Tridimensional analysis of the developing human motor and peripheral nervous systems – 3D-HUMAN

Congenital defects afflict more than 3% of births. These developmental diseases are a major public healthcare burden and today the molecular bases of the tissue-tissue interactions that ensure correct tissue morphogenesis in humans remain largely unknown. This is also the case for three-dimensional cellular interactions in humans, which specify the development of neurons in the peripheral nervous system and the growth of their axons. We have recently been able to combine whole-mount immunostaining and 3D imaging of solvent-cleared organs with light-sheet fluorescence microscopy, to study the 3D organization of several organs and systems in transparent human embryos and fetuses. The aim of this project is to form a consortium whose research will improve current knowledge of the development of the peripheral nervous system, particularly neuromuscular, in humans during the first trimester of gestation. This will allow us to establish a unique interactive database for neuroscientists and clinicians. We will focus on the cephalic region, the limbs and some internal organs, the development of which is poorly understood, and will improve 3D imaging methods for whole organs. Currently available scaling immunohistochemical detection to large tissue volumes has limitations due to restricted multiplexing capability of antibody labels. Moreover, human specimens in good condition are rare, especially pathological cases. Therefore, optimizing their exploitation is a priority and this aim seeks improving 3D-labeling methods. We will develop novel immunohistochemistry (IHC) multiplexed strategy to visualize up 8-10 antigens within the same sample. We will also further seek to develop whole-body IHC using single-chain variable fragments of conventional antibodies, which could outperform conventional antibodies when it comes to tissue penetration. This approach will thus improve immunostaining of very large samples, such as fetal organs and/or whole fetuses at gestational weeks 10-15 (GW10-GW15). In parallel, we will use multiplexed single-molecule in situ hybridization methods (MSM-FISH) combined with IHC on both tissue sections and intact human embryos, to dissect human fetal tissues in more detail in terms of their cellular composition and high-resolution architecture.
Our second objective is to revisit through 3D imaging the embryonic development of peripheral, sensory, motor and autonomic human nerves using a large battery of markers (largely already validated) specific to different populations. We will astudy the expression pattern of the main axonal guidance molecules and their receptors in the peripheral nervous system. Another main objective is to decipher the phenotype of human motor neurons (MNs), according to their topographical distribution. MN phenotyping relies upon knowledge accumulated in mice, and the genetic fate map of MN pools and subtype is largely unknown in human embryos. We will characterize with a large panel of markers the topographic distribution of MNs in human embryos and fetuses. We will delineate the period of developmental cell death of human MNs. Last ,we will map the development of motor nerves in correlation with their target muscles. Finally, we will study the development of autonomic innervation (vegetative system) of the pancreas, as there is evidence to suggest that abnormal development of this innervation may promote diabetes. We will compare the pancreatic innervation of embryos from lean or obese women.
Theiscombination of novel strategies and approaches, focused on human embryos, will enable us to address important questions in the field of human embryogenesis and ultimately to better understand the etiology of certain neurodevelopmental and muscular diseases. The two partners, Dr Chédotal (Paris) and Dr Giacobini (Lille) are recognized experts in their field and have all the required technical know-how to perform the planned experiments, as demonstrated by their collaborative track-record.