Olfactory sensory neurons (OSNs) project a single axon from the olfactory epithelium to the olfactory bulb. OSNs initially target large, distinct, individually identifiable neuropils called protoglomeruli in the zebrafish embryo. Here we examine the contributions Robo axonal guidance receptors make to OSN axon targeting of protoglomeruli. We show that OSNs that project to the DZ protoglomerulus express higher levels of robo2 than those that project to the CZ protoglomerulus, and concordant with this observation, DZ-projecting axons are more often misrouted by loss of robo2 than are CZ-projecting axons. Further, we demonstrate that in the absence of robo2 , robo1 contributes to DZ-targeting but not to CZ-targeting. The loss of either robo1 or robo3 by themselves do not affect targeting to either the CZ or DZ protoglomeruli. These findings identify OSN subtype-dependent contributions of Robo receptors to vertebrate olfactory circuit assembly. In the absence of repellent Slit/Robo signaling, we propose that Netrin1b steers OSN axons to ectopic ventral midline locations where Slit1a and Netrin1b are both expressed.

To understand how neural circuits are assembled, it is essential to identify and characterize the axonal guidance cues and receptors that determine the axonal trajectories and connections between neurons. We performed single-cell RNA sequencing of olfactory sensory neurons from zebrafish to identify candidate axonal guidance-related genes that are differentially expressed according to sensory axon target location in the olfactory bulb. Among the candidates we identified were several members of the non-clustered delta-protocadherin family of adhesion molecules. We found that two members of the delta1-protocadherin family, pcdh7b and pcdh11, are most highly expressed in sensory neurons that project to a specific identifiable neuropil in the early olfactory bulb called the DZ protoglomerulus. Knocking down either one of these protocadherins impairs the ability of sensory axons to terminate within the DZ protoglomerulus. Knockdown does not affect the ability of other sensory axons from terminating normally in a separate neuropil called the CZ protoglomerulus. In contrast, two members of the delta2-protocadherin family, pcdh10b and pcdh17, are most highly expressed in sensory neurons that project to the CZ protoglomerulus. Knocking down pcdh10b induces ectopic terminations of CZ projecting sensory axons. Knocking down pcdh17 induces substantial ectopic axonal trajectories and impairs CZ projecting sensory axons from finding and terminating in the CZ protoglomerulus. Knockdowns of either pcdh10b or pcdh17 do not affect DZ projecting sensory axons. We conclude that delta1-protocadherins help DZ projecting sensory axons enter and remain within the DZ protoglomerulus, while delta2-protocadherins help CZ projecting sensory axons navigate to the CZ protoglomerulus.

Olfactory sensory neurons that express related odorant receptors specifically target large identifiable neuropils called protoglomeruli when they first reach the olfactory bulb in the zebrafish. This crude odorant receptor-related mapping is further refined as odorant receptor-specific glomeruli segregate from protoglomeruli later in development. Netrins are a prominent class of axon guidance molecules whose contribution to olfactory circuit formation is poorly studied. Morpholino knock down experiments have suggested that Netrin/Dcc signaling is involved in normal protoglomerular targeting. Here we extend these findings with more detailed characterization and modeling of netrin expression, and by examining protoglomerular targeting in mutant lines fornetrin1a (ntn1a), netrin1b (ntn1b), and their receptorsunc5b,dcc, andneo1a. We confirm thatntn1a,ntn1b, anddccare required for normal protoglomerular guidance of a subset of olfactory sensory neurons that are labeled with the Tg(or111-7:IRES:Gal4) transgene. We also observe errors in the targeting of these axons inunc5bmutants, but not inneo1a mutants. Our findings are consistent with ntn1a andntn1bacting primarily as attractants for olfactory sensory neurons targeting the central zone protoglomerulus.

Background Olfactory Sensory Neuron (OSN) axons project from the zebrafish olfactory epithelium to reproducible intermediate target locations in the olfactory bulb called protoglomeruli at early stages in development. Two classes of OSNs expressing either OMP or TRPC2 exclusively target distinct, complementary protoglomeruli. Using RNAseq, we identified axon guidance receptors nrp2a and nrp2b , and their ligand sema3fa , as potential guidance factors that are differentially expressed between these two classes of OSNs. Methods To investigate their role in OSN axon guidance, we assessed the protoglomerular targeting fidelity of OSNs labeled by OMP:RFP and TRPC2:Venus transgenes in nrp2a , nrp2b , or sema3fa mutants. We used double mutant and genetic interaction experiments to interrogate the relationship between the three genes. We used live time-lapse imaging to compare the dynamic behaviors of OSN growth cones during protoglomerular targeting in heterozygous and mutant larvae. Results The fidelity of protoglomerular targeting of TRPC2-class OSNs is degraded in nrp2a , nrp2b , or sema3fa mutants, as axons misproject into OMP-specific protoglomeruli and other ectopic locations in the bulb. These misprojections are further enhanced in nrp2a;nrp2b double mutants suggesting that nrp2s work at least partially in parallel in the same guidance process. Results from genetic interaction experiments are consistent with sema3fa acting in the same biological pathway as both nrp2a and nrp2b . Live time-lapse imaging was used to examine the dynamic behavior of TRPC2-class growth cones in nrp2a mutants compared to heterozygous siblings. Some TRPC2-class growth cones ectopically enter the dorsal-medial region of the bulb in both groups, but in fully mutant embryos, they are less likely to correct the error through retraction. The same result was observed when TRPC2-class growth cone behavior was compared between sema3fa heterozygous and sema3fa mutant larvae. Conclusions Our results suggest that nrp2a and nrp2b expressed in TRPC2-class OSNs help prevent their mixing with axon projections in OMP-specific protoglomeruli, and further, that sema3fa helps to exclude TRPC2-class axons by repulsion from the dorsal-medial bulb.

Semaphorins are a large and important family of signaling molecules conserved in Bilateria. An important determinant of the biological function of their largest class, the secreted class 3 semaphorins, is the specificity of their binding to neuropilins, a key component of a larger holoreceptor complex. We compared these binding specificities in mice and zebrafish, species whose most recent common ancestor was more than 400 million years in the past. We also compared the binding specificities of zebrafish class 3 semaphorins that were duplicated very early within the teleost lineage. We found a surprising conservation of neuropilin binding specificities when comparing both paralogous zebrafish semaphorin pairs and orthologous zebrafish and mouse semaphorin pairs. This finding was further supported by a remarkable conservation of binding specificities in cross-species pairings of semaphorins and neuropilins. Our results suggest that the qualitative specificities with which particular semaphorins bind to particular neuropilins has remained nearly invariant over approximately 400 million years of evolution.

Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.

BACKGROUND:The axons of Olfactory Sensory Neurons (OSNs) project to reproducible target locations within the Olfactory Bulb (OB), converting odorant experience into a spatial map of neural activity. We characterized the initial targeting of OSN axons in the zebrafish, a model system suitable for studying axonal targeting early in development. In this system the initial targets of OSN axons are a small number of distinct, individually identifiable neuropilar regions called protoglomeruli. Previously, Olfactory Marker Protein-expressing and TRPC2-expressing classes of OSNs were shown to project to specific, non-overlapping sets of protoglomeruli, indicating that particular subsets of OSNs project to specific protoglomerular targets. We set out to map the relationship between the classical Odorant Receptor (OR) an OSN chooses to express and the protoglomerulus its axon targets.METHODS:A panel of BACs were recombineered so that the axons of OSNs choosing to express modified ORs were fluorescently labeled. Axon projections were followed into the olfactory bulb to determine the protoglomeruli in which they terminated.RESULTS:RNA-seq demonstrates that OSNs express a surprisingly wide variety of ORs and Trace Amine Associated Receptors (TAARs) very early when sensory axons are arriving in the bulb. Only a single OR is expressed in any given OSN even at these early developmental times. We used a BAC expression technique to map the trajectories of OSNs expressing specific odorant receptors. ORs can be divided into three clades based upon their sequence similarities. OSNs expressing ORs from two of these clades project to the CZ protoglomerulus, while OSNs expressing ORs from the third clade project to the DZ protoglomerulus. In contrast, OSNs expressing a particular TAAR project to multiple protoglomeruli. Neither OR choice nor axonal targeting are related to the position an OSN occupies within the olfactory pit.CONCLUSIONS:Our results demonstrate that it is not the choice of a particular OR, but of one from a category of ORs, that is related to initial OSN target location within the olfactory bulb. These choices are not related to OSN position within the olfactory epithelium.

Olfactory sensory axons target well-defined intermediate targets in the zebrafish olfactory bulb called protoglomeruli well before they form odorant receptor-specific glomeruli. A subset of olfactory sensory neurons are labeled by expression of the or111-7:IRES: GAL4 transgene whose axons terminate in the central zone (CZ) protoglomerulus. Previous work has shown that some of these axons misproject to the more dorsal and anterior dorsal zone (DZ) protoglomerulus in the absence of Netrin 1/Dcc signaling. In search of additional cues that guide these axons to the CZ, we found that Semaphorin 3D (Sema3D) is expressed in the anterior bulb and acts as a repellent that pushes them towards the CZ. Further analysis indicates that Sema3D signaling is mediated through Nrp1a, while Nrp2b also promotes CZ targeting but in a Sema3D-independent manner. nrp1a, nrp2b and dcc transcripts are detected in or111-7 transgene-expressing neurons early in development and both Nrp1a and Dcc act cell-autonomously in sensory neurons to promote accurate targeting to the CZ. dcc and nrp1a double mutants have significantly more DZ misprojections than either single mutant, suggesting that the two signaling systems act independently and in parallel to direct a specific subset of sensory axons to their initial protoglomerular target.

A given olfactory sensory neuron (OSN) in the olfactory epithelium chooses to express only one odorant receptor (OR) from the OR gene repertoire. OSNs expressing the same receptor project axons to the same reproducibly positioned glomerulus in the olfactory bulb. As a consequence, specific odorants evoke distinctive patterns of glomerular activation in the bulb. The human odorant receptor repertoire contains 400 genes while the zebrafish repertoire contains approximately 143 genes. Our objective is to correlate the odorant specificity of each zebrafish OR with the associated glomerulus they target in the bulb. To achieve this we are cloning ORs into an expression vector which is then transfected into Hana3A cells. OR activation is monitored via a high‐throughput luciferase reporter assay in response to a large library of approximately 850 individual and defined odorants. In our first preliminary studies we have successfully cloned three ORs (111‐7, 111‐11, and 128‐5) and have identified specific candidate odorants that activate each of them. We subsequently cloned 9 more ORs into the expression vector for further analysis and characterization of odorant activation. The heterologous expression system was found to be suitable for matching odorants to zebrafish ORs. In future studies, we will seek to determine a functional relationship between odorant‐specific responses of the odorant receptors with the target locations within the bulb of the olfactory sensory neurons that express them.

Growing axons navigate a complex environment as they respond to attractive and repellent guidance cues. Axons can modulate their responses to cues through a G-protein-coupled, cAMP-dependent signaling pathway. To examine the role of G-protein signaling in axon guidance in vivo, we used the GAL4/UAS system to drive expression of dominant-negative heterotrimeric G-proteins (DNG) in retinal ganglion cells (RGCs) of embryonic zebrafish. Retinal axons normally cross at the ventral midline and project to the contralateral tectum. Expression of DNGα(S) in RGCs causes retinal axons to misproject to the ipsilateral tectum. These errors resemble misprojections in adcy1, adcy8, nrp1a, sema3D, or sema3E morphant embryos, as well as in sema3D mutant embryos. nrp1a is expressed in RGCs as their axons extend toward and across the midline. sema3D and sema3E are expressed adjacent to the chiasm, suggesting that they facilitate retinal midline crossing. We demonstrate synergistic induction of ipsilateral misprojections between adcy8 knockdown and transgenic DNGα(S) expression, adcy8 and nrp1a morphants, or nrp1a morphants and transgenic DNGα(S) expression. Using qPCR analysis, we show that either transgenic DNGα(S)-expressing embryos or adcy8 morphant embryos have decreased levels of nrp1a and nrp1b mRNA. Ipsilateral misprojections in adcy8 morphants are corrected by the expression of an nrp1a rescue construct expressed in RGCs. These findings are consistent with the idea that elevated cAMP levels promote Neuropilin1a expression in RGCs, increasing the sensitivity of retinal axons to Sema3D, Sema3E, or other neuropilin ligands at the midline, and consequently facilitate retinal axon crossing in the chiasm.

Olfactory sensory neurons expressing particular olfactory receptors project to specific reproducible locations within the bulb. The axonal guidance cues that organize this precise projection pattern are only beginning to be identified. To aid in their identification and characterization, we generated a transgenic zebrafish line, OR111-7:IRES:Gal4, in which a small subset of olfactory sensory neurons is labeled. Most sensory neurons expressing the OR111-7 transgene project to a specific location within the bulb, the central zone protoglomerulus, while a smaller number project to the lateral glomerulus 1 protoglomerulus. Inhibiting Netrin/DCC (deleted in colorectal cancer) signaling perturbs the ability of OR111-7-expressing axons to enter the olfactory bulb and alters their patterns of termination within the bulb. The Netrin receptor DCC is expressed in olfactory sensory neurons around the time that they elaborate their axons, netrin1a is expressed near the medial-most margin of the olfactory bulb, and netrin1b is expressed within the ventral region of the bulb. Loss of Netrin/DCC signaling components causes some OR111-7-expressing sensory axons to wander posteriorly after exiting the olfactory pit, away from netrin-expressing areas in the bulb. OR111-7-expressing axons that enter the bulb target the central zone less precisely than normal, spreading away from netrin-expressing regions. These pathfinding errors can be corrected by the reexpression of DCC within OR111-7 transgene-expressing neurons in DCC morphant embryos. These findings implicate Netrins as the only known attractants for olfactory sensory neurons, first drawing OR111-7-expressing axons into the bulb and then into the ventromedially positioned central zone protoglomerulus.

SDF1 reduces the responsiveness of axonal growth cones to repellent guidance cues in a pertussis-toxin-sensitive, cAMP-dependent manner. Here, we show that SDF1's antirepellent effect can be blocked in embryonic chick dorsal root ganglia (DRGs) by expression of peptides or proteins inhibiting either Gα(i), Gα(q), or Gβγ. SDF1 antirepellent activity is also blocked by pharmacological inhibition of PLC, a common effector protein for Gα(q). We also show that SDF1 antirepellent activity can be mimicked by overexpression of constitutively active Gα(i), Gα(q), or Gα(s). These results suggest a model in which multiple G protein components cooperate to produce the cAMP levels required for SDF1 antirepellent activity.

Axons follow highly stereotyped and reproducible trajectories to their targets. In this review we address the properties of the first pioneer neurons to grow in the developing nervous system and what has been learned over the past several decades about the extracellular and cell surface substrata on which axons grow. We then discuss the types of guidance cues and their receptors that influence axon extension, what determines where cues are expressed, and how axons respond to the cues they encounter in their environment.

The chemokine SDF1 activates a cAMP-mediated signaling pathway that antagonizes retinal responses to the midline repellent slit. We show that knocking down the calmodulin-activated adenylate cyclase ADCY8 makes retinal axons insensitive to SDF1. Experiments in vivo using male and female zebrafish (Danio rerio) confirm a mutual antagonism between slit signaling and ADCY8-mediated signaling. Unexpectedly, knockdown of ADCY8 or another calmodulin-activated cyclase, ADCY1, induces ipsilateral misprojections of retinal axons that would normally cross the ventral midline. We demonstrate a cell-autonomous requirement for ADCY8 in retinal neurons for normal midline crossing. These findings are the first to show that ADCY8 is required for axonal pathfinding before axons reach their targets. They support a model in which ADCY8 is an essential component of a signaling pathway that opposes repellent signaling. Finally, they demonstrate that ADCY8 helps regulate retinal sensitivity to midline guidance cues.

Growth cone guidance during development, as well as axonal extension in neural repair and plasticity, is strongly regulated by both attractive (growth-promoting) and repulsive (growth-inhibiting) guidance molecules. The growth cone collapse assay has been widely and successfully used for the identification and purification of molecules that are repulsive to growth cones or inhibit axonal outgrowth. Here we provide a detailed description of the assay, which uses the morphology of the growth cone after exposure to a test protein as the readout. With the modifications detailed in this protocol, this assay can be used for the biochemical enrichment of proteins with a collapsing activity and for the identification of a collapsing activity of a known protein or gene. This assay does not require very specialized equipment and can be established by every lab with experience in neuronal cell culture. It can be completed in 3 d.

Retinal ganglion cell axons exit the eye, enter the optic stalk, cross the ventral midline at the optic chiasm, and terminate in the optic tectum of the zebrafish. While in the optic stalk, they grow immediately adjacent to cells expressing the powerful retinal axon repellent slit2. The chemokine stromal cell-derived factor-1 (SDF1) is expressed within the optic stalk and its receptor CXCR4 is expressed in retinal ganglion cells. SDF1 makes cultured retinal axons less responsive to slit2. Here, we show that reducing SDF1 signaling in vivo rescues retinal axon pathfinding errors in zebrafish mutants that have a partial functional loss of the slit receptor robo2. In contrast, reducing SDF1 signaling in animals that completely lack the robo2 receptor does not rescue retinal guidance errors. These results demonstrate that endogenous levels of SDF1 antagonize the repellent effects of slit/robo signaling in vivo and that this antagonism is important during axonal pathfinding.

Neural crest cells migrate along two discrete pathways within the trunk of developing embryos. In the chick, early migrating crest cells are confined to a ventral pathway medial to the dermamyotome while later cells migrate on a dorsal pathway lateral to the dermamyotome. Here we show that Slits are expressed in the dermamyotome, that early migrating crest cells express the Slit receptors Robo 1 and Robo 2, that Slit2 repels migrating crest cells in an in vitro assay, and that the misexpression of a dominant-negative Robo1 receptor induces a significant fraction of early crest cells to migrate ectopically in the dorso-lateral pathway. These findings suggest that Slits, most likely those expressed in the dermamyotome, help to confine the migration of early crest cells to the ventral pathway.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. Here, we propose a new role for this neurotransmitter in the developing nervous system. We show that glutamate or the metabotropic class I agonist S-3,5-dihydroxyphenyl glycine, acting through the metabotropic glutamate receptor 1 (mGluR1), can reduce the activity of multiple axonal repellents in vitro. This effect is mediated by a pertussis toxin-sensitive activation of protein kinase A and the subsequent inactivation of Rho. This signaling pathway appears to be identical to the one we described previously for stromal derived factor-1-induced reduction of axonal repellent activities. Activation of mGluR1 can also promote increased survival of embryonic retinal ganglion cells in culture. We propose that neurotransmitter-induced modulation of repellent strength provides a novel mechanism by which activity can influence neuronal morphology.

Altering the concentrations of cyclic nucleotides within nerve cells can dramatically change their responses to axonal guidance cues, but the physiological signals that might induce such alterations are unknown. Here we show that the chemokine stromal cell-derived factor 1 (SDF-1) reduces the repellent activities of slit-2 on cultured retinal ganglion cell axons, of semaphorin 3A on dorsal root ganglion sensory axons, and of semaphorin 3C on sympathetic axons. This is a modulatory effect because SDF-1 has no detectable attractive or repellent effects on retinal or DRG axons by itself. This modulation is mediated through CXCR4, the receptor of SDF-1, and a pertussis toxin-sensitive G-protein-coupled signaling pathway that induces an elevation of cAMP. The spinal cords of CXCR4 mutant mice contain hyperfasciculated and aberrantly projecting axons. These results suggest that SDF-1 plays an essential role in modulating axonal responsiveness to various known guidance cues through a cyclic nucleotide-dependent signaling pathway.