Biomedical subjects

Catherina G Becker

Publications and source records attributed to Catherina G Becker.

17 recordsLinked to original sources

PubMed2007

Growth and pathfinding of regenerating axons in the optic projection of adult fish.

In contrast to mammals, teleost fish are able to regrow severed long-range projection axons in the central nervous system (CNS), leading to recovery of function. The optic projection in teleost fish is used to study neuron-intrinsic and environmental molecular factors that determine successful axon regrowth and navigation through a complex CNS pathway back to original targets. Here we review evidence for regeneration-specific regulation and robust expression of growth- and pathfinding-associated genes in regenerating retinal ganglion cell (RGC) axons of adult fish. The environment of the CNS in fish appears to contain few inhibitory molecules and at the same time a number of promoting molecules for axon regrowth. Finally, some environmental cues that are used as guidance cues for developing RGC axons are also present in continuously growing adult animals. These molecules may serve as guidance cues for the precise navigation of axons from newly generated RGCs in adult animals as well as of regenerating RGC axons after a lesion. The application of new molecular techniques especially to adult zebrafish, is likely to produce new insights into successful axonal regeneration in the CNS of fish and the absence thereof in mammals.

Animals↗

PubMed2005

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 co-injecting 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.

Animals↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Differences in the regenerative response of neuronal cell populations and indications for plasticity in intraspinal neurons after spinal cord transection in adult zebrafish.

In zebrafish, the capacity to regenerate long axons varies among different populations of axotomized neurons after spinal cord transection. In specific brain nuclei, 84-92% of axotomized neurons upregulate expression of the growth-related genes GAP-43 and L1.1 and 32-51% of these neurons regrow their descending axons. In contrast, 16-31% of spinal neurons with axons ascending to the brainstem upregulate these genes and only 2-4% regrow their axons. Dorsal root ganglion (DRG) neurons were not observed to regrow their ascending axons or to increase expression of GAP-43 mRNA. Expression of L1.1 mRNA is high in unlesioned and axotomized DRG neurons. In the lesioned spinal cord, expression of growth-related molecules is increased in a substantial population of non-axotomized neurons, suggesting morphological plasticity in the spinal-intrinsic circuitry. We propose that locomotor recovery in spinal-transected adult zebrafish is influenced less by recovery of ascending pathways, but more by regrowth of descending tracts and rearrangement of intraspinal circuitry.

Animals↗

PubMed2005

Expression of collapsin response mediator proteins in the nervous system of embryonic zebrafish.

Collapsin response mediator proteins (CRMPs also known as TUC, Drp, Ulip, TOAD-64) are cytosolic phosphoproteins that are involved in signal transduction during axon growth and in cytoskeletal dynamics. Here we report cloning and mRNA expression patterns of CRMP-1, -2, -3, -4 and, owing to a genome duplication in teleosts, two homologs of CRMP-5 (CRMP-5a and -5b) in embryonic zebrafish at 16 and 24h post-fertilization (hpf). CRMPs are evolutionarily conserved and zebrafish CRMPs show amino acid identities of 76-90% with their homologs in humans, with the exception of CRMP-3, which shows only 67% homology. Between 16 and 24hpf, expression of CRMPs generally increased in many regions of the CNS undergoing neuronal differentiation and axonogenesis, but not in the proliferative ventricular zone. Structures that were typically labeled by most, but not all the CRMP probes were the telencephalon, the nucleus of the tract of the post-optic commissure, the epiphysis, the nucleus of the medial longitudinal fascicle, clusters of hindbrain neurons, cranial ganglia, as well as Rohon-Beard neurons. No expression of CRMP mRNAs was observed outside the nervous system. Thus, expression patterns of different CRMP family members correlate with neuronal differentiation and axonogenesis in embryonic zebrafish.

Amino Acid Sequence↗

PubMed2004

L1.1 is involved in spinal cord regeneration in adult zebrafish.

Adult zebrafish, in contrast to mammals, regrow axons descending from the brainstem after spinal cord transection. L1.1, a homolog of the mammalian recognition molecule L1, is upregulated by brainstem neurons during axon regrowth. However, its functional relevance for regeneration is unclear. Here, we show with a novel morpholino-based approach that reducing L1.1 protein expression leads to impaired locomotor recovery as well as reduced regrowth and synapse formation of axons of supraspinal origin after spinal cord transection. This indicates that L1.1 contributes to successful regrowth of axons from the brainstem and locomotor recovery after spinal cord transection in adult zebrafish.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

Expression and mapping of duplicate neuropilin-1 and neuropilin-2 genes in developing zebrafish.

Previously, we described the isolation and characterization of the first zebrafish neuropilin gene, which we now call nrp1a, and found its protein to be a mediator of vascular endothelial growth factor (VEGF)-dependent angiogenesis [Proc. Natl Acad. Sci. USA 99 (2002) 10470]. Subsequently, we have isolated three other full-length neuropilin genes (nrp1b, nrp2a, and nrp2b) and find that they map to independent zebrafish linkage groups. The nrp1s and nrp2s had differential spatio-temporal gene expression profiles with nrp1a being most prominent in the gut, brain, retina, hypochord, motorneurons, fin bud and mandibular cartilage, nrp1b in the brain, dorsal aorta, melanophores, ventral fin, and heart, nrp2a in the brain, retina, heart, and caudal vessels, and nrp2b in the brain, retina, gut, fin bud, melanophores, heart, and caudal vessels. In addition, we have identified an alternatively-spliced transcript of the nrp1b gene (denoted as nrp1b(s)) which is predicted to encode a soluble form of Nrp1b, containing only the a, b, and c extracellular domains. Transcript expression of nrp1b(s) was different from full-length nrp1b transcript, with prominence in the brain, developing mouth, heart, and fin bud. The NRP1s were tested for VEGF-binding ability. Both 125 kDa Nrp1a and 145 kDa Nrp1b bound 125I-labelled VEGFA165. In summary, two nrp1 and two nrp2 genes, with expression patterns similar to higher vertebrates, have been isolated from zebrafish.

Alternative Splicing↗

PubMed2003

Tenascin-R as a repellent guidance molecule for developing optic axons in zebrafish.

To investigate the role of tenascin-R in nervous system development, we studied axon pathfinding in the developing optic system of zebrafish. Zebrafish tenascin-R has the same domain structure as tenascin-R in amniotes. Amino acid sequence identity with human tenascin-R is 60%. In 3-d-old larvae, tenascin-R mRNA is expressed in scattered cells throughout the periventricular cell layer of the diencephalon and tectum. Tenascin-R immunoreactivity is not detectable in the optic nerve, optic tract, or tectal optic neuropil but immediately borders the optic tract caudally. Reducing expression of tenascin-R in 3-d-old larvae in vivo by injecting morpholinos into fertilized eggs led to excessive branching of the optic tract in 86% of all injected larvae compared with 20-37% in controls. Branches were almost exclusively caudal, where tenascin-R immunoreactivity normally borders the optic tract, suggesting a role for tenascin-R in guiding optic axons in the ventral diencephalon by a contact-repellent mechanism.

Animals↗

PubMed2003

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.

Age Factors↗

PubMed2003

Integrin antagonists affect growth and pathfinding of ventral motor nerves in the trunk of embryonic zebrafish.

Integrins are thought to be important receptors for extracellular matrix (ECM) components on growing axons. Ventral motor axons in the trunk of embryonic zebrafish grow in a midsegmental pathway through an environment rich in ECM components. To test the role of integrins in this process, integrin antagonists (the disintegrin echistatin in native and recombinant form, as well as the Arg-Gly-Asp-Ser peptide) were injected into embryos just prior to axon outgrowth at 14-16 h postfertilization (hpf). All integrin antagonists affected growth of ventral motor nerves in a similar way and native echistatin was most effective. At 24 hpf, when only the three primary motor axons per trunk hemisegment had grown out, 80% (16 of 20) of the embryos analyzed had abnormal motor nerves after injection of native echistatin, corresponding to 19% (91 of 480) of all nerves. At 33 hpf, when secondary motor axons were present in the pathway, 100% of the embryos were affected (24 of 24), with 20% of all nerves analyzed (196 of 960) being abnormal. Phenotypes comprised abnormal branching (64% of all abnormal nerves) and truncations (36% of all abnormal nerves) of ventral motor nerves at 24 hpf and mostly branching of the nerves at 33 hpf (94% of all abnormal nerves). Caudal branches were at least twice as frequent as rostral branches. Surrounding trunk tissue and a number of other axon fascicles were apparently not affected by the injections. Thus integrin function contributes to both growth and pathfinding of axons in ventral motor nerves in the trunk of zebrafish in vivo.

Animals↗

PubMed2003

Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish.

A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.

Animals↗

PubMed2003

Comparing protein stabilities during zebrafish embryogenesis.

The stabilities of many key proteins are regulated, e.g. via ubiquitination and proteasomal degradation, with important biological consequences. We present a convenient method that allows the analysis and comparison of protein stabilities during embryogenesis using early zebrafish development as a model system. Basically, this method involves ectopic overexpression of epitope-tagged proteins via mRNA injections in one-to-four-cell stage embryos and subsequent protein detection after various time points. Indeed, the protein stability of the ubiquitin ligase RLIM, which is able to autoubiquitinate and target itself for proteasomal degradation, was much shorter when compared to a protein consisting of a Myc epitope-tag and a nuclear localization domain. Thus, this method may be used more widely for the study of developmental protein stability.

Animals↗

PubMed2002

Repellent guidance of regenerating optic axons by chondroitin sulfate glycosaminoglycans in zebrafish.

We analyzed the role of chondroitin sulfate (CS) glycosaminoglycans, putative inhibitors of axonal regeneration in mammals, in the regenerating visual pathway of adult zebrafish. In the adult, CS immunoreactivity was not detectable before or after an optic nerve crush in the optic nerve and tract but was constitutively present in developing and adult nonretinorecipient pretectal brain nuclei, where CSs may form a boundary preventing regenerating optic fibers from growing into these inappropriate locations. Enzymatic removal of CSs by chondroitinase ABC after optic nerve crush significantly increased the number of animals showing erroneous growth of optic axons into the nonretinorecipient magnocellular superficial/posterior pretectal nucleus (83% vs 42% in controls). In vitro, a substrate border of CSs, but not heparan sulfates, strongly repelled regenerating retinal axons from adult zebrafish. We conclude that CSs contribute to repellent axon guidance during regeneration of the optic projection in zebrafish.

Animals↗

PubMed2002

Multiple functions of LIM domain-binding CLIM/NLI/Ldb cofactors during zebrafish development.

The crucial involvement of CLIM/NLI/Ldb cofactors for the exertion of the biological activity of LIM homeodomain transcription factors (LIM-HD) has been demonstrated. In this paper we show that CLIM cofactors are widely expressed during zebrafish development with high protein levels in specific neuronal cell types where LIM-HD proteins of the Isl class are synthesized. The overexpression of a dominant-negative CLIM molecule (DN-CLIM) that contains the LIM interaction domain (LID) during early developmental stages of zebrafish embryos results in an impairment of eye and midbrain-hindbrain boundary (MHB) development and disturbances in the formation of the anterior midline. On a cellular level we show that the outgrowth of peripheral but not central axons from Rohon Beard (RB) and trigeminal sensory neurons is inhibited by DN-CLIM overexpression. We demonstrate a further critical role of CLIM cofactors for axonal outgrowth of motor neurons. Additionally, DN-CLIM overexpression causes an increase of Isl-protein expression levels in specific neuronal cell types, likely due to a protection of the DN-CLIM/LIM-HD complex from proteasomal degradation. Our results demonstrate multiple roles of the CLIM cofactor family for the development of entire organs, axonal outgrowth of specific neurons and protein expression levels.

Animals↗

PubMed2002

Expression of the zebrafish recognition molecule F3/F11/contactin in a subset of differentiating neurons is regulated by cofactors associated with LIM domains.

We have identified a zebrafish homolog of the F3/F11/contactin (F3) recognition molecule. The gene shares 55% amino acid identity with F3 molecules in other vertebrates. Expression of F3 mRNA is first detectable at 16 h post-fertilization (hpf) in trigeminal and Rohon-Beard neurons. At 18-24 hpf, additional weaker expression is present in discrete cell clusters in the hindbrain, in the anterior lateral line/acoustic ganglion and in spinal motor neurons. Transcription factors of the LIM homeodomain class (LIM-HD) and their associated cofactors CLIM/NLI/Ldb (CLIM) have been implicated in the development of peripheral axons of trigeminal and Rohon-Beard neurons. We demonstrate that ectopic overexpression of a dominant-negative CLIM molecule early during zebrafish development strongly reduces expression of F3 mRNA in these neurons indicating regulation of F3 by the LIM-HD protein network. These results and the spatiotemporal correlation of F3 expression with axonal differentiation in a subset of primary neurons suggest an important role of F3 for axon growth.

Amino Acid Sequence↗

PubMed2002

Expression of the zebrafish recognition molecule F3/F11/contactin in a subset of differentiating neurons is regulated by cofactors associated with LIM domains.

Animals↗