspended in 50 µL cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Igepal CA-630, Sigma-Aldrich, I8896) and incubated for 2 minutes on ice, followed by stopping the reaction by dilution with 300 µl ATAC resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl 2 ) and 10 minutes centrifugation step (500g, 4°C). After centrifugation supernatant was used for RNA cytosolic fraction by adding to 1 ml of TRIZOL and isolated nuclei of CMs were incubated in 30 µl and of EC in 10 µl transposase mixture with 5% Nextera Tagment DNA Enzyme TDE (Illumina #15027916) in transposition buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 20% Dimethylformamide) under shaking for 30 minutes at 37 °C. Immediately following the transposition reaction, the mixture was purified using ChIP DNA Clean and Concentrator Kit (Zymo D5205). Library amplification together with indexing was performed by a 5 cycle preamplification step, a SYBR green qPCR side reaction with 1/10 of the preamplified solution to determine the extra cycle number and a final PCR with individually adapted cycle numbers to avoid duplicates and ensure similar final concentrations using NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs, M0541S) and Nextera Index Kit (Illumina, 15055290). Magnetic bead purification with two-sided size selection using 57 undiluted Agencourt AMPure XP Beads (Beckman Coulter, A63881) ensured library sizes between 150 and 1000 bp, detected by Bioanalyzer High Sensitivity DNA analysis (Agilent,. Libraries were mixed in equimolar ratios and sequencing was performed on a BGISEQ-500 platform.

ATAC-Sequencing data analysis

ATAC-Seq paired-end sequencing reads were trimmed of adapters using Trimmomatic-0.39 and then mapped to mm10 mouse genome with Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF].

Following removal of unmapped reads by SAMtools (-F 1804 -f 2) [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF], PCR artefacts were excluded by the MarkDuplicates.jar from Picard-tools-1.119 [START_REF] Tools | By Broad Institute[END_REF]. Read quality was controlled by the MultiQC tool [START_REF] Ewels | MultiQC: summarize analysis results for multiple tools and samples in a single report[END_REF]. BamCoverage function of deepTools [START_REF] Ramírez | deepTools: a flexible platform for exploring deep-sequencing data[END_REF] with normalization to the reads per genomic content was used to generate the bigwig files for visualization of ATAC-Seq reads in the genome browser IGV (-bs 20 --smoothLength 40 --normalizeUsing RPGC --effectiveGenomeSize 2150570000 -e). Therefore, bam files of each 3 replicates were merged by BamTools [START_REF] Barnett | BamTools: a C++ API and toolkit for analyzing and managing BAM files[END_REF] and processing was performed like described. Peak calling was applied from merged bamfiles with MACS14 (-p 1e-3 -g 1.87e9 --nomodel --shiftsize 100) [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF]. Called peaks from merged files and individual bam files were further used to quantify and normalize counts as well as calculating differentially accessible chromatin regions upon the different treatments using the R package DiffBind (Stark and Brown) with an edgeR based TMM normalization using read counts and effective library size and an edgeR method to perform differential binding affinity analysis. Peaks were annotated by a custom R script combining ChIPseeker [START_REF] Yu | ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization[END_REF] and rtracklayer packages [START_REF] Lawrence | rtracklayer: an R package for interfacing with genome browsers[END_REF]. Promoter regions were defined as ±3 kb around the gene transcription start site. Differentially accessible peaks were filtered by R for a p-value of 0.05 and a Log2(FoldChange) of ≥ 0.58 and ≤ -0.58.

Principle component analysis (PCA) and heatmap of 10 000 most significant peaks were generated by the DiffBind package. Volcano plots are based on the EnhancedVolcano

Figure 1 .

 1 Figure 1. Ablation of Rnf20 in Isl1+ cardiovascular progenitors results in defects in SHF development.

  (D) Frontal views of E10.5 control and Isl1 cre/+ Rnf20 -/fl hearts, showing short outflow tract (OFT) and a smaller right ventricle (RV). Scale bars, 100 µm. (E) H&E stainings of transverse sections of control and Isl1 cre/+ Rnf20 -/fl embryos at E10.5 showing shortening of the OFT. Scale bars, 200 µm. (F) Histological analysis of control and Rnf20 knockout hearts at E11.5 showing a smaller RV, hypoplastic ventricular wall and abnormal trabeculation. Magnified images of indicated RV regions (square) are shown in the right panel. Scale bars, 200 µm (left), 50 µm (right). (G) RV and left ventricle (LV) wall thickness (top) and trabecular layer thickness (bottom) in control (n=8 for E11.5, n=6 for E12.5) and Isl1 cre/+ Rnf20 -/fl embryos (n=6 for E11.5, n=5 for E12.5) of E11.5 and E12.5 hearts. (H, I) Immunostaining with EdU, anti-MF20 (CMs), anti-IB4 (ECs) and DAPI (nucleus) (H) and quantification of EdU-labeled CMs (EdU+/MF20+ cells) in RV and LV of E12.5 control and Isl1 cre/+ Rnf20 -/fl embryos (n=4) (I), showing a marked reduction in the number of proliferating CMs upon Rnf20 ablation. Scale bars, 100 µm. 31 (J, K) Immunostaining for H2Bub1 catalyzed by Rnf20 and MF20 in RV and LV of E11.5 control and Isl1 cre/+ -Rnf20 -/fl embryos (J) and magnified areas (K). Arrows point to H2Bub1-positive CMs in the LV; asterisks indicate H2Bub1-negative endocardial cells. Scale bars, 50 µm. For this and all other figures, error bars represent mean ±SD. *p<0.05 **p<0.01 ***p<0.001.

Figure 2 .

 2 Figure 2. Rnf20 controls ECM dynamics and signaling in SHF development.

(

  H) Co-immunoprecipitation with N1ICD and Rnf20, showing that Rnf20 interacts with N1ICD. (I) Overlap between genes differentially expressed in Rnf20 and Rbpj knockout (Klaus et al., 2012) compared to control hearts. (J) Relative expression of genes associated with cardiac jelly protein degradation in control and Isl1 cre/+ Rnf20 -/fl RV and OFT. 34 (K) Images of Alcian blue and nuclear fast red staining of transverse sections of control and Isl1 cre/+ Rnf20 -/fl hearts at E12.5. Magnified images of indicated regions are shown in the right panels. Scale bars, 200 µm (whole heart), 50 µm (magnified regions).

Figure 3 .

 3 Figure 3. Endothelial Rnf20 is essential for heart development and function.

Figure 4 .

 4 Figure 4. Endothelial Rnf20 instructs CM behavior in an in vitro co-differentiation system.

(

  H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). 40 (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the codifferentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4).(M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.

Figure 5 .

 5 Figure 5. Endothelial Rnf20 inhibits EndMT, CM maturation and cell cycle withdrawal.

Figure 6 .

 6 Figure 6. Rnf20 safeguards endothelial function by controlling TF inputs.

Figure 7 .

 7 Figure 7. Rnf20-dependent EC-derived factors regulate CM proliferation, binucleation and contractility

  FBS in PBS: CD31-APC with mouse specificity(1:500, Thermo Fisher Scientific, together with its IgG2a kappa APC-Isotype control (1:500, Thermo Fisher Scientific, 17-4321-81), CD31-PE/Cyanine7 with mouse specificity(1:500, Thermo Fisher Scientific, together with its IgG2a kappa PE/Cyanine 7-Isotype control (1:500, Thermo Fisher Scientific, 25-4321-81), CD31-APC with human specificity for HUVEC experiments (1:500, Thermo Fisher Scientific, 17-0319-42) together with its IgG1 kappa APC-Isotype control (1:500, Thermo Fisher Scientific, 17-4714-82) and Vybrant DyeCycle Green Stain (1:500, Thermo Fisher Scientific, V35004). CD31 antibodies incubated for 30 minutes at 4°C or 15 minutes at room temperature. Vybrant Dye Cycle Green Stain was added for 30 minutes at 37°C followed by direct FACS measurement without further washing steps. Edu incorporation and staining with the Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific, C10634) was performed according to the manufacturer's instructions with the use of the fluorescent dye from Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye (Thermo Fisher Scientific, C10337). Stained cells were analyzed using an FACS Canto II Flow Cytometer (BD). Cell sorting with the FACS Aria IIu High Speed Cell Sorter (BD) was performed by the FlowCore Mannheim. Data were analyzed using FlowJo 10.7.2 software (BD).10x Genomics scRNA-Sequencing E14.5 control and Cdh5 CreERT2 -Rnf20 fl/fl KO hearts were digested with DNase I and Collagenase I according to the isolation technique described before. Whole heart cells were FACS sorted for living cells by staining with SYTOX™ Blue Dead Cell Stain (1:2000, Thermo Fisher Scientific, S34857) and then diluted at a density of 1000 cells/µl in 0.4% BSA. 8000 cells were used for library preparation according to the Chromium Next GEM Single Cell 3' Kit v3.1 (10xGenomics, 1000269) with the Chromium Next GEM Chip G Single Cell Kit (10xGenomics, 1000127) and sequenced on a BGISEQ-500 platform.

Figure S5 .

 S5 Figure S5. Effect of angiocrine factors, receptors on endothelial cells and extracellular matrix proteins on CM behavior, Related to Figure 7.

Acknowledgements

We thank Dalyan Enver Devran and Alexandra Buse for technical assistance. Further, we like to thank Stefanie Uhlig, the FlowCore Mannheim, the Institute of Transfusion Medicine and Immunology and the NGS Core Facility Single Cell Services, for excellent support. We are grateful to Boyan Garvalov for careful reading of the manuscript and the constructive comments. This work was supported by the CRC 1366 (Projects A03, A06), the CRC 873 (Project A16), the CRC1550 (Project A03) funded by the DFG and the DZHK (81Z0500202), funded by BMBF.

Author contributions

LK, RG, NT-E, OL, AZ, FT, JC, YD, YW, YR, EC, JAK, PLS, CCW performed the experiments.

LK performed bioinformatic analysis, JC provided bioinformatics input. CCW, GB, TW, RO and JH provided reagents and valuable intellectual input. LK and GD designed the experiments, analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Declaration of interests

The authors declare no competing interests.

Methods

RESOURCE AVAILABILITY

Cell lines and cell culture HEK293T cells were purchased from ATCC (CRL-3216) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, 10270106), 1% penicillinstreptomycin (Thermo Fisher Scientific, 15140122) and 2 mM L-glutamine (Thermo Fisher Scientific, 25030123).

HUVEC cells and cultured in Endothelial Cell Growth Medium MV 2 (PromoCell, C-22022), supplemented with 1% penicillin-streptomycin.

One day old (P1) rat CMs were isolated via Langendorff perfusion technique and cultured in PAN BIOTECH M199 media (Biolab, P04-07500), supplemented with 10% FBS.

Murine E14-Nkx2-5-EmGFP ESCs [START_REF] Hsiao | Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter[END_REF] were maintained on mitomycin (Sigma-Aldrich, M4287) treated mouse embryonic fibroblasts (MEF) in KnockOut TM DMEM (Thermo Fisher Scientific, 10829018) supplemented with 10% KnockOut serum replacement (Thermo Fisher Scientific, 10828028), 2 mM L-glutamine, 0.04 mM 2-mercaptoethanol (Sigma-Aldrich, M3148), 0.1 mM non-essential amino acids (Thermo Fisher Scientific, 11140035), 1 mM sodium pyruvate (Thermo Fisher Scientific, 11360070), 4.5 mg/ml D-glucose, 1% penicillinstreptomycin and 1000 U/ml leukemia inhibitory factor (LIF ESGRO, Millipore, ESG1107).

Mouse lines

Isl1 knockout mouse line (Isl1tm1(cre)Sev-C57BL/6) was a kind gift from Sylvia Evans. The Tg(Tek-cre)12Flv line was obtained from Jackson Laboratory. The Tg(Cdh5-cre/ERT2)1Rha was obtained from Prof. Ralf H. Adams. Cre deleter lines were crossed with Rnf20tm1c(EUCOMM)Wtsi mice to induce specific deletion of Rnf20 in Isl1 positive, Tie2 positive and Cdh5 positive cells following the presented in the figures tamoxifen injection schemes. All animal experiments were performed according to the regulations issued by the package [START_REF] Blighe | EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling[END_REF]. Gene ontology pathway analysis was performed using WEB-based GEne SeT AnaLysis Toolkit [START_REF] Liao | WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs[END_REF]. Average ATAC-Seq tag intensities in the ATAC-Seq normalized mapped reads were calculated using ngs.plot.r [START_REF] Shen | ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases[END_REF] by filtering the ATAC-Seq peaks by the genes of interest. TOBIAS Footprinting motif analysis (Bentsen et al., 2020b) of the filtered peaks was performed with the TOBIAS BINDetect function using the merged bigwig files of the treatments and the JASPAR 2020 open-access database of curated, non-redundant transcription factor binding profiles [START_REF] Fornes | JASPAR 2020: update of the open-access database of transcription factor binding profiles[END_REF]. Spearman correlation analysis of the normalized counts from all individual replicates of ATAC-Seq and RNA-Seq was based on the calculation of the spearman correlation factor r using the cor.test function in R. Visualization of the correlating genes filtered by r ≥ 0.5 and ≤ -0.5 was done by the R package ggpubr [START_REF] Kassambara | ggpubr: 'ggplot2' Based Publication Ready Plots[END_REF].

ChIP-Sequencing data analysis of public data sets

RNA Polymerase II (Pol II) ChIP-Seq from E14.5 hearts [START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF] was analyzed based on the ATAC-Seq data processing. Peak calling was applied from merged bam-files with MACS14 (-c $Input -p 1e-2 -g 1.87e9 --nomodel --shiftsize 100) considering the ChIP input as a background. Pol II ChIP-Seq peaks were filtered by differentially expressed genes upon Isl1-mediated Rnf20 deletion as well as non-changed genes (NC) and pausing index was calculated by the ratio of -50bp to +300bp Pol II signal divided by the Pol II signal within +300bp to +2kb for these peaks. The pausing index was then classified into high (> 3 rd quartile), medium (between 3 rd quartile and median) and low (< median).

Quantification and statistical analysis

All the statistical analyses were performed using OriginPro 2020b and R programming software. All experiments were performed at least three independent times and the respective data were used for statistical analyses. Differences between groups were assessed using an unpaired two-tailed Student's t-test. Error bars represent mean ± SD. P-values represent statistical significance *p<0.05 **p<0.01 ***p<0.001.

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Supplemental information