Julio A. Barbas

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk

FGF acts as a positional cue that prevents premature neural crest cell specification and EMT caudally while, at the same time, retinoic acid promotes EMT rostrally.

SOX5 controls cell cycle progression in neural progenitors by interfering with the WNT–β‐catenin pathway

Genes of the SOX family of high‐mobility group transcription factors are essential during nervous system development. In this study, we show that SOX5 is expressed by neural progenitors in the chick spinal cord and is turned off as differentiation proceeds. The overexpression of SOX5 in neural progenitors causes premature cell cycle exit and prevents terminal differentiation. Conversely, knocking down SOX5 protein extends the proliferative period of neural progenitors and causes marked cell death in a dorsal interneuron (dI3) population. Furthermore, SOX5 reduces WNT–β‐catenin signalling, thereby triggering the expression of the negative regulator of the pathway axin2. We propose that SOX5 regulates the timing of cell cycle exit by opposing WNT–β‐catenin activity on cell cycle progression.

Dynamic Sox5 protein expression during cranial ganglia development

Sox5 is a member of the SoxD group of HMG‐box transcription factors that, during the early stages of development, promotes neural crest generation. However, little is known about Sox5 function in neural crest derivatives such as the peripheral sensory nervous system. We have analysed the embryonic expression of Sox5 during chick cranial ganglia development, from the stages of ganglia condensation to those of differentiation. During this period, Sox5 expression is maintained in the crest‐derived satellite glial cells in all the cranial ganglia. In contrast, Sox5 is only transiently expressed in a subpopulation of differentiating neurons of both neural crest and placode origin. This detailed analysis provides a good base to dissect the possible role of Sox5 in neural cell fate determination by future functional approaches. Developmental Dynamics 236:2702–2707, 2007.

Antibodies against Drosophila potassium channels identify membrane proteins across species

Shaker is a complex locus (ShC) in Drosophila that encodes components of the K+ channel responsible for the IA current. We have raised antibodies against synthetic peptides of selected sequences from the Sh products. One of the antisera identifies a 71 kDa protein band in immunoblots from Drosophila neural membrane proteins. We demonstrate that this protein is encoded within the viable (V) region of the ShC since deletions and breakpoints in this part of the complex eliminate this band from the immunoblots. Certain Sh mutations abolish the production of this product while other do not seem to interfere with it. The same antiserum identifies bands of different apparent molecular weight (Mr) in membrane extracts of nervous systems of a variety of organisms including vertebrates.

A new method to study permeation of β-lactam antibiotics into reconstituted vesicles from the outer membrane of Pseudomonas aeruginosa NCTC 10662

An impairment of antibiotic permeation through the outer membrane of Gram-negative bacteria is considered to be one of the main causes of bacterial resistance to fl-lactam antibiotics [1]. It is particularly marked in Pseudomonas aeruginosa. Kinetic data on the permeation of fl-lactams have been mathematically analysed in [2]. These authors took advantage of an inducible fl-lactamase located within the periplasmic space of Escherichia coli and measured the degradation of fl-lactams after permeation of the antibiotic through the outer membrane. Other workers have studied the kinetics of permeation of solutes using model membrane vesicles made either from outer membranes or from some of their constituents [3,4]. Methods for studying permeation have included the use of radiolabelled solutes [3] and light-scattering measurements based on the change in volume of the vesicles upon permeation of the solute [5]. Here, we describe a new, simple spectrophotometric method for studying fl-lactam permeation into vesicles containing an enveloped fl-lactamase. These vesicles were made from the outer membrane fraction of P. aeruginosa and from purified F porin. A new method for isolation of homogeneous F porin is also reported.

Sox5 controls dorsal progenitor and interneuron specification in the spinal cord

The basic organization of somatosensory circuits in the spinal cord is already setup during the initial patterning of the dorsal neural tube. Extrinsic signals, such as Wnt and TGF‐β pathways, activate combinatorial codes of transcription factors that are responsible for generating a pattern of discrete domains of dorsal progenitors (dp). These progenitors will give rise to distinct dorsal interneurons (dI). The Wnt/ βcatenin signaling pathway controls specification of dp/dI1–3 progenitors and interneurons. According to the current model in the field, Wnt/βcatenin activity seems to act in a graded fashion in the spinal cord, as different relative levels determine the identity of adjacent progenitors. However, it is not clear how this activity gradient is controlled and how the identities of dI1–3 are differentially regulated by Wnt signalling. We have determined that two SoxD transcription factors, Sox5 and Sox6, are expressed in restricted domains of dorsal progenitors in the neural tube. Using gain‐ and loss‐of function approaches in chicken embryos, we have established that Sox5 controls cell fate specification of dp2 and dp3 progenitors and, as a result, controls the correct number of the corresponding dorsal interneurons (dI2 and dI3). Furthermore, Sox5 exerts its function by restricting dorsally Wnt signaling activity via direct transcriptional induction of the negative Wnt pathway regulator Axin2. By that way, Sox5 acts as a Wnt pathway modulator that contributes to sharpen the dorsal gradient of Wnt/βcatenin activity to control the distinction of two functionally distinct types of interneurons, dI2 and dI3 involved in the somatosensory relay.

An unusual linkage between polysaccharides and some major proteins from the outer membrane of Escherichia coli.

(E. coli) Outer membrane OmpA Lipopolysaccharide

Final steps of the maturation of Omp F, a major protein from the outer membrane of Escherichia coli

Pulse‐labelling experiments with E. coli cells allowed us to follow the incorporation of de novo proteins into the outer membrane of the cell envelope. Labelled membrane samples containing increasingly different levels of newly synthesized Omp F protein were subjected to chemical cross‐linking with a bifunctional cleavable reagent in order to investigate the process of trimer formation of the protein. From the results obtained, we conclude that the formation of functional Omp F trimers is substantially delayed to, and can be distinguished from, the incorporation of Omp F monomers to the outer membrane.

Sox5 controls cell cycle progression in neural progenitors by interfering with Wnt/β-catenin pathway

Every year, a significant numbers of Canadians are affected by psychiatric and neurological disorders such as depression, autism and schizophrenia. The possibility of promoting neurogenesis following the onset of these diseases is an extremely appealing therapeutic option. The functionally intertwined Retinoblastoma (pRb) and E2F protein families are well known for their essential roles in regulating cell cycle entry; however we have described expanded roles for these proteins in regulating multiple aspects of neural development. Determining the mechanism that regulates these processes is essential for our understanding of how neural development proceeds. We show that the absence of the cell cycle regulatory protein E2F4 leads to a deficit in neural stem cell numbers and a severe impairment of self renewal. Additionally, E2F4 deficiency results in a loss of ventral telencephalic structures, a phenotype with striking similarity to animals lacking the Sonic Hedgehog (Shh) gene. We have previously shown that loss of both Shh expression and ventral telencephalic structures are rescued by interbreeding E2F4 s mutant with mice heterozygous for Gli3, a negative regulator of the Shh pathway. Preliminary data using ChIP-on Chip has identified Gli3 as a regulatory target for the E2F4 transcription factor. These findings suggest that E2F4 negatively regulates Gli3 expression and that in the absence of E2F4, Gli3 aberrantly represses Shh expression, consequently impeding ventral telencephalic patterning. In conclusion, these findings suggest that E2F4 is an essential regulator of neural stem cell renewal and telencephalic patterning by regulating the activity of the Shh pathway through repression of Gli3 expression. This work was funded by CMM travel grants, OGSST, FGPS to DDT, CIHR grant to RSS.