Functionalization of titanium based metallic biomaterials for implant applications.

Surface immobilization with active functional molecules (AFMs) on a nano-scale is a main field in the current biomaterial research. The functionalization of a vast number of substances and molecules, ranging from inorganic calcium phosphates, peptides and proteins, has been investigated throughout recent decades. However, in vitro and in vivo results are heterogeneous. This may be attributed partially to the limits of the applied immobilization methods. Therefore, this paper highlights the advantages and limitations of the currently applied methods for the biological nano-functionalization of titanium-based biomaterial surfaces. The second part describes a newer immobilization system, using the nanomechanical fixation of at least partially single-stranded nucleic acids (NAs) into an anodic titanium oxide layer as an immobilization principle and their hybridization ability for the functionalization of the surface with active functional molecules conjugated to the respective complementary NA strands.

Knockdown of ephrin-A5 expression by 40% does not affect motor axon growth or migration into the chick hindlimb.

Bidirectional signaling between Eph receptor tyrosine kinases and their cell-surface protein signals, the ephrins, comprises one mechanism for guiding motor axons to their proper targets. During projection of motor axons from the lateral motor column (LMC) motor neurons of the spinal cord to the hindlimb muscles in chick embryos, ephrin-A5 has been shown to be expressed in the LMC motor axons until they reach the base of the limb bud and initiate sorting into their presumptive dorsal and ventral nerve trunks, at which point expression is extinguished. We tested the hypothesis that this dynamic pattern of ephrin-A5 expression in LMC motor axons is important for the growth and guidance of the axons to, and into, the hindlimb by knocking down endogenous ephrin-A5 expression in the motor neurons and their axons. No perturbation of LMC motor axon projections was observed in response to this treatment, suggesting that ephrin-A5 is not needed for LMC motor axon growth or guidance.

The Wnt and BMP families of signaling morphogens at the vertebrate neuromuscular junction.

The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.

Identification of responsive cells in the developing somite supports a role for beta-catenin-dependent Wnt signaling in maintaining the DML myogenic progenitor pool.

Somitic beta-catenin is involved in both maintaining a stem cell population and controlling myogenic differentiation. It is unclear how beta-catenin-dependent Wnt signaling accomplishes these disparate roles. The present study shows that only dorsal cells in the early somite respond to beta-catenin-dependent Wnt signaling and as the somites compartmentalize to form the dermomyotome and myotome, responding cells are detected primarily in the dorsomedial lip (DML). Forced activation of Wnt target genes in DML cells prevents their progeny from entering the myotome, while blocking activation allows myotomal entry. This suggests a role for beta-catenin-dependent/Wnt signaling in maintaining progenitor cells in the DML and that if beta-catenin-dependent/Wnt signaling is required to induce myogenesis, the response is transitory and rapidly down-regulated.

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb.

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.

Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse.

Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.

Ephrin-A5 exerts positive or inhibitory effects on distinct subsets of EphA4-positive motor neurons.

Eph receptor tyrosine kinases and ephrins are required for axon patterning and plasticity in the developing nervous system. Typically, Eph-ephrin interactions promote inhibitory events; for example, prohibiting the entry of neural cells into certain embryonic territories. Here, we show that distinct subsets of motor neurons that express EphA4 respond differently to ephrin-A5. EphA4-positive LMC(l) axons avoid entering ephrin-A5-positive hindlimb mesoderm. In contrast, EphA4-positive MMC(m) axons extend through ephrin-A5-positive rostral half-sclerotome. Blocking EphA4 activation in MMC(m) neurons or expanding the domain of ephrin-A5 expression in the somite results in the aberrant growth of MMC(m) axons into the caudal half-sclerotome. Moreover, premature expression of EphA4 in MMC(m) neurons leads to a portion of their axons growing into novel ephrin-A5-positive territories. Together, these results indicate that EphA4-ephrin-A5 signaling acts in a positive manner to constrain MMC(m) axons to the rostral half-sclerotome. Furthermore, we show that Eph activation localizes to distinct subcellular compartments of LMC(l) and MMC(m) neurons, consistent with distinct EphA4 signaling cascades in these neuronal subpopulations.

Embryological and genetic manipulation of chick development.

The ability to combine embryological manipulations with gene function analysis makes the chick a valuable system for the vertebrate developmental biologist. We describe methods for those unfamiliar with the chick wishing to initiate chick experiments in their lab. After outlining how to prepare chick embryos, we provide protocols for introducing beads or cells expressing secreted factors into the embryo and for culturing tissue explants as a means of assessing development in vitro. Chick gain-of-function and loss-of-function (RNAi and morpholino oligonucleotide) approaches are outlined, and methods for introducing these reagents by electroporation are detailed.

Neural crest cells and motor axons in avians: Common and distinct migratory molecules.

It has long been thought that the same molecules guide both trunk neural crest cells and motor axons as these cell types grow and extend to their target regions in developing embryos. There are common territories that are navigated by these cell types: both cells grow through the rostral portion of the somitic sclerotomes and avoid the caudal half of the sclerotomes. However, these cell types seem to use different molecules to guide them to their target regions. In this review, I will talk about the common and distinct methods of migration taken by trunk neural crest cells and motor axons as they grow and populate their target regions through chick embryos at the level of the trunk.

Mechanisms of growth cone repulsion.

Research conducted in the last century suggested that chemoattractants guide cells or their processes to appropriate locations during development. Today, we know that many of the molecules involved in cellular guidance can act as chemorepellents that prevent migration into inappropriate territories. Here, we review some of the early seminal experiments and our current understanding of the underlying molecular mechanisms.

Transfecting neural crest cells in avian embryos using in ovo electroporation.

INTRODUCTIONThis protocol outlines a method for introducing genes into the neural crest cells of avian embryos at vagal/cardiac and trunk levels. Electroporation offers the unique opportunity to misexpress genes or "knock down" the expression of their protein products using short hairpin RNAs (shRNAs) within targeted populations of cells. Electroporated cells can be visualized by using DNA plasmids that contain enhanced green fluorescent protein (EGFP) or other fluorescent reporter proteins.

Using in ovo electroporation to transfect cells in avian somites.

INTRODUCTIONIn ovo electroporation is an efficient approach for manipulating gene expression in multiple tissues of the chicken embryo, which greatly facilitates functional analysis of the role of specific genes during development. A successful protocol to deliver plasmid DNA into a specific tissue has to meet three basic criteria: high efficiency of transfection, low mortality of embryos, and localization of transfected cells. In this protocol, we describe an in ovo electroporation procedure for targeting muscle precursor cells in the lateral dermomyotome and axial skeletal precursor cells in the sclerotome of the somites of stage 15 chicken embryos. At this stage, the microinjection of DNA solution into individual somites is relatively easy, and the orientation of the electrodes directs the negatively charged DNA into the target tissues. The range of electrical current used is adjusted to maximize the transfection but minimize the mortality of the embryos.

Transfecting avian motor neurons and their axons using in ovo electroporation.

INTRODUCTIONIn ovo electroporation of half of the avian neural tube is a simple procedure in which one places the electrodes parallel to the neural tube, flanking the intended axial region of transfection. It is possible to modify this technique to target the ventral quadrant of the neural tube that contains motor neurons in the lateral motor column (LMC) and their axons by positioning the electrodes in an offset dorsal/ventral configuration, instead of the standard parallel position. If the electroporation is performed in the neural tube of stage 15 chick embryos, the medial portion of the LMC is targeted primarily; however, if neural tubes of stage 17 embryos are electroporated, the entire LMC will be transfected. This technique can be used to examine the behavior of motor axons as they project into the developing limb when genes are misexpressed, overexpressed, or knocked down via RNAi (using short hairpin RNA [shRNA]). The un-electroporated half of the neural tube serves as an internal control, or an enhanced green fluorescent protein (EGFP) reporter construct (pCAX) serves as a control for the electroporation and for EGFP expression. By electroporating a DNA construct that contains EGFP, or co-electroporating the DNA of interest with a GFP reporter construct, it is possible to verify the success of the electroporation in ovo.

PINCH-1 expression during early avian embryogenesis: implications for neural crest and heart development.

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. We previously had performed four modified differential display (DD) screens in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. We show here for the first time that chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration.

A primer on using in ovo electroporation to analyze gene function.

The chicken embryo has served as a classic model system for developmental studies due to its easy access for surgical manipulations and a wealth of data about chicken embryogenesis. Notably, the mechanisms controlling limb development have been explored best in the chick. Recently, the method of in ovo electroporation has been used successfully to transfect particular cells/tissues during embryonic development, without the production or infectivity associated with retroviruses. With the sequencing of the chicken genome near completion, this approach will provide a powerful opportunity to examine the function of chicken genes and their counterparts in other species. In ovo electroporation has been most effectively used to date for ectopic or overexpression analyses. However, recent studies indicate that this approach can be used successfully for loss-of-function analyses, including protein knockdown experiments with morpholinos and RNAi. Here, I will discuss parameters for using in ovo electroporation successfully to study developmental processes.

EphA4 signaling promotes axon segregation in the developing auditory system.

Precision of synaptic connections within neural circuits is essential for the accurate processing of sensory information. Specificity is exemplified at cellular and subcellular levels in the chick auditory brainstem, where nucleus magnocellularis (NM) neurons project bilaterally to nucleus laminaris (NL). Dorsal dendrites of NL neurons receive input from ipsilateral, but not contralateral, branches of NM axons whereas ventral dendrites are innervated by contralateral NM axons. This organization is analogous to that of the mammalian medial superior olive (MSO) and represents an important component of the circuitry underlying sound localization. However, the molecular mechanisms that establish segregated inputs to individual regions of NL neurons have not been identified. During synapse formation in NL, the EphA4 receptor is expressed in dorsal, but not ventral NL, neuropil, suggesting a potential role in targeting synapses to appropriate termination zones. Here, we directly tested this role by ectopically expressing EphA4 and disrupting EphA4 signaling using in ovo electroporation. We found that both misexpression of EphA4 and disruption of EphA4 signaling resulted in an increase in the number of NM axons that grow aberrantly across NL cell bodies into inappropriate regions of NL neuropil. EphA4 signaling is thus essential for targeting axons to distinct subsets of dendrites. Moreover, loss of EphA4 function resulted in morphological abnormalities of NL suggestive of errors in cell migration. These results suggest that EphA4 has multiple roles in the formation of auditory brainstem nuclei and their projections.

Targeting the EphA4 receptor in the nervous system with biologically active peptides.

EphA4 is a member of the Eph family of receptor tyrosine kinases and has important functions in the developing and adult nervous system. In the adult, EphA4 is enriched in the hippocampus and cortex, two brain structures critical for learning and memory. To identify reagents that can discriminate between the many Eph receptors and selectively target EphA4, we used a phage display approach. We identified three 12-amino acid peptides that preferentially bind to EphA4. Despite lack of a common sequence motif, these peptides compete with each other for binding to EphA4 and antagonize ephrin binding and EphA4 activation at micromolar concentrations, indicating that they bind with high affinity to the ephrin-binding site. Furthermore, one of the peptides perturbs the segmental migration of EphA4-positive neural crest cells in chick trunk organotypic explants. Hence, this peptide can disrupt the physiological function of endogenous EphA4 in situ. We also identified additional peptides that bind to EphA5 and EphA7, two other receptors expressed in the nervous system. This panel of peptides may lead to the development of pharmaceuticals that differentially target Eph receptors to modulate neuronal function in specific regions of the nervous system.