SINGLE-CELL LINEAGE ANALYSIS IN OVO

SANCHEZ, MARIO; FIGUERES OÑATE, MARÍA; LÓPEZ MASCARAQUE, LAURA; OLMOS CARREÑO, CINDY; SCICOLONE, GABRIEL

Asturias

Congreso; VII Olfatory Meeting. Red Olfativa Española.; 2018

Red Olfativa Española.

SINGLE-CELL LINEAGE ANALYSIS IN OVOCINDY LORENA OLMOS CARREÑO1, MARIA FIGUERES-OÑATE2, MARIO SANCHEZ2 , GABRIEL SCICOLONE1, LAURA LÓPEZ-MASCARAQUE2. 1. Instituto de Biología Celular y Neurociencias ?Profesor Eduardo De Robertis? (UBA-CONICET), Facultad de Medicina, UBA, Buenos Aires, Argentina. 2. Instituto Cajal-CSIC, Madrid, Spain. Clonal cell analysis defines the potential of single cells, allowing todecode the neural heterogeneity of the different cell lineages and their clonal relationships. At this respect, we have adapted the mouse genetic tracing strategy, UbC-StarTrack (Figueres-Oñate et al., 2016), to a chick embryo model. UbC-StarTrack is based on gene transfection of different fluorescent reporter proteins, six in the cytoplasm and six in the nucleus, driven by an ubiquitous promoter in PiggyBac-based vectors. This will allow producing inheritable marks that enable the long-term in vivo tracing the progenitor cell and all its descendants. In this way this strategy attributes a specific and unique color-code to single neural precursors, to determine to which extent a single cell has a multi-lineage potential or whether those progenitors comprise several pools generating different neural types.To evaluate the efficency of this system in chicken model, we first analyzed the expression of two plasmids, an integrable plasmid containing eGFP under the control of the Ubiquitin C (UbC) promoter and other non integrable containing mCherry in transfected neurospheres obtained from dissociated cells of the retinal ciliary margin (CM). Then, UbC-StarTtrack constructs were in ovo co-electroporated into the embryonic retinal CM at 3.5 days (E3.5), to label the cell progeny of progenitor/stem cells at different post-electroporation days (two, five and eight-days). This allowed us to determine the spatial dispersion, migration and differentiation of CM stem/progenitor cells in the retina at the selected developmental chick stages. Moreover, we will be able to determine both the cell types originated from single cells and their clonal relationships within the retina. In conclusion: 1) UbC-StarTrack is useful in chicken model and derivatives of single-labelled cells were found in retina, tectum and optic thalamus. 2) Cell clones formed columnar associations of cells between the inner and outer limitants. 3) Cell clones displayed a large dispersion along the dorso-ventral axis, whereas their dispersion was limited by the anterior-posterior axis. 4) At these experimental ages, mixed cell clones were not detected. Supported by Research Grants: BFU2016-75207-R from MINECO and MHE-200016, PIP 0441 from CONICET and UBACYT 0526.Key words: progenitors/stem cells, Ciliary margin, Clonal analysis, UbC-StarTrack, chick embryo.