Astrocytes are now distinguished as major regulators of neuronal growth and synaptic development. Recently, they have been identified as key players in the progression of a number of developmental disorders; however, in fragile X syndrome (FXS), the role of astrocytes is not known. Using a coculture design, we found that hippocampal neurons exhibited abnormal dendritic morphology and a decreased number of presynaptic and postsynaptic protein aggregates when they were grown on astrocytes from a fragile X mouse. Moreover, we found that normal astrocytes could prevent the development of abnormal dendrite morphology and preclude the reduction of presynaptic and postsynaptic protein clusters in neurons from a fragile X mouse. These experiments are the first to establish a role for astrocytes in the altered neurobiology of FXS. Our results support the notion that astrocytes contribute to abnormal dendrite morphology and the dysregulated synapse development in FXS.

Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development.Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis.These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.

BackgroundFragile X syndrome is the most common inherited form of mental impairment characterized by cognitive impairment, attention deficit and autistic behaviours. The mouse model of Fragile X is used to study the underlying neurobiology associated with behavioral deficiencies. The effect of Fragile X glial cells on the development of neurons has not been studied. We used a co-culture technique in combination with morphometrics on immunostained neurons to investigate the role of astrocytes in the development delays associated with hippocampal neuron development.ResultsWe found that hippocampal neurons grown on Fragile X astrocytes exhibited a significant difference from the neurons grown with normal astrocytes after 7 days in vitro for many parameters including increases in dendritic branching and in area of the cell body. However, after 21 days in culture, the neurons grown on Fragile X astrocytes exhibited morphological characteristics that did not differ significantly from the neurons grown on normal astrocytes. With antibodies to the pre-synaptic protein, synapsin, and to the excitatory post-synaptic protein, PSD-95, we quantified the number of developing excitatory synapses on the dendrites. In addition to the delays in dendritic patterning, the development of excitatory synapses was also delayed in the hippocampal neurons.ConclusionsThese experiments are the first to establish a role for astrocytes in the delayed growth characteristics and abnormal morphological features in dendrites and synapses that characterize the Fragile X syndrome.

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.

Post-translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase-mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild-type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre-mediated replacement of the wild-type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild-type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post-translational modifications in development and disease.