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 ...
Highlights d GABAergic and glutamatergic cerebellar neurons are generated from Sox2 + progenitors d Single Sox2 + ECPs can give rise to both excitatory and inhibitory cerebellar neurons d Notch activity mediates GABAergic versus glutamatergic cell fates in Sox2 + ECPs
14The bewildering diversity of brain neurons arises from relatively few pluripotent progenitors 15 through poorly understood mechanisms. The cerebellum is an attractive model to investigate 16 mechanisms of neuronal diversification because the different subtypes of excitatory and 17 inhibitory neurons are well described 1,2 . The cerebellum is a hub for control of motor function 18 and contributes to a number of higher brain functions such as reward-related cognitive 19 processes 3 . Deficits in cerebellar development lead to severe neurological disorders such as 20 cerebellar ataxias 4 and medulloblastomas 5 , a heterogeneous and severe groups of childhood 21 brain tumors, thus underlying the importance of understanding the cellular and molecular 22 control of cerebellar development. In contrast to text book models, we report that excitatory 23 and inhibitory cerebellar neurons derive from the same pluripotent embryonic cerebellar stem 24 cells (eCSC). We find that the excitatory versus inhibitory fate decision of a progenitor is 25 regulated by Notch signaling, whereby the cell with lower Notch activity adopts the excitatory 26 fate, while the cell with higher Notch activity adopts the inhibitory fate. Thus, Notch-mediated 27 binary cell fate choice is a conserved strategy for generating neuronal diversity from common 28 progenitors that is deployed at different developmental time points in a context specific manner. 29 30 31 Body text 32 Cerebellar anlagen 33 Text book models suggest that different cerebellar neurons arise from two distinct progenitor 34 pools (Fig.S1A) located either dorsally in the rhombic lip (RL) or ventrally in the ventricular 35 zone (VZ). Two basic helix-loop-helix (bHLH) transcription factors called Atonal homologue 36 1 (Atoh1; RL) and Pancreas transcription factor 1 alpha (Ptf1a; VZ), mark these progenitors. 37 Atoh1 + RL progenitors give rise to glutamatergic neurons, while Ptf1a + VZ progenitors give 38 rise to GABAergic neurons 6 . However, pseudo-time trajectory analysis of single-cell RNAseq 39 (scRNAseq) embryonic mouse cerebellum data suggests that a common pool of progenitors 40 branches into either a glutamatergic fate or a GABAergic fate 7 . In addition, cell fate in the two 41 germinal niches can be switched when Atoh1 and Ptf1a are ectopically expressed in the VZ 42 and RL, respectively 8,9 . Finally, the classic neural stem cell marker Sox2 is an early VZ marker, 43 but cells expressing both Sox2 and the RL marker Atoh1 were observed in human cerebellar 44 organoids 10 . Whether this means that common pluripotent progenitors competent to generate 45 both excitatory and inhibitory lineages exist, and how this binary fate decision is regulated, are 46 unknown. 47 52 Sox2 CreERT2 /Gt(ROSA)26Sor tdTomato /Atoh1 GFP mice using low doses of Tamoxifen (TM: 0.1mg, 53 0.03mg). Under both conditions, in all mice examined, Sox2 + cells and their progeny labelled 54 with tdTomato (tdTomato+). We observed Ptf1a + /tdTomato + and Atoh1 + /tdTomato + cells at 55 E12.5 in the VZ a...
Mosaic Analysis with Double Markers (MADM) offers a unique approach to visualize and concomitantly manipulate genetically-defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage; single-cell morphology and physiology; genomic imprinting phenotypes; and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM could only be applied to <25% of all mouse genes on select chromosomes thus far. To overcome this limitation, we generated transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validated their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond proof-of-principle, we applied our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We found striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.
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