Pioneer neuron pathfinding from normal and ectopic locations in vivo after removal of the basal lamina.

The contribution of the basal lamina to Ti1 pioneer axon guidance in grasshopper limb buds was investigated by allowing growth cones to migrate in 30%-31% stage limbs from which the basal lamina had been removed by enzymatic treatment. When the Ti1 axons extended from their normal location, the pathways established in the absence of basal lamina were normal. This indicates that the basal lamina is not required for initial proximal axon outgrowth, recognition of limb segment boundaries, or selective interaction with neuronal somata. Removal of the basal lamina from slightly older (32% stage) embryos resulted in displacement of the Ti1 somata to ectopic locations in approximately 50% of the limbs. Pathfinding from ectopic locations was aberrant in 45% of the cases observed. This demonstrates that if orienting information is present in the basal lamina-free epithelium at this stage, it is not the predominant factor in determining growth cone orientation from ectopic locations.

Extracellular matrix and neurite outgrowth.

The complex relationship between neuronal cells and the extracellular matrix molecules with which they interact both positively and negatively is currently being investigated on many fronts. Major areas of experimental emphasis include the characterization of an increasing number of extracellular matrix and cell surface associated molecules, the identification of receptors for these molecules, and the analysis of the function of extracellular matrix molecules with respect to neuronal process outgrowth.

Neural development: axon regeneration derailed by dendrites.

Maturing neurons gradually lose the ability to regenerate axons. In the retina, signals from neighboring cells have been found to induce a perinatal switch from extension of axons to extension of dendrites, a change that may contribute to regeneration failure.

Adult neuronal regeneration induced by transgenic integrin expression.

In a variety of adult CNS injury models, embryonic neurons exhibit superior regenerative performance when compared with adult neurons. It is unknown how young neurons extend axons in the injured adult brain, in which adult neurons fail to regenerate. This study shows that cultured adult neurons do not adapt to conditions that are characteristic of the injured adult CNS: low levels of growth-promoting molecules and the presence of inhibitory proteoglycans. In contrast, young neurons readily adapt to these same conditions, and adaptation is accompanied by an increase in the expression of receptors for growth-promoting molecules (receptors of the integrin family). Surprisingly, the regenerative performance of adult neurons can be restored to that of young neurons by gene transfer-mediated expression of a single alpha-integrin.

Embryonic neurons adapt to the inhibitory proteoglycan aggrecan by increasing integrin expression.

The primary mediators of cell migration during development, wound healing and metastasis, are receptors of the integrin family. In the developing and regenerating nervous system, chondroitin sulfate proteoglycans (CSPGs) inhibit the integrin-dependent migration of neuronal growth cones. Here we report that embryonic sensory neurons cultured on the growth-promoting molecule laminin in combination with the inhibitory CSPG aggrecan rapidly adapt to inhibition. Adaptation is associated with a two- to threefold increase in the levels of RNA and surface protein for two laminin receptors, integrin alpha6beta1 and alpha3beta1, indicating that integrin expression is regulated by aggrecan. Increased integrin expression is associated both with increases in neuronal cell adhesion/outgrowth and with decreases in the ability of aggrecan to inhibit cell adhesion. Directly increasing integrin expression by adenoviral infection is sufficient to eliminate the inhibitory effects of aggrecan, indicating that upregulation of integrin receptors may promote neuronal regeneration in the presence of inhibitory matrix components.

Integrins and cAMP mediate netrin-induced growth cone collapse.

Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal that netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance.

Alternative sources of pluripotent stem cells: altered nuclear transfer.

Altered nuclear transfer (ANT) is one of several methods that have been suggested for obtaining pluripotent stem cells without destroying human embryos. ANT proposes to alter the nucleus of a somatic cell and/or the cytoplasm of an enucleated oocyte such that when the two are combined, they do not produce a zygote, but rather they form a cell capable of producing pluripotent stem cells without being an embryo. The ANT proposal raises the serious question of whether it is possible to know with confidence that this procedure generates a non-embryo, rather than merely an embryo with a deficiency. Here I address the question of how embryos can be distinguished from non-embryos using scientific criteria and apply these criteria to the two forms of ANT proposed thus far: ANT combined with oocyte-assisted reprogramming (ANT-OAR) or with gene deletion (ANT-GD). I propose that the first globally coordinated event in human development, the formation of trophoblast and inner cell mass (ICM) lineages via Cdx2-Oct3/4 mutual cross-repression, is the earliest act of the embryo qua embryo; it is an operation intrinsic to an embryo as such, and entities lacking the power (potentia) for such an act cannot be considered embryos. Thus, I will argue that formation of trophoblast-ICM lineages is a both necessary and sufficient criterion for determining whether ANT produces an embryo or a non-embryonic entity.

Neural crest motility on fibronectin is regulated by integrin activation.

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although recent work has shown that receptor recycling plays an important role in NCC motility on laminin, the molecular mechanisms regulating NCC motility on fibronectin remain unclear. One mechanism by which cells regulate motility is by modulating the affinity of integrin receptors. Here, we provide evidence that cranial and trunk NCCs rely on functional regulation of integrins to migrate efficiently on fibronectin (FN) in vitro. For NCCs cultured on fibronectin, velocity decreases after Mn2+ application (a treatment that activates all surface integrins) while velocity on laminin (LM) is not affected. The distribution of activated integrin beta 1 receptors on the surface of NCCs is also substratum-dependent. Integrin activation affects cranial and trunk NCCs differently when cultured on different concentrations of FN substrata; only cranial NCCs slow in a FN concentration-dependent manner. Furthermore, Mn2+ treatment alters the distribution and number of activated integrin beta 1 receptors on the surface of cranial and trunk NCCs in different ways. We provide a hypothesis whereby a combination of activated surface integrin levels and the degree to which those receptors are clustered determines NCC motility on fibronectin.

Combined integrin activation and intracellular cAMP cause Rho GTPase dependent growth cone collapse on laminin-1.

Cyclic nucleotides regulate the response of both developing and regenerating growth cones to a wide range of guidance molecules through poorly understood mechanisms. It is not clear how cAMP levels are regulated or how they translate into altered growth cone behavior. Here, we show that intracellular cAMP levels are influenced by substrata and integrin receptors. We also show that growth cones require a substratum-specific balance between cAMP levels, integrin function and Rho GTPases to maintain motility and prevent collapse. Embryonic chick dorsal root ganglion neurons plated on different concentrations of laminin extend growth cones at similar speeds, yet have distinct levels of integrin expression, integrin activation and intracellular cAMP levels. Either increasing cAMP signaling or activating integrins enhances the rate of growth cone motility, but only on substrata where these two factors are endogenously low (i.e. low concentrations of laminin). Surprisingly, combining these two positive manipulations induces growth cone collapse and retraction on laminin but not on fibronectin. Collapse and retraction on laminin are Rho and Rac1 GTPase dependent and are associated with internalization of integrins, the primary receptors responsible for adhesion. These observations define a novel pathway through which cAMP influences growth cone motility and establish a link between integrins, cAMP and Rho GTPases in growth cones.

Removal of the basal lamina in vivo reveals growth cone-basal lamina adhesive interactions and axonal tension in grasshopper embryos.

The Ti1 afferent neurons are the first cells to undergo axonogenesis in embryonic grasshopper limbs. The Ti1 growth cones migrate between the limb epithelium and its basal lamina. We have investigated the nature of growth conebasal lamina interactions in vivo by removing the basal lamina with mild enzymatic digestion. Treatment with elastase, ficin, or papain removes the basal lamina when viewed in scanning electron microscopy. Trypsin and chymotrypsin leave the basal lamina intact. If the basal lamina is removed after the Ti1 growth cones have extended over intrasegmental epithelium but are not yet in contact with either differentiated segment boundaries or neurons, the growth cones retract to the cell somata. If the basal lamina is removed by elastase, and the Ti1 neurons are allowed to extend axons after treatment, a second elastase digestion does not cause the axons to retract. It is therefore unlikely that axon retraction is due to general proteolysis. These results suggest that if Ti1 growth cones have initially extended in the presence of an intact basal lamina, they are dependent on the lamina to remain extended over this region of the limb. The retraction of the Ti1 axons after removal of the basal lamina is inhibited by cytochalasin D, suggesting that microfilament-based cytoskeletal components underlie this event. This result indicates that the axons are under tension in vivo. The ability of the Ti1 growth cones to resist axonal tension suggests that adhesive interactions between the growth cones and the basal lamina underlie normal axon outgrowth in vivo.

Pioneer growth cone adhesion in vivo to boundary cells and neurons after enzymatic removal of basal lamina in grasshopper embryos.

The Ti1 pioneer neurons of embryonic grasshopper limbs extend axons between the limb epithelium and its basal lamina. Their growth cones exhibit high affinity for both limb segment boundaries and immature neurons. We have investigated the role of the basal lamina in growth cone adhesion to neurons and segment boundaries by removing the basal lamina with mild enzymatic digestion when the Ti1 growth cones are in contact with different cellular substrates. If the basal lamina is removed when the Ti1 growth cones are in contact with other neurons, the growth cones remain in contact with the neuronal somata, and the Ti1 cell bodies in contact with the neuronal somata, and the Ti1 cell bodies reposition proximally. This suggests that the basal lamina is involved in the adhesion of the Ti1 somata to the substrate but not in growth cone-neuronal adhesion. This is the first direct evidence that growth cones establish adhesive cell-cell interactions with other neurons in vivo. Enzymatic treatments that remove the basal lamina also cause embryonic limbs to elongate. If the Ti1 axons are strongly apposed to 2 segment boundaries prior to protease treatment, their somata reposition to the nearest segment boundary, yet their axons do not retract off of the segment boundaries, despite severe stretching by the enzyme-induced limb expansion. These results indicate that the affinity of the Ti1 cells for segment boundaries is due at least in part to adhesive cell-cell interactions that are resistant to proteolytic digestion and independent of the basal lamina.

Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth.

Receptors of the integrin family are expressed by every cell type and are the primary means by which cells interact with the extracellular matrix. The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, cell morphogenesis and cell migration. Here we show that the concentration of substratum-bound ligand (laminin) post-translationally regulates the amount of receptor (alpha6beta1, integrin) expressed on the surface of sensory neurons. When ligand availability is low, surface amounts of receptor increase, whereas integrin RNA and total integrin protein decrease. Ligand concentration determines surface levels of integrin by altering the rate at which receptor is removed from the cell surface. Furthermore, increased expression of integrin at the cell surface is associated with increased neuronal cell adhesion and neurite outgrowth. These results indicate that integrin regulation maintains neuronal growth-cone motility over a broad range of ligand concentrations, allowing axons to invade different tissues during development and regeneration.

Neural crest motility and integrin regulation are distinct in cranial and trunk populations.

The neural crest is a transient cell population that travels long distances through the embryo to form a wide range of derivatives. The extensive migration of the neural crest is highly unusual and incompletely understood. We examined the ability of neural crest cells (NCCs) to migrate under different conditions in vitro. Unlike most motile cell types, avian NCCs migrate efficiently on a wide range of fibronectin concentrations. Strikingly, the migration of NCCs on laminin depends on the axial level from which the crest is derived. On high concentrations of laminin, cranial NCCs migrate at approximately twice the rate of trunk NCCs and show greater persistence, a higher percentage of migratory cells, and a less organized cytoskeleton. The difference in migration between cranial and trunk neural crest is not due to transcriptional differences in integrin mRNA, but rather to differences in posttranslational regulation. Overexpression of a single integrin is sufficient to significantly slow the migration velocity of cranial neural crest cultured on high laminin densities. These results demonstrate that neural crest cells accommodate a wide range of ECM concentrations in vitro and suggest that differences in integrin regulation along the anterior-posterior axis may contribute to differences in neural crest migration and cell fate.

Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism.

Imaginal discs of Drosophila are simple epithelial tissues that undergo dramatic changes in shape during metamorphosis, including elongation to form adult appendages such as legs and wings. We have examined the cellular basis of leg disc morphogenesis by staining filamentous actin to outline cell boundaries in discs and observing cell shapes with scanning confocal laser microscopy (SCLM). Surprisingly, we found that prior to the onset of morphogenesis, cells in the dorsal-lateral regions of leg discs are compressed in the proximal-distal axis and greatly elongated circumferentially. These cells are also asymmetric in the apical-basal axis, being more elongated in the apical-most region of the cell than they are subapically, and frequently contacting different sets of neighbors apically and basally. Elongated cells were first observed in early third instar discs, and persisted through several rounds of cell division as the discs matured. During appendage elongation in vivo and trypsin-accelerated elongation in vitro, these highly asymmetric cells became isometric. As the apical cell profiles changed shape, apical and basal cell contacts came into register. Measurements of apical cell dimensions suggest that changes in cell shape account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6 h after puparium formation (AP). The conversion of a stable population of anisometric cells to isometric dimensions constitutes a novel mechanism for altering the proportions of an epithelial sheet during development.

Selective recognition between embryonic afferent neurons of grasshopper appendages in vitro.

Selective affinity between afferent neurons has been proposed as a major mechanism underlying the assembly of the insect peripheral nervous system during development. Afferent insect neurons establish adhesive interactions in vivo that are resistant to proteolytic degradation by elastase and independent of the basal lamina. We have tested whether afferent neurons express selective affinity for one another under more simplified and controlled conditions in vitro. We report here that (1) afferent neurons from dissociated embryonic tissue selectively aggregate within 80 min when incubated with rotation, (2) afferent axons establish and maintain fasciculation in vitro, and (3) afferent neuronal processes in vitro preferentially contact the somata of other afferent neurons in a mixed field of cells.

Spatially regulated translation in embryos: asymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus.

Transition from symmetry to asymmetry is a central theme in cell and developmental biology. In Xenopus embryos, dorsal-ventral asymmetry is initiated by a microtubule-dependent cytoplasmic rotation during the first cell cycle after fertilization. Here we show that the cytoplasmic rotation initiates differential cytoplasmic polyadenylation of maternal Xwnt-11 RNA, encoding a member of the Wnt family of cell-cell signaling factors. Translational regulation of Xwnt-11 mRNA along the dorsal-ventral axis results in asymmetric accumulation of Xwnt-11 protein. These results demonstrate spatially regulated translation of a maternal cell-signaling factor along the vertebrate dorsal-ventral axis and represent a novel mechanism for Wnt gene regulation. Spatial regulation of maternal RNA translation, which has been established in invertebrates, appears to be an evolutionarily conserved mechanism in the generation of intracellular asymmetry and the consequential formation of the multicellular body pattern.

Site-specific cleavage of basement membrane collagen IV during Drosophila metamorphosis.

Breakdown of basement membranes is an important step in the controlled rearrangement of cells during metamorphosis, cell migration, and metastatic spread of tumor cells. One of our two laboratories found a unique collagenous peptide that only appears during metamorphosis of Drosophila melanogaster. The other laboratory previously reported that during 20-hydroxyecdysone-induced eversion of Drosophila imaginal discs a glycoprotein named gp125 arises (Birr et al., 1990). We show that these two peptides are identical and that they are formed from basement membrane collagen IV. Cleavage occurs at an imperfection of this homotrimeric collagen helix between residues 755/756 in the sequence CALDE/IKMPAK. The peptide is the carboxyl fragment, 100,647 M(r), as derived from the amino acid sequence of the collagen alpha 1(IV) chain. The corresponding amino fragment was also recovered from a disulfide-linked aggregate. This specific cleavage supports the concept of highly targeted, controlled breakdown of basement membranes during metamorphosis. Furthermore, these cuts occur at strategic sites of the predicted supramolecular network of collagen IV molecules of Drosophila basement membranes.