Catherina G. Becker

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.

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons ability to negotiate novel pathways in the spinal cord gray matter. J. Comp. Neurol. 433:131–147, 2001.

Gradients of ephrin-A2 and ephrin-A5b mRNA during retinotopic regeneration of the optic projection in adult zebrafish.

Regeneration of optic axons in the continuously growing optic system of adult zebrafish was analyzed by anterograde tracing and correlated with the mRNA expression patterns of the recognition molecules ephrin‐A2 and ephrin‐A5b in retinal targets. The optic tectum and diencephalic targets are all reinnervated after a lesion. However, the rate of erroneous pathway choices was increased at the chiasm and the bifurcation between the ventral and dorsal brachium of the optic tract compared to unlesioned animals. Tracer application to different retinal positions revealed retinotopic reinnervation of the tectum within 4 weeks after the lesion. In situ hybridization analysis indicated the presence of rostral‐low to caudal‐high gradients of ephrin‐A2 and ephrin‐A5b mRNAs in unlesioned control tecta and after a unilateral optic nerve lesion. By contrast, the parvocellular superficial pretectal nucleus showed retinotopic organization of optic fibers but no detectable expression of ephrin‐A2 and ephrin‐A5b mRNAs. However, a row of cells delineating the terminal field of optic fibers in the dorsal part of the periventricular pretectal nucleus was intensely labeled for ephrin‐A5b mRNA and may thus provide a stop signal for ingrowing axons. Ephrin‐A2 and ephrin‐A5b mRNAs were not detectable in the adult retina, despite their prominent expression during development. Thus, given a complementary receptor system in retinal ganglion cells, expression of ephrin‐A2 and ephrin‐A5b in primary targets of optic fibers in adult zebrafish may contribute to guidance of optic axons that are continuously added to the adult projection and of regenerating axons after optic nerve lesion. J. Comp. Neurol. 427:469–483, 2000.

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.

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.

Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish

The HNK-1 glycoepitope, carried by many cell recognition molecules, is present in the developing posterior lateral line nerve and on other primary axons of zebrafish. To elucidate the function of HNK-1 in vivo, the antibody 412 to HNK-1 was injected into zebrafish embryos at 16 h post fertilization (hpf). The injected antibody bound specifically to axons carrying HNK-1. This treatment selectively affected the growth of either one or both posterior lateral line nerves in 39% of the experimental cases (13 of 33 animals), which was significantly more (P<0.0002) than in uninjected, vehicle injected, and non-immune IgG injected controls (1.2% of the animals; one of 85 animals), as assessed at 27 or 33 hpf. Other HNK-1 immunoreactive nerves, such as the ventral motor nerves were unaffected, indicating that antibody binding per se did not interfere with axon growth. The primordium of the posterior lateral line was not affected in its caudal migration and in depositing differentiating neuromasts along the trunk, showing that injections did not retard development and that initial formation of lateral line organs is probably independent of contact with nerve fibers. We suggest that the HNK-1 glycoepitope is an important modulator of embryonic nerve growth.

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.

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.