Vision-dependent specification of cell types and function in the developing cortex.

The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.

Synaptic Specificity, Recognition Molecules, and Assembly of Neural Circuits.

Developing neurons connect in specific and stereotyped ways to form the complex circuits that underlie brain function. By comparison to earlier steps in neural development, progress has been slow in identifying the cell surface recognition molecules that mediate these synaptic choices, but new high-throughput imaging, genetic, and molecular methods are accelerating progress. Over the past decade, numerous large and small gene families have been implicated in target recognition, including members of the immunoglobulin, cadherin, and leucine-rich repeat superfamilies. We review these advances and propose ways in which combinatorial use of multifunctional recognition molecules enables the complex neuron-neuron interactions that underlie synaptic specificity.

Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila.

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity.

The shape of things to come.

Surface receptors can link binding of ligands to changes in the actin-based cell cytoskeleton. Chia et al. and Chen et al. provide evidence for direct binding between the cytoplasmic tails of receptors and the WAVE complex, a regulator of the actin nucleator Arp2/3 complex, which might help to explain how environmental signals are translated into changes in morphology and motility.

Synaptic gradients transform object location to action.

To survive, animals must convert sensory information into appropriate behaviours1,2. Vision is a common sense for locating ethologically relevant stimuli and guiding motor responses3-5. How circuitry converts object location in retinal coordinates to movement direction in body coordinates remains largely unknown. Here we show through behaviour, physiology, anatomy and connectomics in Drosophila that visuomotor transformation occurs by conversion of topographic maps formed by the dendrites of feature-detecting visual projection neurons (VPNs)6,7 into synaptic weight gradients of VPN outputs onto central brain neurons. We demonstrate how this gradient motif transforms the anteroposterior location of a visual looming stimulus into the fly's directional escape. Specifically, we discover that two neurons postsynaptic to a looming-responsive VPN type promote opposite takeoff directions. Opposite synaptic weight gradients onto these neurons from looming VPNs in different visual field regions convert localized looming threats into correctly oriented escapes. For a second looming-responsive VPN type, we demonstrate graded responses along the dorsoventral axis. We show that this synaptic gradient motif generalizes across all 20 primary VPN cell types and most often arises without VPN axon topography. Synaptic gradients may thus be a general mechanism for conveying spatial features of sensory information into directed motor outputs.

Probabilistic splicing of Dscam1 establishes identity at the level of single neurons.

The Drosophila Dscam1 gene encodes a vast number of cell recognition molecules through alternative splicing. These exhibit isoform-specific homophilic binding and regulate self-avoidance, the tendency of neurites from the same cell to repel one another. Genetic experiments indicate that different cells must express different isoforms. How this is achieved is unknown, as expression of alternative exons in vivo has not been shown. Here, we modified the endogenous Dscam1 locus to generate splicing reporters for all variants of exon 4. We demonstrate that splicing does not occur in a cell-type-specific fashion, that cells sharing the same anatomical location in different individuals express different exon 4 variants, and that the splicing pattern in a given neuron can change over time. We conclude that splicing is probabilistic. This is compatible with a widespread role in neural circuit assembly through self-avoidance and is incompatible with models in which specific isoforms of Dscam1 mediate homophilic recognition between processes of different cells.

A global timing mechanism regulates cell-type-specific wiring programmes.

The assembly of neural circuits is dependent on precise spatiotemporal expression of cell recognition molecules1-5. Factors controlling cell type specificity have been identified6-8, but how timing is determined remains unknown. Here we describe induction of a cascade of transcription factors by a steroid hormone (ecdysone) in all fly visual system neurons spanning target recognition and synaptogenesis. We demonstrate through single-cell sequencing that the ecdysone pathway regulates the expression of a common set of targets required for synaptic maturation and cell-type-specific targets enriched for cell-surface proteins regulating wiring specificity. Transcription factors in the cascade regulate the expression of the same wiring genes in complex ways, including activation in one cell type and repression in another. We show that disruption of the ecdysone pathway generates specific defects in dendritic and axonal processes and synaptic connectivity, with the order of transcription factor expression correlating with sequential steps in wiring. We also identify shared targets of a cell-type-specific transcription factor and the ecdysone pathway that regulate specificity. We propose that neurons integrate a global temporal transcriptional module with cell-type-specific transcription factors to generate different cell-type-specific patterns of cell recognition molecules regulating wiring.

Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila.

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in Drosophila, we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Octß1R, Octß2R, two isoforms of OAMB, and mGluR. The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show expression patterns for these receptors in female brains, and that 5-HT1A and 5-HT2B localize to the mushroom bodies and central complex respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Octß1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their functions at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the mushroom bodies as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor.Significance Statement In Drosophila, despite remarkable advances in both connectomic and genomic studies, antibodies to many aminergic GPCRs are not available. We have overcome this obstacle using evolutionary conservation to identify loci in GPCRs amenable to epitope-tagging, and CRISPR/Cas9 genome editing to generate eight novel lines. This method may also be applied to other GPCRs and allows cell-specific expression of the tagged receptor. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and postsynaptic sites that express the same receptor, and the use of relatively distant targets by presynaptic release sites that may employ volume transmission as well as standard synaptic signaling.

Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly.

The chemoaffinity hypothesis for neural circuit assembly posits that axons and their targets bear matching molecular labels that endow neurons with unique identities and specify synapses between appropriate partners. Here, we focus on two intriguing candidates for fulfilling this role, Drosophila Dscams and vertebrate clustered protocadherins (Pcdhs). In each, a complex genomic locus encodes large numbers of neuronal transmembrane proteins with homophilic binding specificity, individual members of which are expressed combinatorially. Although these properties suggest that Dscams and Pcdhs could act as specificity molecules, they may do so in ways that challenge traditional views of how neural circuits assemble.

A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms.

Drosophila Dscam encodes a vast family of immunoglobulin (Ig)-containing proteins that exhibit isoform-specific homophilic binding. This diversity is essential for cell recognition events required for wiring the brain. Each isoform binds to itself but rarely to other isoforms. Specificity is determined by "matching" of three variable Ig domains within an approximately 220 kD ectodomain. Here, we present the structure of the homophilic binding region of Dscam, comprising the eight N-terminal Ig domains (Dscam(1-8)). Dscam(1-8) forms a symmetric homodimer of S-shaped molecules. This conformation, comprising two reverse turns, allows each pair of the three variable domains to "match" in an antiparallel fashion. Structural, genetic, and biochemical studies demonstrate that, in addition to variable domain "matching," intramolecular interactions between constant domains promote homophilic binding. These studies provide insight into how "matching" at all three pairs of variable domains in Dscam mediates isoform-specific recognition.

The molecular basis of self-avoidance.

Self-avoidance, the tendency of neurites of the same cell to selectively avoid each other, is a property of both vertebrate and invertebrate neurons. In Drosophila, self-avoidance is mediated by a large family of cell recognition molecules of the immunoglobulin superfamily encoded, via alternative splicing, by the Dscam1 locus. Dscam1 promotes self-avoidance in dendrites, axons, and prospective postsynaptic elements. Expression analysis suggests that each neuron expresses a unique combination of isoforms. Identical isoforms on sister neurites exhibit isoform-specific homophilic recognition and elicit repulsion between processes, thereby promoting self-avoidance. Although any isoform can promote self-avoidance, thousands are necessary to ensure that neurites readily discriminate between self and nonself. Recent studies indicate that a large family of cadherins in the mouse, i.e., the clustered protocadherins, functions in an analogous fashion to promote self-avoidance. These studies argue for the evolution of a common molecular strategy for self-avoidance.

Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms.

Down Syndrome cell adhesion molecule (Dscam) genes encode neuronal cell recognition proteins of the immunoglobulin superfamily. In Drosophila, Dscam1 generates 19,008 different ectodomains by alternative splicing of three exon clusters, each encoding half or a complete variable immunoglobulin domain. Identical isoforms bind to each other, but rarely to isoforms differing at any one of the variable immunoglobulin domains. Binding between isoforms on opposing membranes promotes repulsion. Isoform diversity provides the molecular basis for neurite self-avoidance. Self-avoidance refers to the tendency of branches from the same neuron (self-branches) to selectively avoid one another. To ensure that repulsion is restricted to self-branches, different neurons express different sets of isoforms in a biased stochastic fashion. Genetic studies demonstrated that Dscam1 diversity has a profound role in wiring the fly brain. Here we show how many isoforms are required to provide an identification system that prevents non-self branches from inappropriately recognizing each other. Using homologous recombination, we generated mutant animals encoding 12, 24, 576 and 1,152 potential isoforms. Mutant animals with deletions encoding 4,752 and 14,256 isoforms were also analysed. Branching phenotypes were assessed in three classes of neurons. Branching patterns improved as the potential number of isoforms increased, and this was independent of the identity of the isoforms. Although branching defects in animals with 1,152 potential isoforms remained substantial, animals with 4,752 isoforms were indistinguishable from wild-type controls. Mathematical modelling studies were consistent with the experimental results that thousands of isoforms are necessary to ensure acquisition of unique Dscam1 identities in many neurons. We conclude that thousands of isoforms are essential to provide neurons with a robust discrimination mechanism to distinguish between self and non-self during self-avoidance.

Coordinate control of synaptic-layer specificity and rhodopsins in photoreceptor neurons.

How neurons make specific synaptic connections is a central question in neurobiology. The targeting of the Drosophila R7 and R8 photoreceptor axons to different synaptic layers in the brain provides a model with which to explore the genetic programs regulating target specificity. In principle this can be accomplished by cell-type-specific molecules mediating the recognition between synaptic partners. Alternatively, specificity could also be achieved through cell-type-specific repression of particular targeting molecules. Here we show that a key step in the targeting of the R7 neuron is the active repression of the R8 targeting program. Repression is dependent on NF-YC, a subunit of the NF-Y (nuclear factor Y) transcription factor. In the absence of NF-YC, R7 axons terminate in the same layer as R8 axons. Genetic experiments indicate that this is due solely to the derepression of the R8-specific transcription factor Senseless (Sens) late in R7 differentiation. Sens is sufficient to control R8 targeting specificity and we demonstrate that Sens directly binds to an evolutionarily conserved DNA sequence upstream of the start of transcription of an R8-specific cell-surface protein, Capricious (Caps) that regulates R8 target specificity. We show that R7 targeting requires the R7-specific transcription factor Prospero (Pros) in parallel to repression of the R8 targeting pathway by NF-YC. Previous studies demonstrated that Sens and Pros directly regulate the expression of specific rhodopsins in R8 and R7. We propose that the use of the same transcription factors to promote the cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity provides a simple and general mechanism for ensuring that transmission of sensory information is processed by the appropriate specialized neural circuits.

Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome.

Neurons express various combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here, we use epitope-tagged endogenous NR subunits, expansion light-sheet microscopy, and electron microscopy (EM) connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion-sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type-specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determine patterns of synaptic inputs. In support of this model, we identify a transmembrane protein selectively associated with a subset of spatially restricted synapses and demonstrate its requirement for synapse formation through genetic analysis. We propose that mechanisms that regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

Dscam2 mediates axonal tiling in the Drosophila visual system.

Sensory processing centres in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. Here we show that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. Our data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. We propose that Dscam2 is a tiling receptor for L1 neurons.

Dscam diversity is essential for neuronal wiring and self-recognition.

Neurons are thought to use diverse families of cell-surface molecules for cell recognition during circuit assembly. In Drosophila, alternative splicing of the Down syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 closely related transmembrane proteins of the immunoglobulin superfamily, each comprising one of 19,008 alternative ectodomains linked to one of two alternative transmembrane segments. These ectodomains show isoform-specific homophilic binding, leading to speculation that Dscam proteins mediate cell recognition. Genetic studies have established that Dscam is required for neural circuit assembly, but the extent to which isoform diversity contributes to this process is not known. Here we provide conclusive evidence that Dscam diversity is essential for circuit assembly. Using homologous recombination, we reduced the entire repertoire of Dscam ectodomains to just a single isoform. Neural circuits in these mutants are severely disorganized. Furthermore, we show that it is crucial for neighbouring neurons to express distinct isoforms, but that the specific identity of the isoforms expressed in an individual neuron is unimportant. We conclude that Dscam diversity provides each neuron with a unique identity by which it can distinguish its own processes from those of other neurons, and that this self-recognition is essential for wiring the Drosophila brain.

Vision sculpts a continuum of L2/3 cell types in the visual cortex during the critical period.

We previously reported that vision specifies Layer 2/3 (L2/3) glutamatergic cell-type identity in the primary visual cortex (V1). Using unsupervised clustering of single-nucleus RNA-sequencing data, we identified molecularly distinct L2/3 cell types in normal-reared (NR) and dark-reared (DR) mice, but the two sets exhibited poor correspondence. Here, we show that classification of cell types was confounded in DR by vision-dependent gene programs that are orthogonal to gene programs underlying cell-type identity. A focused clustering analysis successfully matches cell types between DR and NR, suggesting that cell identity-defining gene programs persist under vision deprivation but are overshadowed by vision-dependent transcriptomic variation. Using multi-tasking theory we show that L2/3 cell types form a continuum between three cell-archetypes. Visual deprivation markedly shifts this distribution along the continuum. Thus, dark-rearing markedly influences cell states thereby masking cell-type-identities and changes the distribution of L2/3 types along a transcriptomic continuum.

Brain wiring determinants uncovered by integrating connectomes and transcriptomes.

Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits.1,2,3,4,5 Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites.6,7,8 Many CAM families have been shown to contribute to brain wiring in different ways.9,10 It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. Here, we integrated the synapse-level connectome of the neural circuit11,12 with the developmental expression patterns7 and binding specificities of CAMs6,13 on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, we focus on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit,14 closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil.12 This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. We propose that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.