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Highlights d Genome-wide resource for genetic mosaic analysis with double markers in mice d Resource for dissection of cell-autonomous gene function of >96% of the mouse genome d Resource for genome-wide analysis of genomic imprinting phenotypes d MADM chromosomes reveal non-random mitotic sister chromatid segregation in vivo

The synaptotrophic hypothesis posits that synapse formation stabilizes dendritic branches, yet this hypothesis has not been causally tested in vivo in the mammalian brain. Presynaptic ligand cerebellin-1 (Cbln1) and postsynaptic receptor GluD2 mediate synaptogenesis between granule cells and Purkinje cells in the molecular layer of the cerebellar cortex. Here we show that sparse but not global knockout of GluD2 causes under-elaboration of Purkinje cell dendrites in the deep molecular layer and overelaboration in the superficial molecular layer. Developmental, overexpression, structure-function, and genetic epistasis analyses indicate that dendrite morphogenesis defects result from competitive synaptogenesis in a Cbln1/GluD2-dependent manner. A generative model of dendritic growth based on competitive synaptogenesis largely recapitulates GluD2 sparse and global knockout phenotypes. Our results support the synaptotrophic hypothesis at initial stages of dendrite development, suggest a second mode in which cumulative synapse formation inhibits further dendrite growth, and highlight the importance of competition in dendrite morphogenesis.

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 CreER T2 , 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 CreER T2 driver is present. 18 , with approximately 1/6 of neurogenic RGPs also producing glia 11. Currently, the genetic and epigenetic factors regulating temporal progression of a stem cell along its lineage are mostly unknown. Temporal patterns of gene expression may have substantial impact on lineage decisions in RGPs 20,21,22,23,24. How this tightly knit relationship between temporal and spatial patterning leads to the molecular diversity of adult neuronal types across cortical areas is not known. Likewise, how the individual stem cell potential and its cellular output is modulated at the cellular and molecular level is an important unanswered question. Future studies will hopefully address some of these questions, ultimately expanding our understanding of functional cortical circuit formation. Developmental neurobiology seeks to understand the lineage relationship that cells in the brain share with one another. Initially, very few research tools were available for this, and many early studies relied on visual observations of division patterns in transparent organisms such as Caenorhabditis elegans 25. Recent decades have seen a dramatic increase in the number and sophistication ...