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Programmable proteins to detect, visualize, modulate, or eliminate proteins of selection in vitro and in vivo are essential to study the targets recognized and the biology that follows. The specificity of programmable proteins can be easily altered by designing their sequences and structures. The flexibility and modularity of these proteins are currently pivotal for synthetic biology and various medical applications. There exist numerous reviews of the concept and application of individual programmable proteins, such as programmable nucleases, single-domain antibodies, and other protein scaffolds. This review proposes an expanded conceptual framework of such programmable proteins based on their programmable principle and target specificity to biomolecules (nucleic acids, proteins, and glycans) and overviews their advantages, limitations, and future directions.

Fasting initiates a multitude of adaptations to allow survival. Activation of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent release of glucocorticoid hormones is a key response that mobilizes fuel stores to meet energy demands. Despite the importance of the HPA axis response, the neural mechanisms that drive its activation during energy deficit are unknown. Here, we show that hypothalamic agouti-related peptide (AgRP)-expressing neurons trigger and are essential for fasting-induced HPA axis activation. AgRP neurons stimulate activity of the HPA axis via projections to the paraventricular hypothalamus (PVH), where, in a mechanism not previously described for AgRP neurons, they presynaptically inhibit the terminals of tonically active GABAergic afferents from the bed nucleus of the stria terminalis (BNST) that otherwise restrain activity of corticotrophin-releasing hormone (CRH)-expressing neurons. This disinhibition of PVH-Crh neurons requires GABA (γ-aminobutyric acid)/GABA-B receptor signaling and potently activates the HPA axis. Importantly, stimulation of the HPA axis by AgRP neurons is independent of their induction of hunger, showing that these canonical ‘hunger neurons’ drive multiple parallel adaptations to the fasted state. Together our findings identify the neural basis for fasting-induced HPA axis activation and uncover a unique means by which AgRP neurons activate downstream neurons – via presynaptic inhibition of GABAergic afferents. Given the potency of this disinhibition of tonically active BNST afferents, other activators of the HPA axis such as psychological stress may work via a similar mechanism. NIH R01DK096010, NIH R00HL144923, AHA 935362 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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Scientists write research articles, process ethics reviews, evaluate proposals and research, and seek funding. Several strategies have been proposed to optimize these operations and to decentralize access to research resources and opportunities. For instance, we previously proposed the trinity review method, combining registered reports with financing and research ethics assessments. However, previously proposed systems have a number of shortcomings, including how to implement them, e.g., who manages them, how incentives for reviewers are paid, etc. Various solutions have been proposed to address these issues, employing decentralized science (DeSci) in an independent organization built on the blockchain. Decentralized approaches that exploit these developments offer potentially profound improvements to the troubled scientific ecosystem. Here, we propose a system that integrates ethics reviews, peer reviews, and funding in a decentralized manner, based on Web3 technology. This new method, named ABCDEF publishing, would enhance the speed, fairness, and transparency of scientific research and publishing.

The Tabula Gallus is a proposed project that aims to create a map of every cell type in the chicken body and chick embryos. Chickens (Gallus gallus) are one of the most recognized model animals that recapitulate the development and physiology of mammals. The Tabula Gallus will generate a compendium of single-cell transcriptome data from Gallus gallus, characterize each cell type, and provide tools for the study of the biology of this species, similar to other ongoing cell atlas projects (Tabula Muris and Tabula Sapiens/Human Cell Atlas for mice and humans, respectively). The Tabula Gallus will potentially become an international collaboration between many researchers. This project will be useful for the basic scientific study of Gallus gallus and other birds (e.g., cell biology, molecular biology, developmental biology, neuroscience, physiology, oncology, virology, behavior, ecology, and evolution). It will eventually be beneficial for a better understanding of human health and diseases.

Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca(2+) indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices. SIGNIFICANCE STATEMENT A new technique for simultaneous optogenetic stimulation and calcium imaging across wide areas of brain slice enables high-throughput mapping of neuronal excitability and synaptic transmission.

The abilities to mark and manipulate specific cell types are essential for an increasing number of functional, structural, molecular, and developmental analyses in model organisms. In a few species, this can be accomplished by germline transgenesis; in other species, other methods are needed to selectively label somatic cells based on the genes that they express. Here, we describe a method for CRISPR-based somatic integration of reporters or Cre recombinase into specific genes in the chick genome, followed by visualization of cells in the retina and midbrain. Loci are chosen based on an RNA-seq-based cell atlas. Reporters can be soluble to visualize the morphology of individual cells or appended to the encoded protein to assess subcellular localization. We call the method eCHIKIN for electroporation- and CRISPR-mediated Homology-instructed Knock-IN.

SUMMARYRetinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. From ∼40,000 single cell transcriptomes, we identified 136 cell types distributed among the six classes conserved across vertebrates – photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and glial cells. To match molecular profiles to morphology, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse and primate retinal cell classes and types. Taken together, our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.

Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.

Sensitive and specific antibodies are essential for detecting molecules in cells and tissues. However, currently used polyclonal and monoclonal antibodies are often less specific than desired, difficult to produce, and available in limited quantities. A promising recent approach to circumvent these limitations is to employ chemically defined antigen-combining domains called "nanobodies," derived from single-chain camelid antibodies. Here, we used nanobodies to prepare sensitive unimolecular detection reagents by genetically fusing cDNAs encoding nanobodies to enzymatic or antigenic reporters. We call these fusions between a reporter and a nanobody "RANbodies." They can be used to localize epitopes and to amplify signals from fluorescent proteins. They can be generated and purified simply and in unlimited amounts and can be preserved safely and inexpensively in the form of DNA or digital sequence.

The ventral midline of the embryonic neural tube, the floor plate, has a profound role in guiding axons during embryonic development. Floor plate-derived guidance cues attract or repel axons, depending on the neuronal subtype and developmental stage. Netrin-1 and its receptor, Deleted in Colon Carcinoma (DCC), are the key constituents of commissurral axons guidance cues toward the floor plate. Recent studies have implicated Down Syndrome Cell Adhesion Molecule (Dscam) as an additional Netrin-1 receptor. In this study, we examined the role of Dscam in guiding defined spinal dorsal interneuron populations. In vivo knockdown and ectopic expression of Dscam were performed in the dorsal dI1, dI2 and dI3 interneurons of chick embryos, by separately increasing or decreasing Dscam expression in each of these three specific interneuronal populations. Neuron-specific gain and loss of function of Dscam had no effect on the axonal trajectories of dI1-3 neurons. The commissural neurons, dI1c and dI2, crossed the midline, and the ipsilaterally projecting neurons, dI1i and dI3, projected ipsilaterally. However, the fasciculation of dI1 axons was diminished when Dscam expression was attenuated. Dscam is not required for either attraction to or repulsion from the floor plate. In contrast, Dscam is required for the fasciculation of axons, probably via homophilic interaction.

Processes of >100 types of interneurons (bipolar and amacrine cells) and projection neurons (retinal ganglion cells, RGCs) form specific and stereotyped patterns of connections in the inner plexiform layer (IPL) of the mouse retina. Four closely related homophilic immunoglobulin superfamily recognition molecules (Sidekick [Sdk] 1, Sdk 2, Dscam, and DscamL1) have been shown to play roles in patterning neuronal arbors and connections in chick retina, and all but Sdk1 have been shown to play related roles in mice. Here, we compare patterns of Sdk1 and Sdk2 expression in mouse retina and use genetic methods to assess roles of Sdk1. In adult retina, 3 neuronal types express sdk1 but not sdk2 at detectable levels, 5 express sdk2 but not sdk1 and 3 express both. Patterns of gene expression and protein localization at or near synapses are established during the first postnatal week. Dendrites of amacrine cells and RGCs that express sdk1 but not sdk2 arborize in the same narrow stratum in the center of the IPL. In the absence of Sdk1, this laminar restriction is degraded. Overexpression of sdk1 in developing cells that normally express sdk2 reorients their dendrites to resemble those of endogenously Sdk1-positive cells, indicating that Sdk1 plays an instructive role in patterning the IPL. Sdk1 fails to affect arbors when introduced after they are mature, suggesting that it is required to form but not maintain laminar restrictions. The effect of ectopically expressed sdk1 requires the presence of endogenous Sdk1, suggesting that the effect requires homophilic interactions among Sdk1-positive neurites. Together with previous results on Sdk2, Dscam, DscamL1, as well as the related Contactins, our results support the idea that an elaborate immunoglobulin superfamily code plays a prominent role in establishing neural circuits in the retina by means of tightly regulated cell type-specific expression and homophilically restricted intercellular interactions.

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection and synaptic transmission, but they are challenging to visualize because existing techniques1,2,3 have insufficient sensitivity and/or specificity. Here we report split horseradish peroxidase (sHRP) as a sensitive and specific tool for detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable for studying mechanisms of communication between a variety of cell types.

Summary Distinct neuronal types connect in complex ways to generate functional neural circuits. The molecular diversity required to specify this connectivity could be supplied by multigene families of synaptic recognition molecules, but most studies to date have assessed just one or a few members at a time. Here, we analyze roles of cadherins (Cdhs) in formation of retinal circuits comprising eight neuronal types that inform the brain about motion in four directions. We show that at least 15 classical Cdhs are expressed by neurons in these circuits and at least 6 (Cdh6–10 and 18) act individually or in combinations to promote specific connectivity among the cells. They act in part by directing the processes of output neurons and excitatory interneurons to a cellular scaffold formed by inhibitory interneurons. Because Cdhs are expressed combinatorially by many central neurons, similar interactions could be involved in patterning circuits throughout the brain.

Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 μ m(3) at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 μm and at depths up to 360 μ m can be discriminated.

Each P-RANbody consists of a HRP reporter and a nanobody that binds to antigen(s). This is a protocol to stain cells and tissues with P-RANbodies. The method is similar to common immunostaining protocols. However, this protocol focuses on some important tips.

This is a protocol for cell adhesion assays using 293T cells or N-cadherin-deficient 293NC cells (Yamagata et al., 2018). HEK293T(ATCC CRL-3216) cells are easily transfectable with plasmids. The 293NC cells lack N-cadherin (Cadherin-2), but maintain the property of its parental 293T cells. Thus, this 293T variant is useful to test cell adhesion (Goodman et al., 2016; Yamagata et al., 2018). Here, I describe a protocol and some technical tips.

Classical cadherins, a set of ~20 related recognition and signaling molecules, have been implicated in many aspects of neural development, including the formation and remodeling of synapses. Mechanisms underlying some of these steps have been studied by expressing N-cadherin (cdh2), a Type 1 cadherin, in heterologous cells, but analysis is complicated because widely used lines express cdh2 endogenously. We used CRISPR-mediated gene editing to generate a Human embryonic kidney (HEK)293 variant lacking Cdh2, then compared the behavior of rodent cortical and hippocampal neurons co-cultured with parental, cdh2 mutant and cdh2-rescued 293 lines. The comparison demonstrated that Cdh2 promotes neurite branching and that it is required for three synaptic organizers, neurologin1 (NLGL1), leucine-rich repeat transmembrane protein 2 (LRRtm2), and Cell Adhesion Molecule 1 (Cadm1/SynCAM) to stimulate presynaptic differentiation, assayed by clustering of synaptic vesicles at sites of neurite-293 cell contact. Similarly, Cdh2 is required for a presynaptic organizing molecule, Neurexin1β, to promote postsynaptic differentiation in dendrites. We also show that another Type I cadherin, Cdh4, and a Type II cadherin, Cdh6, can substitute for Cdh2 in these assays. Finally, we provide evidence that the effects of cadherins require homophilic interactions between neurites and the heterologous cells. Together, these results indicate that classical cadherins act together with synaptic organizers to promote synaptic differentiation, perhaps in part by strengthening the intracellular adhesion required for the organizers to act efficiently. We propose that cadherins promote high affinity contacts between appropriate partners, which then enable synaptic differentiation.

In the mammalian retina, processes of approximately 70 types of interneurons form specific synapses on roughly 30 types of retinal ganglion cells (RGCs) in a neuropil called the inner plexiform layer. Each RGC type extracts salient features from visual input, which are sent deeper into the brain for further processing. The specificity and stereotypy of synapses formed in the inner plexiform layer account for the feature-detecting ability of RGCs. Here we analyse the development and function of synapses on one mouse RGC type, called the W3B-RGC. These cells have the remarkable property of responding when the timing of the movement of a small object differs from that of the background, but not when they coincide. Such cells, known as local edge detectors or object motion sensors, can distinguish moving objects from a visual scene that is also moving. We show that W3B-RGCs receive strong and selective input from an unusual excitatory amacrine cell type known as VG3-AC (vesicular glutamate transporter 3). Both W3B-RGCs and VG3-ACs express the immunoglobulin superfamily recognition molecule sidekick 2 (Sdk2), and both loss- and gain-of-function studies indicate that Sdk2-dependent homophilic interactions are necessary for the selectivity of the connection. The Sdk2-specified synapse is essential for visual responses of W3B-RGCs: whereas bipolar cells relay visual input directly to most RGCs, the W3B-RGCs receive much of their input indirectly, via the VG3-ACs. This non-canonical circuit introduces a delay into the pathway from photoreceptors in the centre of the receptive field to W3B-RGCs, which could improve their ability to judge the synchrony of local and global motion.

Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show that SO synapses normally have significantly more mushroom spines and higher-magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered that these differences require the Type II classic cadherins, cadherins-6, -9, and -10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and -10 to regulate mushroom spine density and high-magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower-magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, -9, and -10 are gatekeepers for high-magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions. DOI: http://dx.doi.org/10.7554/eLife.19058.001

Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs. DOI: http://dx.doi.org/10.7554/eLife.15015.001, eLife digest Neurons connect with each other to form complex circuits that underlie mental activities. Mapping these connections to obtain a so-called wiring diagram is an essential step in learning how the brain works. The only way to do this precisely enough is by using electron microscopy. However, this technique is so time-consuming that thousands of hours of work are typically required to image even the smallest of tissue samples. Electron microscopes fire beams of electrons at tissue samples, and detect the scattering of the electrons. Stains are used to make specific neurons less permeable to electrons, or more “electron dense”. Labeled cells scatter more electrons, which increases the contrast of the images. In an approach called serial-section electron microscopy, a tissue sample is first cut into extremely thin sections. These are imaged individually, and the images are then pieced together to reconstruct the sample. Joesch et al. have now developed a new procedure – named ARTEMIS – that uses a combination of multiple techniques to speed up the mapping of neurons and their connections. ARTEMIS makes use of genetic engineering, serial-scanning electron microscopy, an enhanced chemical staining procedure and a new image processing approach. First, gene technology is used to selectively stain specific types of neurons in mice and flies. Then, a tissue sample is collected and treated with a chemical that enhances the electron density of the stained neurons, without disrupting the tissue’s structure. Next, a labeled target neuron is imaged at relatively low resolution to reveal its overall structure. Small areas of that neuron are then re-imaged at higher resolution to map the connections between neurons. Lastly, an algorithm pieces together the individual images to produce a reconstruction of the cell. This pipeline of steps reduces the time required to map the shapes and connectivity of neurons with electron microscopy by some two orders of magnitude. This should enable neuroscientists to obtain more rapid insights into the roles of specific neural circuits in the brains of healthy animals, and to identify cases where this wiring goes awry and leads to disease. DOI: http://dx.doi.org/10.7554/eLife.15015.002

Bipolar, amacrine, and retinal ganglion cells elaborate arbors and form synapses within the inner plexiform layer (IPL) of the vertebrate retina. Specific subsets of these neuronal types synapse in one or a few of the ≥10 sublaminae of the IPL. Four closely related Ig superfamily transmembrane adhesion molecules—Sidekick1 (Sdk1), Sdk2, Dscam, and DscamL—are expressed by non-overlapping subsets of chick retinal neurons and promote their lamina-specific arborization (Yamagata and Sanes, 2008). Here, we asked whether contactins (Cntns), six homologs of Sdks and Dscams, are expressed by and play roles in other subsets.In situhybridization showed thatcntn1–5were differentially expressed by subsets of amacrine cells. Immunohistochemistry showed that each Cntn protein was concentrated in a subset of IPL sublaminae. To assess roles of Cntns in retinal development, we focused on Cntn2. Depletion of Cntn2 by RNA interference markedly reduced the ability of Cntn2-positive cells to restrict their arbors to appropriate sublaminae. Conversely, ectopic expression ofcntn2redirected neurites of transduced neurons to the Cntn2-positive sublaminae. Thus, both loss- and gain-of-function strategies implicate Cntn2 in lamina-specific neurite targeting. Studies in heterologous cells showed that Cntn2 mediates homophilic adhesion, but does not bind detectably to Sdks, Dscams, or other Cntns. Overexpression analysis showed that Cntns1 and 3 can also redirect neurites to appropriate sublaminae. We propose that Cntns, Sdks, and Dscams comprise an Ig superfamily code that uses homophilic interactions to promote lamina-specific targeting of retinal dendrites in IPL.

The retina contains ganglion cells (RGCs) that respond selectively to objects moving in particular directions. Individual members of a group of ON-OFF direction-selective RGCs (ooDSGCs) detect stimuli moving in one of four directions: ventral, dorsal, nasal, or temporal. Despite this physiological diversity, little is known about subtype-specific differences in structure, molecular identity, and projections. To seek such differences, we characterized mouse transgenic lines that selectively mark ooDSGCs preferring ventral or nasal motion as well as a line that marks both ventral- and dorsal-preferring subsets. We then used the lines to identify cell surface molecules, including Cadherin 6, CollagenXXVα1, and Matrix metalloprotease 17, that are selectively expressed by distinct subsets of ooDSGCs. We also identify a neuropeptide, CART (cocaine- and amphetamine-regulated transcript), that distinguishes all ooDSGCs from other RGCs. Together, this panel of endogenous and transgenic markers distinguishes the four ooDSGC subsets. Patterns of molecular diversification occur before eye opening and are therefore experience independent. They may help to explain how the four subsets obtain distinct inputs. We also demonstrate differences among subsets in their dendritic patterns within the retina and their axonal projections to the brain. Differences in projections indicate that information about motion in different directions is sent to different destinations.

Four transmembrane adhesion molecules—Sidekick-1, Sidekick-2, Down's syndrome cell adhesion molecule (Dscam), and Dscam-like—are determinants of lamina-specific synapse formation in the vertebrate retina. Their C termini are predicted to bind postsynaptic density (PSD)-95/Discs Large/ZO-1 (PDZ) domains, which are present in many synaptic scaffolding proteins. We identify members of the membrane-associated guanylate kinase with inverted orientation (MAGI) and PSD-95 subfamilies of multi-PDZ domain proteins as binding partners for Sidekicks and Dscams. Specific MAGI and PSD-95 family members are present in distinct subsets of retinal synapses, as are Sidekicks and Dscams. Using Sidekick-2 as an exemplar, we show that its PDZ-binding C terminus is required for both its synaptic localization in photoreceptors and its ability to promote lamina-specific arborization of presynaptic and postsynaptic processes in the inner plexiform layer. In photoreceptor synapses that contain both MAGI-1 and PSD-95, Sidekick-2 preferentially associates with MAGI-1. Depletion of MAGI-1 from photoreceptors by RNA interference blocks synaptic localization of Sidekick-2 in photoreceptors without affecting localization of PSD-95. Likewise, depletion of MAGI-2 from retinal ganglion cells and interneurons interferes with Sidekick-2-dependent laminar targeting of processes. These results demonstrate that localization and function of Sidekick-2 require its incorporation into a MAGI-containing synaptic scaffold.

The selectivity with which proprioceptive sensory neurons innervate their central and peripheral targets implies that they exhibit distinctions in muscle-type identity. The molecular correlates of proprioceptor identity and its origins remain largely unknown, however. In screens to define muscle-type proprioceptor character we find all-or-none differences in gene expression for proprioceptors that control antagonistic muscles at a single hindlimb joint. Analysis of three of these genes, cadherin13 (cdh13), semaphorin5a (sema5a) and cartilage-acidic protein-1 (crtac1), reveals expression in proprioceptor subsets that supply muscle-groups located at restricted dorso-ventral and proximo-distal domains of the limb. Genetically altering the dorso-ventral character of the limb mesenchyme elicits a change in the profile of proprioceptor cdh13, sema5a and crtac1 expression. These findings indicate that proprioceptors acquire aspects of their muscle-type identity in response to mesenchymal signals expressed in restricted proximo-distal and dorso-ventral domains of the developing limb.

Axons of retinal ganglion cells (RGCs) carry visual information to the brain. In most vertebrates, the major synaptic target of RGCs is the optic tectum. In the chick, RGC axons form synapses in just 4 of 16 histologically recognizable laminae (the retinorecipient laminae [RRLs]), and arbors of individual RGCs are confined to a single RRL. To analyze the development and function of these parallel pathways, markers are required that selectively label them. Here, we have identified molecular markers for individual RRLs and for RGCs that project to them. Some of the markers may mediate or modulate signaling through the separate pathways: neuropeptides (substance P, neuromedin B, somatostatin-I and -II) and their receptors (substance P receptor), neurotransmitter synthetic enzymes (choline acetyltransferase) and the corresponding receptors (acetylcholine receptor beta2) and calcium-binding proteins (parvalbumin and calbindin). Other markers are adhesive proteins that could mediate selective connectivity of RGC subsets within specific RRLs (cadherin-7, cadherin-11, reelin and neuropilin-1). We further show that RGC subsets whose axons project to specific RRLs are heterogeneous with respect to the retinal sublaminae within which their dendrites arborize. Our results define laminar-specified circuits from retina to brain and support a model in which RGCs transmit information from multiple sources to single central laminae, where it can be integrated.

Chondroitin sulfate proteoglycans (CSPGs) of the extracellular matrix help stabilize synaptic connections in the postnatal brain and impede regeneration after injury. Here, we show that a CSPG of the lectican family, versican, also promotes presynaptic maturation in the developing brain. In the embryonic chick optic tectum, versican is expressed selectively by subsets of interneurons confined to the retinorecipient laminae, in which retinal axons arborize and form synapses. It is a major receptor for theVicia villosaB4lectin (VVA), shown previously to inhibit invasion of the retinorecipient lamina by retinal axons (Inoue and Sanes, 1997).In vitro, versican promotes enlargement of presynaptic varicosities in retinal axons. Depletion of versicanin ovo, by RNA interference, results in retinal arbors with smaller than normal varicosities. We propose that versican provides a lamina-specific cue for presynaptic maturation and discuss the related but distinct effects of versican depletion and VVA blockade.

Formation, differentiation and plasticity of synapses, the specialized cell-cell contacts through which neurons communicate, all require interactions between pre- and post-synaptic partners. Several synaptically localized adhesion molecules potentially capable of mediating these interactions have been identified recently. Functional studies suggest roles for some of them in target recognition (e.g. SYG-1 and sidekicks), formation and alignment of synaptic specializations (e.g. SynCAM, neuroligin and neurexin), and regulation of synaptic structure and function (e.g. cadherins and syndecan).

A major determinant of specific connectivity in the central nervous system is that synapses made by distinct afferent populations are restricted to particular laminae in their target area. We identify Sidekick (Sdk)-1 and -2, homologous transmembrane immunoglobulin superfamily molecules that mediate homophilic adhesion in vitro and direct laminar targeting of neurites in vivo. sdk-1 and -2 are expressed by nonoverlapping subsets of retinal neurons; each sdk is expressed by presynaptic (amacrine and bipolar) and postsynaptic (ganglion) cells that project to common inner plexiform (synaptic) sublaminae. Sdk proteins are concentrated at synaptic sites, and Sdk-positive synapses are restricted to the 2 (of ≥10) sublaminae to which sdk-expressing cells project. Ectopic expression of Sdk in Sdk-negative cells redirects their processes to a Sdk-positive sublamina. These results implicate Sdks as determinants of lamina-specific synaptic connectivity.

Lamina-specific neuronal connections are a fundamental feature in many parts of the vertebrate central nervous system. In the chick, the optic tectum is the primary visual centre, and it has a multilaminated structure consisting of 15 laminae, of which only three or four receive retinal projections. Each of the retinorecipient laminae establishes synaptic connections selectively from one of a few subsets of retinal ganglion cells (RGCs). We have generated a series of monoclonal antibodies that appear to stain only one of the retinorecipient laminae. One of these, TB4, stained lamina F which receives inputs from a subpopulation of approximately 10-20% of RGCs which express the presynaptic acetylcholine receptor beta2-subunit. TB4 recognized a single 79-kDa protein on immunoblotting. cDNA cloning and immunochemical analysis revealed that the TB4 antigen molecule was ezrin, a cytoskeletal-membrane linker molecule belonging to the ezrin-radixin-moesin family. Unilateral enucleation of the eye, both prior to and after the establishment of retinotectal projections, attenuated the lamina-selective staining with TB4 in the contralateral tectum, suggesting that ezrin is anterogradely transported from RGCs to lamina F. Ezrin was thus expressed in a subset of RGCs that project to lamina F. Similar subset-selective expression and resultant lamina-selective distribution of ezrin were also observed in the lamina-specific central projections from the dorsal root ganglia. The staining pattern with TB4 in the dorsal root ganglia and spinal cord indicated that high expression of ezrin was restricted in cutaneous sensory neurons, but not in muscle sensory neurons. Since ezrin modulates cell morphology and cell adhesion profiles by linking membrane proteins with the cytoskeleton, it was suggested that ezrin is involved in the formation and/or maintenance of lamina-specific connections for neuronal subpopulations in the visual and somatosensory systems.

In many parts of the vertebrate central nervous system, inputs of distinct types confine their synapses to individual laminae. Such laminar specificity is a major determinant of synaptic specificity. Recent studies of several laminated structures have begun to identify some of the cells (such as guidepost neurons in hippocampus), molecules (such as N-cadherin in optic tectum, semaphorin/collapsin in spinal cord, and ephrins in cerebral cortex), and mechanisms (such as activity-dependent refinement in lateral geniculate) that combine to generate laminar specificity.