ER stress transforms random olfactory receptor choice into axon targeting precision.

Olfactory sensory neurons (OSNs) convert the stochastic choice of one of >1,000 olfactory receptor (OR) genes into precise and stereotyped axon targeting of OR-specific glomeruli in the olfactory bulb. Here, we show that the PERK arm of the unfolded protein response (UPR) regulates both the glomerular coalescence of like axons and the specificity of their projections. Subtle differences in OR protein sequences lead to distinct patterns of endoplasmic reticulum (ER) stress during OSN development, converting OR identity into distinct gene expression signatures. We identify the transcription factor Ddit3 as a key effector of PERK signaling that maps OR-dependent ER stress patterns to the transcriptional regulation of axon guidance and cell-adhesion genes, instructing targeting precision. Our results extend the known functions of the UPR from a quality-control pathway that protects cells from misfolded proteins to a sensor of cellular identity that interprets physiological states to direct axon wiring.

Development of the Binocular Circuit.

Seeing in three dimensions is a major property of the visual system in mammals. The circuit underlying this property begins in the retina, from which retinal ganglion cells (RGCs) extend to the same or opposite side of the brain. RGC axons decussate to form the optic chiasm, then grow to targets in the thalamus and midbrain, where they synapse with neurons that project to the visual cortex. Here we review the cellular and molecular mechanisms of RGC axonal growth cone guidance across or away from the midline via receptors to cues in the midline environment. We present new views on the specification of ipsi- and contralateral RGC subpopulations and factors implementing their organization in the optic tract and termination in subregions of their targets. Lastly, we describe the functional and behavioral aspects of binocular vision, focusing on the mouse, and discuss recent discoveries in the evolution of the binocular circuit.

Axon-axon interactions determine modality-specific wiring and subcellular synaptic specificity in a somatosensory circuit.

Synaptic connections between neurons are often formed in precise subcellular regions of dendritic arbors with implications for information processing within neurons. Cell-cell interactions are widely important for circuit wiring; however, their role in subcellular specificity is not well understood. We studied the role of axon-axon interactions in precise targeting and subcellular wiring of Drosophila somatosensory circuitry. Axons of nociceptive and gentle touch neurons terminate in adjacent, non-overlapping layers in the central nervous system (CNS). Nociceptor and touch receptor axons synapse onto distinct dendritic regions of a second-order interneuron, the dendrites of which span these layers, forming touch-specific and nociceptive-specific connectivity. We found that nociceptor ablation elicited extension of touch receptor axons and presynapses into the nociceptor recipient region, supporting a role for axon-axon interactions in somatosensory wiring. Conversely, touch receptor ablation did not lead to expansion of nociceptor axons, consistent with unidirectional axon-axon interactions. Live imaging provided evidence for sequential arborization of nociceptive and touch neuron axons in the CNS. We propose that axon-axon interactions and modality-specific timing of axon targeting play key roles in subcellular connection specificity of somatosensory circuitry.

A cell-ECM mechanism for connecting the ipsilateral eye to the brain.

Information about features in the visual world is parsed by circuits in the retina and is then transmitted to the brain by distinct subtypes of retinal ganglion cells (RGCs). Axons from RGC subtypes are stratified in retinorecipient brain nuclei, such as the superior colliculus (SC), to provide a segregated relay of parallel and feature-specific visual streams. Here, we sought to identify the molecular mechanisms that direct the stereotyped laminar targeting of these axons. We focused on ipsilateral-projecting subtypes of RGCs (ipsiRGCs) whose axons target a deep SC sublamina. We identified an extracellular glycoprotein, Nephronectin (NPNT), whose expression is restricted to this ipsiRGC-targeted sublamina. SC-derived NPNT and integrin receptors expressed by ipsiRGCs are both required for the targeting of ipsiRGC axons to the deep sublamina of SC. Thus, a cell-extracellular matrix (ECM) recognition mechanism specifies precise laminar targeting of ipsiRGC axons and the assembly of eye-specific parallel visual pathways.

Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

Rewiring the future: drugs abused in adolescence may predispose to mental illness in adult life by altering dopamine axon growth.

Adolescence is a period of increased exploration and novelty-seeking, which includes new social behaviors, as well as drug experimentation, often spurred on by peer pressure. This is unfortunate, as the immature state of the adolescent brain makes it particularly susceptible to the negative developmental impact of drug use. During adolescence, dopamine terminals, which have migrated from the ventral tegmental area, pause in the nucleus accumbens, before segregating by either forming local connections or growing towards the prefrontal cortex (PFC). This developmentally late and lengthy process renders adolescent dopamine axon pathfinding vulnerable to disruption by substance use. Indeed, exposure to stimulant drugs in adolescent male mice, but not females, triggers dopamine axons to mistarget the nucleus accumbens and to grow ectopically to the PFC. Some evidence suggests that at this novel site, the functional organization of the ectopic dopamine axons mirrors that of the intended target. The structural rewiring dysregulates local synaptic connectivity, leading to poor impulse control ability, deficits of which are a core symptom of substance-use disorders. In the present commentary, we argue that different substances of abuse induce dopamine mistargeting events with the off-target trajectory prescribed by the type of drug, leading to psychiatric outcomes later in life.

Rorß regulates selective axon-target innervation in the mammalian midbrain.

Developmental control of long-range neuronal connections in the mammalian midbrain remains unclear. We explored the mechanisms regulating target selection of the developing superior colliculus (SC). The SC is a midbrain center that directs orienting behaviors and defense responses. We discovered that a transcription factor, Rorß, controls establishment of axonal projections from the SC to two thalamic nuclei: the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP). A genetic strategy used to visualize SC circuits revealed that in control animals Rorß+ neurons abundantly innervate the dLGN but barely innervate the LP. The opposite phenotype was observed in global and conditional Rorb mutants: projections to the dLGN were strongly decreased, and projections to the LP were increased. Furthermore, overexpression of Rorb in the wild type showed increased projections to the dLGN and decreased projections to the LP. In summary, we identified Rorß as a key developmental mediator of colliculo-thalamic innervation. Such regulation could represent a general mechanism orchestrating long-range neuronal connections in the mammalian brain.

Guidance of retinal axons in mammals.

In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face 'choices' in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision.

Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli.

The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. Here we show that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation.

Neuronal territory formation by the atypical cadherins and clustered protocadherins.

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.

The cellular prion protein promotes olfactory sensory neuron survival and axon targeting during adult neurogenesis.

The cellular prion protein (PrPC) has been associated with diverse biological processes including cell signaling, neurogenesis, and neuroprotection, but its physiological function(s) remain ambiguous. Here we determine the role of PrPC in adult neurogenesis using the olfactory system model in transgenic mice. Olfactory sensory neurons (OSNs) within the olfactory sensory epithelium (OSE) undergo neurogenesis, integration, and turnover even into adulthood. The neurogenic processes of proliferation, differentiation/maturation, and axon targeting were evaluated in wild type, PrP-overexpressing, and PrP-null transgenic mice. Our results indicate that PrPC plays a role in maintaining mature OSNs within the epithelium: overexpression of PrPC resulted in greater survival of mitotically active cells within the OSE, whereas absence of prion protein resulted in fewer cells being maintained over time. These results are supported by both quantitative PCR analysis of gene expression and protein analysis characteristic of OSN differentiation. Finally, evaluation of axon migration determined that OSN axon targeting in the olfactory bulb is PrPC dose-dependent. Together, these findings provide new mechanistic insight into the neuroprotective role for PrPC in adult OSE neurogenesis, whereby more mature neurons are stably maintained in animals expressing PrPC.

Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.

Neurons display a striking degree of functional and morphological diversity, and the developmental mechanisms that underlie diversification are of significant interest for understanding neural circuit assembly and function. We find that the morphology of Drosophila sensory neurons is diversified through a series of suppressive transcriptional interactions involving the POU domain transcription factors Pdm1 (Nubbin) and Pdm2, the homeodomain transcription factor Cut, and the transcriptional regulators Scalloped and Vestigial. Pdm1 and Pdm2 are expressed in a subset of proprioceptive sensory neurons and function to inhibit dendrite growth and branching. A subset of touch receptors show a capacity to express Pdm1/2, but Cut represses this expression and promotes more complex dendritic arbors. Levels of Cut expression are diversified in distinct sensory neurons by selective expression of Scalloped and Vestigial. Different levels of Cut impact dendritic complexity and, consistent with this, we show that Scalloped and Vestigial suppress terminal dendritic branching. This transcriptional hierarchy therefore acts to suppress alternative morphologies to diversify three distinct types of somatosensory neurons.

The Cytokine CXCL12 Promotes Basket Interneuron Inhibitory Synapses in the Medial Prefrontal Cortex.

Prenatally, the cytokine CXCL12 regulates cortical interneuron migration, whereas its postnatal functions are poorly understood. Here, we report that CXCL12 is expressed postnatally in layer V pyramidal neurons and localizes on their cell bodies in the medial prefrontal cortex (mPFC), while its receptors CXCR4/CXCR7 localize to the axon terminals of parvalbumin (PV) interneurons. Conditionally eliminating CXCL12 in neonatal layer V pyramidal neurons led to decreased axon targeting and reduced inhibitory perisomatic synapses from PV+ basket interneurons onto layer V pyramidal neurons. Consequently, the mPFC of Cxcl12 conditional mutants displayed attenuated inhibitory postsynaptic currents onto layer V pyramidal neurons. Thus, postnatal CXCL12 signaling promotes a specific interneuron circuit that inhibits mPFC activity.

Corticothalamic Axons Are Essential for Retinal Ganglion Cell Axon Targeting to the Mouse Dorsal Lateral Geniculate Nucleus.

UNLABELLED: Retinal ganglion cells (RGCs) relay information about the outside world to multiple subcortical targets within the brain. This information is either used to dictate reflexive behaviors or relayed to the visual cortex for further processing. Many subcortical visual nuclei also receive descending inputs from projection neurons in the visual cortex. Most areas receive inputs from layer 5 cortical neurons in the visual cortex but one exception is the dorsal lateral geniculate nucleus (dLGN), which receives layer 6 inputs and is also the only RGC target that sends direct projections to the cortex. Here we ask how visual system development and function changes in mice that develop without a cortex. We find that the development of a cortex is essential for RGC axons to terminate in the dLGN, but is not required for targeting RGC axons to other subcortical nuclei. RGC axons also fail to target to the dLGN in mice that specifically lack cortical layer 6 projections to the dLGN. Finally, we show that when mice develop without a cortex they can still perform a number of vision-dependent tasks. SIGNIFICANCE STATEMENT: The dorsal lateral geniculate nucleus (dLGN) is a sensory thalamic relay area that receives feedforward inputs from retinal ganglion cells (RGCs) in the retina, and feed back inputs from layer 6 neurons in the visual cortex. In this study we examined genetically manipulated mice that develop without a cortex or without cortical layer 6 axonal projections, and find that RGC axons fail to project to the dLGN. Other RGC recipient areas, such as the superior colliculus and suprachiasmatic nucleus, are targeted normally. These results provide support for a new mechanism of target selection that may be specific to the thalamus, whereby descending cortical axons provide an activity that promotes feedforward targeting of RGC axons to the dLGN.

Human von Economo neurons express transcription factors associated with Layer V subcerebral projection neurons.

The von Economo neurons (VENs) are large bipolar Layer V projection neurons found chiefly in the anterior cingulate and frontoinsular cortices. Although VENs have been linked to prevalent illnesses such as frontotemporal dementia, autism, and schizophrenia, little is known about VEN identity, including their major projection targets. Here, we undertook a developmental transcription factor expression study, focusing on markers associated with specific classes of Layer V projection neurons. Using mRNA in situ hybridization, we found that VENs prominently express FEZF2 and CTIP2, transcription factors that regulate the fate and differentiation of subcerebral projection neurons, in humans aged 3 months to 65 years. In contrast, few VENs expressed markers associated with callosal or corticothalamic projections. These findings suggest that VENs may represent a specialized Layer V projection neuron for linking cortical autonomic control sites to brainstem or spinal cord regions.

Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions.

Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that αPS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia.

Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring.

Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. Here, we examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. We found that synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.

Neuronal miR-17-5p contributes to interhemispheric cortical connectivity defects induced by prenatal alcohol exposure.

Structural and functional deficits in brain connectivity are reported in patients with fetal alcohol spectrum disorders (FASDs), but whether and how prenatal alcohol exposure (PAE) affects axonal development of neurons and disrupts wiring between brain regions is unknown. Here, we develop a mouse model of moderate alcohol exposure during prenatal brain wiring to study the effects of PAE on corpus callosum (CC) development. PAE induces aberrant navigation of interhemispheric CC axons that persists even after exposure ends, leading to ectopic termination in the contralateral cortex. The neuronal miR-17-5p and its target ephrin type A receptor 4 (EphA4) mediate the effect of alcohol on the contralateral targeting of CC axons. Thus, altered microRNA-mediated regulation of axonal guidance may have implications for interhemispheric cortical connectivity and associated behaviors in FASD.

Single-nucleus RNA sequencing of developing superior colliculus identifies neuronal diversity and candidate mediators of circuit assembly.

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment of neural connectivity. Here we perform single-nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Using these data, we find that Pax7 expression is restricted to a subset of GABAergic neurons. Our data provide a valuable resource for interrogating the mechanisms of circuit development and identifying markers for manipulating specific SC neuronal populations and circuits.

To Stick or Not to Stick: The Multiple Roles of Cell Adhesion Molecules in Neural Circuit Assembly.

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.