Biology subjects

Granato, M.

Publications and source records attributed to Granato, M..

18 recordsLinked to original sources

bioRxiv2026

Huntingtin and Celsr3 Orchestrate Spontaneous Long-Distance Spinal Cord Regeneration

Spontaneous spinal cord regeneration is an endogenous process operational in diverse vertebrate and select mammalian species. Yet despite resulting in functional regeneration, axonal regrowth dynamics as well as underlying molecular programs are largely unknown. Live-cell imaging following laser axotomy of individual zebrafish spinal cord M-cell axons reveals a time-sensitive multistep process. From a genetic screen we uncover previously unrecognized roles for two evolutionary conserved genes, celsr3 and huntingtin (htt) in spontaneous spinal cord regeneration. Specifically, we find that htt is critical to initiate a transcriptional injury response in regenerating M-cells, enabling their axon to traverse the injury site. Once axons cross the injury site celsr3 functions to increase growth rates, while both celsr3 and htt function to sustain long-range axonal growth. Moreover, we show that htt is dispensable for optic nerve and peripheral motor neuron regeneration, and that reducing soma-to-injury site distance in htt mutant M-cell axons partially restores regeneration, demonstrating a selective role in long-range CNS axon regeneration. Consistent with a transport-based mechanism, genetic knockdown of htt-associated axonal transport proteins recapitulates the htt mutant regeneration phenotype. Combined, our results provide compelling evidence for a time and distance-sensitive multistep molecular framework in which celsr3 dependent growth rate modulation and htt dependent axonal transport promote spontaneous, long-range spinal cord regeneration.

neuroscience↗

bioRxiv2026

Unique and overlapping behavioral effects of isoform-specific NRXN1 deletions across development

Mutations in the human neurexin 1 (NRXN1) gene are associated with neurodevelopmental disorders including autism and schizophrenia. Vertebrate NRXN1 produces three major NRXN1 isoforms, named , {beta}, and{gamma} . Human genetic data suggests that deletions located at the 5 region of the gene disrupting the isoform associate mostly with clinical behavioral deficits. Yet, 3 deletions that disrupt multiple isoforms have also been identified in clinical cases. Whether deletions that selectively affect specific NRXN1 isoforms result in specific behavioral deficits remains unclear. Here, we show that zebrafish harboring gene deletions that differentially encompass nrxn1a-, -{beta}, and/or -{gamma} display both unique and overlapping locomotion, sensorimotor behavior, and social behavior deficits. Combined, our results strongly support a model by which domain selective nrxn1 deletions predict behavioral phenotypes. Moreover, our results demonstrate compelling relationships between genotype and resulting behavioral phenotypes, providing functional insights into the complexity of NRXN1-associated neuropsychiatric behaviors.

neuroscience↗

bioRxiv2024

Glial cell derived pathway directs regenerating optic nerve axons toward the CNS midline

After optic nerve injury, several retinal ganglion cell (RGC) intrinsic signaling pathways have been shown to enhance RGC survival and RGC axonal growth. In contrast, few extrinsic cues have been identified that guide regenerating RGC axons toward and across the optic chiasm. Here, we use live-cell imaging in larval zebrafish to show that regrowing RGC axons initiate growth toward the midline and extend along a trajectory similar to their original projection. From a candidate genetic screen, we identify the glycosyltransferase Lh3 (also referred to as Plod3) to be required to direct regrowing RGC axons toward the midline during active regeneration. Moreover, we show that transgenic lh3 expression in sox10+ presumptive olig2+ oligodendrocytes located near the optic chiasm restores directed axonal growth in lh3 mutants. Finally, we find that mutants in collagen 18a1 (col18a1), a putative Lh3 substrate, display RGC axonal misguidance phenotypes similar to lh3 mutants, suggesting that lh3 may act through col18a1 during regeneration. Combined, these data identify lh3 as part of a glial derived molecular pathway critical for guiding in vivo regenerating RGC axons toward and across the optic chiasm.

neuroscience↗

bioRxiv2024

Serotonin neuromodulation directs optic nerve regeneration

Optic nerve (ON) regeneration in mammalian systems is limited by an overshadowing dominance of inhibitory factors. This has severely hampered the identification of pro- regenerative pathways. Here, we take advantage of the regenerative capacity of larval zebrafish to identify pathways that promote ON regeneration. From a small molecule screen, we identified modulators of serotonin (5-HT) signaling that inhibit ON regeneration. We find several serotonin type-1 receptor genes are expressed in RGC neurons during regeneration and that inhibiting 5- HT1 receptors or components of the 5-HT pathway selectively impedes ON regeneration. We show that 5-HT1 receptor signaling is dispensable during ON development yet is critical for regenerating axons to emerge from the injury site. Blocking 5-HT receptors once ON axons have crossed the chiasm does not inhibit regeneration, suggesting a selective role for 5-HT receptor signaling early during ON regeneration. Finally, we show that agonist-mediated activation of 5-HT1 receptors leads to enhanced and ectopic axonal regrowth. Combined, our results provide evidence for mechanisms through which serotonin-dependent neuromodulation directs ON regeneration in vivo. Summary Statement5-HT1 receptors selectively promote early optic nerve regrowth in zebrafish but are dispensable during development. Serotonin receptor-dependent modulation instructs regenerating optic nerve axons toward the brain in vivo.

developmental biology↗

bioRxiv2024

Celsr3 drives development and connectivity of the acoustic startle hindbrain circuit

In the developing brain, groups of neurons organize into functional circuits that direct diverse behaviors. One such behavior is the evolutionarily conserved acoustic startle response, which in zebrafish is mediated by a well-defined hindbrain circuit. While numerous molecular pathways that guide neurons to their synaptic partners have been identified, it is unclear if and to what extent distinct neuron populations in the startle circuit utilize shared molecular pathways to ensure coordinated development. Here, we show that the planar cell polarity (PCP)-associated atypical cadherins Celsr3 and Celsr2, as well as the Celsr binding partner Frizzled 3a/Fzd3a, are critical for axon guidance of two neuron types that form synapses with each other: the command-like neuron Mauthner cells that drive the acoustic startle escape response, and spiral fiber neurons which provide excitatory input to Mauthner cells. We find that Mauthner axon growth towards synaptic targets is vital for Mauthner survival. We also demonstrate that symmetric spiral fiber input to Mauthner cells is critical for escape direction, which is necessary to respond to directional threats. Moreover, we identify distinct roles for Celsr3 and Celsr2, as Celsr3 is required for startle circuit development while Celsr2 is dispensable, though Celsr2 can partially compensate for loss of Celsr3 in Mauthner cells. This contrasts with facial branchiomotor neuron migration in the hindbrain, which requires Celsr2 while we find that Celsr3 is dispensable. Combined, our data uncover critical and distinct roles for individual PCP components during assembly of the acoustic startle hindbrain circuit. HighlightsO_LIThe PCP cadherin Celsr3 regulates startle circuit development in zebrafish C_LIO_LICelsr3 and other PCP-associated proteins promote Mauthner axon growth and guidance C_LIO_LICelsr3 is required for spiral fiber and glia targeting to the Mauthner axon cap C_LIO_LISymmetric spiral fiber input to Mauthners is critical for escape direction C_LI

neuroscience↗

bioRxiv2023

A single base pair substitution on Chromosome 25 in zebrafish distinguishes between development and acute regulation of behavioral thresholds.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animals life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate largely independently from those regulating acute threshold regulation.

neuroscience↗

bioRxiv2023

A glia cell dependent mechanism at a peripheral nerve plexus critical for target-selective axon regeneration

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from three nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

neuroscience↗

bioRxiv2023

Mitochondrial genes in the 22q11.2 deleted region regulate neural stem and progenitor cell proliferation

Microdeletion of a 3Mbp region encompassing 45 protein-coding genes at chromosome 22q11.2 (22q11.2DS) predisposes to multiple neurodevelopmental disorders and is one of the greatest genetic risk factors for schizophrenia. Defective mitochondrial function has been hypothesized to contribute to 22q11.2DS pathogenesis; however, which of the six mitochondrial genes contribute to neurodevelopmental phenotypes and their underlying mechanisms remain unresolved. To systematically test 22q11.2DS genes for functional roles in neurodevelopment and behavior, we generated genetic mutants for each of the 37 conserved zebrafish orthologs and performed high throughput behavioral phenotyping using seven behavioral assays. Through this unbiased approach, we identified five single-gene mutants with partially overlapping behavioral phenotypes. Two of these genes, mrpl40 and prodha, encode for mitochondrial proteins and, similar to what we observed in mrpl40 and prodha mutants, pharmacologic inhibition of mitochondrial function during development results in microcephaly. Finally, we show that both mrpl40 and prodha mutants display neural stem and progenitor cell phenotypes, with each gene regulating different neural stem cell populations. Combined, our results demonstrate a critical role for mitochondrial function in neural stem and progenitor cell populations in the developing vertebrate brain and provide compelling evidence that mitochondrial dysfunction during neurodevelopment is linked to brain volume and behavioral phenotypes observed in models of 22q11.2DS.

neuroscience↗

bioRxiv2022

Molecular and functional mechanisms of long-term visual habituation in larval zebrafish

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt, or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small molecule-screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including: Melatonin, Estrogen and GABA signaling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

neuroscience↗

bioRxiv2022

Pharmacogenetic and whole-brain activity analyses uncover integration of distinct molecular and circuit programs that drive learning

Habituation is a foundational learning process critical for animals to adapt their behavior to changes in their sensory environment. Although habituation is considered a simple form of learning, the identification of a multitude of molecular pathways including several neurotransmitter systems that regulate this process suggests an unexpected level of complexity. How the vertebrate brain integrates these various pathways to accomplish habituation learning, whether they act independently or intersect with one another, and whether they act via divergent or overlapping neural circuits has remained unclear. To address these questions, we combined pharmacogenetic pathway analysis with unbiased whole-brain activity mapping using the larval zebrafish. This approach revealed five distinct molecular and circuit modules that regulate habituation learning and identified a set of molecularly defined brain regions associated with four of the five modules. Moreover, we find that in module 1 the palmitoyltransferase Hip14 cooperates with dopamine and NMDA signaling to drive plasticity, while in module 3 the adaptor protein complex subunit Ap2s1 drives habituation by antagonizing dopamine signaling, revealing two distinct and opposing roles for dopaminergic neuromodulation in the regulation of learning. Combined, our results define a core set of distinct modules that act in concert to regulate learning-associated plasticity, and provide compelling evidence that even seemingly simple learning behaviors in a compact vertebrate brain are regulated by a complex and overlapping set of molecular and circuit mechanisms.

neuroscience↗

bioRxiv2021

cacna2d3, a voltage-gated calcium channel subunit, functions in vertebrate habituation learning and the startle sensitivity threshold

The ability to filter sensory information into relevant versus irrelevant stimuli is a fundamental, conserved property of the central nervous system and is accomplished in part through habituation learning. Synaptic plasticity that underlies habituation learning has been described at the cellular level, yet the genetic regulators of this plasticity remain poorly understood, as do circuits that mediate sensory filtering. A forward genetic screen for zebrafish genes that control habituation learning identified a mutant allele doryp177 that caused reduced habituation of the acoustic startle response. Whole-genome sequencing identified the calcium voltage-gated channel auxiliary subunit alpha-2/delta-3 (cacna2d3) as a candidate gene affected in doryp177 mutants. Behavioral characterization of larvae homozygous for two additional, independently derived mutant alleles of cacna2d3, together with failure of these alleles to complement doryp177, confirmed a critical role for cacna2d3 in habituation learning. Notably, detailed analyses of the acoustic response in mutant larvae also revealed increased startle sensitivity to acoustic stimuli, suggesting a broader role for cacna2d3 in controlling innate response thresholds to acoustic stimuli. Taken together, our data demonstrate a critical role for cacna2d3 in sensory filtering, a process that is disrupted in human CNS disorders, e.g. ADHD, schizophrenia, and autism.

neuroscience↗

bioRxiv2021

Agrin/Lrp4 signal constrains MuSK activity during neuromuscular synapse development in appendicular muscle

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway critical in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that like axial muscle, developing appendicular muscles develop aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. Surprisingly, we find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve endings and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in contrast to axial muscle in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK activity to organize neuromuscular synapses. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

neuroscience↗

bioRxiv2021

Central nervous system hypomyelination disrupts axonal conduction and behaviour in larval zebrafish

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, due to their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behaviour. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias towards reorientation behaviour instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.

neuroscience↗

bioRxiv2020

Robo2 drives target selective peripheral nerve regeneration in response to glia derived signals

Peripheral nerves are divided into multiple branches leading to divergent synaptic targets. This poses a remarkable challenge for regenerating axons as they select their original trajectory at nerve branch-points. Despite implications for functional regeneration, the molecular mechanisms underlying target selectivity are not well characterized. Zebrafish motor nerves are composed of a ventral and a dorsal branch that diverge at a choice point, and we have previously shown that regenerating axons faithfully select their original branch and targets. Here we identify Robo2 as a key regulator of target selective regeneration. We demonstrate that Robo2 function in regenerating axons is required and sufficient to drive target selective regeneration, and that Robo2 acts in response to glia located precisely where regenerating axons select the branch-specific trajectory to prevent and correct axonal errors. Combined our results reveal a glia derived mechanism that acts locally via axonal Robo2 to promote target selective regeneration.

neuroscience↗

bioRxiv2020

A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. This identified mutants in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry. Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. Combined, our results identify a diverse set of eight genes all associated with human disorders that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway. Author summaryUnderlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study these molecular pathways that control behavior, we use a simple vertebrate behavior, the acoustic startle response, in the larval zebrafish. In response to an intense noise, larval zebrafish will quickly turn and swim away to escape. From a genetic screen, we have identified a number of mutants that behave in abnormal ways in response to an acoustic stimulus. We cloned these mutants and identified eight genes that regulate startle behavior. All eight genes are associated with human disorders, and here we focus on two genes, dolk and kcna1a, encoding Dolk, a key regulator of protein glycosylation, and the potassium channel Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements and that Dolk is required for Kv1.1 protein localization to axons of neurons throughout the nervous system, providing strong evidence that dolk and kcna1a act in a common molecular pathway. Combined, our studies provide new insights into the genetic regulation of startle behavior.

neuroscience↗

bioRxiv2019

Acute regulation of habituation learning via posttranslational palmitoylation

Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in the environment. Despite its well documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Hip14 as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified neuron and provide compelling evidence that Hip14 palmitoylates the Shaker-like channel subunit Kv1.1, thereby regulating Kv1.1 subcellular localization. Furthermore, we show that loss of either Kv1.1 or Hip14 leads to habituation deficits, and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, our results uncover a previously unappreciated role for acute post-translational palmitoylation at defined circuit components to regulate learning.

neuroscience↗

bioRxiv2018

Distributed plasticity drives visual habituation learning in larval zebrafish

Habituation is a simple form of learning, where animals learn to reduce their responses to repeated innocuous stimuli. While habituation is simple in concept, its exact implementation in the vertebrate brain is not clear. It could occur via a single plasticity event at a singular site in the circuit, or alternatively via more complex strategies that combine multiple mechanisms at various processing stages and sites. Here, we use a visual habituation assay in larval zebrafish, where larvae habituate to sudden reductions in illumination (dark flashes). We find that 8 different components of this response habituate, including the probability of executing a response, its latency, and measures of its magnitude. Through behavioural analyses, we find that habituation of these different behavioural components occurs independently of each other and at different locations in the circuit. Further, we use genetic and pharmacological manipulations to show that habituation of different behavioural components are molecularly distinct. These results are consistent with a model by which visual habituation originates from the combination of multiple independent processes, which each act to adapt specific components of behaviour. This may allow animals to more specifically habituate behaviour based on stimulus context or internal state.

neuroscience↗

bioRxiv2017

A genetic basis for molecular asymmetry at vertebrate electrical synapses

Neural network function is based upon the patterns and types of connections made between neurons. Neuronal synapses are adhesions specialized for communication and they come in two types, chemical and electrical. Communication at chemical synapses occurs via neurotransmitter release whereas electrical synapses utilize gap junctions for direct ionic and metabolic coupling. Electrical synapses are often viewed as symmetrical structures, with the same components making both sides of the gap junction. By contrast, we show that a broad set of electrical synapses in zebrafish, Danio rerio, require two gap-junction-forming Connexins for formation and function. We find that one Connexin functions presynaptically while the other functions postsynaptically in forming the channels. We also show that these synapses are required for the speed and coordination of escape responses. Our data identify a genetic basis for molecular asymmetry at vertebrate electrical synapses and show they are required for appropriate behavioral performance.

neuroscience↗