Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Hospital for Sick Children

University of Toronto

Spatial patterns of neuronal connectivity are critical for neural circuit function and information processing. For many neuron types, the development of stereotyped dendritic and axonal territories involves reiterative contacts between neurites and successive re-calibration of branch outgrowth and directionality. Here I review emerging roles for members of the atypical cadherins (Fmi/Celsrs) and the clustered Protocadherins (Pcdhs) in neurite patterning. These cell-surface molecules have shared functions: they engage in homophilic recognition and mediate dynamic and contact-dependent interactions to establish reproducible and space-filling arborization patterns. As shown in genetic and molecular studies, the atypical cadherins and clustered Pcdhs serve in multiple contexts and signal diverse actions such as neurite repulsion or selective adhesion. In some cell types, they regulate the non-overlapping arrangement of branches achieved through homotypic interactions, such as in self-avoidance or tiling. In others, they promote dendritic complexity through cell-cell interactions. With critical roles in both the fine-scale arrangement of axonal and dendritic branching and the large-scale organization of axon tracts and neuronal networks, the atypical cadherins and clustered Pcdhs are key regulators of neural circuit assembly and function.

Neuronal territory formation by the atypical cadherins and clustered protocadherins.

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, -α, -β, and -γ (, , and , respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between  and  clusters. To circumvent neonatal lethality associated with deletion of , we used Crispr-Cas9 genome editing in mice to combine a constitutive  mutant allele with a conditional  allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both  and  clusters led to far more dramatic defects in survival and self-avoidance than  deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development. The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called -α, -β, and -γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between -α and -γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.

Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins  from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of  from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in  mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain. A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the  in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Neurons develop dendritic morphologies that bear cell type-specific features in dendritic field size and geometry, branch placement and density, and the types and distributions of synaptic contacts. Dendritic patterns influence the types and numbers of inputs a neuron receives, and the ways in which neural information is processed and transmitted in the circuitry. Even subtle alterations in dendritic structures can have profound consequences on neuronal function and are implicated in neurodevelopmental disorders. In this chapter, I review how growing dendrites acquire their exquisite patterns by drawing examples from diverse neuronal cell types in vertebrate and invertebrate model systems. Dendrite morphogenesis is shaped by intrinsic and extrinsic factors such as transcriptional regulators, guidance and adhesion molecules, neighboring cells and synaptic partners. I discuss molecular mechanisms that regulate dendrite morphogenesis with a focus on five aspects of dendrite patterning: (1) Dendritic cytoskeleton and cellular machineries that build the arbor; (2) Gene regulatory mechanisms; (3) Afferent cues that regulate dendritic arbor growth; (4) Space-filling strategies that optimize dendritic coverage; and (5) Molecular cues that specify dendrite wiring. Cell type-specific implementation of these patterning mechanisms produces the diversity of dendrite morphologies that wire the nervous system.

Julie L. Lefebvre

Program for Neuroscience and Mental Health

Hospital for Sick Children

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Julie L. Lefebvre

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Program for Neuroscience and Mental Health

Hospital for Sick Children

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Program for Neuroscience and Mental Health