Normal and pathological human embryology

A tridimensional atlas of the developing human head

3D visualization of the tongue and larynx muscles and their innervation in a PCW7.5 human embryo; 3D visualization of extraocular muscles and oculomotor nerves in a PCW8.4 human fetus; 3D visualization of extraocular muscles, oculomotor nerves and lacrimal gland in a PCW11.3 human fetus; 3D visualization of the external, middle, and inner ear in a PCW7.5 human embryo. Blain et al., 2024 Cell

HuDeCa

HuDeCA (Human Developmental Cell Atlas) is a 5-year program initiated by Inserm to build a reference atlas of embryonic and fetal human cell types. The project has been designed from the outset to enable contributions to the Human Cell Atlas (HCA), an ambitious international initiative to map all normal cell states of the body at every human life stages.

Transparent Human Embryo

The Collection of Immunolabeled Transparent Human Embryos and Fetuses is the most advanced molecular embryology tool. Building upon the pioneering ‘DISCO’ clearing methods developed for rodent embryos, we have shifted gears to another level, where direct knowledge of molecular and cellular ontogenesis may prove critical to improve Human pregnancy.

• Alain CHEDOTAL — Professor, UCBL, HDR

1. A tridimensional atlas of the developing human head.
   Blain R, Couly G, Shotar E, Blévinal J, Toupin M, Favre A, Abjaghou A, Inoue M, Hernández-Garzón E, Clarençon F, Chalmel F, Mazaud-Guittot S, Giacobini P, Gitton Y, Chédotal A.
   Cell (2023) — Show abstract

   The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

2. A human embryonic limb cell atlas resolved in space and time.
   Zhang B, He P, Lawrence JEG, Wang S, Tuck E, Williams BA, Roberts K, Kleshchevnikov V, Mamanova L, Bolt L, Polanski K, Li T, Elmentaite R, Fasouli ES, Prete M, He X, Yayon N, Fu Y, Yang H, Liang C, Zhang H, Blain R, Chedotal A, FitzPatrick DR, Firth H, Dean A, Bayraktar OA, Marioni JC, Barker RA, Storer MA, Wold BJ, Zhang H, Teichmann SA.
   Nature (2023) — Show abstract

   Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

3. Yolk sac cell atlas reveals multiorgan functions during human early development.
   Goh I, Botting RA, Rose A, Webb S, Engelbert J, Gitton Y, Stephenson E, Quiroga Londoño M, Mather M, Mende N, Imaz-Rosshandler I, Yang L, Horsfall D, Basurto-Lozada D, Chipampe NJ, Rook V, Lee JTH, Ton ML, Keitley D, Mazin P, Vijayabaskar MS, Hannah R, Gambardella L, Green K, Ballereau S, Inoue M, Tuck E, Lorenzi V, Kwakwa K, Alsinet C, Olabi B, Miah M, Admane C, Popescu DM, Acres M, Dixon D, Ness T, Coulthard R, Lisgo S, Henderson DJ, Dann E, Suo C, Kinston SJ, Park JE, Polanski K, Marioni J, van Dongen S, Meyer KB, de Bruijn M, Palis J, Behjati S, Laurenti E, Wilson NK, Vento-Tormo R, Chédotal A, Bayraktar O, Roberts I, Jardine L, Göttgens B, Teichmann SA, Haniffa M.
   Science (2023) — Show abstract

   The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

4. Single-cell roadmap of human gonadal development.
   Garcia-Alonso L, Lorenzi V, Mazzeo CI, Alves-Lopes JP, Roberts K, Sancho-Serra C, Engelbert J, Marečková M, Gruhn WH, Botting RA, Li T, Crespo B, van Dongen S, Kiselev VY, Prigmore E, Herbert M, Moffett A, Chédotal A, Bayraktar OA, Surani A, Haniffa M, Vento-Tormo R.
   Nature (2022) — Show abstract

   Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

5. Bilateral visual projections exist in non-teleost bony fish and predate the emergence of tetrapods.
   Vigouroux RJ, Duroure K, Vougny J, Albadri S, Kozulin P, Herrera E, Nguyen-Ba-Charvet K, Braasch I, Suárez R, Del Bene F, Chédotal A.
   Science (2021) — Show abstract

   In most vertebrates, camera-style eyes contain retinal ganglion cell neurons that project to visual centers on both sides of the brain. However, in fish, ganglion cells were thought to innervate only the contralateral side, suggesting that bilateral visual projections appeared in tetrapods. Here we show that bilateral visual projections exist in non-teleost fishes and that the appearance of ipsilateral projections does not correlate with terrestrial transition or predatory behavior. We also report that the developmental program that specifies visual system laterality differs between fishes and mammals, as the Zic2 transcription factor, which specifies ipsilateral retinal ganglion cells in tetrapods, appears to be absent from fish ganglion cells. However, overexpression of human ZIC2 induces ipsilateral visual projections in zebrafish. Therefore, the existence of bilateral visual projections likely preceded the emergence of binocular vision in tetrapods.

6. Revisiting the role of Dcc in visual system development with a novel eye clearing method.
   Vigouroux RJ, Cesar Q, Chédotal A, Nguyen-Ba-Charvet KT.
   Elife (2020) — Show abstract

   The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as Dcc knockouts die at birth. To circumvent this drawback, we generated an eye-specific Dcc mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of Dcc, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, Dcc-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.

7. Clearing method for 3-dimensional immunofluorescence of osteoarthritic subchondral human bone reveals peripheral cholinergic nerves.
   Courties A, Belle M, Senay S, Cambon-Binder A, Sautet A, Chédotal A, Berenbaum F, Sellam J.
   Sci Rep (2020) — Show abstract

   The cholinergic system plays a major anti-inflammatory role in many diseases through acetylcholine (Ach) release after vagus nerve stimulation. Osteoarthritis (OA) is associated with local low-grade inflammation, but the regulatory mechanisms are unclear. Local Ach release could have anti-inflammatory activity since articular cells express Ach receptors involved in inflammatory responses. Using the 3DISCO clearing protocol that allows whole-sample 3-dimensional (3D) analysis, we cleared human OA cartilage-subchondral bone samples to search for cholinergic nerve fibres able to produce Ach locally. We analysed 3 plugs of knee cartilage and subchondral bone from 3 OA patients undergoing arthroplasty. We found no nerves in the superficial and intermediate articular cartilage layers, as evidenced by the lack of Peripherin staining (a peripheral nerves marker). Conversely, peripheral nerves were found in the deepest layer of cartilage and in subchondral bone. Some nerves in the subchondral bone samples were cholinergic because they coexpressed peripherin and choline acetyltransferase (ChAT), a specific marker of cholinergic nerves. However, no cholinergic nerves were found in the cartilage layers. It is therefore feasible to clear human bone to perform 3D immunofluorescence. Human OA subchondral bone is innervated by cholinergic fibres, which may regulate local inflammation through local Ach release.

8. Decoding human fetal liver haematopoiesis
   Popescu DM, Botting RA, Stephenson E, Green K, Webb S, Jardine L, Calderbank EF, Polanski K, Goh I, Efremova M, Acres M, Maunder D, Vegh P, Gitton Y, Park JE, Vento-Tormo R, Miao Z, Dixon D, Rowell R, McDonald D, Fletcher J, Poyner E, Reynolds G, Mather M, Moldovan C, Mamanova L, Greig F, Young MD, Meyer KB, Lisgo S, Bacardit J, Fuller A, Millar B, Innes B, Lindsay S, Stubbington MJT, Kowalczyk MS, Li B, Ashenberg O, Tabaka M, Dionne D, Tickle TL, Slyper M, Rozenblatt-Rosen O, Filby A, Carey P, Villani AC, Roy A, Regev A, Chédotal A, Roberts I, Göttgens B, Behjati S, Laurenti E, Teichmann SA, Haniffa M.
   Nature (2019) — Show abstract

   Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

9. Tridimensional Visualization and Analysis of Early Human Development
   Belle M, Godefroy D, Couly G, Malone SA, Collier F, Giacobini P, Chédotal A.
   Cell (2017) — Show abstract

   Generating a precise cellular and molecular cartography of the human embryo is essential to our understanding of the mechanisms of organogenesis in normal and pathological conditions. Here, we have combined whole-mount immunostaining, 3DISCO clearing, and light-sheet imaging to start building a 3D cellular map of the human development during the first trimester of gestation. We provide high-resolution 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 of paramount importance to understanding human development in health and disease.

10. Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains.
    Casoni F, Malone SA, Belle M, Luzzati F, Collier F, Allet C, Hrabovszky E, Rasika S, Prevot V, Chédotal A, Giacobini P.
    Development (2016) — Show abstract

    Fertility in mammals is controlled by hypothalamic neurons that secrete gonadotropin-releasing hormone (GnRH). These neurons differentiate in the olfactory placodes during embryogenesis and migrate from the nose to the hypothalamus before birth. Information regarding this process in humans is sparse. Here, we adapted new tissue-clearing and whole-mount immunohistochemical techniques to entire human embryos/fetuses to meticulously study this system during the first trimester of gestation in the largest series of human fetuses examined to date. Combining these cutting-edge techniques with conventional immunohistochemistry, we provide the first chronological and quantitative analysis of GnRH neuron origins, differentiation and migration, as well as a 3D atlas of their distribution in the fetal brain. We reveal not only that the number of GnRH-immunoreactive neurons in humans is significantly higher than previously thought, but that GnRH cells migrate into several extrahypothalamic brain regions in addition to the hypothalamus. Their presence in these areas raises the possibility that GnRH has non-reproductive roles, creating new avenues for research on GnRH functions in cognitive, behavioral and physiological processes.

11. Human Cell Atlas Developmental Biological Network. A roadmap for the Human Developmental Cell Atlas
    Haniffa M, Taylor D, Linnarsson S, Aronow BJ, Bader GD, Barker RA, Camara PG, Camp JG, Chédotal A, Copp A, Etchevers HC, Giacobini P, Göttgens B, Guo G, Hupalowska A, James KR, Kirby E, Kriegstein A, Lundeberg J, Marioni JC, Meyer KB, Niakan KK, Nilsson M, Olabi B, Pe'er D, Regev A, Rood J, Rozenblatt-Rosen O, Satija R, Teichmann SA, Treutlein B, Vento-Tormo R, Webb S
    Nature (2021) — Show abstract

    The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

12. Tissue clearing and its applications in neuroscience
    Ueda HR, Ertürk A, Chung K, Gradinaru V, Chédotal A, Tomancak P, Keller PJ.
    Nat Rev Neurosci (2020) — Show abstract

    State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.