Direct Lineage Conversion of Adult Mouse Liver Cells and B Lymphocytes to Neural Stem Cells

SummaryOverexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors

Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet

Extensions of MADM (Mosaic Analysis with Double Markers) in Mice

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM

Transsynaptic Teneurin Signaling in Neuromuscular Synapse Organization and Target Choice. Nature. 2012; (this issue

Synapse assembly requires trans-synaptic signals between the preand postsynapse 1 , but our understanding of the essential organizational molecules involved in this process remains incomplete 2 . Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains Vertebrate teneurins are enriched in the developing brain Both Ten-m and Ten-a were enriched at the larval NMJ ( The localization of Ten-a and Ten-m suggested their trans-synaptic interaction. To examine this, we co-expressed Myc-tagged Ten-a in nerves using the Q system 14 and haemagglutinin (HA)-tagged Ten-m in muscles using GAL4. Muscle Ten-m was able to coimmunoprecipitate nerve Ten-a from larval synaptosomes To determine Teneurin function at the NMJ, we examined the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55

Structural consequences of replacement of an α- helical pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein α -helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein α -helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 Å . In wild-type thioredoxin, P40 is located in the interior of helix two, a long a-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

Structural consequences of replacement of an \alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein $\alpha$-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein $\alpha$-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1–1.5 kcal/mol in the temperature range 280–330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 \AA. In wild-type thioredoxin, P40 is located in the interior of helix two, a long $\alpha$-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

Targeted knock-in approach to create new <i>Rosa26</i> MADM with <i>GT</i> and <i>TG</i> cassettes.

<p><b>A</b>) Schematic representation of new alleles: <i>R26^GT</i> and <i>R26^TG</i>. <b>B), C) and D)</b> Representative confocal images from tissues indicated on the bottom and genotypes indicated on top. Expected labeling was observed only when Cre was present (compare <b>B</b> with <b>C</b> and <b>D</b>). Bright cellular labeling observed in <b>C</b> and <b>D</b> originates from native tdT and GFP fluorescence (no additional immunostaining was performed). Some sections were stained with DAPI to label nuclei (blue). Scale bars, 50 µm.</p