Clonal analysis of neural progenitors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 28-10-2016Esta tesis tiene embargado el acceso al texto completo hasta el 28-04-2018A key question in developmental neurobiology is how a pool of progenitors proliferates and differentiates to create an adult brain of appropriate size and cellular composition. So important is the control of the final size, as the appropriate distribution of cells with different embryonic origins. Each neural progenitor should produce a certain number of neuronal/glial cells encompassing a clone, and all these clones together will result in the adult functional nervous system. The overall goal of this thesis was aimed to develop an in vivo lineage-tracing genetic method to trace all the neural progeny derived from a single cell, UbC-StarTrack. This will allow to follow cell dispersion of the progeny from embryonic and postnatal mice neural progenitors, independently of their lineage. To validate the method we selected as an experimental system the olfactory bulb, since it is one of the main regions of embryonic and adult neurogenesis. Then, the main experimental approach was based on gene transfection by electroporation with a combination of diverse fluorescent reporter proteins. This produced inheritable marks that enabled the long-term in vivo tracing of the different neural cells from its generation, during embryonic development, to its final fate in the adult brain. First, we analyzed the fate of embryonic progenitors lining either the ventricular surface or the ependymal layer of the olfactory bulbs. Second, we addressed the fate of postnatal progenitors form the dorsolateral region of the ventricular surface, to finally perform a clonal analysis of newly generated olfactory bulb cells from those postnatal SVZ progenitors. This thesis will advance the understanding of cell heterogeneity that can be decoded by studying their ontogenetic origin, and could yield to track cell lineages to understand their functional clonal relationships.Una de las preguntas más importantes en Neurobiología del Desarrollo es como un grupo de progenitores prolifera y se diferencia para crear un cerebro con el tamaño y composición celular adecuado. Tan importante es el control del tamaño final, como la correcta distribución de células con distinto origen embrionario. Cada progenitor debe generar un determinado numero de células gliales/neuronales y estos clones resultarán en el sistema nervioso central adulto funcional. El objetivo general del trabajo presentado en esta Tesis Doctoral fue generar un método de análisis clonal ubicuo con el propósito de trazar toda la progenie de células individuales, seguir clones pertenecientes a distintos linajes neurales provenientes de progenitores tanto embrionarios como postnatales. El método desarrollado se basa en la identificación de células hermanas mediante el análisis del especifico código de colores que expresan, generado por la combinación aleatoria de distintos reporteros fluorescentes en las células progenitoras y transmitido de manera equitativa a toda la descendencia. Los estudios de análisis clonal de los distintos linajes neurales se han centrado en el sistema olfativo del ratón, ya que es uno de los centros donde tiene lugar la neurogénesis tanto en estadios embrionarios como postnatales. El principal enfoque experimental llevado a cabo, se basa en la transfección mediante electroporación de distintos constructos genómicos que expresan proteínas fluorescentes diferentes bajo un promotor ubicuo. Esto produce marcas estables hereditables que permiten el marcaje in vivo a largo plazo de células neurales desde su generación en el desarrollo embrionario hasta su destino final en el cerebro adulto. El presente trabajo de tesis contiene los detalles de la generación de un método ubicuo de análisis clonal al que hemos denominado UbC-StarTrack, en primer lugar haciendo una comparativa de la expresión de vectores integrados en el genoma vs. vectores que permanecen sin integrar. En segundo lugar, hemos analizado la descendencia de progenitores embrionarios, posicionados en la superficie del ventrículo lateral o de la zona ependimaria intrabulbar, que convergen en el bulbo olfativo adulto. Seguidamente, examinamos el destino de progenitores postnatales localizados en la región dorso-lateral adyacente a los ventrículos laterales. Para concluir, mostramos los resultados clonales preliminares obtenidos con el método de análisis clonal desarrollado en las células generadas a partir de progenitores postnatales, implicadas en la neurogénesis en adulto que tiene lugar en el sistema olfativo del ratón. Con todo esto, este trabajo de Tesis Doctoral exhibe un avance en el conocimiento de la heterogeneidad celular, siendo ésta decodificada a través de su ontogenia. Además, abre un nuevo campo al estudio de los distintos tipos celulares no sólo por su linaje sino por su relación clonal, haciendo posible el análisis de la interrelación entre los clones obtenidos y sus implicaciones fisiológicas

Spatiotemporal analyses of neural lineages after embryonic and postnatal progenitor targeting combining different reporters

Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.This work was supported by research Grant BFU2013-48807-R from the Spanish Ministry of Economy and Competitiveness.Peer reviewedPeer Reviewe

Lineage Tracing and Cell Potential of Postnatal Single Progenitor Cells In Vivo.

Understanding the contribution of adult neural progenitor cells (NPCs) and their lineage potential is a great challenge in neuroscience. To reveal progenitor diversity and cell-lineage relationships of postnatal NPCs in the subventricular zone (SVZ), we performed in vivo lineage-tracing genetic analysis using the UbC-StarTrack. We determined the progeny of single SVZ-NPCs, the number of cells per clone, the dispersion of sibling cells, and the cell types within clones. Long-term analysis revealed that both the cell-dispersion pattern and number of cells comprising clones varied depending on the glial/neuronal nature of sibling cells. Sibling-olfactory interneurons were primarily located within the same layer, while sibling-glial cells populated SVZ-adjacent areas. Sibling astrocytes and interneurons did not form big clones, whereas oligodendroglial-lineage clones comprised the largest clones originated in adult brains. These results demonstrate the existence of SVZ postnatal bipotential progenitors that give rise to clones widely dispersed across the olfactory bulb and SVZ-adjacent areas.We are thankful to the Animal Facility staff for their assistance. We are also very grateful to Carmen Hernandez and Belen Garcia from the Imaging and Microscopy Facility for their support with ImageJ macro design and confocal microscopy, along with Emilio Tejera for his technical assistance. The authors also thank Hayley Colman, Michael Kawaja, and Rebekah Jordan Barnett for their English review of the manuscript. This work was supported by research grant number BFU2016-75207-R from the Spanish Ministry of Economy and Competitiveness

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells

Development of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor

The adult mouse brain contains an extensive neuro-genic niche in the lateral walls of the lateral ventricles.This epithelium, which has a unique pinwheel organi-zation, contains multiciliated ependymal (E1) cellsand neural stem cells (B1). This postnatal germinalepithelium develops from the embryonic ventricularzone, but the lineage relationship between E1 andB1 cells remains unknown. Distinct subpopulationsof radial glia (RG) cells in late embryonic and earlypostnatal development either expand their apicaldomain >11-fold to form E1 cells or retain small api-cal domains that coalesce into the centers of pin-wheels to form B1 cells. Using independent methodsof lineage tracing, we show that individual RG cellscan give rise to clones containing E1 and B1 cells.This study reveals key developmental steps in theformation of the postnatal germinal niche and theshared cellular origin of E1 and B1 cells.The authors would like to thank members of the Alvarez-Buylla laboratory forhelpful discussions, William Walantus for technical expertise, and KennethXavier Probst of Xavier Studio for cell illustration designs. This study was sup-ported by funding from the Howard Hughes Medical Institute/Helen Hay Whit-ney Foundation (to L.C.F.); Becas Chile, Ministerio de Educación (to J.I.P.); Deutsche Forschungsgemeinschaft (to K.O.); NIH grant F32 NS103221 (toS.A.R.); MINECO (BFU2016-80360-R; to M.F.-O.); and NIH grants R37HD32116 and R01 NS28478, the Heather and Melanie Muss Endowed Chair,and a generous gift from the John G. Bowes Research Fund (to A.A.-B.)

Multiple origins and modularity in the spatiotemporal emergence of cerebellar astrocyte heterogeneity.

The morphological, molecular, and functional heterogeneity of astrocytes is under intense scrutiny, but how this diversity is ontogenetically achieved remains largely unknown. Here, by quantitative in vivo clonal analyses and proliferation studies, we demonstrate that the major cerebellar astrocyte types emerge according to an unprecedented and remarkably orderly developmental program comprising (i) a time-dependent decline in both clone size and progenitor multipotency, associated with clone allocation first to the hemispheres and then to the vermis(ii) distinctive clonal relationships among astrocyte types, revealing diverse lineage potentials of embryonic and postnatal progenitors; and (iii) stereotyped clone architectures and recurrent modularities that correlate to layer-specific dynamics of postnatal proliferation/differentiation. In silico simulations indicate that the sole presence of a unique multipotent progenitor at the source of the whole astrogliogenic program is unlikely and rather suggest the involvement of additional committed components

Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes

A broad molecular framework of how neural stem cells are specified toward astrocyte fate during brain development has proven elusive. Here we perform comprehensive and integrated transcriptomic and epigenomic analyses to delineate gene regulatory programs that drive the developmental trajectory from mouse embryonic stem cells to astrocytes. We report molecularly distinct phases of astrogliogenesis that exhibit stage- and lineage-specific transcriptomic and epigenetic signatures with unique primed and active chromatin regions, thereby revealing regulatory elements and transcriptional programs underlying astrocyte generation and maturation. By searching for transcription factors that function at these elements, we identified NFIA and ATF3 as drivers of astrocyte differentiation from neural precursor cells while RUNX2 promotes astrocyte maturation. These transcription factors facilitate stage-specific gene expression programs by switching the chromatin state of their target regulatory elements from primed to active. Altogether, these findings provide integrated insights into the genetic and epigenetic mechanisms steering the trajectory of astrogliogenesis