Branchiomotor Neurons Research Articles

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.

Early development of respiratory motor circuits in larval zebrafish (Danio rerio).

Rhythm-generating circuits in the vertebrate hindbrain form synaptic connections with cranial and spinal motor neurons, to generate coordinated, patterned respiratory behaviors. Zebrafish provide a uniquely tractable model system to investigate the earliest stages in respiratory motor circuit development in vivo. In larval zebrafish, respiratory behaviors are carried out by muscles innervated by cranial motor neurons-including the facial branchiomotor neurons (FBMNs), which innervate muscles that move the jaw, buccal cavity, and operculum. However, it is unclear when FBMNs first receive functional synaptic input from respiratory pattern-generating neurons, and how the functional output of the respiratory motor circuit changes across larval development. In the current study, we used behavior and calcium imaging to determine how early FBMNs receive functional synaptic inputs from respiratory pattern-generating networks in larval zebrafish. Zebrafish exhibited patterned operculum movements by 3days postfertilization (dpf), though this behavior became more consistent at 4 and 5dpf. Also by 3dpf, FBMNs fell into two distinct categories ("rhythmic" and "nonrhythmic"), based on patterns of neural activity. These two neuron categories were arranged differently along the dorsoventral axis, demonstrating that FBMNs have already established dorsoventral topography by 3dpf. Finally, operculum movements were coordinated with pectoral fin movements at 3dpf, indicating that the operculum behavioral pattern was driven by synaptic input. Taken together, this evidence suggests that FBMNs begin to receive initial synaptic input from a functional respiratory central pattern generator at or prior to 3dpf. Future studies will use this model to study mechanisms of normal and abnormal respiratory circuit development.

Celsr1 suppresses Wnt5a-mediated chemoattraction to prevent incorrect rostral migration of facial branchiomotor neurons.

In the developing hindbrain, facial branchiomotor (FBM) neurons migrate caudally from rhombomere 4 (r4) to r6 to establish the circuit that drives jaw movements. Although the mechanisms regulating initiation of FBM neuron migration are well defined, those regulating directionality are not. In mutants lacking the Wnt/planar cell polarity (PCP) component Celsr1, many FBM neurons inappropriately migrate rostrally into r3. We hypothesized that Celsr1 normally blocks inappropriate rostral migration of FBM neurons by suppressing chemoattraction towards Wnt5a in r3 and successfully tested this model. First, FBM neurons in Celsr1; Wnt5a double mutant embryos never migrated rostrally, indicating that inappropriate rostral migration in Celsr1 mutants results from Wnt5a-mediated chemoattraction, which is suppressed in wild-type embryos. Second, FBM neurons migrated rostrally toward Wnt5a-coated beads placed in r3 of wild-type hindbrain explants, suggesting that excess Wnt5a chemoattractant can overcome endogenous Celsr1-mediated suppression. Third, rostral migration of FBM neurons was greatly enhanced in Celsr1 mutants overexpressing Wnt5a in r3. These results reveal a novel role for a Wnt/PCP component in regulating neuronal migration through suppression of chemoattraction.

Hindbrain defects induced by Di-butyl phthalate (DBP) in developing zebrafish embryos

Di-butyl phthalate (DBP) is a globally used plasticizer found in alarmingly high concentrations in soil and water ecosystems. As phthalates are non-covalently bound to plastic polymers, phthalates easily leach into the aquatic environment. The effects of DBP on aquatic organisms is concerning, most notably, studies have focused on the endocrine-disrupting effects. However, reports on the developmental neurotoxicity of DBP are rare. Using the zebrafish vertebrate model system, we treated pre-gastrulation staged embryos with 2.5 μM DBP, a concentration environmentally noted. We find that general hindbrain structure and rhombomere patterning is disrupted at 72 h post fertilization (hpf). We investigated hindbrain specific neural patterning of cranial motor neurons and find defects in branchiomotor neuron patterning and migration. Furthermore, defects in r4 specific Mauthner neuron development were also noted. Thus, we conclude that DBP exposure during embryonic development induces defects to the hindbrain and concomitantly the neurons that are born and differentiate there.

Zebrafish gata2 Orthologs Are Necessary but Not Sufficient for Developmental Hematopoiesis

Germline mutations in the GATA2 gene have been identified in patients with a variety of hematological disorders unified by their with predisposition to myelodysplastic syndromes (MDS) and defective immunity. The GATA2 gene encodes a transcription factor that plays a major role in hematopoiesis. However, the precise effects of GATA2 haploinsufficiency in developmental hematopoiesis and the impact of this in MDS predisposition remain unknown. We have developed zebrafish mutants of gata2 to model this and demonstrate that heterozygosity results in defective primitive and definitive granulopoiesis.Zebrafish carry two orthologues to the mammalian GATA2 gene: gata2a and gata2b . gata2a and gata2b demonstrate distinct expression patterns. By 32 hours post fertilization (hpf), expression of gata2a is seen throughout the dorsal aorta (DA) and cardinal vein, primitive erythrocytes and the nervous system. In contrast, gata2b expression is restricted to the ventral DA (VDA) and branchiomotor neurons. We hypothesised that this division of expression may translate into a requirement for both ohnologs as necessary but not sufficient for normal developmental hematopoiesis.Homozygous gata2a mutants carrying an 11bp deletion (gata2aum27 ) have been previously described. These mutants have abnormal vascular development in the aorta resulting in defective circulation. We analysed developmental hematopoiesis in these mutants. gata2aum27/um27 and gata2aum27 /+ showed normal hemangioblasts, and primitive hematopoiesis before 18 somites. At 22hpf however myeloid transcription factor cebpa showed a dose dependent decrease in expression resulting from gata2a loss, while pu.1 expression remained normal. This suggests that Gata2a may be a direct regulator of cebpa in primitive myeloid cells, or that cebpa expression defines a sub-population of myeloid cells. We then assessed hematopoietic stem cells (HSCs) in gata2aum27 mutants. gata2aum27/um27 showed severely decreased or absent HSCs in the caudal hematopoietic tissue (CHT, fetal liver equivalent) at 52hpf, likely due to absent blood flow in the aorta. gata2aum27/+ mutants also showed a decrease in HSCs at this time point, however this decrease was transient, resolving by 72hpf (A). Further assessment of definitive hematopoiesis showed that gata2um27/um27 lacked mature granulocytes and thymocytes, in keeping with the observed HSC defect. By contrast myeloid cells in gata2aum27/+ at 4dpf (assessed by Sudan Black (SB) stain) were unaffected (B,C) . Interestingly lck-expressing thymocytes showed a range of phenotypes, from normal, through reduced and some with a complete absence of thymocytes in one of their thymii.We next used CRISPR to generate a gata2b mutant line. This line carries a 4bp deletion leading to a truncated protein lacking both zinc fingers (gata2bU5008 ). In contrast to gata2aum27/um27 , gata2bu5008/5008 have normal circulation and were viable beyond 5dpf. Loss of gata2b by morpholino knockdown results in absence of HSC, however gata2b5008/+ showed no difference in HSC numbers in the CHT at 60hpf. Myeloid cells were unaffected in gata2b5008 mutants by SB at 48hpf, however, by 4dpf gata2b5008 show a dose dependent reduction in myeloid cells (D). This suggests that Gata2b regulates myeloid cell proliferation or differentiation after the emergence of HSCs.We next studied haematopoiesis in crosses of gata2aum27/+ to gata2bu5008/+ . Quantification of granulocytes showed normal numbers at 48hpf in all resulting genotypes. By 4dpf gata2b5008/+ have a reduced number of SB expressing granulocytes regardless of their gata2a genotype, suggesting that Gata2a is dispensable for definitive myelopoiesis (E). Quantification of HSCs show HSC numbers are unaffected in gata2aum27/+ ; gata2bu5008/+ at 48hpf. Assessment of thymocytes by contrast shows a greater than additive effect on thymocyte numbers than in single mutants alone, suggesting that both Gata2a and Gata2b contribute to lymphoid development in an additive manner. Further characterisation of the effects of gata2aum27/+ ; gata2bu5008/+ in combination is ongoing.In conclusion our results support the notion at that haploinsufficient levels of Gata2a and Gata2b are necessary but not sufficient for normal developmental hematopoiesis and that combined heterozygous loss of these genes models the haploinsufficieny observed in patients in GATA2 haploinsufficient diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Defective Neuronal Positioning Correlates With Aberrant Motor Circuit Function in Zebrafish.

Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can cause a myriad of neurological disorders. To investigate the functional consequences of defective neuronal positioning on circuit function, we studied a zebrafish frizzled3a (fzd3a) loss-of-function mutant off-limits (olt) where the facial branchiomotor (FBM) neurons fail to migrate out of their birthplace. A jaw movement assay, which measures the opening of the zebrafish jaw (gape), showed that the frequency of gape events, but not their amplitude, was decreased in olt mutants. Consistent with this, a larval feeding assay revealed decreased food intake in olt mutants, indicating that the FBM circuit in mutants generates defective functional outputs. We tested various mechanisms that could generate defective functional outputs in mutants. While fzd3a is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles was unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, motor axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data show that aberrant positioning of FBM neurons in olt mutants is correlated with subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs.

Development and migration of the zebrafish rhombencephalic octavolateral efferent neurons.

In vertebrate animals, motor and sensory efferent neurons carry information from the central nervous system (CNS) to peripheral targets. These two types of efferent systems sometimes bear a close resemblance, sharing common segmental organization, axon pathways, and chemical messengers. Here, we focus on the development of the octavolateral efferent neurons (OENs) and their interactions with the closely-related facial branchiomotor neurons (FBMNs) in zebrafish. Using live-imaging approaches, we investigate the birth, migration, and projection patterns of OENs. We find that OENs are born in two distinct groups: a group of rostral efferent neurons (RENs) that arises in the fourth segment, or rhombomere (r4), of the hindbrain and a group of caudal efferent neurons (CENs) that arises in r5. Both RENs and CENs then migrate posteriorly through the hindbrain between 18 and 48 hrs postfertilization, alongside the r4-derived FBMNs. Like the FBMNs, migration of the r4-derived RENs depends on function of the segmental identity gene hoxb1a; unlike the FBMNs, however, both OEN populations move independently of prickle1b. Further, we investigate whether the previously described "pioneer" neuron that leads FBMN migration through the hindbrain is an r4-derived FBMN/REN or an r5-derived CEN. Our experiments verify that the pioneer is an r4-derived neuron and reaffirm its role in leading FBMN migration across the r4/5 border. In contrast, the r5-derived CENs migrate independently of the pioneer. Together, these results indicate that the mechanisms OENs use to navigate the hindbrain differ significantly from those employed by FBMNs.

Dpysl2 (CRMP2) is required for the migration of facial branchiomotor neurons in the developing zebrafish embryo.

Dihydropyrimidinase-like family proteins (Dpysls) are relevant in several processes during nervous system development; among others, they are involved in axonal growth and cell migration. Dpysl2 (CRMP2) is the most studied member of this family; however, its role in vivo is still being investigated. Our previous studies in zebrafish showed the requirement of Dpysl2 for the proper positioning of caudal primary motor neurons and Rohon-Beard neurons in the spinal cord.In the present study, we show that Dpysl2 is necessary for the proper migration of facial branchiomotor neurons during early development in zebrafish. We generated a dpysl2 knock-out (KO) zebrafish mutant line and used different types of antisense morpholino oligonucleotides (AMO) to analyze the role of Dpysl2 in this process. Both dpysl2 KO mutants and morphants exhibited abnormalities in the migration of these neurons from rhombomers (r) 4 and 5 to 6 and 7. The facial branchiomotor neurons that were expected to be at r6 were still located at r4 and r5 hours after the migration process should have been completed. In addition, mutant phenotypes were rescued by injecting dpysl2 mRNA into the KO embryos. These results indicate that Dpysl2 is involved in the proper migration of facial branchiomotor neurons in developing zebrafish embryos.

The Role of Nesprin in the Facial Branchiomotor Neuron (FBMN) Migration of Danio rerio (zebrafish) development

Nuclear envelope spectrin‐repeat proteins (Nesprins) are part of the LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, which plays a vital role in the binding of F‐actin cytoskeleton to the nuclear envelope. Nesprins are crucial in maintaining the integrity of the nuclear membrane. Nuclear membrane movement is a fundamental aspect in cell migration and muscle development. Previous work has demonstrated that Nesprins may be associated with myosin‐dependent tension, which has implications in muscular dystrophy due to the role that nesprins play in muscle cells. We hypothesize that expressing dominant‐negative nesprin in Danio rerio (zebrafish) embryos would result in defects in the migration of facial branchiomotor neurons and also cause disruptions in nuclear positioning in multinucleated muscle cells. To test this hypothesis, we use transient transgenic expression of dominant Nesprin in cranial motor neurons using the islet1 promoter. We quantify the extent of migration in neurons with inactive Nesprin. In addition, we express dominant‐negative Nesprin in muscle cells using a heat shock promoter and explore the positioning of nuclei in developing skeletal muscle myofibrils. Our preliminary data shows that transgenic zebrafish embryos with expression of dominant‐negative nesprin have disruptions and defects in tangential FBMN migration. However, with further experimentation and data collection in progress, we hope to analyze our findings for statistical significance. Our goal is to gain insight into the function of Nesprins in embryo development to better devise therapeutic strategies for patients with mutations in Nesprins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct roles for the cell adhesion molecule Contactin2 in the development and function of neural circuits in zebrafish

Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.

The Postmigratory Alar Topography of Visceral Cranial Nerve Efferents Challenges the Classical Model of Hindbrain Columns

The classic columnar model of cranial nerve central representation assumes that all motor and sensory hindbrain neurons develop within four radial migration domains, held to be separated by a sulcal alar-basal boundary (sulcus limitans). This essay reviews a number of developmental data that challenge these concepts. These results are interpreted within the framework of present day neuromeric conception of the brainstem (the prosomeric model). Advances in dorsoventral patterning of the spinal cord and hindbrain now show that there exist up to eight alar microzones and five basal microzones (molecularly and histogenetically distinct longitudinal progenitor domains). This reveals that the classic tetracolumnar model is excessively simplistic. There is both older and recent data revealing that the visceral efferent neurons of the cranial nerves (preganglionic and branchiomotor neurons) are generated next to the floor plate and later migrate dorsalwards before adopting their final topography in the mantle, contrary to the purely radial migration assumed in the classic model. Moreover, various results support the conclusion that at least the branchiomotor neurons end their migration and mature within the alar region of the mantle. Evidence on this point obtained in chick embryos is reviewed in detail, and novel evidence in mouse embryos is presented. Anat Rec, 302:485-504, 2019. © 2018 Wiley Periodicals, Inc.

Role of branchiomotor neurons in controlling food intake of zebrafish larvae

The physical act of eating or feeding involves the coordinated action of several organs like eyes and jaws, and associated neural networks. Moreover, the activity of the neural networks controlling jaw movements (branchiomotor circuits) is regulated by the visual, olfactory, gustatory and hypothalamic systems, which are largely well characterized at the physiological level. By contrast, the behavioral output of the branchiomotor circuits and the functional consequences of disruption of these circuits by abnormal neural development are poorly understood. To begin to address these questions, we sought to evaluate the feeding ability of zebrafish larvae, a direct output of the branchiomotor circuits, and developed a qualitative assay for measuring food intake in zebrafish larvae at 7 days post-fertilization. We validated the assay by examining the effects of ablating the branchiomotor neurons. Metronidazole-mediated ablation of nitroreductase-expressing branchiomotor neurons resulted in a predictable reduction in food intake without significantly affecting swimming ability, indicating that the assay is robust. Laser-mediated ablation of trigeminal motor neurons resulted in a significant decrease in food intake, indicating that the assay is sensitive. Importantly, in larvae of a genetic mutant with severe loss of branchiomotor neurons, food intake was abolished. These studies establish a foundation for dissecting the neural circuits driving a motor behavior essential for survival.

Key Features of Structural and Functional Organization of Zebrafish Facial Motor Neurons Are Resilient to Disruption of Neuronal Migration

The location of neurons early in development can be critical for their ability to differentiate and receive normal synaptic inputs. Indeed, disruptions in neuronal positioning lead to a variety of neurological disorders. Neurons have, however, shifted their positions across phylogeny, suggesting that changes in location do not always spell functional disaster. Toinvestigate the functional consequences of abnormal positioning, we leveraged previously reported genetic perturbations to disrupt normal neuronal migration-and thus positioning-in a population of cranial motor neurons, the facial branchiomotor neurons (FBMNs). We used a combination of topographical, morphological, physiological, and behavioral analyses to determine whether key functional features of FBMNs were still established in migration mutants, in spite of a dramatic rostrocaudal repositioning of these neurons in hindbrain. We discovered that FBMNs seem remarkably resilient to a disruption in positioning, suggesting that they may not rely heavily on rostrocaudal positioning to guide their functional development. Thus, the role of positioning may vary across the developing nervous system, with some populations-like facial motor neurons-exhibiting greater resilience to abnormal positioning that permits them to shift location as a part of evolutionary change.

ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons.

LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development.

Cadherin-2 Is Required Cell Autonomously for Collective Migration of Facial Branchiomotor Neurons.

Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs.

The atypical cadherin Celsr1 functions non-cell autonomously to block rostral migration of facial branchiomotor neurons in mice

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3–r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons.

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.

PCP Signaling between Migrating Neurons and their Planar-Polarized Neuroepithelial Environment Controls Filopodial Dynamics and Directional Migration.

The planar cell polarity (PCP) pathway is a cell-contact mediated mechanism for transmitting polarity information between neighboring cells. PCP “core components” (Vangl, Fz, Pk, Dsh, and Celsr) are essential for a number of cell migratory events including the posterior migration of facial branchiomotor neurons (FBMNs) in the plane of the hindbrain neuroepithelium in zebrafish and mice. While the mechanism by which PCP signaling polarizes static epithelial cells is well understood, how PCP signaling controls highly dynamic processes like neuronal migration remains an important outstanding question given that PCP components have been implicated in a range of directed cell movements, particularly during vertebrate development. Here, by systematically disrupting PCP signaling in a rhombomere-restricted manner we show that PCP signaling is required both within FBMNs and the hindbrain rhombomere 4 environment at the time when they initiate their migration. Correspondingly, we demonstrate planar polarized localization of PCP core components Vangl2 and Fzd3a in the hindbrain neuroepithelium, and transient localization of Vangl2 at the tips of retracting FBMN filopodia. Using high-resolution timelapse imaging of FBMNs in genetic chimeras we uncover opposing cell-autonomous and non-cell-autonomous functions for Fzd3a and Vangl2 in regulating FBMN protrusive activity. Within FBMNs, Fzd3a is required to stabilize filopodia while Vangl2 has an antagonistic, destabilizing role. However, in the migratory environment Fzd3a acts to destabilize FBMN filopodia while Vangl2 has a stabilizing role. Together, our findings suggest a model in which PCP signaling between the planar polarized neuroepithelial environment and FBMNs directs migration by the selective stabilization of FBMN filopodia.

2- and 6-O-sulfated proteoglycans have distinct and complementary roles in cranial axon guidance and motor neuron migration.

The correct migration and axon extension of neurons in the developing nervous system is essential for the appropriate wiring and function of neural networks. Here, we report that O-sulfotransferases, a class of enzymes that modify heparan sulfate proteoglycans (HSPGs), are essential to regulate neuronal migration and axon development. We show that the 6-O-sulfotransferases HS6ST1 and HS6ST2 are essential for cranial axon patterning, whilst the 2-O-sulfotransferase HS2ST (also known as HS2ST1) is important to regulate the migration of facial branchiomotor (FBM) neurons in the hindbrain. We have also investigated how HS2ST interacts with other signals in the hindbrain and show that fibroblast growth factor (FGF) signalling regulates FBM neuron migration in an HS2ST-dependent manner.

Investigation of the Cellular and Molecular Mechanisms of Facial Branchiomotor Neuron Migration

A fundamental feature of brain development is the migration of neurons from their place of birth to the location where they will assemble and integrate into a neural circuit. We have recently reported that one facial branchiomotor neuron (FBMN) can influence the migration of another FBMN, defining this cell movement as a collective migration. As with any collective migration, cell‐to‐cell contact is required for one cell's influence on another. To determine if this was the case, we imaged the migration of membrane‐mCherry‐expressing FBMNs in live zebrafish embryos. We find that some neurons display persistent, sustained migration in one direction (posterior), whereas other FBMNs stall for short periods of time. We noticed that FBMNs stalled in migration shortly after cell‐to‐cell contact with neuron in their migratory path. This is reminiscent of contact inhibition of locomotion (CIL) observed for many other cell types including neural crest cells. We therefore, quantified the speed and direction of neuron movement before and after neuron‐to‐neuron cell contacts. Consistent with this, centrosomes, which often re‐orient in the direction of migration, were often found on the anterior side of migrating FBMNs. Previous work has shown that planar cell polarity (PCP) genes are essential for FBMN migration. Here we show that PCP mutant neurons migrate in random directions suggesting that PCP proteins are not required for migration per se, but are required for persistent directed movements. Taken together these data suggest the possibility that PCP proteins control contact‐dependent cell polarization in FBMN migration.