Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system.

Wiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching.

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75(NTR) antagonists.

LINGO-1 is a potent negative regulator of oligodendrocyte differentiation and hence may play a pivotal restrictive role during remyelination in demyelinating diseases such as multiple sclerosis. However, little is known as to which stages of oligodendrocyte differentiation are inhibited by LINGO-1, which domains of the protein are involved and whether accessory proteins are required. Here, we show that LINGO-1 expression in the human oligodendroglial cell line MO3.13 inhibited process extension and this was reversed by an anti-LINGO-1 antibody or the antagonist LINGO-1-Fc. LINGO-1 expression was also found to inhibit myelin basic protein transcription in the rat oligodendroglial cell line CG4. Both of these inhibitory actions of LINGO-1 were abrogated by deletion of the entire ectodomain or cytoplasmic domains but, surprisingly, were unaffected by deletion of the leucine-rich repeats (LRRs). As in neurons, LINGO-1 physically associated with endogenous p75(NTR) in MO3.13 cells and, correspondingly, its inhibition of process extension was reversed by antagonists of p75(NTR). Thus, LINGO-1 inhibits multiple aspects of oligodendrocyte differentiation independently of the LRRs via a process that requires p75(NTR) signalling.

An extremely rare case of indirect hernia type co-existing with testicular ectopia.

We present an extremely rare case of inguinal hernia coexisting with testicular ectopia in a child. Male infant 9.5 month old presented with an empty scrotum and the ipsilateral intravaginal testis lying in a high iliac crest position. When crying a moving right inguinal bulge appeared on clinical examination. This grew bigger in moments of increased abdominal pressure and seemed to move upwards towards the right ileac crest. No abdominal wall defect could be palpated. At operation a large hernia sac fixed in the area of the right iliac crest was identified. Adjacent was the fixation point of the gubernaculum and the testis was found in an ectopic location. We removed the large sac after separating the vas and vessels and the testis and we strengthened the dorsal inguinal wall and fixed the testis in a subdartos scrotal pouch. No postoperative complications happened. An undescended testis may present as an iliac crest ectopy, coexisting with moving inguinal hernia. In our case we propose that the higher position of the aponeurosis of the external oblique in combination with ectopia of gubernacular fixation in the ipsilateral scrotum may have caused the ectopic fixation of the sac in the ipsilateral inguinal crest.

Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord.

Dorsal commissural axons in the developing spinal cord cross the floor plate, then turn rostrally and grow along the longitudinal axis, close to the floor plate. We used a subtractive hybridization approach to identify guidance cues responsible for the rostral turn in chicken embryos. One of the candidates was the morphogen Sonic hedgehog (Shh). Silencing of the gene SHH (which encodes Shh) by in ovo RNAi during commissural axon navigation demonstrated a repulsive role in post-commissural axon guidance. This effect of Shh was not mediated by Patched (Ptc) and Smoothened (Smo), the receptors that mediate effects of Shh in morphogenesis and commissural axon growth toward the floor plate. Rather, functional in vivo studies showed that the repulsive effect of Shh on postcommissural axons was mediated by Hedgehog interacting protein (Hip).

Screening for gene function in chicken embryo using RNAi and electroporation.

In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Gene functions cannot be determined by high-throughput methods but require analysis in the context of the entire organism. This is particularly true in the developing vertebrate nervous system. Because of its easy accessibility in the egg, the chicken embryo has been the model of choice for developmental in vivo studies. However, its usefulness has been hampered by a lack of methods for genetic manipulation. Here we describe an approach that could compensate for this disadvantage. By combining gene silencing by dsRNA (through RNA interference, RNAi) with in ovo electroporation, we developed an efficient method to induce loss of gene function in vivo during the development of the chicken CNS. This method opens new possibilities for studying gene function not only by gain-of-function but also by loss-of-function approaches and therefore represents a new tool for functional genomics.

New tools for gene manipulation in chicken embryos.

Genomics has changed the pace by which genes are analyzed. Rather than looking at genes one by one, gene expression today is studied at the genome level. Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes. Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates. With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo has become an efficient in vivo system to study gene function during development. A major advantage of in ovo RNAi is the fact that the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes. Thus, this new approach is a valuable tool for functional genomics.

Modulation of the Na(+)-H(+) antiport activity by adrenaline on erythrocytes from normal and obese individuals.

The effect of adrenaline on normal and obese human Na(+)-H(+) antiport (NHE 1) erythrocyte activity has been studied. Adrenaline increased both intracellular pH (pHi) and Na(+) influx in erythrocyte suspensions. This effect of adrenaline was inhibited by amiloride or EIPA, indicating that adrenaline stimulated NHE 1. Phorbol myristicate ester (PMA), a protein kinase C (PKC) stimulator, increased the activity of NHE 1 whereas calphostin C, a PKC inhibitor, partially inhibited NHE 1 activation induced by adrenaline. The effect of adrenaline to NHE 1 was counteracted by prazocin and by propranolol as well indicating the involvement of both alpha and beta 2 adrenergic receptors. The effect of adrenaline on erythrocyte NHE 1 activity was significantly more profound in obese compared to normal subjects. These data indicate that adrenaline induces an increase of pHi and Na(+) uptake of human erythrocytes through stimulation of NHE 1 activity. The significantly more profound stimulation of NHE 1 activity by adrenaline in obese as compared to normal subjects is discussed.

The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis elegans.

BACKGROUND: The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however. RESULTS: We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects. CONCLUSION: We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly.