Neuro-developpement embryonnaire et pathologies pediatriques associées

Pour le grand Public

Comment nos connexions nerveuses se construisent-elles ? le neurone nouveau-né élabore un prolongement, l'axone, voué à s'engager dans un incroyable voyage, à la recherche des cellules avec lesquelles il va établir une communication. Ainsi au cours du développement embryonnaire et post-natal, des millions d’axones partent à la recherche de leurs partenaires, certains restant confinés dans le cerveau ou la moelle épinière, d’autres colonisant tout l’organisme pour innerver les muscles, la peau, les viscères. Les signaux qui permettent aux axones de se repérer dans l’espace, qu’on appelle les signaux topographiques ou de guidage, sont également utilisés par des cellules qui se déplacent pour construire des structures nerveuses. Amener chaque axone et chaque cellule à bon port est une véritable gageure et c'est ce sont ces processus d'adressage que notre équipe étudie.

Les axones et cellules peuvent-ils se perdre en chemin ou se tromper de destination ? Diverses pathologies infantiles se traduisent par des altérations de navigation des cellules et axones. Nombre reste encore à découvrir, en raison de leur occurrence précoce et des difficultés d'étude qui en découlent. Par ailleurs, ce phénomène physiologique de migration a également un corrélat pathologique, celui des cancers pédiatriques d’origine embryonnaire qui surviennent en pleine construction de l’organisme. Notre équipe étudient différents contextes de pathologie et cherche également à comprendre si les cellules malignes utilisent les signaux topographiques de l'embryogenèse de façon opportuniste, pour disséminer et établir des métastases à distance.

Offre Post-doc

Childhood malignancies of the nervous system: exploring the dissemination of tumoral cells in light of the mechanisms of the embryonic development.

The project aims at characterizing shared and unshared behaviors of malignant cells with their cells of origin labeled by specific fluorescent reporters, focusing on migratory processes. The objectives are to compare cell behaviors using imaging techniques and transcriptomic analyses to identify signaling pathways governing the pathfinding strategies of malignant cells during their dissemination.

Start Up

Oncofactory est une spin-off du laboratoire de V. Castellani co-fondée par C. Delloye-Bourgeois et V. Castellani, qui exploite une plateforme technologique innovante permettant de créer des répliques miniaturisées de cancers de patients à partir de biopsies et chirurgies, dans un organisme embryonnaire aviaire, d’administrer des thérapies, d’imager les réponses tumorales par microscopie 3D dans l’organisme entier, et de réaliser des analyses moléculaires à large échelle sur les répliques tumorales. Oncofactory utilise sa plateforme pour accompagner les industries pharmaceutiques dans leurs programmes précliniques en oncologie.

REACT4KIDS

Notre équipe appartient au réseau REACT4KIDS (REsearchers in oncology ACTing for kids), un réseau collaboratif de recherche fondamentale en oncologie pédiatrique, dont les missions sont de favoriser les échanges entre les acteurs de ce secteur et de répondre à des problématiques communes. REACT4KIDS a un seul objectif: permettre de mieux comprendre les bases moléculaires des cancers des enfants et adolescents, afin de pouvoir dans l’avenir mieux les soigner.

• Valérie CASTELLANI — DR1 CNRS, HDR – valerie.castellani@univ-lyon1.fr
• Céline DELLOYE-BOURGEOIS — CRCN, CNRS – celine.delloye-bourgeois@univ-lyon1.fr
• Servane TAUSZIG-DELAMASURE — DR2, CNRS, HDR – servane.tauszig-delamasure@univ-lyon1.fr
• Julien FALK — CRCN, CNRS, HDR – julien.falk@univ-lyon1.fr
• Frédéric MORET — MCU, UCBL, HDR – frederic.moret@univ-lyon1.fr
• Muriel BOZON — AI, CNRS – muriel.bozon@univ-lyon1.fr
• Karine THOINET — AI, CNRS – karine.thoinet@univ-lyon1.fr
• Emy THEOULE — IE, UCBL – emy.theoulle@univ-lyon1.fr
• Florie REYNAUD — IR, UCBL – florie.reynaud@univ-lyon1.fr
• Caroline IMBERT — IE, UCBL – caroline.imbert@univ-lyon1.fr
• Franck BOISMOREAU — Post-doctorant, UCBL – franck.boismoreau@univ-lyon1.fr
• Sophie CALVET — Post-doctorante, CNRS – sophie.calvet@univ-lyon1.fr
• Maëlys ANDRE — Doctorante, UCBL – maelys.andre@univ-lyon1.fr
• Benjamin VILLALARD — Doctorant, CNRS – benjamin.villalard@univ-lyon1.fr
• Marion MALLET — M2
• Raphael GURY — M2
• Sofia DJAMAI — M1
• Laurine FIGEAC — M1

1. An avian embryo patient-derived xenograft model for preclinical studies of human breast cancers.
   Jarrosson L, Costechareyre C, Gallix F, Ciré S, Gay F, Imbaud O, Teinturier R, Marangoni E, Aguéra K, Delloye-Bourgeois C, Castellani V.
   iScience (2021) — Résumé

   Lack of preclinical patient-derived xenograft cancer models in which to conduct large-scale molecular studies seriously impairs the development of effective personalized therapies. We report here an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large-scale RNA-seq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple-negative breast cancer (TNBC) and validated using TNBC standards of care and an investigational therapeutic agent.

2. X-linked partial corpus callosum agenesis with mild intellectual disability: identification of a novel L1CAM pathogenic variant.
   Bousquet I, Bozon M, Castellani V, Touraine R, Piton A, Gérard B, Guibaud L, Sanlaville D, Edery P, Saugier-Veber P, Putoux A.
   Neurogenetics (2021) — Résumé

   athogenic variants in L1CAM, the gene encoding the L1 cell adhesion molecule, are responsible for a wide clinical spectrum including X-linked hydrocephalus with stenosis of the Sylvius aqueduct, MASA syndrome (mental retardation, aphasia, shuffling gait, adducted thumbs), and a form of spastic paraplegia (SPG1). A moderate phenotype with mild intellectual disability (ID) and X-linked partial corpus callosum agenesis (CCA) has only been related to L1CAM in one family. We report here a second family, including 5 patients with mild to moderate ID and partial CCA without signs usually associated with L1CAM pathogenic variations (such as hydrocephalus, pyramidal syndrome, thumb adductus, aphasia). We identified a previously unreported c.3226A > C transversion leading to a p.Thr1076Pro amino acid substitution in the fifth fibronectin type III domain (FnIII) of the protein which co-segregates with the phenotype within the family. We performed in vitro assays to assess the pathogenic status of this variation. First, the expression of the novel p.Thr1076Pro mutant in COS7 cells resulted in endoplasmic reticulum (ER) retention and reduced L1CAM cell surface expression, which is expected to affect both L1CAM-mediated cell-cell adhesion and neurite growth. Second, immunoblotting techniques showed that the immature form of the L1CAM protein was increased, indicating that this variation led to a lack of maturation of the protein. ID associated with CCA is not a common clinical presentation of L1CAM pathogenic variants. Genome-wide analyses will identify such variations and it is important to acknowledge this atypical phenotype.

3. SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate.
   Ducuing H, Gardette T, Pignata A, Kindbeiter K, Bozon M, Thoumine O, Delloye-Bourgeois C, Tauszig-Delamasure S, Castellani V.
   Elife (2020) — Résumé

   Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.

4. A Spatiotemporal Sequence of Sensitization to Slits and Semaphorins Orchestrates Commissural Axon Navigation.
   Pignata A, Ducuing H, Boubakar L, Gardette T, Kindbeiter K, Bozon M, Tauszig-Delamasure S, Falk J, Thoumine O, Castellani V.
   Cell Reports (2019) — Résumé

   Accurate perception of guidance cues is crucial for cell and axon migration. During initial navigation in the spinal cord, commissural axons are kept insensitive to midline repellents. Upon midline crossing in the floor plate, they switch on responsiveness to Slit and Semaphorin repulsive signals and are thus propelled away and prevented from crossing back. Whether and how the different midline repellents control specific aspects of this navigation remain to be elucidated. We set up a paradigm for live-imaging and super-resolution analysis of PlexinA1, Neuropilin-2, and Robo1/2 receptor dynamics during commissural growth cone navigation in chick and mouse embryos. We uncovered a remarkable program of sensitization to midline cues achieved by unique spatiotemporal sequences of receptor allocation at the growth-cone surface that orchestrates receptor-specific growth-cone behavior changes. This reveals post-translational mechanisms whereby coincident guidance signals are temporally resolved to allow the generation of specific guidance responses.

5. Hijacking of Embryonic Programs by Neural Crest-Derived Neuroblastoma: From Physiological Migration to Metastatic Dissemination.
   Delloye-Bourgeois C, Castellani V.
   Front Mol Neurosci. (2019) — Résumé

   In the developing organism, complex molecular programs orchestrate the generation of cells in adequate numbers, drive them to migrate along the correct pathways towards appropriate territories, eliminate superfluous cells, and induce terminal differentiation of survivors into the appropriate cell-types. Despite strict controls constraining developmental processes, malignancies can emerge in still immature organisms. This is the case of neuroblastoma (NB), a highly heterogeneous disease, predominantly affecting children before the age of 5 years. Highly metastatic forms represent half of the cases and are diagnosed when disseminated foci are detectable. NB arise from a transient population of embryonic cells, the neural crest (NC), and especially NC committed to the establishment of the sympatho-adrenal tissues. The NC is generated at the dorsal edge of the neural tube (NT) of the vertebrate embryo, under the action of NC specifier gene programs. NC cells (NCCs) undergo an epithelial to mesenchymal transition, and engage on a remarkable journey in the developing embryo, contributing to a plethora of cell-types and tissues. Various NCC sub-populations and derived lineages adopt specific migratory behaviors, moving individually as well as collectively, exploiting the different embryonic substrates they encounter along their path. Here we discuss how the specific features of NCC in development are re-iterated during NB metastatic behaviors.

6. Septin functions during neuro-development, a yeast perspective
   Falk J, Boubakar L, Castellani V.
   Curr Opin Neurobiol (2019) — Résumé

   Septins, discovered almost half a century ago in yeast, have prominent contributions in a broad range of morphological and functional processes from yeast to human. Septins now emerge as key players of neurodevelopment and more specifically of the mechanisms driving the complex morphological differentiation and compartmentalization of neurons that are fundamental to their function. We review here recent advances in Septin-mediated processes of neuron differentiation, which enlighten similarities and differences between neuron and yeast polarity programs.

7. Commissural axon navigation in the spinal cord: A repertoire of repulsive forces is in command.
   Ducuing H, Gardette T, Pignata A, Tauszig-Delamasure S, Castellani V.
   Semin Cell Dev Biol. (2019) — Résumé

   The navigation of commissural axons in the developing spinal cord has attracted multiple studies over the years. Many important concepts emerged from these studies which have enlighten the general mechanisms of axon guidance. The navigation of commissural axons is regulated by a series of cellular territories which provides the diverse guidance information necessary to ensure the successive steps of their pathfinding towards, across, and away from the ventral midline. In this review, we discuss how repulsive forces, by propelling, channelling, and confining commissural axon navigation, bring key contributions to the formation of this neuronal projection.

8. Microenvironment-Driven Shift of Cohesion/Detachment Balance within Tumors Induces a Switch toward Metastasis in Neuroblastoma.
   Delloye-Bourgeois C, Bertin L, Thoinet K, Jarrosson L, Kindbeiter K, Buffet T, Tauszig-Delamasure S, Bozon M, Marabelle A, Combaret V, Bergeron C, Derrington E, Castellani V.
   Cancer Cell (2017) — Résumé

   Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.

9. Molecular Memory of Morphologies by Septins during Neuron Generation Allows Early Polarity Inheritance.
   Boubakar L, Falk J, Ducuing H, Thoinet K, Reynaud F, Derrington E, Castellani V.
   Neuron (2017) — Résumé

   Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity.

10. Genetic specification of left-right asymmetry in the diaphragm muscles and their motor innervation.
    Charoy C, Dinvaut S, Chaix Y, Morlé L, Sanyas I, Bozon M, Kindbeiter K, Durand B, Skidmore JM, De Groef L, Seki M, Moons L, Ruhrberg C, Martin JF, Martin DM, Falk J, Castellani V.
    Elife (2017) — Résumé

    The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

11. Cerebrospinal fluid-derived Semaphorin3B orients neuroepithelial cell divisions in the apicobasal axis.
    Arbeille E, Reynaud F, Sanyas I, Bozon M, Kindbeiter K, Causeret F, Pierani A, Falk J, Moret F, Castellani V.
    Nature Communications (2015) — Résumé

    The spatial orientation of cell divisions is fundamental for tissue architecture and homeostasis. Here we analysed neuroepithelial progenitors in the developing mouse spinal cord to determine whether extracellular signals orient the mitotic spindle. We report that Semaphorin3B (Sema3B) released from the floor plate and the nascent choroid plexus in the cerebrospinal fluid (CSF) controls progenitor division orientation. Delivery of exogenous Sema3B to neural progenitors after neural tube opening in living embryos promotes planar orientation of their division. Preventing progenitor access to cues present in the CSF by genetically engineered canal obstruction affects the proportion of planar and oblique divisions. Sema3B knockout phenocopies the loss of progenitor access to the CSF. Sema3B binds to the apical surface of mitotic progenitors and exerts its effect via Neuropilin receptors, GSK3 activation and subsequent inhibition of the microtubule stabilizer CRMP2. Thus, extrinsic control mediated by the Semaphorin signalling orients progenitor divisions in neurogenic zones.

12. PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments.
    Delloye-Bourgeois C, Jacquier A, Charoy C, Reynaud F, Nawabi H, Thoinet K, Kindbeiter K, Yoshida Y, Zagar Y, Kong Y, Jones YE, Falk J, Chédotal A, Castellani V.
    Nature Neuroscience (2014) — Résumé

    Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1-SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors.

Financements passés

• European Research Council

Offre Post-doc