Jörn Schweitzer

Expression of the paralogous tyrosine hydroxylase encoding genes th1 and th2 reveals the full complement of dopaminergic and noradrenergic neurons in zebrafish larval and juvenile brain

The development of dopaminergic and noradrenergic neurons has received much attention based on their modulatory effect on many behavioral circuits and their involvement in neurodegenerative diseases. The zebrafish (Danio rerio) has emerged as a new model organism with which to study development and function of catecholaminergic systems. Tyrosine hydroxylase is the entry enzyme into catecholamine biosynthesis and is frequently used as a marker for catecholaminergic neurons. A genome duplication at the base of teleost evolution resulted in two paralogous zebrafish tyrosine hydroxylase‐encoding genes, th1 and th2, the expression of which has been described previously only for th1. Here we investigate the expression of th2 in the brain of embryonic and juvenile zebrafish. We optimized whole‐mount in situ hybridization protocols to detect gene expression in the anatomical three‐dimensional context of whole juvenile brains. To confirm whether th2‐expressing cells may indeed use dopamine as a neurotransmitter, we also included expression of dopamine beta hydroxylase, dopa decarboxylase, and dopamine transporter in our analysis. Our data provide the first complete account of catecholaminergic neurons in the zebrafish embryonic and juvenile brain. We identified four major th2‐expressing neuronal groups that likely use dopamine as transmitter in the zebrafish diencephalon, including neurons of the posterior preoptic nucleus, the paraventricular organ, and the nuclei of the lateral and posterior recesses in the caudal hypothalamus. th2 Expression in the latter two groups resolves a previously reported discrepancy, in which strong dopamine but little tyrosine hydroxylase immunoreactivity had been detected in the caudal hypothalamus. Our data also confirm that there are no mesencephalic DA neurons in zebrafish. J. Comp. Neurol. 518:423–438, 2010.

Expression of protein zero is increased in lesioned axon pathways in the central nervous system of adult zebrafish

The immunoglobulin superfamily molecule protein zero (P0) is important for myelin formation and may also play a role in adult axon regeneration, since it promotes neurite outgrowth in vitro. Moreover, it is expressed in the regenerating central nervous system (CNS) of fish, but not in the nonregenerating CNS of mammals. We identified a P0 homolog in zebrafish. Cell type‐specific expression of P0 begins in the ventromedial hindbrain and the optic chiasm at 3–5 days of development. Later (at 4 weeks) expression has spread throughout the optic system and spinal cord. This is consistent with a role for P0 in CNS myelination during development. In the adult CNS, glial cells constitutively express P0 mRNA. After an optic nerve crush, expression is increased within 2 days in the entire optic pathway. Expression peaks at 1 to 2 months and remains elevated for at least 6 months postlesion. After enucleation, P0 mRNA expression is also upregulated but fails to reach the high levels observed in crush‐lesioned animals at 4 weeks postlesion. Spinal cord transection leads to increased expression of P0 mRNA in the spinal cord caudal to the lesion site. The glial upregulation of P0 mRNA expression after a lesion of the adult zebrafish CNS suggests roles for P0 in promoting axon regeneration and remyelination after injury. GLIA 41:301–317, 2003.

Genetic dissection of dopaminergic and noradrenergic contributions to catecholaminergic tracts in early larval zebrafish

The catecholamines dopamine and noradrenaline provide some of the major neuromodulatory systems with far‐ranging projections in the brain and spinal cord of vertebrates. However, development of these complex systems is only partially understood. Zebrafish provide an excellent model for genetic analysis of neuronal specification and axonal projections in vertebrates. Here, we analyze the ontogeny of the catecholaminergic projections in zebrafish embryos and larvae up to the fifth day of development and establish the basic scaffold of catecholaminergic connectivity. The earliest dopaminergic diencephalospinal projections do not navigate along the zebrafish primary neuron axonal scaffold but establish their own tracts at defined ventrolateral positions. By using genetic tools, we study quantitative and qualitative contributions of noradrenergic and defined dopaminergic groups to the catecholaminergic scaffold. Suppression of Tfap2a activity allows us to eliminate noradrenergic contributions, and depletion of Otp activity deletes mammalian A11‐like Otp‐dependent ventral diencephalic dopaminergic groups. This analysis reveals a predominant contribution of Otp‐dependent dopaminergic neurons to diencephalospinal as well as hypothalamic catecholaminergic tracts. In contrast, noradrenergic projections make only a minor contribution to hindbrain and spinal catecholaminergic tracts. Furthermore, we can demonstrate that, in zebrafish larvae, ascending catecholaminergic projections to the telencephalon are generated exclusively by Otp‐dependent diencephalic dopaminergic neurons as well as by hindbrain noradrenergic groups. Our data reveal the Otp‐dependent A11‐type dopaminergic neurons as the by far most prominent dopaminergic system in larval zebrafish. These findings are consistent with a hypothesis that Otp‐dependent dopaminergic neurons establish the major modulatory system for somatomotor and somatosensory circuits in larval fish. J. Comp. Neurol. 518:439–458, 2010.

Tenascin-R as a repellent guidance molecule for newly growing and regenerating optic axons in adult zebrafish.

In adult fish, in contrast to mammals, new optic axons are continuously added to the optic projection, and optic axons regrow after injury. Thus, pathfinding of optic axons during development, adult growth, and adult regeneration may rely on the same guidance cues. We have shown that tenascin-R, a component of the extracellular matrix, borders the optic pathway in developing zebrafish and acts as a repellent guidance molecule for optic axons. Here we analyze tenascin-R expression patterns along the unlesioned and lesioned optic pathway of adult zebrafish and test the influence of tenascin-R on growing optic axons of adult fish in vitro. Within intraretinal fascicles of optic axons and in the optic nerve, newly added optic axons grow in a tenascin-R immunonegative pathway, which is bordered by tenascin-R immunoreactivity. In the brain, tenascin-R expression domains in the ventral diencephalon, in non-retinorecipient pretectal nuclei and in some tectal layers closely border the optic pathway in unlesioned animals and during axon regrowth. We mimicked these boundary situations with a sharp substrate border of tenascin-R in vitro. Optic axons emanating from adult retinal explants were repelled by tenascin-R substrate borders. This is consistent with a function of tenascin-R as a repellent guidance molecule in boundaries for adult optic axons. Thus, tenascin-R may guide newly added and regenerating optic axons by a contact-repellent mechanism in the optic pathway of adult fish.

Evolution of myelin proteolipid proteins: Gene duplication in teleosts and expression pattern divergence

The coevolution of neurons and their supporting glia to the highly specialized axon-myelin unit included the recruitment of proteolipids as neuronal glycoproteins (DMbeta, DMgamma) or myelin proteins (DMalpha/PLP/DM20). Consistent with a genome duplication at the root of teleosts, we identified three proteolipid pairs in zebrafish, termed DMalpha1 and DMalpha2, DMbeta1 and DMbeta2, DMgamma1 and DMgamma2. The paralogous amino acid sequences diverged remarkably after gene duplication, indicating functional specialization. Each proteolipid has adopted a distinct spatio-temporal expression pattern in neural progenitors, neurons, and in glia. DMalpha2, the closest homolog to mammalian PLP/DM20, is coexpressed with P0 in oligodendrocytes and upregulated after optic nerve lesion. DMgamma2 is expressed in multipotential stem cells, and the other four proteolipids are confined to subsets of CNS neurons. Comparing protein sequences and gene structures from birds, teleosts, one urochordate species, and four invertebrates, we have reconstructed major steps in the evolution of proteolipids.

Neuropilin-1a is involved in trunk motor axon outgrowth in embryonic zebrafish

Neuropilin‐1, a receptor for axon‐repellent semaphorins and vascular endothelial growth factor (VEGF), functions both in angiogenesis and axon growth. Here, we show strong expression of neuropilin‐1a in primary motor neurons in the trunk of embryonic zebrafish. Reducing the expression of neuropilin‐1a using antisense morpholino oligonucleotides induced aberrant branching of motor nerves, additional exit points of motor nerves from the spinal cord, and migration of neurons out of the spinal cord along the motor axon pathway in a dose‐dependent manner. These phenotypes could be partially rescued by coinjecting neuropilin‐1a mRNA. Other axons in the spinal cord and head appeared unaffected by the morpholino treatment. In addition, neuropilin‐1a morpholino treatment disturbed normal formation of blood vessels in the trunk of 24 hours postfertilization embryos, as shown by microangiography. Morpholinos to VEGF also disturbed formation of blood vessels but did not affect motor axons, indicating that correct formation of blood vessels is not needed for the growth of primary motor axons. Morpholinos to the semaphorin 3A homologs semaphorin 3A1 and semaphorin 3A2 also had no effect on motor axon growth. However, combined injections of neuropilin‐1a morpholino, at a concentration that did not elicit axonal aberrations when injected alone, with VEGF, semaphorin 3A1, or semaphorin 3A2 morpholinos synergistically increased the proportion of embryos showing aberrant motor axon growth. Thus, neuropilin‐1a in primary motor neurons may integrate signals from several ligands and is needed for proper segmental growth of primary motor nerves in zebrafish. Developmental Dynamics 234:535–549, 2005.

Dopaminergic and noradrenergic circuit development in zebrafish

Dopaminergic and noradrenergic neurons constitute some of the major far projecting systems in the vertebrate brain and spinal cord that modulate the activity of circuits controlling a broad range of behaviors. Degeneration or dysfunction of dopaminergic neurons has also been linked to a number of neurological and psychiatric disorders, including Parkinsons disease. Zebrafish (Danio rerio) have emerged over the past two decades into a major genetic vertebrate model system, and thus contributed to a better understanding of developmental mechanisms controlling dopaminergic neuron specification and axonogenesis. In this review, we want to focus on conserved and dynamic aspects of the different catecholaminergic systems, which may help to evaluate the zebrafish as a model for dopaminergic and noradrenergic cellular specification and circuit function as well as biomedical aspects of catecholamine systems.

PlexinA3 Restricts Spinal Exit Points and Branching of Trunk Motor Nerves in Embryonic Zebrafish

The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons.

Development of the Dopamine Systems in Zebrafish

Dopaminergic neurons develop in several distinct regions of the vertebrate brain and project locally or send long axonal projections to distant parts of the CNS to modulate the activity of a variety of circuits, controlling aspects of physiology, behavior and movement. The molecular control of dopaminergic differentiation and the evolution of the various dopaminergic systems are not well understood, as research has mostly focused on ascending mammalian dopaminergic systems of the substantia nigra and ventral tegmental area. Zebrafish have evolved as an excellent genetic and experimental embryological model to study specification and axonal projectivity of dopaminergic neurons. The large evolutionary distance between fish and mammals provides the opportunity to identify conserved core regulatory mechanisms that control differentiation and projection behavior of the various dopaminergic groups in vertebrates. Here, we present an overview of the formation of dopaminergic groups and their projections in zebrafish. We will further review the results from genetic analyses, which have revealed insights on signals as well as transcription factors contributing to dopaminergic differentiation. Together with recently established paradigms for behavioral analysis, dopaminergic systems are studied at all levels in zebrafish, from molecular and cellular to systems and behavioral.

Tenascin-C is involved in motor axon outgrowth in the trunk of developing zebrafish

Motor axons in the trunk of the developing zebrafish exit from the ventral spinal cord in one ventral root per hemisegment and grow on a common path toward the region of the horizontal myoseptum, where they select their specific pathways. Tenascin‐C, a component of the extracellular matrix, is concentrated in this choice region. Adaxial cells and other myotomal cells express tenascin‐C mRNA, suggesting that these cells are the source of tenascin‐C protein. Overexpressing an axon repellent fragment containing the cysteine‐rich region and the epidermal growth factor‐like repeats of tenascin‐C led to retarded growth of ventral motor nerves between their spinal exit point and the horizontal myoseptum. Injection of a protein fragment containing the same part of tenascin‐C also induced slower growth of motor nerves. Conversely, knock down of tenascin‐C protein resulted in abnormal lateral branching of ventral motor nerves. In the zebrafish unplugged mutant, in which axons display pathfinding defects in the region of the horizontal myoseptum, tenascin‐C immunoreactivity was not detectable in this region, indicating an abnormal extracellular matrix in unplugged. We conclude that tenascin‐C is part of a specialized extracellular matrix in the region of the horizontal myoseptum that influences the growth of motor axons. Developmental Dynamics 234:550–566, 2005.