Morgane Belle

David Godefroy

Gérard Couly

Samuel A Malone

Francis Collier

Paolo Giacobini

Alain Chédotal
email: alain.chedotal@inserm.fr

Tridimensional visualization and analysis of early human development

Generating a precise cellular and molecular cartography of the human embryo is essential to understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we combined whole-mount immunostaining, 3DISCO clearing and light-sheet imaging to start building a three-dimensional cellular map of the human development during the first trimester of gestation. We provide highresolution 3D images of the developing peripheral nervous, muscular, vascular, cardiopulmonary and urogenital systems. We found that the adult-like pattern of skin innervation is established before the end of the first trimester, showing important intra-and inter-individual variations in nerve branches. We also present evidence for a differential vascularization of the male and female genital tracts concomitant with sex determination. This work paves the way for a cellular and molecular reference atlas of human cells, which will be paramount for understanding human development in health and disease.

Introduction

For centuries, the intricacies of human development have remained an enigma and its study unexplored due to sociocultural, religious and moral reasons [START_REF] Morgan | Icons of Life: A Cultural History of Human Embryos[END_REF]. In the second half of the twentieth century, a growing interest for experimental embryology and technological developments [START_REF] King | A brief historical note on staining by hematoxylin and eosin[END_REF] led to the first scientific descriptions of human embryo development [START_REF] His | Anatomie menschlicher Embryonen[END_REF][START_REF] Keibel | Normentafeln zur Entwicklungsgeschichte der Wirbeltiere[END_REF]. In the mid of the 20 th century, Franklin Mall and colleagues at the Carnegie Institute collected, sectioned and stained thousands of human embryos and fetuses [START_REF] Morgan | Icons of Life: A Cultural History of Human Embryos[END_REF]. Wax reconstructions and 3D embryo models were generated [START_REF] Norman | Methods and technique of reconstruction[END_REF] to define stages of human embryogenesis, still in use today (Keibel and Mall, 1910;[START_REF] O'rahilly | Developmental stages in human embryos[END_REF]. Similar collections of human embryos were initiated in other countries [START_REF] Blechschmidt | The Beginnings of Human Life[END_REF][START_REF] Fujimoto | Nishimura's collection of human embryos and related publications[END_REF]. These data still appear in all modern textbooks and atlases of embryogenesis and developmental biology. More recently, histological sections were digitized and reconstructed in 3D [START_REF] De Bakker | An interactive three-dimensional digital atlas and quantitative database of human development[END_REF]. Immunohistochemical studies have also been performed but lack the 3D information which is essential to understand organogenesis. Ex vivo 3D images of paraformaldehyde (PFA)-fixed human embryos were recently obtained using magnetic resonance imaging or phase-contrast-X-ray radiographic computed tomography, however, the resolution is still far from being at the cellular level [START_REF] Kanahashi | A Novel Strategy to Reveal the Latent Abnormalities in Human Embryonic Stages from a Large Embryo Collection[END_REF][START_REF] Yamada | Developmental atlas of the early first trimester human embryo[END_REF].

Whole mouse brains and embryos rendered transparent with various procedures, can be imaged with excellent cell resolution [START_REF] Richardson | Clarifying Tissue Clearing[END_REF].

Solvent-based clearing methods such as 3DISCO (Three-dimensional imaging of solvent cleared organs) [START_REF] Ertürk | Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury[END_REF] and its derivatives iDISCO [START_REF] Renier | iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging[END_REF], iDiSCO+ [START_REF] Renier | Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes[END_REF] and uDISCO [START_REF] Pan | Shrinkagemediated imaging of entire organs and organisms using uDISCO[END_REF] are fully compatible with whole-mount immunofluorescence [START_REF] Belle | A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System[END_REF][START_REF] Renier | iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging[END_REF]. The rise of light sheet fluorescence microscopy (LSFM) has led to the development of imaging setups compatible with large field objectives and thick (>1 cm) specimens [START_REF] Dodt | Ultramicroscopy : three-dimensional visualization of neuronal networks in the whole mouse brain[END_REF]. We set out to develop a protocol which combines antibody labeling and tissue clearing for better analyses in human embryology.

Here we have performed whole-mount immunostaining on 36 human embryos and fetuses ranging from 6 to 14 weeks of gestation with over 70 antibodies most of which had never been used in humans (see STAR methods and Table S1). We generated 3D images of human embryos at an unprecedented cellular resolution. This work offers a unique opportunity to start building a molecular and cellular atlas for the study of human development. The possible applications of this new method in the field of embryology are countless. They open new avenues for the study of molecular mechanisms regulating the development of human embryos in physiological and pathological conditions.

Results and Discussion

Development of the human peripheral nervous system

We used a protocol for whole-mount immunostaining and 3DISCO clearing to analyze the development of human embryos (first 8 weeks of gestation) and fetuses (from 8.5 to 14 weeks, see methods). We first studied the development of the peripheral nervous system (PNS) using antibodies against the neuron-specific intermediate filament protein peripherin (Prph), which in rodents is expressed by sensory and autonomic axons [START_REF] Parysek | A type III intermediate filament gene is expressed in mature neurons[END_REF]. In human fetuses, Prph labels auditory and vomeronasal nerves [START_REF] Casoni | Development of the neurons controlling fertility in humans: New insights from 3D imaging and transparent fetal brains[END_REF][START_REF] Locher | Neurosensory development and cell fate determination in the human cochlea[END_REF].

We first used embryos at gestation week 6 (GW6 or Carnegie stage CS17, n=1; see STAR Methods and experimental procedures for stage determination), GW7 (CS19, n=2), GW7.5 (CS21, n=1) and GW8 (CS23, n=3). As previously reported for mice [START_REF] Belle | A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System[END_REF][START_REF] Renier | iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging[END_REF], tissue-shrinkage was observed (20-40% of its original size) and remained homogeneous throughout the embryo. This shrinkage can be avoided using iDISCO+ [START_REF] Renier | Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes[END_REF], however, the cleared embryos produced by this method are more fragile and their fluorescence less stable than with 3DISCO. In addition, the methanol treatment needed for iDISCO+ is not compatible with some antibodies. Last, the size reduction after 3DISCO allowed whole embryos to be imaged using LSFM and Prph+ peripheral nerves to be visualized from the brainstem and spinal cord to their distal extremities S1B and Movie S1).

We next tested specific markers of sensory and motor axons, focusing primarily on the innervation of the developing feet and hands from GW6-GW12. Motor and sensory axons, were respectively labeled with antibodies against choline acetyl transferase (ChAT; n=14 cases) and transient adhesion glycoprotein-1 (Tag-1/Contactin 2; n=22 cases) [START_REF] Karagogeos | Expression of the Cell Adhesion Proteins BEN / SC1 / DM-GRASP and TAG-1 Defines Early Steps of Axonogenesis in the Human Spinal Cord[END_REF]. Double staining for Prph and Tag-1 (n=14) confirmed that both markers overlapped in peripheral nerves, although from GW9 the thinnest axonal branches were better seen with Tag-1 (Figure 1D). In contrast, there was no overlap between ChAT+ and Prph+ (Figure 1E; n=6) or ChAT+ and Tag-1+ (Figure 1F and S1C; n=7) immunoreactive axons. Motor nerve roots were not immunoreactive for Prph (data not shown), suggesting that in humans, Prph is differentially expressed between spinal cord and cranial motor axons.

3D virtual dissection of organ innervation with Imaris segmentation plugins was performed to trace, artificially color and reconstruct individual nerve fascicles (STAR Methods). This method was applied to GW7 (CS19; n=2) and GW8 (CS23; n=1; Figure 1G) cranial nerves (Figure 1G and S1A and Movie S1). Fetuses older than GW8, too large for our LSFM microscope, were dissected prior to processing to image limbs and other large organs.

Segmentation-based tracing was used to reconstruct the sensory innervation of the hand from GW6 to GW11.5. The human hand contains the ulnar (or cubital), median and radial nerves, which innervate a different hand areas in a pattern highly conserved between individuals [START_REF] Dogan | Communications between the palmar digital branches of the median and ulnar nerves: A study in human fetuses and a review of the literature[END_REF].

Sensory nerves and their branches in the hands of 14 embryos and fetuses were reconstructed in 3D using Prph (GW7-GW11, n=11) or Tag-1 (GW6-9.5, n=3) immunostaining (Figures 2A,2B and S1D), and used to create a developmental timelapse of sensory innervation in the human hand (Figure 2B and Movie S2).

Furthermore, we reconstructed the motor (ChAT+) and sensory (Tag-1+) innervation of two GW8.5 and GW9 fetuses (Figures 2C,2D and S1C and Movie S2) to determine where and how the two types of nerves diverge.

We next compared the nerve branching patterns in 11 hands and found, in all individuals analyzed, striking heterogeneity between the left and right hands. At all ages, the median nerve was the most similar (Figure S2A and not shown), whereas noticeable differences in the number and length of branches existed between the left and right ulnar or radial nerves (Figures S2A,S2B and Movie S2). The low number of cases analyzed did not allow detecting any correlation with hand laterality.

Interestingly, unlike in adult, the musculocutaneaous nerve extended into the hand, at least until GW11 (Figure 2B).

The adult cutaneous innervation is topographically organized with each of the three nerves innervating non-overlapping skin territories. To determine how this map is established, we pseudo-colored the domains overlying Tag-1+ axons in hands at GW6, GW8.5 and GW11.5 (Figure S2C and Movie S2). At GW8.5, most of the hand surface was already covered with nerves whose topography appeared mature by GW11.5. The middle and index fingers are particularly interesting as their dorsal surfaces are shared by several nerves. The median nerve which normally only arborizes ventrally, projects to the dorsal side of these fingers. At GW6 the median nerve had not yet reached the tip of the index on the palmar (ventral) side but started to branch dorsally, ahead of the radial nerve, between future phalanges (Figure S2D). By GW8 the median nerve reached the tip of the index finger ventrally and started to cover the dorsal and distal part, whereas the radial nerve only innervated the caudal part of the dorsal side of the index finger. This pattern was unchanged at later ages (Figure S2D). Together, our novel protocol allows 3D visualization of the development of the peripheral innervation of early human fetal stages (first trimester of life) in an unprecedented level of precision and detail and revealed several unknown features of the development of limb innervation. First, an adult-like pattern of the domains of the skin covered by each nerve is already established during the late embryonic-early fetal period. However, within each domain the pattern of nerve branching can be highly divergent between individuals but also within the same individual. This is particularly striking at the level of the hands, where the arborization of the ulnar and radial nerves significantly differ between the right and left hands. This suggests that the development of the PNS innervation is, at least to some extent, stochastic and not profoundly influenced by specific guidance cues distributed in the developing limbs and extremities [START_REF] Hassan | Beyond Molecular Codes: Simple Rules to Wire Complex Brains[END_REF]. Moreover, in digits that are shared by multiple nerves there is no significant overlap of the branches originating from the different nerves. Our data infer that median nerves grow faster and start invading the dorsal part of some digits prior to the other nerves and that this may prevent them from entering these territories. Finally, significant remodeling of the sensory innervation occurs. The length of human embryogenesis, compared to rodents, will facilitate the analysis of tissue remodeling during development.

Next, we further validated our method by testing a large palette of antibodies against proteins expressed by many cell types in developing mouse and human embryos (STAR Methods).

3D analysis of human muscle development using LSFM

The current knowledge of skeletal and head muscle development in humans is based on histological studies conducted during the first half of the twentieth century [START_REF] Gasser | The development of the facial muscles in man[END_REF][START_REF] Gilbert | The Origin and Development of the Human Extrinsic Ocular Muscles[END_REF], and immunostaining with a few muscle cell markers [START_REF] Abe | Expression of intermediate filaments at muscle insertions in human fetuses[END_REF][START_REF] De | Development of the platysma muscle and the superficial musculoaponeurotic system (human specimens at 8-17 weeks of development)[END_REF]. The distribution of muscle cell progenitors in the embryo, however, has never been reported. The transcription factor (TF) Pax7, is a muscle stem cell marker and key initiator of myogenesis in jawed vertebrates [START_REF] Bryson-Richardson | The genetics of vertebrate myogenesis[END_REF]. We observed Pax7 immunoreactive nuclei homogenously distributed within each developing muscle of GW9.5 and GW10.5 arms and legs (Figures 3A,3B and Movie S3;n=1). As expected, all developing Pax7+ muscles were contacted by ChAT+ motor branches (Figure 3B). GW9 leg and arm (n=1) were also triple-immunostained for ChAT, Tag-1 and myogenin (Myog), a myogenic TF expressed by differentiating myoblasts [START_REF] Bryson-Richardson | The genetics of vertebrate myogenesis[END_REF] (Figure 3C and Movie S3). This immunostaining shows that clusters of Myog expressing cells are found at the extremities of each motor nerve branch. Doublecortin (Dcx) is an X-linked gene which encodes a microtubule-associated [START_REF] Gleeson | doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein[END_REF][START_REF] Des Portes | Doublecortin is the major gene causing X-linked subcortical laminar heterotopia (SCLH)[END_REF]. Mutations in DCX impede neuronal migration and cause lissencephaly. Dcx may also influence the development of the neuromuscular junction in humans [START_REF] Bourgeois | A critical and previously unsuspected role for doublecortin at the neuromuscular junction in mouse and human[END_REF]. Dcx immunostaining on limbs at GW8 (n=1) GW9.5 (n=1), GW10 (n=1), GW10.5 (n=1) and GW13.5 (n=1) showed that developing muscles express Dcx (Figure 3D and data not shown).

Sensory nerves were also immunoreactive for Dcx (Figure 3D). Myosin heavy chain (MHC) was previously detected in skeletal, heart and craniofacial muscles from human fetuses [START_REF] Abe | Expression of intermediate filaments at muscle insertions in human fetuses[END_REF]. Here we studied the distribution of MHC+ muscles throughout the body at GW8-GW14 (Figure 3E and Movie S3; n=9 cases). All muscles in the upper half of a GW8 embryo (n=1) were labeled and identified with an unprecedented resolution (Figure 3E and Movie S3). Whole-mount MHC muscle staining was also performed on GW11 and GW14 arms and legs (Figures S3B,3C).

Developing muscles and motor innervation could be simultaneously visualized following MHC/ChAT double immunostaining (Figure 3F and not shown), and importantly, the muscles within each hand and foot of a GW9.5 fetus could be segmented (Figures 3F and S3A,S3D and Movie S3). Moreover, the shape of the developing bones, which appear as black areas on optical sections, could also be extracted and visualized in 3D (Figure S3D).

3D analysis of the vasculature in human embryos

Existing data based on the filling of large arteries and veins [START_REF] Mall | On the Development of the blood-vessels of the brain in the human embryo[END_REF], 3D reconstructions of histological sections [START_REF] De Bakker | An interactive three-dimensional digital atlas and quantitative database of human development[END_REF] and vascular corrosion casting [START_REF] Zawiliński | Vascular system of the human spinal cord in the prenatal period: A dye injection and corrosion casting study[END_REF] provide an incomplete view of the early development of human vasculature. In chick and mouse, blood vessels arise from endothelial cell progenitors (angioblasts) which aggregate to form a dense vascular meshwork covering the embryo, a process known as vasculogenesis [START_REF] Chung | Developmental and Pathological Angiogenesis[END_REF]. Functional continuity occurs later through the remodeling of this initial endothelial network. As the embryo grows, new vessels sprout from existing ones, in a process called angiogenesis. Here, we first performed whole-mount labeling with antibodies against plasmalemma vesicle-associated protein (Plvap; also known as PV-1 or PAL-E), a transmembrane glycoprotein expressed by fenestrated microvascular endothelial cells [START_REF] Elima | Molecular identification of PAL-E, a widely used endothelial cell marker[END_REF]. Whole embryos at GW8 (n=2), or various organs from GW9.5-GW14 fetuses (n=8) were labeled and imaged with LSFM. At GW8, an extremely dense Plvap+ vascular network of endothelial cells extended throughout the body (Figure 4A and Movie S4), except in developing bones and corneas (Figure 4B and Movie S4). This supports the existence of a phase of vasculogenesis preceding angiogenesis in human embryos. Among other endothelial cell markers tested, Collagen IV also labels capillaries, but less efficiently than Plvap (Figure 4C). The 3D organization of developing arteries on GW9-GW14 organs (n=6 fetuses) were be assessed with immunostaining for smooth muscle-specific α-actin (SMA; Figure 4D and Movie S4).

Mammalian lymphatic vasculature development is documented for several species [START_REF] Yang | Development of the mammalian lymphatic vasculature[END_REF] but not much is known about human lymphangiogenesis [START_REF] Sabin | The lymphatic system in human embryos, with a consideration of the morphology of the system as a whole[END_REF]. The lymphatic system is crucial for immune response and understanding its development might be relevant to gain some insights into some human diseases. To start investigating lymphangiogenesis in human fetuses, we next labeled the gastrointestinal tract of a GW14 fetus with antibodies against the glycoprotein podoplanin, a lymphatic cell marker [START_REF] Breiteneder-Geleff | Angiosarcomas Express Mixed Endothelial Phenotypes of Blood and Lymphatic Capillaries[END_REF][START_REF] Schuster | Development of Blood and Lymphatic Endothelial Cells in Embryonic and Fetal Human Skin[END_REF] and found many isolated podoplanin+ cells covering the gut and stomach (Figure 4E). Lymphatic cells could also be seen using Prox-1 staining (data not shown). The lack of podoplanin+ capillaries at this age suggests that, as in other mammals, the human lymphatic system might mature later than the vascular system.

Lung development in human embryos

In human embryos, the lung primordia and the trachea emerge from the foregut endoderm around CS11/GW4 (O' Rahilly and Müller, 1987) and airway branching proceeds from GW6 to GW16 during the pseudoglandular stage [START_REF] Galambos | Molecular mechanisms of pulmonary vascular development[END_REF]. New bronchial branches, which bud from existing ones, further divide and lead to an expansion of the tree. Only a few immunohistological studies of human embryo lungs have been conducted [START_REF] Sparrow | Development of the innervation and airway smooth muscle in human fetal lung[END_REF]. Here we performed whole-mount immunostaining with 11 antibodies on lungs from 8 embryos and fetuses at GW8-GW14.

In the mouse, the sex determining region Y (SRY)-box 9 (Sox9) TF is expressed in alveolar epithelial cell progenitors at the distal part of each lung branch [START_REF] Rockich | Sox9 plays multiple roles in the lung epithelium during branching morphogenesis[END_REF], whereas Sox2 is restricted to the proximal part [START_REF] Maeda | Transcriptional control of lung morphogenesis[END_REF]. We observed a similar result in human lungs where all terminal bronchial buds were Sox9+ (Figures 5A-5D, 5H, and S4A and Movie S5), whereas Sox2 was expressed in a complementary pattern (Figures S4A andS4D). Strikingly, double staining for Sox9 and Dcx, which was not yet reported to be present in the lung epithelium, showed that Dcx was also expressed in the proximal epithelial portion of each airway (Figures 5A and5B). We also visualized in 3D the vasculature of the lung with Plvap (Figures

5A-5C and S4B

). The small capillary plexuses in the lung mesenchyme wrapping around terminal buds at GW8 are not isolated but form a continuous network with the larger vessels (Figures 5A-5C and S4B), thereby supporting an angiogenesis mode of lung vascularization as observed in mice [START_REF] Parera | Distal angiogenesis: a new concept for lung vascular morphogenesis[END_REF].

The complete reconstruction of the mouse airway lineage revealed that the branching pattern is highly stereotyped with new branches budding according to 3 modes: domain branching (daughter branches form in a row), planar and orthogonal bifurcation budding [START_REF] Metzger | The branching programme of mouse lung development[END_REF]. We performed such a lineage analysis in human whole-lungs using the segmentation of Sox9/Dcx (or Sox9/Sox2) bronchioles (Figures 5D and 5E and Movie S5). Similarly to the mouse, we observed three branching modes in humans (Figure 5E), but with some evidence of asymmetric bifurcations (Figure 5E). We next performed immunolabeling for smooth muscle markers SMA and myosin smooth muscle (MyoSM). In both cases, smooth muscles surrounding the bronchi and the proximal part of airway epithelial tubules were labeled (Figures 5F and 5G and S4A). The absence of smooth muscles on distal buds was confirmed using Sox9 immunostaining (Figure 5H). Incidentally, smooth muscle staining also allowed to visualization of lung arteries and arterioles (Figures 5F and5G and Movie S5).

The lung is primarily innervated by sympathetic and parasympathetic axons traveling through the vagus nerve. Parasympathetic ganglia also contribute to the innervation of the trachea and main bronchi [START_REF] Aven | Mechanisms of respiratory innervation during embryonic development[END_REF]. Due to the complexity of the airway tree, understanding its innervation can only be achieved in 3D. We visualized lung innervation using immunostaining against Prph, Tag-1 and ßIII-Tubulin.

Immunoreactive vagus axons extended to the most distal portion of the airway branches (Figures S4C andS4D). Preliminary results indicate that the development of the vasculature of the human heart and its innervation (using immunostaining for Tyrosine hydroxylase) can also be studied with this 3D method (Figures 5I and 5K and S4E and Movie S5).

3D Analysis of urogenital system development in human embryos

In adult mammals, the urogenital system is comprised of the kidneys, urinary tracts gonads and reproductive ducts. Before GW7-GW8, the human gonadal ridges are undifferentiated. Genital ducts appear morphologically similar in both sexes with two paired structures: the Wolffian ducts (WD) which are derived from the mesonephros and the Müllerian ducts (MD) whose differentiation is induced and guided by the WD.

A major reorganization of the genital tracts accompanies sex determination [START_REF] Jacob | Development , Differentiation and Derivatives of the Wolffian and Müllerian Ducts[END_REF][START_REF] Orvis | Cellular mechanisms of Müllerian duct formation in the mouse[END_REF]. In males, MD regress under the influence of an anti-Müllerian hormone produced by the testis. In females, the WD degenerate and the MD transform into the female reproductive ducts [START_REF] Georgas | An illustrated anatomical ontology of the developing mouse lower urogenital tract[END_REF][START_REF] O'rahilly | The timing and sequence of events in the development of the human reproductive system during the embryonic period proper[END_REF]. Knowledge of urogenital system development is primarily based on histological and electron microscopy analysis [START_REF] Fritsch | Molecular characteristics and alterations during early development of the human vagina[END_REF][START_REF] Hashimoto | Development of the human Müllerian duct in the sexually undifferentiated stage[END_REF], and has not yet been studied at a cellular and molecular level in humans unlike in mice [START_REF] Georgas | An illustrated anatomical ontology of the developing mouse lower urogenital tract[END_REF][START_REF] Little | A high-resolution anatomical ontology of the developing murine genitourinary tract[END_REF]. We studied the development of the human urogenital system using antibodies against Pax2 TF. Pax2 mRNA was previously detected in the mesonephros and WD at GW6-7 [START_REF] Tellier | Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome[END_REF]. We used embryos and fetuses of either sex, including 11 males (GW8-GW14) and 9 females (GW7-GW14).

All major components of the urogenital system were visualized in 3D (Figures 6 and7, and S5 and Movie S6), and individually segmented. In the youngest male embryo (GW8), the caudal tip of the MD was in close contact with the WD but had not yet fully elongated (Figures 6A and6B) and mesonephric tubules stemming from the WD covered the testis primordium. Kidneys still occupied a ventral position adjacent to the genital ridges (Figure 6A). At GW9.5 (n=1), the MD further elongated along the WD but did not yet join (Figures 6C and6D). In GW10 male fetuses (n=2), the two MD were fused and extended medially between the two WD up to the urogenital sinus. However, in both cases, the MD started to fragment dorsally (Figure 6E, F and Figure S5A), and this remnant of the fused MD might give rise to the prostatic utricle.

At older ages, such as GW14 (n=1), the mesonephric nephrons of the WD have regressed while the epididymis ducts and vas deferens have emerged (Figure 6G).

As previously reported [START_REF] Jacob | Development , Differentiation and Derivatives of the Wolffian and Müllerian Ducts[END_REF], an apical MD remnant is still present next to the rete testis by GW14 (Figures 6G and 6J and Movie S6). Sox9 is essential to testes differentiation in males and its mRNA was first detected around CS19-CS21 (GW7-7.5) [START_REF] Hanley | SRY, SOX9, and DAX1 expression patterns during human sex determination and gonadal development[END_REF]. Using whole-mount immunostaining for Sox9, on GW10, GW11, GW13.5 and GW14 fetuses (n=1 for each) we visualized the 3D organization of the testis cords, containing Sox9+ Sertoli cells (Figure 6H and 6J and Movie S6 and not shown). We also stained GW10.5-GW13 female fetuses with Pax2 (n=6) and followed in 3D the reorganization of the urogenital tract (Figures 7 andS5).

In our youngest female fetus (GW10.5) the MD have already fused to form the uterovaginal canal (Figure 7A). The WD were still continuous but mesonephric tubules began to regress. At GW11.5, the regression of the mesonephros and WD was more pronounced and the length of the two MD increased (Figure 7B). At GW13, the distal part of the WD had dissolved whereas its cranial part transformed into the Fallopian tubes (Figures 7C and S5B and Movie S6). The vascularization of the developing gonads is thought to play a role in their maturation [START_REF] Brennan | Divergent vascular mechanisms downstream of Sry establish the arterial system in the XY gonad[END_REF][START_REF] Coveney | Four-dimensional analysis of vascularization during primary development of an organ, the gonad[END_REF]. We performed double staining for Pax2 and Plvap to study the interaction between the vasculature and the genital tracts. Interestingly, at GW8 and GW10, the developing testes and WD were embedded in a dense capillary meshwork but the MD was avascular (Figures 7D and7E). In contrast, in a GW10.5 female fetus, both the MD and the WD were vascularized (Figures 7E and7F) and a dense vascular network ensheathed the MD by GW13 (Figures 7F and7G and Movie S6). While we could not study younger female embryos, these results suggest that MD angiogenesis may be sex dependent. This differential vascularization of the developing MD in males and females at GW10 suggest that their regression in males, which starts around this stage could be linked to or facilitated by the lack of vascularization. By contrast, the WD are vascularized in both sexes. Interestingly, the distal fused part of the Müllerian ducts does not seem to regress and likely gives rise to the prostatic utricle as previously proposed [START_REF] Jacob | Development , Differentiation and Derivatives of the Wolffian and Müllerian Ducts[END_REF].

Information on the early development of human nephrons is scarce [START_REF] Ludwig | Early development of the human mesonephros[END_REF]. Staining of GW10-GW13.5 kidneys (n=5) showed that Pax2 and Sox9 are both expressed in the epithelium of the ureteric tree (Figure S6). Pax2, together with another TF, Six2, was also found in nephron precursors of the cap mesenchyme surrounding the ureteric tips in the nephrogenic zone of the developing kidney [START_REF] O'brien | Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators[END_REF]. Staining of the endothelial cells with Plvap revealed the presence of developing glomeruli and their morphology (Figures S6D andS6E).

Transcription factors: new tools to study human embryology

During development, cell differentiation toward specific lineages is controlled by a cascade of transcription factors. We used antibodies against 19 transcription factors to follow the development of multiple organs (see also above).

In mouse and human embryos, Sox10 is expressed by oligodendrocytes, neural crest cells and some of their derivatives [START_REF] Betters | Analysis of early human neural crest development[END_REF][START_REF] Bondurand | Expression of the SOX10 gene during human development[END_REF].

SOX10 mutations cause various diseases such as neurocristopathies, melanoma and peripheral demyelinating neuropathies [START_REF] Bondurand | The role of SOX10 during enteric nervous system development[END_REF]. Here we performed double immunostaining for Sox10 and Prph on limbs from five fetuses, at GW9-GW12. Sox10+ cells were widely distributed throughout the PNS, at the level of the dorsal root ganglia and all along Prph-labeled nerves and branches (Figure S7A and Movie S7). These Sox10+ cells most likely correspond to Schwann cell precursors, which in the mouse migrate along PNS axons [START_REF] Espinosa-Medina | Neurodevelopment. Parasympathetic ganglia derive from Schwann cell precursors[END_REF].

In the developing tongue, taste bud primordia and papillae have been identified at GW9-GW10 using electron microscopy [START_REF] Hersch | Scanning electron microscopy of developing papillae on the tongue of human embryos and fetuses[END_REF]. No markers of developing human taste buds are known. In order to visualize tongue papillae, we performed immuno-labeling with antibodies against Prox1 and Sox2, both of which control mouse taste bud differentiation [START_REF] Nakayama | During development intense Sox2 expression marks not only Prox1-expressing taste bud cell but also perigemmal cell lineages[END_REF][START_REF] Okubo | Sox2 is required for development of taste bud sensory cells[END_REF]. Both Prox1 and Sox2 were detected in lingual papillae (including circumvallate papillae) covering the surface of the tongue (Figure S7B and S7C and Movie S7) in a similar pattern, suggesting that they are co-expressed in taste bud precursors, as observed in mice.

Whole-mount immunostaining for Sox2, which is known to be expressed by many types of cell progenitors [START_REF] Sarkar | The Sox Family of Transcription Factors: Versatile Regulators of Stem and Progenitor Cell Fate[END_REF], was also performed on other tissues from GW9.5, GW10 and GW12 fetuses. Strikingly, Sox2 expression was detected at the tip of each digit, in a narrow ring of cells lining the edge of the developing nails (Figure S7D and Movie S7). This indicates that Sox2 is a marker of fingernail stem cells in human fetuses, suggesting a role for this TF in nail development. Sox2 was also highly expressed in the ventricular zone of the developing brain (Figure S7E). Finally, we focused on the digestive system, which was collected in embryos and fetuses from GW7 to GW14 (n=13). Staining with Sox9 clearly revealed the 3D organization of the developing villi of the gut epithelium where stem cells express this TF (Figure S7F and Movie S7) [START_REF] Bastide | Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium[END_REF]. The enteric nervous system (ENS) has recently been the focus of many studies in part because of its frequent involvement in several genetic diseases [START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF]. ENS neurons are derived from neural crest cells which colonize the gut around GW4. Double labeling for Phox2b (paired-like homeobox 2b) and Prph revealed the dense network of ENS neurons, which formed interconnected clusters covering the surface of the gut (Figure S7G and Movie S7). Prph staining also revealed the parasympathetic innervation of the digestive system (Figure S7G).

Conclusion

Here, we have combined immunolabeling with whole-body and whole-organ 3D imaging to provide a new comprehensive and unbiased method to study the early development of multiple human biological systems.

Non-invasive medical imaging of embryos and fetuses in utero has made remarkable progress. This includes 3D/4D obstetrical ultrasonography which can generate holographic images of the embryo [START_REF] Baken | Design and validation of a 3D virtual reality desktop system for sonographic length and volume measurements in early pregnancy evaluation[END_REF][START_REF] Pooh | Imaging of the human embryo with magnetic resonance imaging microscopy and high-resolution transvaginal 3dimensional sonography: Human embryology in the 21st century[END_REF] but mostly provides information about surface features and cavities, and 3D power Doppler which visualizes the embryo vasculature [START_REF] Weisstanner | MRI of the Fetal Brain[END_REF]. In addition, in utero magnetic resonance imaging (MRI) provides a good appreciation of the developing CNS in the fetus [START_REF] Weisstanner | MRI of the Fetal Brain[END_REF] and diffusion tensor imaging (DTI) tractography is now used as a prenatal diagnostic of callosal dysgenesis as early as GW20 [START_REF] Jakab | Disrupted developmental organization of the structural connectome in fetuses with corpus callosum agenesis[END_REF]. Whilst these techniques represent valuable tools to visualize the gross anatomy of the fetus, they lack in resolution and are insufficient to resolve the molecular signature of individual cells or to know their organization within organs. Recently, whole-organ histology and 3D-reconstruction analysis based on the digital alignment of embryo sections has been performed (de Bakker et al., 2016), but it is extremely time consuming. Our method, which preserves the 3D organization of the organs while achieving a great cellular resolution, is rapid, highly reproducible and should provide clinicians with a reliable spatial framework for the correlation of in utero and postmortem 3D images of embryos and fetuses.

The simplicity of this method, its robustness (for example, we could label endothelial cells in a fresh frozen GW5 embryo kept for 11 years at -80°C, unpublished data) and sensitivity will facilitate its transfer to human embryology laboratories. Its use for the analysis of embryos and fetuses with genetic diseases and malformations will improve our understanding of their etiology.

Currently, the main limitations of our method are the availability of human embryos, the number of antibody combinations (a maximum of four at this time), the compatibility of the antibodies with our protocol (Table S1) and the storage of large size light-sheet image datasets. Nevertheless, the spectrum of future investigations and applications of this method in the field of embryology and fetology are countless.

For instance, defining how many cells give rise to an individual organ and understanding how cell numbers are regulated during development is essential to understanding the process of organogenesis. Here we show that whole-mount immunostaining with transcription factors and proliferation markers (KI67, H3P) expressed in specific cell types (such as stem cells and muscle precursors) can be efficiently carried out in cleared embryos and fetal organs. This opens the possibility to obtain the number of cells expressing these factors, as well as mapping their localization in the body and estimating the rate of cell proliferation during the first trimester of human gestation --a critical period for various diseases in which tissue growth is perturbed. The recent surge of Zika virus infection and reports of its deleterious effects in the brain [START_REF] Oliveira Melo | Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: Tip of the iceberg?[END_REF] has demonstrated how incomplete our understanding of human embryo development is, and how little we know about the mechanisms through which pathogens, toxins and mutations impact embryogenesis. Our work shows that it should be possible in the near future to build a reference 3D atlas of the developing human. As a first step in this direction, all our 3D datasets are made available on a dedicated website (https://transparent-humanembryo.com/) which will also serve as a repository for additional embryology 3D data generated from our laboratory and others. This reference 3D atlas of the developing human and specific organs and systems not only represents a powerful educational online tool for researchers, educators and students worldwide, but will allow 3D printing of anatomical models for didactic purposes in health sciences education programs. 3 andS3.

Related to Figures

Movie S4

3D imaging of the developing vasculature.

Related to Figure 4.

Movie S5

3D imaging of the developing cardiopulmonary system. Related to Figures 5 andS4.

Movie S6

3D imaging of the developing urogenital system. 6 and7 andS5.

Related to Figures

Movie S7

3D analysis of transcription factor expression in human embryos.

Related to Figure S7.

METHOD DETAILS

Tissue collection and processing

For embryos younger than 8 gestation weeks (GW), the developmental age was assessed using the Carnegie stages (CS) [START_REF] O'rahilly | Developmental stages in human embryos[END_REF]. Gestation weeks of each embryo or fetus studied were estimated based on the information about: 1) menstrual weeks, 2) morphology, 3) length (crown-rump), 4) craniofacial structures, and 5) position of the tongue. Embryos and fetuses, were fixed by immersion in 4% PFA at 4°C for 1 to 5 days depending on size. For fetuses between GW9-14, tissues were dissected after fixation and post-fixed in 4% PFA at 4°C overnight. Embryos and fetuses, did not present any malformations.

Sex determination

Sex determination was obtained by isolating DNA from extracted tissues using lysis buffer containing 0.1mg/ml proteinase K, 5M Sodium Chloride, 20% Sodium dodecyl sulfate, 1M Tris, pH 8.0 solution in water and stored at 54°C over night. DNA was precipitated with isopropanol (1:1) and re-suspended in RNase/DNase-free water for 3 hr at 65°C. A PCR was performed in a thermocycler (Biorad) using the following steps: 94°C for 3 min and 35 cycles of 94°C for 1 min; 56°C for 30 s; 72°C for 30 s and 72°C for 5 min. For genotyping the following primers were used: SRY sense 5'-AGCGATGATTACAGTCCAGC-3' and antisense 5'-CCTACAGCTTTGTCCAGTGG-3'; FGF16 sense 5'-CGGGAGGGATACAGGACTAAAC-3' and antisense 5'-CTGTAGGTAGCATCTGTGGC-3'. Only 20 embryos could be assessed, as prior tissue processing prevented DNA extraction for other cases.

Bleaching

To remove pigmentation and reduce signal-to-noise ratio related to hematomas, the tissue bleaching was carried out [START_REF] Renier | iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging[END_REF]. The samples were dehydrated for 1hr at RT in ascending concentrations of methanol in 1XPBS (50%, 80%, 100%).

The samples were then treated overnight at 4°C with a 6% hydrogen peroxide solution in 100% methanol. The following day, samples were re-hydrated for 1hr at RT in descending concentrations of methanol (100% twice, 80%, 50%) and washed in 1XPBS during 1hr. Samples were kept at 4°C for further processing.

Whole-mount immunostaining

Samples were permeabilized and blocked by rotation at 70 rpm in 1XPBS containing 0.2% gelatin (Prolabo), and 0.5% Triton X-100 (Sigma-Aldrich) (PBSGT) at RT [START_REF] Belle | A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System[END_REF]. For immunostaining, samples were transferred to a solution containing 0.1% saponin (10mg/mL) in PBSGT together with the primary antibodies (listed in the Key Resource Table and in Table S1) and placed at 37°C (Benchmark, Incu-Shaker Mini), with rotation at 70 rpm, for 7 to 14 days depending on tissue size and density. This was followed by six washes of 30 min in PBSGT at RT. Next, secondary antibodies (listed in the Key Resource Table ) were diluted in a solution containing 0.1% saponin (10mg/ml) in PBSGT and passed through a 0.22 µm filter. Samples were incubated at 37°C (Benchmark, Incu-Shaker Mini) in the secondary antibody solution overnight or for 2 days depending on sample size and density. After six washes of 30 min in PBSGT at RT, samples were stored in the dark at 4°C in until tissue clearing. The protocol was similar for single and multiple labeling.

Agarose embedding

As previously described [START_REF] Belle | A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System[END_REF][START_REF] Renier | iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging[END_REF] small samples were embedded in agarose prior to clearing and processing with the LSFM. To embed samples, 1.5% agarose (Roth) was prepared in TAE 1X (Invitrogen).

Tissue clearing

3DISCO

For all tissue clearing, a modified 3DISCO clearing protocol was used. All incubation steps were performed in dark conditions at RT in a fume hood, on a tube rotator (SB3, Stuart) at 14 rpm, using a 15 ml centrifuge tube (TPP, Dutscher). Samples were first dehydrated in ascending concentrations (50%, 80%, and 100%) of tetrahydrofuran (THF; anhydrous, containing 250 ppm butylated hydroxytoluene inhibitor, Sigma-Aldrich) diluted in H2O. The initial 50% THF bath was done overnight while the 80% and 100% THF incubations were left for 1.5 hr each. Samples next underwent a delipidation step of 30 min in dichloromethane (DCM; Sigma-Aldrich) followed by an overnight clearing step in dibenzyl ether (DBE; Sigma-Aldrich). The next day, samples were stored in individual light-absorbing glass vials (Rotilabo, Roth) at RT. In these conditions, samples could be stored and imaged for up to 9 months without any significant fluorescence loss.

Methanol clearing

For large tissues, methanol clearing was used to achieve higher transparency using a modification from the iDISCO+ protocol [START_REF] Renier | Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes[END_REF]. Whole embryos (GW8) and tissues (>GW8) were dehydrated in methanol/1XPBS series (n=7) : 20%, 40%, 60%, 80%, 100% x2 for 1 hr each at RT on a tube rotator (SB3, Stuart) at 14 rpm, using a 15 ml centrifuge tube (TPP, Dutscher) covered with aluminum foil to avoid contact with light. Then samples were incubate overnight in 2/3 DCM/ 1/3 Methanol. After 30 min in 100% DCM, samples were transferred to DBE.

3D imaging and image processing

3D imaging was performed as previously described. Acquisitions were performed by using an ultramicroscope I (LaVision BioTec) with the ImspectorPro software (LaVision BioTec). The light sheet was generated by a laser (wavelength 488, 561 or 640nm, Coherent Sapphire Laser, LaVision BioTec) and focused using two cylindrical lenses. Two adjustable protective lenses were applied for small and large working distances. A binocular stereomicroscope (MXV10, Olympus) with a 2x objective (MVPLAPO, Olympus) was used at different magnifications (0.63x, 1x, 1.25x, 1.6×, 2x, 2.5x, 3.2x, 4x, 5x, and 6.3x). The corresponding zoom factors and numerical apertures are available at http://lavisionbiotec.com/ultramicroscope-iispecifications.html.

Samples were placed in an imaging reservoir made of 100% quartz (LaVision BioTec) filled with DBE and illuminated from the side by the laser light. A PCO Edge SCMOS CCD camera (2,560 × 2,160 pixel size, LaVision BioTec) was used to acquire images. The step size between each image was fixed at 1 and 2 µm. All tiff images are generated in 16-bit. For large samples, a platform was created using PDMS (Sylgard) fixed to a sample holder from LaVision.

Image processing

Images, 3D volume, and movies were generated using Imaris x64 software (version 8.0.1, Bitplane). Stack images were first converted to imaris file (.ims) using ImarisFileConverter and 3D reconstruction was performed using the "volume rendering" function. To facilitate image processing, images were converted to an 8-bit format. To obtain opaque visualizations, the normal shading view was applied.

Optical slices were obtained using the "orthoslicer" tool. To isolate a specific region of the tissue, the surface tool was used and the mask option was selected. For isolation of smaller structures, i.e. nerve segmentation, the surface tool was manually applied and each nerve was subsequently pseudo-colored. For the hand, the foot and the urogenital system, nerves and organs were initially segmented manually and visualized in 3D using the 3D rendering tool. In order to visualize bone structures in 3D, skin is artificially removed by segmentation and the contrast is modified using normal shading. 3D pictures and movies were generated using the "snapshot" and "animation" tools. Movie reconstruction with .tiff series are done with ImageJ (1.50e, Java 1.8.0_60, 64-bit), titles and transitions addition have been done with iMovie (version 10.1.1).

QUANTIFICATION AND STATISTICAL ANALYSIS

N/A

  Figure legends

Figure 2 3D

 2 Figure2

Figure 3 3D

 3 Figure 3

Figure 4 3D

 4 Figure 4

Figure 5 3D

 5 Figure 5

Figure 6 3D

 6 Figure 6

Figure 7 Comparative

 7 Figure 7

Figure S3 .

 S3 Figure S3. Related to Figure 3

Figure

  Figure S7. Related to Figure 5

Acknowledgements

We thank the midwives of the Gynaecology Department, Jeanne de Flandres Hospital of Lille (Centre d'Orthogénie), France, for their assistance and support.

DATA AND SOFTWARE AVAILABILITY

All movies and files are available through a dedicated website, https://transparenthuman-embryo.com/.

ADDITIONAL RESOURCES

Access to the 3D image datasets is available through https://transparent-humanembryo.com/. The database was created with the help of keen eye technologies.

KEY RESOURCES TABLE

We also thank Robin Vigouroux, Reha Erzurumlu and Peter Kozulin for correcting the manuscript, Yorick Gitton and Stéphane Fouquet (Institut de la Vision, Imaging Facility) for their help with the database, and Olivier Pourquié, Domna Karagogeos, William Stallcup, Carmen Birchmeier and Jean-François Brunet for kindly providing some antibodies. Myog and Pax7 antibodies were obtained from the Developmental Studies Hybridoma Bank. We are also grateful to Olivier Pourquié, José Sahel, Constantino Sotelo and Nicole Le Douarin for helpful discussions. This work was supported by the Institut National de la Santé et de la Recherche Médicale, INSERM, France (grant number U1172), Agence Nationale de la Recherche, ANR, France (ANR-14-CE12-0015-01 to P.G., ANR-14-CE13-0004-01 to A.C.). It was performed in the frame of the LABEX LIFESENSES (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02.

Table S1. Related to Figure 1.

Primary antibodies tested but not working with our protocol

Movie S1.

3D analysis of human cranial nerve development. 1 andS1.

Related to Figures

Movie S2

3D development of hand and foot innervation.

Related to Figures 2 and S1 and S2.

Movie S3

3D imaging of the developing muscles.

Supplementary figure legends

STAR Methods

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr Alain Chédotal (alain.chedotal@inserm.fr).

All data has been deposited in https://transparent-human-embryo.com/ and access to the high-resolution movies and image stacks is regulated via an MTA agreement available online.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Human embryos (<GW8.5) and fetuses (≥GW8.5) were obtained with the parent's written informed consent (Gynaecology Hospital Jeanne de Flandres, Lille, France) with approval of the local ethic committee (protocol N° PFS16-002). Tissues were made available in accordance with the French bylaw (Good practice concerning the conservation, transformation and transportation of human tissue to be used therapeutically, published on December 29, 1998). Permission to utilize human tissues was obtained from the French agency for biomedical research (Agence de la Biomédecine, Saint-Denis La Plaine, France). For this study, we used 7 embryos at Carnegie Stage 17 (CS17) corresponding to 41 days of development (E46) or gestational week 6 (GW6) (n=1)(O'Rahilly and Müller, 1987), CS19/E46/GW7 (n=2), CS21/E51/GW7.5 (n=1), CS23/E56/GW8 (n=3), and 29 fetuses at GW8.5 (n=2), GW9 (n=3), GW9.5 (n=5), GW10 (n=6), GW10.5 (n=2), GW11 (n=3), GW11.5 (n=1), GW12 (n=1), GW13 (n=1), GW13. (n=1) and GW14 (n=4). This included 9 females and 11 males (sex was undetermined for the others).

KEY RESOURCES