Growth cone dynamics in the zebrafish embryonic forebrain are regulated by Brother of Cdo.

During development of the embryonic zebrafish brain, the differential expression of axon guidance molecules directs the growth of axons along defined neuronal tracts. Neurons within the dorsorostral cluster of the presumptive telencephalon project axons ventrally along the supraoptic tract. Brother of Cdo (Boc) is a known axon guidance molecule that is expressed in a broad band lying ventral to the dorsorostral cluster of neurons. Loss of Boc function has previously been shown to perturb the development of the supraoptic tract. We have used live cell imaging of individual growth cones within the living zebrafish embryo to determine how Boc regulates the growth cone dynamics and axon guidance within the supraoptic tract. A plasmid construct encoding elavl3-eGFP was injected into early embryos to selectively label a small number of neurons while the expression of Boc was knocked down by injection of antisense morpholino oligonucleotides. Time-lapse imaging of growth cones within the living embryos revealed that loss of Boc significantly affected the morphology of growth cones in comparison to axons within control embryos. Growth cones navigating along the supraoptic tract in the absence of Boc extended significantly longer filopodia in the rostrocaudal direction. These results indicate that Boc acts to restrict axons and their filopodia within the narrow pathway of the supraoptic tract. The highly selective nature of these pathfinding defects reveal that Boc is likely to be one of many molecules that coordinate the trajectory of axons within the supraoptic tract.

Yap controls stem/progenitor cell proliferation in the mouse postnatal epidermis.

Tissue renewal is an ongoing process in the epithelium of the skin. We have begun to examine the genetic mechanisms that control stem/progenitor cell activation in the postnatal epidermis. The conserved Hippo pathway regulates stem cell turnover in arthropods through to vertebrates. Here we show that its downstream effector, yes-associated protein (YAP), is active in the stem/progenitor cells of the postnatal epidermis. Overexpression of a C-terminally truncated YAP mutant in the basal epidermis of transgenic mice caused marked expansion of epidermal stem/progenitor cell populations. Our data suggest that the C-terminus of YAP controls the balance between stem/progenitor cell proliferation and differentiation in the postnatal interfollicular epidermis. We conclude that YAP functions as a molecular switch of stem/progenitor cell activation in the epidermis. Moreover, our results highlight YAP as a possible therapeutic target for diseases such as skin cancer, psoriasis, and epidermolysis bullosa.

Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth.

Axons of primary olfactory neurons are intimately associated with olfactory ensheathing cells (OECs) from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, little is understood about the nature and role of interactions between OECs and axons during development of the olfactory nerve pathway. We have used high resolution time-lapse microscopy to examine the growth and interactions of olfactory axons and OECs in vitro. Transgenic mice expressing fluorescent reporters in primary olfactory axons (OMP-ZsGreen) and ensheathing cells (S100ß-DsRed) enabled us to selectively analyse these cell types in explants of olfactory epithelium. We reveal here that rather than providing only a permissive substrate for axon growth, OECs play an active role in modulating the growth of pioneer olfactory axons. We show that the interactions between OECs and axons were dependent on lamellipodial waves on the shaft of OEC processes. The motility of OECs was mediated by GDNF, which stimulated cell migration and increased the apparent motility of the axons, whereas loss of OECs via laser ablation of the cells inhibited olfactory axon outgrowth. These results demonstrate that the migration of OECs strongly regulates the motility of axons and that stimulation of OEC motility enhances axon extension and growth cone activity.

OMP-ZsGreen fluorescent protein transgenic mice for visualisation of olfactory sensory neurons in vivo and in vitro.

Research into the biology of the mammalian olfactory system would be greatly enhanced by transgenic reporter mice with cell-specific fluorescence. To this end we previously generated a mouse whose olfactory ensheathing cells (OECs) express DsRed driven by the S100ß promoter. We present here a transgenic reporter mouse whose olfactory sensory neurons express ZsGreen, driven by the olfactory marker protein (OMP) promoter. ZsGreen was very strongly expressed throughout the cytoplasm of olfactory sensory neurons labelling them in living cells and after fixation. Labelled sensory neurons were seen in all olfactory regions in the nose and fluorescent axons coursed through the lamina propria and into the main and accessory bulbs. We developed methods for culturing embryonic and postnatal olfactory sensory neurons using these mice to visualise living cells in vitro. ZsGreen was expressed along the length of axons providing exceptional detail of the growth cones. The ZsGreen fluorescence was very stable, without fading during frequent imaging. The combination of OMP-ZsGreen and S100ß-DsRed transgenic mice is ideal for developmental studies and neuron-glia assays and they can be bred with mutant mice to dissect the roles of various molecules in neurogenesis, differentiation, axon growth and targeting and other aspects of olfactory sensory neuron and glia biology.

Correction of aberrant axon growth in the developing mouse olfactory bulb.

During development of the primary olfactory system, sensory axons project from the nasal cavity to the glomerular layer of the olfactory bulb. In the process axons can branch inappropriately into several glomeruli and sometimes over-shoot the glomerular layer, entering the deeper external plexiform layer. However in the adult, axons are rarely observed within the external plexiform layer. While chemorepulsive cues are proposed to restrict axons to the glomerular layer in the embryonic animal, these cues are clearly insufficient for all axons in the postnatal animal. We hypothesised that the external plexiform layer is initially an environment in which axons are able to grow but becomes increasingly inhibitory to axon growth in later postnatal development. We have determined that rather than having short localised trajectories as previously assumed, many axons that enter the external plexiform layer had considerable trajectories and projected preferentially along the ventro-dorsal and rostro-caudal axes for up to 950 µm. With increasing age, fewer axons were detected within the external plexiform layer but axons continued to be present until P17. Thus the external plexiform layer is initially an environment in which axons can extensively grow. We next tested whether the external plexiform layer became increasingly inhibitory to axon growth by microdissecting various layers of the olfactory bulb and preparing protein extracts. When assayed using olfactory epithelium explants of the same embryonic age, primary olfactory axons became increasingly inhibited by extract prepared from the external plexiform layer of increasingly older animals. These results demonstrate that primary olfactory axons can initially grow extensively in the external plexiform layer, but that during postnatal development inhibitory cues are upregulated that reduce axon growth within the external plexiform layer.

Lamellipodia mediate the heterogeneity of central olfactory ensheathing cell interactions.

The growth and guidance of primary olfactory axons are partly attributed to the presence of olfactory ensheathing cells (OECs). However, little is understood about the differences between the subpopulations of OECs and what regulates their interactions. We used OEC-axon assays and determined that axons respond differently to peripheral and central OECs. We then further purified OECs from anatomically distinct regions of the olfactory bulb. Cell behaviour assays revealed that OECs from the olfactory bulb were a functionally heterogeneous population with distinct differences which is consistent with their proposed roles in vivo. We found that the heterogeneity was regulated by motile lamellipodial waves along the shaft of the OECs and that inhibition of lamellipodial wave activity via Mek1 abolished the ability of the cells to distinguish between each other. These results demonstrate that OECs from the olfactory bulb are a heterogeneous population that use lamellipodial waves to regulate cell-cell recognition.

Motile membrane protrusions regulate cell-cell adhesion and migration of olfactory ensheathing glia.

Olfactory ensheathing cells (OECs) are candidates for therapeutic approaches for neural regeneration due to their ability to assist axon regrowth in central nervous system lesion models. However, little is understood about the processes and mechanisms underlying migration of these cells. We report here that novel lamellipodial protrusions, termed lamellipodial waves, are integral to OEC migration. Time-lapse imaging of migrating OECs revealed that these highly dynamic waves progress along the shaft of the cells and are crucial for mediating cell-cell adhesion. Without these waves, cell-cell adhesion does not occur and migrational rates decline. The activity of waves is modulated by both glial cell line-derived neurotrophic factor and inhibitors of the JNK and SRC kinases. Furthermore, the activity of lamellipodial waves can be modulated by Mek1, independently of leading edge activity. The ability to selectively regulate cell migration via lamellipodial waves has implications for manipulating the migratory behavior of OECs during neural repair.

Genetic manipulation of blood group carbohydrates alters development and pathfinding of primary sensory axons of the olfactory systems.

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood group H carbohydrate is expressed by primary sensory neurons in both the main and accessory olfactory systems while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the absence of blood group H carbohydrate resulted in the delayed maturation of the glomerular layer of the main olfactory bulb. In addition, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice caused mis-routing of axons in the glomerular layer of the main olfactory bulb and led to exuberant growth of vomeronasal axons in the accessory olfactory bulb. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development of the olfactory nerve pathways.

BOC, brother of CDO, is a dorsoventral axon-guidance molecule in the embryonic vertebrate brain.

The early axon scaffolding in the embryonic vertebrate brain consists of a series of ventrally projecting axon tracts that grow into a single major longitudinal pathway connected across the midline by commissures. We have investigated the role of Brother of CDO (BOC), an immunoglobulin (Ig) superfamily member distantly related to the Roundabout (Robo) family of axon-guidance receptors, in the development of this embryonic template of axon tracts in the zebrafish brain. A zebrafish homologue of BOC was isolated and shown to be expressed predominantly in the developing neural plate and later in the neural tube and developing brain. Zebrafish boc was initially highly localized to discrete bands in the mid- and hindbrain, but, as the major brain subdivisions emerged, it became more evenly expressed along the rostrocaudal axis, particularly in dorsal regions. The function of zebrafish boc was examined by a loss-of-function approach. Analysis of embryos injected with antisense morpholinos designed against boc revealed highly selective defects in the development of dorsoventrally projecting axon tracts. Loss of boc caused ventrally projecting axons, particularly those arising from the presumptive telencephalon, to follow aberrant trajectories. These data indicate that boc is an axon-guidance molecule playing a fundamental role in pathfinding during the early patterning of the axon scaffold in the embryonic vertebrate brain.

Complementary and layered expression of Ephs and ephrins in developing mouse inner ear.

The distributions of the Eph-class receptors EphA4 and EphB1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development.