The enteric nervous system(ENS) is essential for digestive function and gut homeostasis. Here we show that the amorphous neuroglia networks of the mouse ENS are composed of overlapping clonal units ...founded by postmigratory neural crest–derived progenitors. The spatial configuration of ENS clones depends on proliferation-driven local interactions of ENS progenitors with lineally unrelated neuroectodermal cells, the ordered colonization of the serosa-mucosa axis by clonal descendants, and gut expansion. Single-cell transcriptomics and mutagenesis analysis delineated dynamic molecular states of ENS progenitors and identified RET as a regulator of neurogenic commitment. Clonally related enteric neurons exhibit synchronous activity in response to network stimulation. Thus, lineage relationships underpin the organization of the peripheral nervous system.
Radial glial progenitors (RGPs) are responsible for producing nearly all neocortical neurons. To gain insight into the patterns of RGP division and neuron production, we quantitatively analyzed ...excitatory neuron genesis in the mouse neocortex using Mosaic Analysis with Double Markers, which provides single-cell resolution of progenitor division patterns and potential in vivo. We found that RGPs progress through a coherent program in which their proliferative potential diminishes in a predictable manner. Upon entry into the neurogenic phase, individual RGPs produce ?8-9 neurons distributed in both deep and superficial layers, indicating a unitary output in neuronal production. Removal of OTX1, a transcription factor transiently expressed in RGPs, results in both deep- and superficial-layer neuron loss and a reduction in neuronal unit size. Moreover, ?1/6 of neurogenic RGPs proceed to produce glia. These results suggest that progenitor behavior and histogenesis in the mammalian neocortex conform to a remarkably orderly and deterministic program.
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we ...developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.
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•Cell type and temporally resolved cell-surface proteomic profiling in intact brains•Proteome-wide coordinated change of neuronal surface landscape over development•New cell-surface regulators of brain wiring from unexpected molecular families•Cell-autonomous control of dendrite targeting by the lipoprotein receptor LRP1
In situ cell-surface proteomic profiling of developing and mature olfactory projection neurons uncovers the temporal evolution of the neuronal surface landscape in development and reveals many new neural wiring molecules belonging to evolutionarily conserved but previously unexpected molecular families.
Mother-infant bonding develops rapidly following parturition and is accompanied by changes in sensory perception and behavior. Here, we study how ultrasonic vocalizations (USVs) are represented in ...the brain of mothers. Using a mouse line that allows temporally controlled genetic access to active neurons, we find that the temporal association cortex (TeA) in mothers exhibits robust USV responses. Rabies tracing from USV-responsive neurons reveals extensive subcortical and cortical inputs into TeA. A particularly dominant cortical source of inputs is the primary auditory cortex (A1), suggesting strong A1-to-TeA connectivity. Chemogenetic silencing of USV-responsive neurons in TeA impairs auditory-driven maternal preference in a pup-retrieval assay. Furthermore, dense extracellular recordings from awake mice reveal changes of both single-neuron and population responses to USVs in TeA, improving discriminability of pup calls in mothers compared with naive females. These data indicate that TeA plays a key role in encoding and perceiving pup cries during motherhood.
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•Temporal association cortex (TeA) is activated in response to pup calls in mothers•TeA’s connectivity is widespread, including robust functional connectivity from A1•TeA is causally related to an auditory-driven maternal behavior by mothers•Spiking response in TeA shows improved USV discrimination in mothers
Tasaka et al. show that the temporal association cortex (TeA) receives monosynaptic inputs from widespread cortical and subcortical regions. TeA shows strong functional connectivity to the primary auditory cortex. In the context of motherhood, TeA plays an important role in encoding and perceiving pup ultrasonic vocalizations by mothers.
This protocol describes a basic method for dissection and immunofluorescence staining of the Drosophila brain at various developmental stages. The Drosophila brain has become increasingly useful for ...studies of neuronal wiring and morphogenesis in combination with techniques such as the 'mosaic analysis with a repressible cell marker' (MARCM) system, where single neurons can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells. Such high-resolution anatomical study of the brain is also important in characterizing the organization of neural circuits using genetic tools such as GAL4 enhancer trap lines, as Drosophila has been intensively used for studying the neural basis of behavior. Advantages of fluorescence immunostaining include compatibility with multicolor labeling and confocal or multiphoton imaging. This brain dissection and immunofluorescence staining protocol requires approximately 2 to 6 d to complete.
Separation effects of sodium polyacrylate (PAAS) and gum Arabic (GA) on flotation of chalcopyrite and magnesium silicate minerals using potassium butyl xanthate (PBX) as collector were investigated ...by micro-flotation experiments, zeta potential, Infrared spectral (IR), SEM-EDS, XPS analysis and copper sulphide ore beneficiation test. The micro-flotation experiments and zeta potential measurements showed that combined depressant consisting of PAAS and GA could efficiently reduce the recoveries of mixed minerals of serpentine and talc more than 25%, while that of chalcopyrite remained above 70% at pH 9.2. Infrared spectral (IR), SEM-EDS and XPS analysis showed that PAAS chemically reacted with Mg on the surface of serpentine, while GA adsorbed on talc surface mainly via physical interaction and hydrogen bond may also play a role. Surface synergism between PAAS and GA was investigated by turbidity test and its depression mechanism was proposed. The technology feasibility of using PAAS and GA to improve the copper sulphide ore flotation performance was verified through artificial mixed ore flotation and laboratory closed-flotation operation.
Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, ...from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.
The nervous system consists of an ensemble of billions of neurons interconnected in a highly specific pattern that allows proper propagation and integration of neural activities. The organization of ...these specific connections emerges from sequential developmental events including axon guidance, target selection, and synapse formation. These events critically rely on cell-cell recognition and communication mediated by cell-surface ligands and receptors. Recent studies have uncovered central roles for leucine-rich repeat (LRR) domain-containing proteins, not only in organizing neural connectivity from axon guidance to target selection to synapse formation, but also in various nervous system disorders. Their versatile LRR domains, in particular, serve as key sites for interactions with a wide diversity of binding partners. Here, we focus on a few exquisite examples of secreted or membrane-associated LRR proteins in Drosophila and mammals and review the mechanisms by which they regulate diverse aspects of nervous system development and function.
Coordinated migration of newly born neurons to their prospective target laminae is a prerequisite for neural circuit assembly in the developing brain. The evolutionarily conserved LIS1/NDEL1 complex ...is essential for neuronal migration in the mammalian cerebral cortex. The cytoplasmic nature of LIS1 and NDEL1 proteins suggest that they regulate neuronal migration cell autonomously. Here, we extend mosaic analysis with double markers (MADM) to mouse chromosome 11 where
Lis1,
Ndel1, and
14-3-3ɛ (encoding a LIS1/NDEL1 signaling partner) are located. Analyses of sparse and uniquely labeled mutant cells in mosaic animals reveal distinct cell-autonomous functions for these three genes.
Lis1 regulates neuronal migration efficiency in a dose-dependent manner, while
Ndel1 is essential for a specific, previously uncharacterized, late step of neuronal migration: entry into the target lamina. Comparisons with previous genetic perturbations of
Lis1 and
Ndel1 also suggest a surprising degree of cell-nonautonomous function for these proteins in regulating neuronal migration.
► Extension of mosaic analysis with double markers (MADM) to mouse chromosome 11 ► Functional analyses of Lis1, Ndel1, and 14-3-3ɛ with single cell resolution ► NDEL1 cell autonomously controls neuronal migration into target laminae ► Extensive cell-nonautonomous or community effects influence neuronal migration
The selective elimination of axons, dendrites, axon and dendrite branches, and synapses, without loss of the parent neurons, occurs during normal development of the nervous system as well as in ...response to injury or disease in the adult. The widespread developmental phenomena of exuberant axonal projections and synaptic connections require both small-scale and large-scale axon pruning to generate precise adult connectivity, and they provide a mechanism for neural plasticity in the developing and adult nervous system, as well as a mechanism to evolve differences between species in a projection system. Such pruning is also required to remove axonal connections damaged in the adult, to stabilize the affected neural circuits, and to initiate their repair. Pruning occurs through either retraction or degeneration. Here we review examples of these phenomena and consider potential cellular and molecular mechanisms that underlie axon retraction and degeneration and how they might relate to each other in development and disease.
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