Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo ( Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons ( up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Polymorphic CAG repeat length in the androgen receptor gene and association with neurodegeneration in a heterozygous female carrier of Kennedy's disease

Kennedy's disease (spinobulbar muscular atrophy) is an X-linked form of motor neuron disease affecting adult males carrying a CAG trinucleotide repeat expansion within the androgen receptor gene. While expression of Kennedy's disease is thought to be confined to males carrying the causative mutation, subclinical manifestations have been reported in a few female carriers of the disease. The reasons that females are protected from the disease are not clear, especially given that all other diseases caused by CAG expansions display dominant expression. In the current study, we report the identification of a heterozygote female carrying the Kennedy's disease mutation who was clinically diagnosed with motor neuron disease. We describe analysis of CAG repeat number in this individual as well as 33 relatives within the pedigree, including two male carriers of the Kennedy's mutation. The female heterozygote carried one expanded allele of the androgen receptor gene with CAG repeats numbering in the Kennedy's disease range (44 CAGs), with the normal allele numbering in the upper-normal range (28 CAGs). The subject has two sons, one of whom carries the mutant allele of the gene and has been clinically diagnosed with Kennedy's disease, whilst the other son carries the second allele of the gene with CAGs numbering in the upper normal range and displays a normal phenotype. This coexistence of motor neuron disease and the presence of one expanded allele and one allele at the upper limit of the normal range may be a coincidence. However, we hypothesize that the expression of the Kennedy's disease mutation combined with a second allele with a large but normal CAG repeat sequence may have contributed to the motor neuron degeneration displayed in the heterozygote female and discuss the possible reasons for phenotypic expression in particular individuals.

Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice

Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (-/-), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4 -/- mice recovered stride length, the ability to walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1-3 months of injury. EphA4 expression was upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned EphA4-/- spinal cords. EphA4 -/- astrocytes failed to respond to the inflammatory cytokines, interferon-gamma or leukemia inhibitory factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4 -/- astrocytes, but longer neurites when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury, axonal inhibition, and astrocytic gliosis.

X-ray microtomography of 410 million-year-old optic capsules from placoderm fishes

The structure of two small ossified optic capsules from mid-Palaeozoic placoderm fishes has been revealed in fine detail, by the use of Xray microtomography analysis and 3D visualisation software. These two specimens are 410 million-year-old; they were collected from an Early Devonian (Lochkovian) limestone in central New South Wales, and are the oldest known optic capsules from jawed fishes. The capsules show attachment areas for seven extrinsic eye muscles, rather than the six until recently deemed universal for gnathostomes. The analysis also revealed structures within the ossified cartilage which covered the medial surface of the eyeball, including nerve tracts, vascular canals, and possibly a choroid rete mirabile. (c) 2005 Elsevier Ltd. All rights reserved.

Gastrointestinal transit in the common brushtail possum measured by gamma scintigraphy

This paper reports an example of the application of pharmaceutical technology to wildlife management, specifically the design of an oral delivery system for the common brushtail possum in New Zealand. Designing an oral delivery system requires a knowledge of the time taken for particulates to reach target sites within the gastrointestinal tract (GIT). The transit time for fluid and indigestible particles of two different size ranges was determined in the common brushtail possum (Trichosurus vulpecula). Technetium-labelled (Tc-99m) anion exchange resin particles (75-125 or 500-700 mu m diameter) or solution (Tc-99m-labelled diethylenetriamine pentaacetic acid, Tc-99m-DTPA) was administered orally. At predetermined times after dosing (3, 6, 12, 24 or 32 h), the distribution of radioactivity throughout excised gastrointestinal tracts was determined by gamma scintigraphy. The transit profile was similar for the three formulations investigated. Unlike other closely related hindgut fermenting marsupials, there was no evidence to support the presence of a colonic separating mechanism in the common brushtail possum. Gastrointestinal transit was independent of body mass, gender and time of day that the dose is given. To target the hindgut for oral delivery of protein and peptide biocontrol agents, the formulation Would need to protect the bioactive for approximately 12 h prior to release. (c) 2005 Elsevier B.V. All rights reserved.

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. (c) 2005 Wiley-Liss, Inc.

Neogenin interacts with RGMa and Netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory. (c) 2006 Elsevier Inc. All rights reserved.

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion. (c) 2006 Australian Society for Parasitology Inc Published by Elsevier Ltd. All fights reserved.

Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome

Recent large-scale analyses of mainly full-length cDNA libraries generated from a variety of mouse tissues indicated that almost half of all representative cloned sequences did flat contain ail apparent protein-coding sequence, and were putatively derived from non-protein-coding RNA (ncRNA) genes. However, many of these clones were singletons and the majority were unspliced, raising the possibility that they may be derived from genomic DNA or unprocessed pre-rnRNA contamination during library construction, or alternatively represent nonspecific transcriptional noise. Here we Show, using reverse transcriptase-dependent PCR, microarray, and Northern blot analyses, that many of these clones were derived from genuine transcripts Of unknown function whose expression appears to be regulated. The ncRNA transcripts have larger exons and fewer introns than protein-coding transcripts. Analysis of the genomic landscape around these sequences indicates that some cDNA clones were produced not from terminal poly(A) tracts but internal priming sites within longer transcripts, only a minority of which is encompassed by known genes. A significant proportion of these transcripts exhibit tissue-specific expression patterns, as well as dynamic changes in their expression in macrophages following lipopolysaccharide Stimulation. Taken together, the data provide strong support for the conclusion that ncRNAs are an important, regulated component of the mammalian transcriptome.

Ryk: A novel Wnt receptor regulating axon pathfinding

Members of the Wnt family and their receptors, the Frizzleds, are key regulators of pivotal developmental processes including embryonic patterning, specification of cell fate, and determination of cell polarity. The versatility and complexity of Wnt signaling has been further highlighted by the emergence of a novel family of Wnt receptors, the Ryk family. In mammals and flies, Ryk is a key chemorepulsive axon guidance receptor responsible for the establishment of important axon tracts during nervous system development. Although the function of Ryk is currently best understood with respect to this role, its widespread expression, both in developing tissues and in the adult, suggests that Ryk may regulate many essential biological processes. This hypothesis is supported by the multiple developmental phenotypes apparent in Ryk loss-of-function mice. These mice display a variety of embryonic abnormalities, including disruption of skeletal, craniofacial and cardiac development. Here we review Ryk structure and function focusing on its activity as an axon guidance receptor. (c) 2006 Elsevier Ltd. All rights reserved.

White and gray matter development in human fetal, newborn and pediatric brains

Brain anatomy is characterized by dramatic growth from the end of the second trimester through the neonatal stage. The characterization of normal axonal growth of the white matter tracts has not been well-documented to date and could provide important clues to understanding the extensive inhomogeneity of white matter injuries in cerebral palsy (CP) patients. However, anatomical studies of human brain development during this period are surprisingly scarce and histology-based atlases have become available only recently. Diffusion tensor magnetic resonance imaging (DTMRI) can reveal detailed anatomy of white matter. We acquired diffusion tensor images (DTI) of postmortem fetal brain samples and in vivo neonates and children. Neural structures were annotated in two-dimensional (2D) slices, segmented, measured, and reconstructed three-dimensionally (3D). The growth status of various white matter tracts was evaluated on cross-sections at 19-20 gestational weeks, and compared with 0-month-old neonates and 5- to 6-year-old children. Limbic, commissural, association, and projection white matter tracts and gray matter structures were illustrated in 3D and quantitatively characterized to assess their dynamic changes. The overall pattern of the time courses for the development of different white matter is that limbic fibers develop first and association fibers last and commissural and projection fibers are forming from anterior to posterior part of the brain. The resultant DTNIRI-based 3D human brain data will be a valuable resource for human brain developmental study and will provide reference standards for diagnostic radiology of premature newborns. (c) 2006 Elsevier Inc. All rights reserved.