Catherine E. Krull

Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration

BACKGROUND In the trunk of avian embryos, neural crest migration through the somites is segmental, with neural crest cells entering the rostral half of each somitic sclerotome but avoiding the caudal half. Little is known about the molecular nature of the cues-intrinsic to the somites-that are responsible for this segmental migration of neural crest cells. RESULTS We demonstrate that Eph-related receptor tyrosine kinases and their ligands are essential for the segmental migration of avian trunk neural crest cells through the somites. EphB3 localizes to the rostral half-sclerotome, including the neural crest, and the ligand ephrin-B1 has a complementary pattern of expression in the caudal half-sclerotome. To test the functional significance of this striking asymmetry, soluble ligand ephrin-B1 was added to interfere with receptor function in either whole trunk explants or neural crest cells cultured on alternating stripes of ephrin-B1 versus fibronection. Neural crest cells in vitro avoided migrating on lanes of immobilized ephrin-B1; the addition of soluble ephrin-B1 blocked this inhibition. Similarly, in whole trunk explants, the metameric pattern of neural crest migration was disrupted by addition of soluble ephrin-B1, allowing entry of neural crest cells into caudal portions of the sclerotome. CONCLUSIONS Both in vivo and in vitro, the addition of soluble ephrin-B1 results in a loss of the metameric migratory pattern and a disorganization of neural crest cell movement. These results demonstrate that Eph-family receptor tyrosine kinases and their transmembrane ligands are involved in interactions between neural crest and sclerotomal cells, mediating an inhibitory activity necessary to constrain neural precursors to specific territories in the developing nervous system.

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 ephexin1s 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.

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. Developmental Dynamics 229:433–439, 2004.

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.

Expression of EphA4, Ephrin-A2 and Ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding

During neural development, spinal motor axons extend in a precise manner from the ventral portion of the developing spinal cord to innervate muscle targets in the limb. Although classical studies in avians have characterized the cellular interactions that influence motor axon pathfinding to the limb, less is known about the molecular mechanisms that mediate this developmental event. Here, we examine the spatiotemporal distributions of the EphA4 receptor tyrosine kinase (RTK) and its cognate ligands, ephrin-A2 and ephrin-A5, on motor neurons, their axons and their pathways to the avian hindlimb to determine whether these molecules may influence axonal projections. The expression patterns of EphA4, ephrin-A2 and ephrin-A5 mRNAs and proteins are highly complex and appear to exhibit some overlap during motor axon outgrowth and pathfinding to the hindlimb, reminiscent of the co-expression of Eph RTKs and ephrins in the retinotectal system. EphA4, similar to the carbohydrate moiety polysialic acid, strikingly marks the main dorsal, but not ventral, nerve trunk after axon sorting at the limb plexus region. Our results suggest that EphA4 RTK and its ligands may influence axon fasciculation and the sorting of axons at the limb plexus, contributing to the correct dorsoventral organization of nerve branches in the hindlimb.

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.

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half‐somite

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.

Embryonic Explant and Slice Preparations for Studies of Cell Migration and Axon Guidance

This chapter describes methods for preparing explants and slice preparations of the embryonic chick and mouse. In addition, it describes methods for culturing explants of embryonic mouse tissue. Each culture system possesses distinct advantages and should be adaptable to many studies of developmental processes that occur over a wide spatiotemporal range. These preparations maintain several features common to the intact embryo, including tissue structural integrity and the correct distribution of various molecular constituents for at least 2-3 days. Of critical importance, the explants and slices retain their tissue architecture for 1-3 days in culture and possess molecular characteristics typical of intact embryos. Furthermore, these types of preparations allow one to disrupt the embryonic environment through the addition of various blocking reagents and observe the subsequent effects of these manipulations via static images and using time-lapse videomicroscopy. These types of paradigms should continue to offer a rich cellular and molecular arena in which to study early developmental events, including cell migration and process outgrowth.

Motor axon pathfinding in the peripheral nervous system.

Functional motor performance is dependent upon the correct assemblage of neural circuitry, a process initiated during embryonic development. How is the complicated neural circuitry that underlies functional behavior formed? During early stages of development, motor neurons extend their axons in a precise manner to their target destinations where they form fine synaptic connections. This process is not random but rather, highly stereotyped and specific. Results of recent studies indicate that positive and negative molecules influence particular steps in the navigation of motor axons to their targets. These molecules include, but are not limited to, members of the Semaphorin family and their receptors, Neuropilins and Plexins, Slits and their Robo receptors, members of the Eph family, extracellular matrix molecules, Hepatocyte Growth Factor/Scatter Factor, peanut agglutinin-binding glycoproteins, and neural cell adhesion molecule. The developing avian peripheral nervous system has served as an excellent model system for many years for studies of the basic cellular interactions that underlie motor axon pathfinding. The principal advantage for the experimental use of the avian embryo is the ease of access to early developmental events. Fine microsurgical manipulations, difficult at best in mouse embryonic development, are readily accomplished in avian embryos and have provided a powerful approach to unraveling the cellular interactions that govern motor axon pathfinding. These approaches, combined in recent years with molecular biology, have begun to produce critical insights into the mechanisms that sculpt cellular architecture during neural development.

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.