A tridimensional atlas of the developing human head.

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.

Development of the Binocular Circuit.

Seeing in three dimensions is a major property of the visual system in mammals. The circuit underlying this property begins in the retina, from which retinal ganglion cells (RGCs) extend to the same or opposite side of the brain. RGC axons decussate to form the optic chiasm, then grow to targets in the thalamus and midbrain, where they synapse with neurons that project to the visual cortex. Here we review the cellular and molecular mechanisms of RGC axonal growth cone guidance across or away from the midline via receptors to cues in the midline environment. We present new views on the specification of ipsi- and contralateral RGC subpopulations and factors implementing their organization in the optic tract and termination in subregions of their targets. Lastly, we describe the functional and behavioral aspects of binocular vision, focusing on the mouse, and discuss recent discoveries in the evolution of the binocular circuit.

Tridimensional Visualization and Analysis of Early Human Development.

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. PAPERCLIP.

A human embryonic limb cell atlas resolved in space and time.

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.

Single-cell roadmap of human gonadal development.

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.

A roadmap for the Human Developmental Cell Atlas.

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.

Publisher Correction: Tissue clearing and its applications in neuroscience.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tissue clearing and its applications in neuroscience.

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.

Decoding human fetal liver haematopoiesis.

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.

A 3D atlas of the human developing pancreas to explore progenitor proliferation and differentiation.

AIMS/HYPOTHESIS: Rodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases. METHODS: We combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules. RESULTS: We detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors. CONCLUSIONS/INTERPRETATION: This work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.

Slit1 inhibits ovarian follicle development and female fertility in mice†.

Previous in vitro studies have suggested that SLIT ligands could play roles in regulating ovarian granulosa cell proliferation and gene expression, as well as luteolysis. However, no in vivo study of Slit gene function has been conducted to date. Here, we investigated the potential role of Slit1 in ovarian biology using a Slit1-null mouse model. Female Slit1-null mice were found to produce larger litters than their wild-type counterparts due to increased ovulation rates. Increased ovarian weights in Slit1-null animals were found to be due to the presence of greater numbers of healthy antral follicles with similar numbers of atretic ones, suggesting both an increased rate of follicle recruitment and a decreased rate of atresia. Consistent with this, treatment of cultured granulosa cells with exogenous SLIT1 induced apoptosis in presence or absence of follicle-stimulating hormone, but had no effect on cell proliferation. Although few alterations in the messenger RNA levels of follicle-stimulating hormone-responsive genes were noted in granulosa cells of Slit1-null mice, luteinizing hormone target gene mRNA levels were greatly increased. Finally, increased phospho-AKT levels were found in granulosa cells isolated from Slit1-null mice, and SLIT1 pretreatment of cultured granulosa cells inhibited the ability of both follicle-stimulating hormone and luteinizing hormone to increase AKT phosphorylation, suggesting a mechanism whereby SLIT1 could antagonize gonadotropin signaling. These findings therefore represent the first evidence for a physiological role of a SLIT ligand in the ovary, and define Slit1 as a novel autocrine/paracrine regulator of follicle development.

Roles of axon guidance molecules in neuronal wiring in the developing spinal cord.

The spinal cord receives, relays and processes sensory information from the periphery and integrates this information with descending inputs from supraspinal centres to elicit precise and appropriate behavioural responses and orchestrate body movements. Understanding how the spinal cord circuits that achieve this integration are wired during development is the focus of much research interest. Several families of proteins have well-established roles in guiding developing spinal cord axons, and recent findings have identified new axon guidance molecules. Nevertheless, an integrated view of spinal cord network development is lacking, and many current models have neglected the cellular and functional diversity of spinal cord circuits. Recent advances challenge the existing spinal cord axon guidance dogmas and have provided a more complex, but more faithful, picture of the ontogenesis of vertebrate spinal cord circuits.

Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.

Netrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.

Non-cell autonomous control of precerebellar neuron migration by Slit and Robo proteins.

During development, precerebellar neurons migrate tangentially from the dorsal hindbrain to the floor plate. Their axons cross it but their cell bodies stop their ventral migration upon reaching the midline. It has previously been shown that Slit chemorepellents and their receptors, Robo1 and Robo2, might control the migration of precerebellar neurons in a repulsive manner. Here, we have used a conditional knockout strategy in mice to test this hypothesis. We show that the targeted inactivation of the expression of Robo1 and Robo2 receptors in precerebellar neurons does not perturb their migration and that they still stop at the midline. The selective ablation of the expression of all three Slit proteins in floor-plate cells has no effect on pontine neurons and only induces the migration of a small subset of inferior olivary neurons across the floor plate. Likewise, we show that the expression of Slit proteins in the facial nucleus is dispensable for pontine neuron migration. Together, these results show that Robo1 and Robo2 receptors act non-cell autonomously in migrating precerebellar neurons and that floor-plate signals, other than Slit proteins, must exist to prevent midline crossing.

Commissural neurons transgress the CNS/PNS boundary in absence of ventricular zone-derived netrin 1.

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in Ntn1 hypomorphic embryos and Dcc knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in Ntn1 and Dcc mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a Ntn1 conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.

Genetic Analysis of the Organization, Development, and Plasticity of Corneal Innervation in Mice.

The cornea has the densest sensory innervation of the body, originating primarily from neurons in the trigeminal ganglion. The basic principles of cornea nerve patterning have been established many years ago using classic neuroanatomical methods, such as immunocytochemistry and electrophysiology. Our understanding of the morphology and distribution of the sensory nerves in the skin has considerably progressed over the past few years through the generation and analysis of a variety of genetically modified mouse lines. Surprisingly, these lines were not used to study corneal axons. Here, we have screened a collection of transgenic and knockin mice (of both sexes) to select lines allowing the visualization and genetic manipulation of corneal nerves. We identified multiple lines, including some in which different types of corneal axons can be simultaneously observed with fluorescent proteins expressed in a combinatorial manner. We also provide the first description of the morphology and arborization of single corneal axons and identify three main types of branching pattern. We applied this genetic strategy to the analysis of corneal nerve development and plasticity. We provide direct evidence for a progressive reduction of the density of corneal innervation during aging. We also show that the semaphorin receptor neuropilin-1 acts cell-autonomously to control the development of corneal axons and that early axon guidance defects have long-term consequences on corneal innervation.SIGNIFICANCE STATEMENT We have screened a collection of transgenic and knockin mice and identify lines allowing the visualization and genetic manipulation of corneal nerves. We provide the first description of the arborization pattern of single corneal axons. We also present applications of this genetic strategy to the analysis of corneal nerve development and remodeling during aging.

Construction and reconstruction of brain circuits: normal and pathological axon guidance.

Perception of our environment entirely depends on the close interaction between the central and peripheral nervous system. In order to communicate each other, both systems must develop in parallel and in coordination. During development, axonal projections from the CNS as well as the PNS must extend over large distances to reach their appropriate target cells. To do so, they read and follow a series of axon guidance molecules. Interestingly, while these molecules play critical roles in guiding developing axons, they have also been shown to be critical in other major neurodevelopmental processes, such as the migration of cortical progenitors. Currently, a major hurdle for brain repair after injury or neurodegeneration is the absence of axonal regeneration in the mammalian CNS. By contrasts, PNS axons can regenerate. Many hypotheses have been put forward to explain this paradox but recent studies suggest that hacking neurodevelopmental mechanisms may be the key to promote CNS regeneration. Here we provide a seminar report written by trainees attending the second Flagship school held in Alpbach, Austria in September 2018 organized by the International Society for Neurochemistry (ISN) together with the Journal of Neurochemistry (JCN). This advanced school has brought together leaders in the fields of neurodevelopment and regeneration in order to discuss major keystones and future challenges in these respective fields.

Slit-Robo signaling.

Slits are secreted proteins that bind to Roundabout (Robo) receptors. Slit-Robo signaling is best known for mediating axon repulsion in the developing nervous system. However, in recent years the functional repertoire of Slits and Robo has expanded tremendously and Slit-Robo signaling has been linked to roles in neurogenesis, angiogenesis and cancer progression among other processes. Likewise, our mechanistic understanding of Slit-Robo signaling has progressed enormously. Here, we summarize new insights into Slit-Robo evolutionary and system-dependent diversity, receptor-ligand interactions, signaling crosstalk and receptor activation.

Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains.

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.