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SummaryUnderstanding 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.

Determining the origin of different glial subtypes is crucial to understand glial heterogeneity, and to enhance our knowledge of glial and progenitor cell behavior in embryos and adults. NG2-glia are homogenously distributed in a grid-like manner in both, gray and white matter of the adult brain. While some NG2-glia in the CNS are responsible for the generation of mature oligodendrocytes (OPCs), most of them do not differentiate and they can proliferate outside of adult neurogenic niches. Thus, NG2-glia constitute a heterogeneous population containing different subpopulations with distinct functions. We hypothesized that their diversity emerges from specific progenitors during development, as occurs with other glial cell subtypes. To specifically target NG2-pallial progenitors and to define the NG2-glia lineage, as well as the NG2-progenitor potential, we designed two new StarTrack strategies using the NG2 promoter. These approaches label NG2 expressing progenitor cells, permitting the cell fates of these NG2 progenitors to be tracked in vivo. StarTrack labelled cells producing different neural phenotypes in different regions depending on the age targeted, and the strategy selected. This specific genetic targeting of neural progenitors in vivo has provided new data on the heterogeneous pool of NG2 progenitors at both embryonic and postnatal ages. NG2-glia are the main proliferative neural cells in the adult brain, representing about 5% of all the CNS cells 1-3. These cells are distributed homogeneously throughout the brain 4 and they are considered to be an independent population of glial cells, otherwise known as oligodendrocyte progenitor cells (OPCs) given that they can differentiate into oligodendrocytes during development, in the adult brain or after injury 5-7. One significant feature of NG2-glia is that they lack gap junction coupling yet they maintain a high input resistance, and they developing voltage-dependent sodium and potassium currents. These cells express ionotropic glutamate and GABA receptors and strikingly, they form direct synapses with neurons 8,9 , raising the possibility that they may modulate the activity of neurons and the operation of neural networks. During development, NG2-glia give rise to cells of the oligodendrocyte lineage and to a few astrocytes 10-12. In addition, these cells generate mature oligodendrocytes, although most of them do not differentiate into oligodendrocytes in the adult CNS 13,14 , rather they remain in a "progenitor" state. Indeed, the role of this undifferentiated population in the mature CNS remains largely unknown 15. What is known is that they increase in number following traumatic insults) and that they contribute to the formation of glial scars, suggesting they help limit neurodegeneration 1,16. Nevertheless, it is not clear whether these features are common to all NG2-glia cells or if there are different subpopulations that fulfil specific functions. Besides oligodendrocytes, NG2-glia have the potential to generate a variety of cell types like astrocytes a...

Astrocytes are organized as communicating cellular networks where each cell is connected to others via gap junctions. These connections are not pervasive and there is evidence for the existence of subgroups composed by preferentially connected cells. Despite being unclear how these are established, we hypothesized lineage might contribute to the establishment of these subgroups. To characterize the functional coupling of clonally related astrocytes, we performed intracellular dye injections in clones of astrocytes labeled with the StarTrack method. This methodology revealed sibling astrocytes are preferentially connected when compared to other surrounding astrocytes. These results suggest the role of the developmental origin in the organization of astrocytes as intercellular networks.

The large phenotypic variation in the olfactory bulb may be related to heterogeneity in the progenitor cells. Accordingly, the progeny of subventricular zone (SVZ) progenitor cells that are destined for the olfactory bulb is of particular interest, specifically as there are many facets of these progenitors and their molecular profiles remain unknown. Using modified StarTrack genetic tracing strategies, specific SVZ progenitor cells were targeted in E12 mice embryos, and the cell fate of these neural progenitors was determined in the adult olfactory bulb. This study defined the distribution and the phenotypic diversity of olfactory bulb interneurons from specific SVZ-progenitor cells, focusing on their spatial pallial origin, heterogeneity, and genetic profile.

NG2-glia, also referred to as oligodendrocyte precursor cells or polydendrocytes, represent a large pool of proliferative neural cells in the adult brain that lie outside of the two major adult neurogenic niches. Although their roles are not fully understood, we previously reported significant clonal expansion of adult NG2-cells from embryonic pallial progenitors using the StarTrack lineage-tracing tool. To define the contribution of early postnatal progenitors to the specific NG2-glia lineage, we used NG2-StarTrack. A temporal clonal analysis of single postnatal progenitor cells revealed the production of different glial cell types in distinct areas of the dorsal cortex but not neurons. Moreover, the dispersion and size of the different NG2 derived clonal cell clusters increased with age. Indeed, clonally-related NG2-glia were located throughout the corpus callosum and the deeper layers of the cortex. In summary, our data reveal that postnatally derived NG2-glia are proliferative cells that give rise to NG2-cells and astrocytes but not neurons. These progenitors undergo clonal cell expansion and dispersion throughout the adult dorsal cortex in a manner that was related to aging and cell identity, adding new information about the ontogeny of these cells. Thus, identification of clonally-related cells from specific progenitors is important to reveal the NG2-glia heterogeneity.

Understanding how an adult brain reaches an appropriate size and cell composition from a pool of progenitors that proliferates and differentiates is a key question in Developmental Neurobiology. Not only the control of final size but also, the proper arrangement of cells of different embryonic origins is fundamental in this process. Each neural progenitor has to produce a precise number of sibling cells that establish clones, and all these clones will come together to form the functional adult nervous system. Lineage cell tracing is a complex and challenging process that aims to reconstruct the offspring that arise from a single progenitor cell. This tracing can be achieved through strategies based on genetically modified organisms, using either genetic tracers, transfected viral vectors or DNA constructs, and even single-cell sequencing. Combining different reporter proteins and the use of transgenic mice revolutionized clonal analysis more than a decade ago and now, the availability of novel genome editing tools and single-cell sequencing techniques has vastly improved the capacity of lineage tracing to decipher progenitor potential. This review brings together the strategies used to study cell lineages in the brain and the role they have played in our understanding of the functional clonal relationships among neural cells. In addition, future perspectives regarding the study of cell heterogeneity and the ontogeny of different cell lineages will also be addressed.

The piriform cortex is a paleocortical area, located in the ventrolateral surface of the rodent forebrain, receiving direct input from the olfactory bulb. The three layers of the PC are defined by the diversity of glial and neuronal cells, marker expression, connections, and functions. However, the glial layering, ontogeny, and sibling cell relationship along the PC is an unresolved question in the field. Here, using multi-color genetic lineage tracing approaches with different StarTrack strategies, we performed a rigorous analysis of the derived cell progenies from progenitors located at the subpallium ventricular surface. First, we specifically targeted E12-progenitors with UbC-StarTrack to analyze their adult derived-cell progeny and their location within the piriform cortex layers. The vast majority of the cell progeny derived from targeted progenitors were identified as neurons, but also astrocytes and NG2 cells. Further, to specifically target single Gsx-2 subpallial progenitors and their derived cell-progeny in the piriform cortex, we used the UbC-(Gsx-2-hyPB)-StarTrack to perform an accurate analysis of their clonal relationships. Our results quantitatively delineate the adult clonal cell pattern from single subpallial E12-progenitors, focusing on glial cells. In summary, there is a temporal pattern in the assembly of the glial cell diversity in the piriform cortex, which also reveals spatio-temporal progenitor heterogeneity.