Sara Wilson

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo

BACKGROUND In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. RESULTS To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensens node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. CONCLUSIONS The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensens node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.

Neural induction : toward a unifying mechanism

Neural induction constitutes the initial step in the generation of the vertebrate nervous system. In attempting to understand the principles that underlie this process, two key issues need to be resolved. When is neural induction initiated, and what is the cellular source and molecular nature of the neural inducing signal(s)? Currently, these aspects of neural induction seem to be very different in amphibian and amniote embryos. Here we highlight the similarities and the differences, and we propose a possible unifying mechanism.

Convergent Inductive Signals Specify Midbrain, Hindbrain, and Spinal Cord Identity in Gastrula Stage Chick Embryos

In the chick embryo, neural cells acquire midbrain, hindbrain, and spinal cord character over a approximately 6 hr period during gastrulation. The convergent actions of four signals appear to specify caudal neural character. Fibroblast growth factors (FGFs) and a paraxial mesoderm-caudalizing (PMC) activity are involved, but neither signal is sufficient to induce any single region. FGFs act indirectly by inducing mesoderm that expresses PMC and retinoid activity and also directly on prospective neural cells, in combination with PMC activity and a rostralizing signal, to induce midbrain character. Hindbrain character emerges from cells that possess the potential to acquire midbrain character upon exposure to higher levels of PMC activity. Induction of spinal cord character appears to involve PMC and retinoid activities.

A Molecular Program for Contralateral Trajectory: Rig-1 Control by LIM Homeodomain Transcription Factors

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Netrin 1 and Dcc signalling are required for confinement of central axons within the central nervous system

The establishment of anatomically stereotyped axonal projections is fundamental to neuronal function. While most neurons project their axons within the central nervous system (CNS), only axons of centrally born motoneurons and peripherally born sensory neurons link the CNS and peripheral nervous system (PNS) together by navigating through specialized CNS/PNS transition zones. Such selective restriction is of importance because inappropriate CNS axonal exit could lead to loss of correct connectivity and also to gain of erroneous functions. However, to date, surprisingly little is known about the molecular-genetic mechanisms that regulate how central axons are confined within the CNS during development. Here, we show that netrin 1/Dcc/Unc5 chemotropism contributes to axonal confinement within the CNS. In both Ntn1 and Dcc mutant mouse embryos, some spinal interneuronal axons exit the CNS by traversing the CNS/PNS transition zones normally reserved for motor and sensory axons. We provide evidence that netrin 1 signalling preserves CNS/PNS axonal integrity in three ways: (1) netrin 1/Dcc ventral attraction diverts axons away from potential exit points; (2) a Dcc/Unc5c-dependent netrin 1 chemoinhibitory barrier in the dorsolateral spinal cord prevents interneurons from being close to the dorsal CNS/PNS transition zone; and (3) a netrin 1/Dcc-dependent, Unc5c-independent mechanism that actively prevents exit from the CNS. Together, these findings provide insights into the molecular mechanisms that maintain CNS/PNS integrity and, to the best of our knowledge, present the first evidence that chemotropic signalling regulates interneuronal CNS axonal confinement in vertebrates.

The expression profile of the tumor suppressor gene Lzts1 suggests a role in neuronal development

Background: Neuronal circuit assembly comprises a number of developmental processes that ultimately underlie function. Identifying the molecular events that dictate these processes can give key insights into how neuronal circuit formation is coordinated. To begin to identify such molecular mechanisms, we have analysed the expression of a candidate gene of entirely unknown function within the nervous system. Here we reveal the spatial and temporal distribution of Lzts1 in mouse and chick embryonic spinal cord and propose potential biological functions. Results: Lzts1 mRNA is transiently expressed at the border of the ventricular and mantle zones in subsets of sensory and motor spinal neurons. The protein is localized to the cell body, axon, and trailing process of motor, commissural, and dorsal root neurons during development. Conclusions: Taken together, the spatial and temporal distribution of Lzts1 is consistent with a potential function(s) in cell cycle regulation, axon growth or guidance, and/or migration of neurons. Developmental Dynamics 241:984–994, 2012.

Commissural axonal corridors instruct neuronal migration in the mouse spinal cord

Unravelling how neurons are guided during vertebrate embryonic development has wide implications for understanding the assembly of the nervous system. During embryogenesis, migration of neuronal cell bodies and axons occurs simultaneously, but to what degree they influence each others development remains obscure. We show here that within the mouse embryonic spinal cord, commissural axons bisect, delimit or preconfigure ventral interneuron cell body position. Furthermore, genetic disruption of commissural axons results in abnormal ventral interneuron cell body positioning. These data suggest that commissural axonal fascicles instruct cell body position by acting either as border landmarks (axon-restricted migration), which to our knowledge has not been previously addressed, or acting as cellular guides. This study in the developing spinal cord highlights an important function for the interaction of cell bodies and axons, and provides a conceptual proof of principle that is likely to have overarching implications for the development of neuronal architecture.

Evolution of the functionally conserved DCC gene in birds

Understanding the loss of conserved genes is critical for determining how phenotypic diversity is generated. Here we focus on the evolution of DCC, a gene that encodes a highly conserved neural guidance receptor. Disruption of DCC in animal models and humans results in major neurodevelopmental defects including commissural axon defects. Here we examine DCC evolution in birds, which is of particular interest as a major model system in neurodevelopmental research. We found the DCC containing locus was disrupted several times during evolution, resulting in both gene losses and faster evolution rate of salvaged genes. These data suggest that DCC had been lost independently twice during bird evolution, including in chicken and zebra finch, whereas it was preserved in many other closely related bird species, including ducks. Strikingly, we observed that commissural axon trajectory appeared similar regardless of whether DCC could be detected or not. We conclude that the DCC locus is susceptible to genomic instability leading to independent disruptions in different branches of birds and a significant influence on evolution rate. Overall, the phenomenon of loss or molecular evolution of a highly conserved gene without apparent phenotype change is of conceptual importance for understanding molecular evolution of key biological processes.