Axon guidance by growth-rate modulation.

Guidance of axons by molecular gradients is crucial for wiring up the developing nervous system. It often is assumed that the unique signature of such guidance is immediate and biased turning of the axon tip toward or away from the gradient. However, here we show that such turning is not required for guidance. Rather, by a combination of experimental and computational analyses, we demonstrate that growth-rate modulation is an alternative mechanism for guidance. Furthermore we show that, although both mechanisms may operate simultaneously, biased turning dominates in steep gradients, whereas growth-rate modulation may dominate in shallow gradients. These results suggest that biased axon turning is not the only method by which guidance can occur.

Bayesian model predicts the response of axons to molecular gradients.

Axon guidance by molecular gradients plays a crucial role in wiring up the nervous system. However, the mechanisms axons use to detect gradients are largely unknown. We first develop a Bayesian "ideal observer" analysis of gradient detection by axons, based on the hypothesis that a principal constraint on gradient detection is intrinsic receptor binding noise. Second, from this model, we derive an equation predicting how the degree of response of an axon to a gradient should vary with gradient steepness and absolute concentration. Third, we confirm this prediction quantitatively by performing the first systematic experimental analysis of how axonal response varies with both these quantities. These experiments demonstrate a degree of sensitivity much higher than previously reported for any chemotacting system. Together, these results reveal both the quantitative constraints that must be satisfied for effective axonal guidance and the computational principles that may be used by the underlying signal transduction pathways, and allow predictions for the degree of response of axons to gradients in a wide variety of in vivo and in vitro settings.

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.

Chemotactic responses of growing neurites to precisely controlled gradients of nerve growth factor.

Chemotaxis plays a key role in many biological systems. In particular in the context of the developing nervous system, growing neurites can respond in vitro to shallow gradients of chemotropic molecules such as nerve growth factor (NGF). However, in such studies the gradient parameters are often not well controlled. Here we present a dataset of ~3500 images of early postnatal rat dorsal root ganglion (DRG) explants growing in 40 different precisely controlled combinations of absolute concentration and gradient steepness of NGF. Each image has been segmented into neurite and explant-body regions. We provide computer code for exploration and quantification of the data, including a Fourier analysis of the outer contour of neurite growth, which allows quantities such as outgrowth and guidance as a function of concentration and gradient steepness to be easily extracted. This is the most comprehensive quantitative dataset of chemotactic responses yet available for any biological system, which we hope will be useful for exploring the biological mechanisms governing chemotaxis.

PNA microarrays for hybridisation of unlabelled DNA samples.

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.

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.

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.

Migrating and circulating breast carcinoma cells are within active phases of the cell cycle.

The emergence of blood-borne epithelial-derived tumour cells in breast cancer patients is generally supposed to be an indicator of cancer cell spread but only a very small percentage of these cells ultimately initiate metastases. In this study, we investigated the presence of the cell cycle marker Ki-67 in mammary tumour cells isolated from patients' blood and SKBR3 breast carcinoma cells by confocal laser scanning microscopy. We also compared the immunostaining patterns for Ki-67 of blood-borne tumour cells from the patients with actively migrating cells within a collagen matrix. Both circulating tumour cells and migrating SKBR3 cells were found to be in G1- or S-phase. These findings will have impact on adjuvant chemotherapy as circulating tumour cells that are within the active part of the cell cycle are highly susceptible to chemotherapeutic agents and therefore can be killed while they migrate to distant organs to build a metastasis.

Assays for eukaryotic cell chemotaxis.

Chemotaxis is essential for many biological processes. Much of our understanding of the mechanisms underlying chemotaxis is based on a variety of in vitro assays. We review these assays, dividing them into groups depending on the process used to generate the gradient. We describe how each method works, its strengths and limitations, and provide some information about the kinds of cells that have been studied with each assay.

Contactin1a expression is associated with oligodendrocyte differentiation and axonal regeneration in the central nervous system of zebrafish.

Contactin1a (Cntn1a) is a zebrafish homolog of contactin1 (F3/F11/contactin) in mammals, an immunoglobulin superfamily recognition molecule of neurons and oligodendrocytes. We describe conspicuous Cntn1a mRNA expression in oligodendrocytes in the developing optic pathway of zebrafish. In adults, this expression is only retained in glial cells in the intraretinal optic fiber layer, which contains 'loose' myelin. After optic nerve lesion, oligodendrocytes re-express Cntn1a mRNA independently of the presence of regenerating axons and retinal ganglion cells upregulate Cntn1a expression to levels that are significantly higher than those during development. After spinal cord lesion, expression of Cntn1a mRNA is similarly increased in axotomized brainstem neurons and white matter glial cells in the spinal cord. In addition, reduced mRNA expression in the trigeminal/anterior lateral line ganglion in erbb3-deficient mutant larvae implies Cntn1a in Schwann cell differentiation. These complex regulation patterns suggest roles for Cntn1a in myelinating cells and neurons particularly in successful CNS regeneration.

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.

Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization.

Cell migration depends mainly on actin polymerization and intracellular organization, which are influenced by a vast variety of actin binding proteins (ABPs). Regulation of ABP activity is mediated by second messengers such as phosphoinositides and calcium. Signaling via these second messengers is initiated and regulated by membrane receptors, e.g., receptor tyrosine kinases (RTKs), and by adhesion molecule interactions (e.g., integrins and selectins) and focal adhesion kinases. A major role in steering second-messenger signaling and thus in actin cytoskeleton reorganization and motility of cancer cells is played by the RTK c-erbB-2. This occurs through a number of signaling pathways which involve mainly enzymes, e.g., phospholipase Cgamma1 and GTPases, which modify signaling molecules. Furthermore large multiprotein complexes including actin-related protein 2/3, Wiskott-Aldrich syndrome protein, profilin, and capping protein among others play an important role in regulating actin reorganization. The complex picture of the mode of actin reorganization, which is involved in tumor cell migration, is slowly emerging from the mists of cellular signaling pathways, but this is still by no means a clear view.

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.