Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present

Enriched Monolayer Precursor Cell Cultures from Micro-Dissected Adult Mouse Dentate Gyrus Yield Functional Granule Cell-Like Neurons

BACKGROUND: Stem cell cultures are key tools of basic and applied research in Regenerative Medicine. In the adult mammalian brain, lifelong neurogenesis originating from local precursor cells occurs in the neurogenic regions of the hippocampal dentate gyrus. Despite widespread interest in adult hippocampal neurogenesis and the use of mouse models to study it, no protocol existed for adult murine long-term precursor cell cultures with hippocampus-specific differentiation potential. METHODOLOGY/PRINCIPAL FINDINGS: We describe a new strategy to obtain serum-free monolayer cultures of neural precursor cells from microdissected dentate gyrus of adult mice. Neurons generated from these adherent hippocampal precursor cell cultures expressed the characteristic markers like transcription factor Prox1 and showed the TTX-sensitive sodium currents of mature granule cells in vivo. Similar to granule cells in vivo, treatment with kainic acid or brain derived neurotrophic factor (BDNF) elicited the expression of GABAergic markers, further supporting the correspondence between the in vitro and in vivo phenotype. When plated as single cells (in individual wells) or at lowest density for two to three consecutive generations, a subset of the cells showed self-renewal and gave rise to cells with properties of neurons, astrocytes and oligodendrocytes. The precursor cell fate was sensitive to culture conditions with their phenotype highly influenced by factors within the media (sonic hedgehog, BMP, LIF) and externally applied growth factors (EGF, FGF2, BDNF, and NT3). CONCLUSIONS/SIGNIFICANCE: We report the conditions required to generate adult murine dentate gyrus precursor cell cultures and to analyze functional properties of precursor cells and their differentiated granule cell-like progeny in vitro

Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry

Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).

Astroglial connexin 43 regulates synaptic vesicle release at hippocampal synapses

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43−/−), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43−/− mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy

Multipotent progenitors instruct ontogeny of the superior colliculus

The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration, attention, and complex behavior (Basso and May, 2017; Cang et al., 2018). The mature SC cytoarchitecture is organized into distinct laminae and composed of a rich variety of neuronal and glial cell types (Ayupe et al., 2023; Edwards et al., 1986; May, 2006; Xie et al., 2021; Zeisel et al., 2018). Precise execution of the developmental programs regulating the generation of SC cell-type diversity is essential, because deficits due to genetic mutations have been associated with neurodevelopmental diseases and SC dysfunction (Jure, 2018; McFadyen et al., 2020). However, the fundamentals directing the ontogeny of the SC are not well understood. Here we pursued systematic lineage tracing at the single progenitor cell level in order to decipher the principles instructing the generation of cell-type diversity in the SC. We combined in silico lineage reconstruction with a novel genetic MADM (Mosaic Analysis with Double Markers)-CloneSeq approach. MADM-CloneSeq enables the unequivocal delineation of cell lineages in situ, and cell identity based on global transcriptome, of individual clonally-related cells. Our systematic reconstructions of cell lineages revealed that all neuronal cell types in SC emerge from local progenitors without any extrinsic source. Strikingly, individual SC progenitors are exceptionally multipotent with the capacity to produce all known excitatory and inhibitory neuron types of the prospective mature SC, with individual clonal units showing no pre-defined composition. At the molecular level we identified an essential role for PTEN signaling in establishing appropriate proportions of specific inhibitory and excitatory neuron types. Collectively, our findings demonstrate that individual multipotent progenitors generate the full spectrum of excitatory and inhibitory neuron types in the developing SC, providing a novel framework for the emergence of cell-type diversity and thus the ontogeny of the mammalian SC