Patrick Charnay

A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories

The induction of long-term potentiation (LTP) in the dentate gyrus of the hippocampus is associated with a rapid and robust transcription of the immediate early gene Zif268. We used a mutant mouse with a targeted disruption of Zif268 to ask whether this gene, which encodes a zinc finger transcription factor, is required for the maintenance of late LTP and for the expression of long-term memory. We show that whereas mutant mice exhibit early LTP in the dentate gyrus, late LTP is absent when measured 24 and 48 hours after tetanus in the freely moving animal. In both spatial and non-spatial learning tasks, short-term memory remained intact, whereas performance was impaired in tests requiring long-term memory. Thus, Zif268 is essential for the transition from short- to long-term synaptic plasticity and for the expression of long-term memories.

Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain

The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.

Biology of hepatitis B virus

Immunochemical investigations of the viral antigens and molecular characterization of the viral DNA have elucidated the nature of the hepatitis B virus infection underlying acute, chronic, and oncogenic disorders of the liver in man. Cloning and sequencing of viral DNA have made possible studies on the structure of the genome and on certain aspects of the biology of the virus, hitherto constrained for a lack of tissue culture systems and laboratory animal models useful in its propagation.

A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells.

Zinc fingers are DNA‐binding domains present in several eukaryotic regulatory proteins. We have identified a mouse gene, Krox‐20, encoding a protein with three zinc fingers and whose expression is activated during G0/G1 transition in cultured cells. Serum stimulation of quiescent cells leads to rapid and transient accumulation of Krox‐20 mRNA, with kinetics similar to those of the c‐fos proto‐oncogene. The induction does not require de‐novo protein synthesis. In the mouse, Krox‐20 is expressed at low levels in tissues which contain rapidly dividing cells. These properties suggest that Krox‐20 encodes a transcription control factor, possibly involved in the modulation of cell proliferation.

Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation.

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.

DETECTION OF HEPATITIS B VIRUS DNA IN LIVER AND SERUM: A DIRECT APPRAISAL OF THE CHRONIC CARRIER STATE

The detection of hepatitis B virus (HBV) DNA in the liver and the serum permits direct study of the interaction between the virus and the liver cell. 40 HBV chronic carriers were studied by the blot technique of Southern to detect HBV DNA, and the results were compared with the serological status and histological status of the patients. It was possible to define two different chronic carrier states. The first is characterised by free viral DNA in the liver, with viral DNA and hepatitis B e antigen (HBeAG) in the serum; integrated HBV DNA is also present, at least in some patients. The second carrier state is characterised by the presence of only integrated HBV DNA sequences in the liver: viral DNA and HBeAg are not present in the serum. In one HBeAg-negative patient, however, free HBV DNA was detected in the liver and HBV DNA was present in the serum. The hybridisation technique appears to be a very sensitive test which could reflect viral multiplication better than HBeAg radioimmunoassay. Since a needle biopsy sample provides sufficient tissue for the Southern blot technique, it should be useful in understanding chronic hepatitis, the selection of patients for antiviral therapy, and the estimation of its efficiency.

The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation

The zinc finger gene Krox20 and many Hox homeobox genes are expressed in segment-restricted domains in the hindbrain. The restricted expression patterns appear before morphological segmentation, suggesting that these transcription factors may play an early role in the establishment and identity of rhombomeric segments. In this paper, we show that the HoxB2 (Hox2.8) gene is normally upregulated in rhombomeres (r) 3, 4, and 5, and we identify an enhancer region upstream of the gene that imposes r3/r5 expression in transgenic mice. This enhancer contains three Krox20-binding sites required in vitro for complex formation with Krox20 protein and in vivo for rhombomere-restricted expression. In transgenic mice, Krox20 expressed in ectopic domains can transactivate a reporter construct containing the HoxB2 r3/r5 enhancer. These data demonstrate that Krox20 is a part of the upstream transcriptional cascade that directly regulates HoxB2 expression during hindbrain segmentation.

Histone H2AX-dependent GABA A receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the ‘DNA damage’ S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine γ-aminobutyric acid (GABA) signalling by means of GABAA receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABAA receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABAA receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline.

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.

Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS

Boundary cap (BC) cells are neural crest derivatives that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various knock-in alleles of the mouse gene Egr2 (also known as Krox20), the expression of which, in trunk regions, is initially restricted to BC cells, we were able to trace BC cell progeny during development and analyze their fate. Trunk BC-derived cells migrated along peripheral axons and colonized spinal nerve roots and dorsal root ganglia (DRG). All Schwann cell precursors occupying the dorsal roots were derived from BC cells. In the DRG, BC-derived cells were the progenitors of both neurons, mainly nociceptive afferents, and satellite cells. These data indicate that BC cells constitute a source of peripheral nervous system (PNS) components that, after the major neural crest ventrolateral migratory stream, feeds a secondary wave of migration to the PNS.