Suresh Jesuthasan

Dynamic microtubules and specification of the zebrafish embryonic axis

BACKGROUND The zebrafish is emerging as an important genetic system for the study of vertebrate development, and many zygotic mutations affecting embryogenesis have been isolated. The early events in development are under the control of maternal genes but are relatively unexplored. Here, the process of axis specification is investigated. RESULTS The vegetal pole of the zygote transiently contains a dense array of parallel microtubules, while microtubules near the equator are disorganized. Irradiation of the zygote with ultraviolet light disrupts the formation of the vegetal microtubule array and causes loss of the axis; brief treatment with nocodazole at this stage also causes defects in the axis. During cleavage stages, yolk cortical microtubules reorganize to form arrays that apparently extend from marginal blastomeres. Prolonged exposure to cold (18 degrees C) or incubation in nocodazole prior to the 32-cell stage disrupts cortical microtubules and causes premature formation of the yolk syncytial layer; these treatments also prevent formation of an axis, as indicated by the absence of goosecoid and forkhead2 expression and of translocation of beta-catenin into nuclei. Cortical microtubule arrays are required for the transport of particles from the vegetal hemisphere into marginal blastomeres, as shown by the movement of polystyrene beads; treatments that prevent axis formation also prevent the entry of beads into blastomeres. CONCLUSIONS To form an organizer, zebrafish blastomeres appear to require substances which are transported from the vegetal hemisphere of the yolk cell by cortical microtubules. Initial asymmetry appears dependent on an array of parallel microtubules at the vegetal pole.

The Habenula Prevents Helpless Behavior in Larval Zebrafish

Animals quickly learn to avoid predictable danger. However, if pre-exposed to a strong stressor, they do not display avoidance even if this causes continued contact with painful stimuli [1, 2]. In rodents, lesioning the habenula, an epithalamic structure that regulates the monoaminergic system, has been reported to reduce avoidance deficits caused by inescapable shock [3]. This is consistent with findings that inability to overcome a stressor is accompanied by an increase in serotonin levels [4]. However, other studies conclude that habenula lesions cause avoidance deficits [5, 6]. These contradictory results may be caused by lesions affecting unintended regions [6]. To clarify the role of the habenula, we used larval zebrafish, whose transparency and amenability to genetic manipulation enables more precise disruption of cells. We show that larval zebrafish learn to avoid a light that has been paired with a mild shock but fail to do so when pre-exposed to inescapable shock. Photobleaching of habenula afferents expressing the photosensitizer KillerRed causes a similar failure in avoidance. Expression of tetanus toxin in dorsal habenula neurons is sufficient to prevent avoidance. We suggest that this region may signal the ability to control a stressor, and that its disruption could contribute to anxiety disorders.

Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc)

Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.

Deletion of the WD40 Domain of LRRK2 in Zebrafish Causes Parkinsonism-Like Loss of Neurons and Locomotive Defect

LRRK2 plays an important role in Parkinsons disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD.

Fear, anxiety, and control in the zebrafish

Emotional responses are triggered by environmental signals and involve profound changes at multiple levels, from molecular to behavior. Much has been learnt about two emotions, fear and anxiety, by studying mammalian models. In particular, neural circuits and the corresponding molecular mechanisms essential for the learning and retention of fear, as well as the activation of anxiety, are well known. In contrast, little is known about how these emotions are terminated. The zebrafish is a newcomer to the world of emotion research. A number of assays for fear and anxiety now exist, but the underlying neural circuitry is largely undefined. Recent experiments, however, appear to provide a hint as to how anxiety is downregulated. In particular, they point to an essential role for a circuit involving the posterior septum, medial habenula, and interpeduncular nucleus. This evolutionarily conserved circuit may fulfill a similar function in mammals.

Asymmetric innervation of the habenula in zebrafish

The habenular complex is a paired structure found in the diencephalon of all vertebrates, linking the forebrain and midbrain. Habenulae are asymmetrical and may contribute to lateralized behavior. Recent studies in zebrafish have characterized molecular pathways that give rise to the habenular asymmetry and the distinct projections of the left and right habenula to the midbrain. However, it is unclear whether there are asymmetries in habenula afferents from the forebrain. By lipophilic dye tracing, we find that axons innervating the habenula derive primarily from a region in the lateral diencephalon containing migrated neurons of the eminentia thalami (EmT). EmT neurons terminate in neuropils in both ipsilateral and contralateral habenula. These axons, together with axons from migrated neurons of the posterior tuberculum and pallial neurons, cross the midline via the habenular commissure. Subsets of pallial neurons terminate only in the medial right habenula, regardless of which side of the brain they originate from. These include an unusual type of forebrain projection: axons that cross the midline twice, at both the anterior and habenular commissures. Our data establish that there is asymmetric innervation of the habenula from the telencephalon, suggesting a mechanism by which habenula asymmetry might contribute to lateralized behavior. J. Comp. Neurol. 502:611–619, 2007.

The Alarm Response in Zebrafish: Innate Fear in a Vertebrate Genetic Model

The alarm response is an antipredator behavior displayed by many fish species and was first described 70 years ago. It is triggered through the olfactory system by substances released from injured skin and is characterized by dramatic, measurable changes in locomotion as well as physiology. We propose that this is an ideal time to revisit this response and to utilize it as an assay for understanding how neural circuits mediate innate fear. A suitable organism for these studies is the zebrafish, a genetic model with a rapidly expanding toolkit for molecular manipulation of the nervous system. Individual neurons mediating the response, ranging from receptor neurons to those in higher brain centers, should first be identified. New tools, specifically transgenic lines that allow spatial and temporal control of neural activity, provide a way to define and test the role of specific neurons, while genetic screens provide a route to identifying individual molecules essential for a normal response. Optical recording, which has proven successful in studies of information processing in the bulb, will provide valuable insights into neural circuitry function during the alarm response. When carried out on mutants, physiological analysis can provide insight into aspects of signal processing that are essential for normal behavior. The alarm response thus provides a paradigm to examine innate fear in a vertebrate system, enabling analysis at multiple levels from genes to the entire neural circuit. Additionally, the context dependency of the response can be utilized to investigate attention and decision making.

The medial habenula as a regulator of anxiety in adult zebrafish.

The habenula consists of a set of nuclei located in the epithalamus. It regulates the release of multiple neuromodulators including serotonin and dopamine, and consists of two major subdivisions—medial and lateral. In all vertebrates, the medial habenula projects to the interpeduncular nucleus (IPN), a midline structure with poorly defined functions (Morley, 1986). Both the medial habenula and the IPN are rich in nicotinic receptors (nAChR). Activity in this pathway, triggered by opioids and nicotine, leads to a rise in dopamine in the nucleus accumbens (Glick et al., 2006; McCallum et al., 2012) and thus underlies the rewarding aspect of substance abuse. Strong activation of nicotinic receptors in the medial habenula or IPN, however, is sufficient to mediate the aversion to high concentration of nicotine (Fowler et al., 2011; Frahm et al., 2011). In contrast, absence of activity in this pathway is critical for the effects of withdrawal (Salas et al., 2009; Baldwin et al., 2011). Hence, depending on the level of activity, the medial habenula-IPN pathway can trigger reward, aversion or the physical and emotional changes that are characteristic of withdrawal.

PHR Regulates Growth Cone Pausing at Intermediate Targets through Microtubule Disassembly

Axonal growth cones use intermediate targets to navigate in the developing nervous system. Encountering these sites is correlated with growth cone pausing. PHR (Phr1, Esrom, Highwire, RPM-1) is a large neuronal ubiquitin ligase that interacts with multiple signaling pathways. Mouse and zebrafish phr mutants have highly penetrant axon pathfinding defects at intermediate targets. Mouse phr mutants contain excessive microtubules in the growth cone, which has been attributed to upregulation of DLK/p38 signaling. Here, we ask whether this pathway and microtubule misregulation are indeed linked to guidance errors in the vertebrate brain, using the zebrafish. By live imaging, we show that loops form when microtubules retract without depolymerizing. JNK, but not p38, phosphorylation is increased in mutant growth cones. However microtubule looping cannot be suppressed by inhibiting JNK. The phr microtubule defect can be phenocopied by taxol, while microtubule destabilization in vitro using nocodazole prevents loop formation. Acute disruption in vivo with nocodazole suppresses the intermediate target guidance defect. Given that microtubule looping is associated with growth cone pausing, we propose that microtubule disassembly, mediated by PHR, is essential for exiting the paused state at intermediate targets.

Baculovirus-Mediated Gene Expression in Zebrafish

In an effort to misexpress genes in zebrafish, we tested the ability of baculovirus to infect and drive gene expression in embryos. By injecting virus into specific tissues and using appropriate promoters, both the location and time of gene expression could be controlled. Using a virus with 2 different promoters, LacZ and GFP could be expressed independently. The efficiency of expression appears to depend on the promoter used. As a test of this system, baculovirus was used to ectopically express ephrinB2a in the presomitic mesoderm. EphrinB2a is normally expressed in the posterior region of developing somites, and baculovirus-mediated misexpression caused abnormal somite boundary formation. Baculovirus can thus be used as a tool for gene misexpression experiments in the zebrafish, especially when localized misexpression is required late in development.