GABA-glycine cotransmitting neurons in the ventrolateral medulla: development and functional relevance for breathing

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern

Novel Mutations in the Asparagine Synthetase Gene (ASNS) Associated With Microcephaly

Microcephaly is a devastating condition defined by a small head and small brain compared to the age- and sex-matched population. Mutations in a number of different genes causative for microcephaly have been identified, e.g., MCPH1, WDR62, and ASPM. Recently, mutations in the gene encoding the enzyme asparagine synthetase (ASNS) were associated to microcephaly and so far 24 different mutations in ASNS causing microcephaly have been described. In a family with two affected girls, we identified novel compound heterozygous variants in ASNS (c.1165G > C, p.E389Q and c.601delA, p.M201Wfs∗28). The first mutation (E389Q) is a missense mutation resulting in the replacement of a glutamate residue evolutionary conserved from Escherichia coli to Homo sapiens by glutamine. Protein modeling based on the known crystal structure of ASNS of E. coli predicted a destabilization of the protein by E389Q. The second mutation (p.M201Wfs∗28) results in a premature stop codon after amino acid 227, thereby truncating more than half of the protein. The novel variants expand the growing list of microcephaly causing mutations in ASNS

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting

Genetic Deletion of Laminin Isoforms β2 and γ3 Induces a Reduction in Kir4.1 and Aquaporin-4 Expression and Function in the Retina

Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells.The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice.These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells

Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation.

BACKGROUND:DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2. METHODOLOGY/PRINCIPAL FINDINGS:In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC(50)-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM. CONCLUSIONS/SIGNIFICANCE:These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters

Immunocytochemical analysis of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A, B: CHO cells cultured in 24-well cell culture plates were transfected with NE+EC (150 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and an expression vector coding for nuclear DsRed (100 ng/well; shown in red). Cells were cultured in the absence (A) or presence of 4OHT (1 µM, B). Recombination is detected by EGFP reporter expression (green; visualized by immunostaining using anti-GFP-antibodies). In blue, DAPI-staining of all nuclei is shown. C–F: Immunocytochemical confirmation of expression of NE and EC and of recombination in NE+EC transfected cells after application of 4OHT. CHO cells were transfected with NE+EC (400 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and cultured in the presence of 4OHT (1 µM). NE (C), EC (D) and EGFP (E) were visualized by immunostaining for Flag-tag, Myc-tag or GFP, respectively, and detected by Cy5-, Cy3- or Cy2-conjugated secondary antibodies. F shows the merged images with NE in magenta, EC in red, EGFP in green and DAPI in blue. Note the additional nuclei stained with DAPI but not by anti-Flag- or anti-Myc-antibodies, which are also negative for EGFP. The bar in F corresponds to 20 µm and applies to all panels.</p

Detailed characterization of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A: CHO cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by luciferase reporter expression. The luminescence of reporter-only transfected cells (1.8±0.3%; n = 15) was subtracted from all values. B: CHO cells were transfected with 100 ng each of NE+EC and cultured in the presence of different concentrations of 4OHT. Luminescence in A and B was normalized to the luminescence observed in N+C transfected cells (100 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. C: PC12 20.4 cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by EGFP reporter expression using flow cytometry. The percentage of EGFP⁺-cells observed in the absence of NE+EC (4.4±0.7%; n = 9) was subtracted from all values. D: Cells were transfected with NE+EC plasmids (400 ng each/well) and cultured in the presence of different concentrations of 4OHT. The number of EGFP-positive cells in C and D was normalized to the number of EGFP-positive cells in N+C transfected cultures (400 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. All panels summarize data from a minimum of 3 independent experiments, each of which was performed at least in triplicate.</p