Equipe corticogenèse

Feret N, Megido AC, Kuony A, Marangoni P, Fichter L, Garcia Llorens S, Attina A, Nhiri N, Jacquet E, Vialaret J, Daien V, David A, Hirtz C, Loulier K, Klein OD, Michon F. Decoding epithelial regeneration in the cornea: multi-omic analysis reveals cellular plasticity as central mechanism. Cell Mol Biol Lett. 2025 Nov 2;30(1):131. doi: 10.1186/s11658-025-00804-9. PMID: 41177882; PMCID: PMC12579813.

Background: Rapid and efficient epithelial regeneration is fundamental for tissue homeostasis and proper function. As the outermost ocular structure, the cornea is transparent, multilayered, and vital for clear vision. Due to its exposed position, the cornea frequently undergoes various forms of injury affecting either the epithelium itself or its surrounding microenvironment, including corneal innervation and the tear film. Corneal abrasion, occurring commonly through trauma or as part of refractive surgical procedures, is typically viewed as a minor event since it usually resolves rapidly. Consequently, the cornea serves as an excellent model for studying epithelial wound healing. However, complications such as persistent epithelial defects or corneal opacity can develop, underscoring critical gaps in understanding the underlying molecular mechanisms. Methods: Utilizing a unilateral corneal abrasion mouse model, we conducted a comprehensive multi-omics analysis, integrating transcriptomics, proteomics, and epitranscriptomics, to dissect the dynamic molecular responses post-injury in both wounded and contralateral tissues. To elucidate the role of the tear film, we performed additional studies involving lacrimal gland ablation combined with corneal injury. We applied RNA sequencing to profile transcriptomic changes in corneal and lacrimal gland tissues, and mass spectrometry to study tear proteomics and epitranscriptomic modifications. Results: We revealed a major modulation of the cornea transcriptome after abrasion, suggesting a regulation of pathways including JAK-STAT, Wnt and TGF-β, and a reduction of nucleoside modifications. The lacrimal gland transcriptome and tears proteome were also significantly affected. Plus, we highlighted a bilateralization, both in the cornea transcriptome and tears proteome. In the tear-deficient conditions, the wound closure rate and molecular responses were altered, and the bilateralization was impacted, with an increased matrix remodeling and a modulation of keratins expression. Conclusions: Our multi-omics analyses revealed extensive epithelial cellular plasticity as a key mechanism driving rapid wound closure, characterized by profound remodeling of transcriptional networks and RNA modifications. Importantly, we uncovered a previously underappreciated role of the lacrimal gland and tear film in mediating bilateral molecular responses following unilateral injury, emphasizing their pivotal roles in tissue regeneration. Additionally, we identified novel regulatory roles for RNA methylation events and critical signaling pathways implicated in epithelial healing.

Feret N, Ter Schiphorst S, Kindermans J, Crowdy H, Fichter L, Vialaret J, Hirtz C, Loulier K, Michon F, Daien V. Temporal Analysis of Tear Fluid Proteome Reveals Critical Corneal Repair Events After Photorefractive Surgery. Invest Ophthalmol Vis Sci. 2025 Oct 1;66(13):49. doi: 10.1167/iovs.66.13.49. PMID: 41147948; PMCID: PMC12577742.

Purpose: To map time-resolved tear proteome changes during corneal epithelial wound healing after photorefractive keratectomy (PRK) and nominate tear-based biomarker panels with translational potential. Methods: Tears from 10 healthy adults were collected before PRK (Pre), ∼30-60 minutes after PRK (D0), and day 3 (D3) using Schirmer strips. Proteins were extracted (SP3), trypsin-digested, and analyzed by DIA on an Evosep One-timsTOF HT platform. DIA-NN (1% FDR) provided identification/quantification. Paired contrasts (Pre vs. D0; Pre vs. D3) defined differentially abundant proteins (adjusted P < 0.05; |fold change|≥2) for pathway enrichment. A stringent screen (adjusted P < 0.001; FC>2.5) and UniProt-based annotation distinguished secreted versus intracellular candidates. Results: Across 2025 identified proteins, 909 (∼45%) were significantly modulated. D0 tears showed an "injury/ECM" program (e.g., TGFBI, lumican, keratocan, fibrinogen chains, complement C8B, APOA1), whereas D3 shifted toward remodeling and epithelial polarity with regulated proteolysis (e.g., SERPINA3, MMP7, THBS1, MUC5B, CXCL17). A subset persisted across time points (e.g., A2M, haptoglobin, OLFML3). Intracellular signatures paralleled phase transitions: D0 was enriched for chromatin/DNA-repair and RNA-binding proteins (histones, XRCC5/Ku80, HNRNPs), whereas D3 highlighted transport/cytoskeletal factors linked to apical remodeling and fluid/ion handling (AQP5, NHERF1/EBP50, ANO1/TMEM16A, ACTN1, RDX). H6PD was elevated at both D0 and D3. Conclusions: Time-stamped, tear-accessible biomarker panels emerge from this analysis: early-injury (TGFBI, lumican/keratocan, fibrinogen/complement), sustained (A2M, haptoglobin, OLFML3), and late-remodeling/epithelial (SERPINA3, MMP7, THBS1, MUC5B, CXCL17), with complementary intracellular markers (AQP5/NHERF1/ANO1). These candidates may support perioperative monitoring, risk stratification (delayed closure, stromal haze), and phase-adapted therapy after PRK, warranting prospective clinical validation.

Dumas L, Durand J and Loulier K (2025) Multicolor Cell Lineage Tracing Using MAGIC Markers Strategies. Methods Mol Biol. 2025;2886:47-63. doi: 10.1007/978-1-0716-4310-5_3. PMID: 39745635

Multicolor MAGIC Markers strategies are useful lineage tracing tools to study brain development at a multicellular scale. In this chapter, we describe an in utero electroporation method to simultaneously label multiple neighboring progenitors and their respective progeny using these multicolor reporters. In utero electroporation enables the introduction of any gene of interest into embryonic neural progenitors lining the brain ventricles through a simple pipeline consisting of a micro-injection followed by the application of electrical pulses. Successful in utero electroporation requires a concise yet complete understanding of each step of the surgical protocol, spanning from the preoperative preparation to the postoperative care, as well as the MAGIC Markers tool outlined in this study. Besides a detailed protocol, we present non-integrative and integrative approaches to demonstrate the range of cell and lineage tracking possibilities of multicolored progenitors and their descent over time.

Alfahel L, Gschwendtberger T, Kozareva V, Dumas L, Gibbs R, Kertser A, Baruch K, Zaccai S, Kahn J, Thau-Habermann N, Eggenschwiler R, Sterneckert J, Hermann A, Sundararaman N, Vaibhav V, Van Eyk JE, Rafuse VF, Fraenkel E, Cantz T, Petri S, Israelson A. Targeting low levels of MIF expression as a potential therapeutic strategy for ALS. Cell Rep Med. 2024 May 21;5(5):101546. doi: 10.1016/j.xcrm.2024.101546. Epub 2024 May 3. PMID: 38703766; PMCID: PMC11148722.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.

Farhat B, Bordeu I, Jagla B, Ibrahim S, Stefanovic S, Blanc H, Loulier K, Simons BD, Beaurepaire E, Livet J, Pucéat M. Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve. Dev Cell. 2024 Feb 5;59(3):339-350.e4. doi: 10.1016/j.devcel.2023.12.006. Epub 2024 Jan 9. PMID: 38198889.

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.

Clavreul S, Dumas L and Loulier K (2022) Astrocyte development in the cerebral cortex:Complexity of their origin, genesis,and maturation.Front. Neurosci. 16:916055. doi: 10.3389/fnins.2022.916055

In the mammalian brain, astrocytes form a heterogeneous population at the morphological, molecular, functional, intra-, and inter-region levels. In the past, a few types of astrocytes have been first described based on their morphology and, thereafter, according to limited key molecular markers. With the advent of bulk and single-cell transcriptomics, the diversity of astrocytes is now progressively deciphered and its extent better appreciated. However, the origin of this diversity remains unresolved, even though many recent studies unraveled the specificities of astroglial development at both population and individual cell levels, particularly in the cerebral cortex. Despite the lack of specific markers for each astrocyte subtype, a better understanding of the cellular and molecular events underlying cortical astrocyte diversity is nevertheless within our reach thanks to the development of intersectional lineage tracing, microdissection, spatial mapping, and single-cell transcriptomic tools. Here we present a brief overview describing recent findings on the genesis and maturation of astrocytes and their key regulators during cerebral cortex development. All these studies have considerably advanced our knowledge of cortical astrogliogenesis, which relies on a more complex mode of development than their neuronal counterparts, that undeniably impact astrocyte diversity in the cerebral cortex.

Dumas, L., Clavreul, S., Michon, F. et al. Multicolor strategies for investigating clonal expansion and tissue plasticity. Cell. Mol. Life Sci. 79, 141 (2022). https://doi.org/10.1007/s00018-021-04077-1

Understanding the generation of complexity in living organisms requires the use of lineage tracing tools at a multicellular scale. In this review, we describe the different multicolor strategies focusing on mouse models expressing several fluorescent reporter proteins, generated by classical (MADM, Brainbow and its multiple derivatives) or acute (StarTrack, CLoNe, MAGIC Markers, iOn, viral vectors) transgenesis. After detailing the multi-reporter genetic strategies that serve as a basis for the establishment of these multicolor mouse models, we briefly mention other animal and cellular models (zebrafish, chicken, drosophila, iPSC) that also rely on these constructs. Then, we highlight practical applications of multicolor mouse models to better understand organogenesis at single progenitor scale (clonal analyses) in the brain and briefly in several other tissues (intestine, skin, vascular, hematopoietic and immune systems). In addition, we detail the critical contribution of multicolor fate mapping strategies in apprehending the fine cellular choreography underlying tissue morphogenesis in several models with a particular focus on brain cytoarchitecture in health and diseases. Finally, we present the latest technological advances in multichannel and in-depth imaging, and automated analyses that enable to better exploit the large amount of data generated from multicolored tissues.

Dumas, L., Clavreul, S., Durand, J., Hernandez-Garzon, E., Abdeladim, L., Barry-Martinet, R., Caballero-Megido, A., Beaurepaire, E., Bonvento, G., Livet, J., Loulier, K. In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes. J. Vis. Exp. (159), e61110, doi:10.3791/61110 (2020).

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

Oriol Ros, Sarah Baudet, Yvrick Zagar, Karine Loulier, Fiona Roche, Sandrine Couvet, Alain Aghaie, Melody Atkins, Alice Louail, Christine Petit, Christine Metin, Yves Mechulam and Xavier Nicol. Cell Reports32, 107934, July 21, 2020.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Takuma Kumamoto, Franck Maurinot, Raphaëlle Barry-Martinet, Célia Vaslin, Sandrine Vandormael-Pournin,Mickaël Le, Marion Lerat, Dragos Niculescu, Michel Cohen-Tannoudji, Alexandra Rebsam, Karine Loulier, Stéphane Nedelec, Samuel Tozer, and Jean Livet. Neuron107, 617–630, August 19, 2020.

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cellstudies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis re-mains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes.Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression uponincorporation into the host genome through transposition, thus enabling rapid and accurate identificationof integration events following transfection with naked plasmids.In vitro, iOn permits rapid drug-free stabletransgenesis of mouse and human pluripotent stem cells with multiple vectors.In vivo, we demonstrate faith-ful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatictransgenesis experiments that reveal neural progenitor potentialities and interaction. These results establishiOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systemsand model organisms by conditioning transgene activation to genomic integration.

Solène Clavreul, Lamiae Abdeladim, Edwin Hernández-Garzón, Dragos Niculescu, Jason Durand, Sio-Hoï Ieng, Raphaëlle Barry, Gilles Bonvento, Emmanuel Beaurepaire, Jean Livet & Karine Loulier
Nature Communications volume 10, Article number: 4884 (2019)

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.

Oriol Ros, Yvrick Zagar, Solène Ribes, Sarah Baudet, Karine Loulier, Sandrine Couvet, Delphine Ladarre, Alain Aghaie, Alice Louail, Christine Petit , Yves Mechulam, Zsolt Lenkei, Xavier Nicol
Cell Rep. 2019 Jun 25;27(13):4003-4012.e6. doi: 10.1016/j.celrep.2019.05.102.

cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation.

Lamiae Abdeladim, Katherine S. Matho, Solène Clavreul, Pierre Mahou, Jean-Marc Sintes, Xavier Solinas, Ignacio Arganda-Carreras, Stephen G. Turney, Jeff W. Lichtman, Anatole Chessel, Alexis-Pierre Bemelmans, Karine Loulier, Willy Supatto, Jean Livet & Emmanuel Beaurepaire
Nature Communicationsvolume 10, Article number: 1662 (2019)

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one‐shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.

Guesmi K, Abdeladim L, Tozer S, Mahou P, Kumamoto T, Jurkus K, Rigaud P,Loulier K, Dray N, Georges P, Hanna M, Livet J, Supatto W, Beaurepaire E, Druon F.
Light Sci Appl. 2018 Jun 6;7:12. doi: 10.1038/s41377-018-0012-2. eCollection 2018.

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.

Saadaoui M, Konno D, Loulier K, Goiame R, Jadhav V, Mapelli M, Matsuzaki F, Morin X
EMBO Rep. 2017 Sep;18(9):1509-1520. doi: 10.15252/embr.201643048. Epub 2017 Jul 6

In many cell types, mitotic spindle orientation relies on the canonical “LGN complex” composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαi in vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species‐specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.

Dumas L, Heitz-Marchaland C, Fouquet S, Suter S, Livet J, Moreau-Fauvarque C, Chédotal A.
Glia. 2015 Apr;63(4):699-717. doi: 10.1002/glia.22779. Epub 2014 Dec 21.

Oligodendrocytes are the myelinating cells of the central nervous system. Multiple markers are available to analyze the populations of oligodendroglial cells and their precursors during development and in pathological conditions. However, the behavior of oligodendrocytes remains poorly characterized in vivo, especially at the level of individual cells. Studying this aspect has been impaired so far by the lack of suitable methods for visualizing single oligodendrocytes, their processes, and their interactions during myelination. Here, we have used multicolor labeling technology to single-out simultaneously many individual oligodendrocytes in the postnatal mouse optic nerve. This method is based on Brainbow, a transgenic system for stochastic expression of multiple fluorescent protein genes through Cre-lox recombination, previously used for visualizing axons and neurons. We used tamoxifen-inducible recombination in myelinating cells of Brainbow transgenic mice to obtain multicolor labeling of oligodendrocytes. We show that the palette of colors expressed by labeled oligodendrocytes is tamoxifen dependent, with the highest doses producing the densest and most colorful labeling. At low doses of tamoxifen, the morphology of single or small clusters of fluorescent oligodendrocytes can be studied during postnatal development and in adult. Internodes are labeled to their extremities, revealing nodes of Ranvier. The new mouse model presented here opens new possibilities to explore the organization and development of the oligodendrocyte network with single-cell resolution.

Loulier K; Raphaëlle Barry; PierreMahou; Yann Le Franc; WillySupatto; Katherine S.Matho; SiohoiIeng; Stéphane Fouquet; Elisabeth Dupin; Ryad Benosman; Alain Chédotal; Emmanuel Beaurepaire; Xavier Morin; JeanLivet
Neuron. 2014 Feb 5;81(3):505-20. doi: 10.1016/j.neuron.2013.12.016

We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor’s descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.

Tabansky I, Lenarcic A, Draft RW, Loulier K, Keskin DB, Rosains J, Rivera-Feliciano J, Lichtman JW, Livet J, Stern JN, Sanes JR, Eggan K
Curr Biol. 2013 Jan 7;23(1):21-31. doi: 10.1016/j.cub.2012.10.054. Epub 2012 Nov 21

Mahou P, Zimmerley M, Loulier K, Matho KS, Labroille G, Morin X, Supatto W, Livet J, Débarre D, Beaurepaire E.
Nat Methods. 2012 Jul 8;9(8):815-8. doi: 10.1038/nmeth.2098.

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

Loulier K, Lathia JD, Marthiens V, Relucio J, Mughal MR, Tang SC, Coksaygan T, Hall PE, Chigurupati S, Patton B, Colognato H, Rao MS, Mattson MP, Haydar TF, Ffrench-Constant C.
PLoS Biol. 2009 Aug;7(8):e1000176. doi: 10.1371/journal.pbio.1000176. Epub 2009 Aug 18.

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin α2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects

Angot E, Loulier K, Nguyen-Ba-Charvet KT, Gadeau AP, Ruat M, Traiffort E
Stem Cells. 2008 Sep;26(9):2311-20. doi: 10.1634/stemcells.2008-0297. Epub 2008 Jul 10.

The adult subventricular zone (SVZ) supports neural stem cell self‐renewal and differentiation and continually gives rise to new neurons throughout adult life. The mechanisms orienting the migration of neuroblasts from the SVZ to the olfactory bulb (OB) via the rostral migratory stream (RMS) have been extensively studied, but factors controlling neuroblast exit from the SVZ remain poorly explored. The morphogen Sonic Hedgehog (Shh) displays proliferative and survival activities toward neural stem cells and is an axonal chemoattractant implicated in guidance of commissural axons during development. We identify here the presence of Shh protein in SVZ extracts and in the cerebrospinal fluid of adult mice, and we demonstrate that migrating neuroblasts in the SVZ and RMS express the Shh receptor Patched. We show that Shh displays a chemoattractive activity in vitro on SVZ‐derived neuronal progenitors, an effect blocked by Cur61414, a Smoothened antagonist. Interestingly, Shh‐expressing cells grafted above the RMS of adult mice exert a chemoattractive activity on migrating neuroblasts in vivo, thus inducing their accumulation and deviation from their normal migratory pathway. Furthermore, the adenoviral transfer of Shh into the lateral ventricle or the blocking of Shh present in the SVZ of adult mice using its physiological antagonist Hedgehog interacting protein or neutralizing Shh antibodies provides in vivo evidence that Shh can retain SVZ‐derived neuroblasts. The ability to modulate the number of neuroblasts leaving the SVZ and reaching the OB through the chemoattractive activity of Shh suggests a novel degree of plasticity in cell migration of this adult stem cell niche.

Loulier K, Ruat M, Traiffort E
J Neurochem. 2006 Jul;98(2):530-42

Sonic hedgehog signaling is required for the maintenance of stem cell niches in the postnatal subventricular zone and the proliferation of neural progenitors in the mature hippocampus. We show here that delivery of Sonic hedgehog protein into the lateral ventricle of adult mice increases cell proliferation in the corpus callosum and cerebral cortex. In this latter area, the number of neural progenitors expressing the proteoglycan NG2 is enhanced 2 days after the injection. In both areas, mRNA up‐regulation of the transcriptional target gene Patched was observed in cells expressing the oligodendroglial transcription factor Olig1. Twenty‐six days following the adenovirus‐mediated delivery of Sonic hedgehog into the lateral ventricle, newly generated cells in the cerebral cortex and in the corpus callosum are influenced towards the initial steps of oligodendrogenesis, as indicated by a 50% increase in the number of cells expressing the oligodendroglial marker DM20. Our experiments demonstrate that the number of oligodendrocyte precursor cells in the cerebral cortex and corpus callosum can be increased upon delivery of Sonic hedgehog proteins and highlight the potential capacity of the adult brain to mobilize a pool of premyelinating cells.

Rossel M, Loulier K, Feuillet C, Alonso S, Carroll P.
Development. 2005 Mar;132(6):1175-85. Epub 2005 Feb 9.

The cytoarchitecture of the hindbrain results from precise and co-ordinated sequences of neuronal migrations. Here, we show that reelin, an extracellular matrix protein involved in neuronal migration during CNS development, is necessary for an early, specific step in the migration of several hindbrain nuclei. We identified two cell populations not previously known to be affected in reeler mutants that show a common migratory defect: the olivocochlear efferent neurons and the facial visceral motor nucleus. In control embryos, these cells migrate first toward a lateral position within the neural tube, and then parallel to the glial cell processes, to a ventral position where they settle close to the pial surface. In reeler mutants, the first migration is not affected, but the neurons are unable to reach the pial surface and remain in an ectopic position. Indeed, this is the first evidence that the migration of specific hindbrain nuclei can be divided into two parts: a reelin-independent and a reelin-dependent migration. We also show that reelin is expressed at high levels at the final destination of the migratory process, while the reelin intracellular effector Dab1 was expressed by cell groups that included the two populations affected. Mice mutant at the Dab1 locus, called scrambler, exhibit the same phenotype, a failure of final migration. However, examination of mice lacking both reelin receptors, ApoER2 and VLDLR, did not reveal the same phenotype, suggesting involvement of an additional reelin-binding receptor. In the hindbrain, reelin signaling might alter the adhesive properties of efferent neurons and their ability to respond to directional cues, as has been suggested for the migration of olfactory bulb precursors.

Hack I, Bancila M, Loulier K, Carroll P, Cremer H.
Nat Neurosci. 2002 Oct;5(10):939-45.

During development, Reelin acts on migrating neuronal precursors and controls correct cell positioning in the cortex and other brain structures by a hitherto unidentified mechanism. Here we show that in the postnatal mouse brain, Reelin acts as a detachment signal for chain-migrating interneuron precursors in the olfactory bulb. Neuronal precursors cultured in Matrigel detached from chains and migrated individually in the presence of exogenously added Reelin protein or Reelin-expressing brain tissues. Furthermore, we found that in reeler mutant mice, neuronal precursors accumulated in the olfactory bulb and remained in clusters, indicating that they did not change from tangential chain-migration to radial individual migration. Our data provide direct evidence that Reelin acts as a detachment signal, but not a stop or guidance cue. We propose that Reelin may have comparable functions during development.