The mean of YFP/CFP ratios for VA8 and VB9 during these periods was considered the averaged activity for VA8 and VB9 for each animal during the indicated mode of locomotion. The difference between the VB9 and VA8 activity level in each animal was normalized by [VB9 − VA8]/[VB9 + VA8]. For correlation analysis, VA8 and VB9 transients of each sample were corrected for photobleaching by dividing fitted linear regression line, normalized by

mean and SD. For correlation analyses of VA8 and VB9 activity change during the transition of directions, eight VA8/VB9 imaging traces were used for correlation analyses. Pearson’s correlation coefficient was calculated by R. MEK activation Detailed procedures for curvature analysis, automated movement analysis, electrophysiology, molecular biology, neuron silencing, ablation and chemical synapse inactivation, immunofluorescent staining, and statistical analysis are provided in Supplemental Experimental Procedures. We thank H. Li and Y. Wang for technical support; A.V. Maricq for akIs11, Z.W. Wang for UNC-9 antisera, C. Bargmann for tetanus toxin cDNA, S. Lockery for exchanging unpublished results, and M. Zhang and H. Suzuki for advice on calcium imaging. We are in debt to L. Avery, C. Bargmann,

J.-L. Bessereau, J. Culotti, C.-Y. Ho, A. Kania, J. Richmond, Q. Wen, J. Woodgett, and anonymous reviewers for critical reading and comments on this manuscript. M. Po was a recipient of a Natural Sciences and selleck kinase inhibitor Engineering Research Council of Canada scholarship. We thank the EJLB foundation, the Canadian Institute of Health Research and the Samuel Lunenfeld Research Institute for supporting this project. “
“Synaptic transmission relies on the fusion of synaptic vesicles (SVs) with the presynaptic plasma membrane (exocytosis) to release neurotransmitters. Digestive enzyme After exocytosis, excess plasma membrane resulting from the addition of SV membrane is rapidly internalized by compensatory endocytosis and used to generate new SVs. Proper nervous system function relies critically on

the efficiency of this membrane-recycling traffic. Clathrin-mediated endocytosis is a major pathway for SV recycling (Dittman and Ryan, 2009 and Heuser and Reese, 1973). In this process, nucleation and growth of the clathrin coat helps gather proteins to be internalized and generates and stabilizes the bilayer curvature required for the formation of the endocytic bud. PI(4,5)P2, a phosphoinositide selectively enriched in the plasma membrane, plays a key role in the recruitment and assembly of the endocytic clathrin adaptors, which, in turn, recruit and promote the assembly of clathrin (Di Paolo and De Camilli, 2006). After a deeply invaginated clathrin-coated pit (CCP) is generated, it undergoes fission with the help of the GTPase dynamin (Ferguson et al., 2007 and Raimondi et al., 2011) and then rapidly loses its coat.

Each goal, of the Strategic Plan addresses a different challenge related to halting biodiversity loss. Strategic Goal A addresses required socio-economic and institutional changes. Strategic Goal B focuses on reducing the direct pressures on biodiversity and ecosystems while Strategic Goal C covers active efforts to improve biodiversity status. Strategic Goal D aims to ensure the

flow of benefits from biodiversity and ecosystems to people, especially to the communities whose subsistence is strongly tied to local ecosystem services. Finally, Strategic Goal E aims at developing the conditions required for implementation of the Strategic Plan as well as developing the knowledge base. Actions to achieve one target may influence other targets; in turn a target may be influenced by actions taken towards http://www.selleckchem.com/products/VX-770.html the attainment of other targets. The

first type of interactions are downstream interactions, while the latter are upstream interactions. Taking actions to achieve targets with a high number of downstream interactions will help achieving progress towards other targets. These can be seen as enabling actions as they can facilitate the achievement of the whole Strategic Plan. A target with a high level of upstream interactions is a target that will benefit from actions taken to achieve several other targets. To determine the potential interactions

among the selleckchem twenty Aichi Targets, a group of 18 experts (composed of GBO-4 Technical Report authors and reviewers) qualitatively assessed how the achievement of any given Aichi Target could influence the achievement of the other targets. The following ordinal scores were used by each expert to qualify all the target interactions, either negative STK38 or positive, in a matrix: (1) low influence, (2) intermediate influence, and (3) high influence. For each entry of the matrix the mode of all the scores was used as the final level of influence (Fig. 1). The relative agreement between all experts was determined by computing, for each entry, the percentage of experts that attributed the mode value to that specific entry. Finally, for each target we calculated the sum of downstream interactions (sum of scores 1, 2 and 3 row-wise), the sum of upstream interactions (sum of scores 1, 2 and 3 column-wise), and the difference between these values (Fig. 2). The analysis was done using R and the packages abind and igraph (Csardi and Nepusz, 2006, Plate and Heiberger, 2011 and R Core Team, 2014). We identified targets under Strategic Goals A and E as having the highest level of net downstream interactions (Fig. 2). Generally, their influence spans all targets (Fig. 1).

In this case also, the result is substantial reduction in regeneration. We find that enforced c-Jun expression in injured Wlds nerves is sufficient to restore axonal regeneration rates to WT values, lending significant support to our model. It is important to note that although the Bungner cells generated in the mutants are dysfunctional, other Schwann cell functions are normal. Thus, mutant cells remyelinated those axons that regenerated, Schwann cell development appeared normal, and Schwann cells and nerve function in uninjured adults were normal. Although 172 genes were disregulated in the distal stump of the c-Jun mutants, the large majority

of the ∼4,000 genes regulated in injured WT nerves remained normally regulated. Therefore, the absence of c-Jun does not have a general impact on the Schwann cell phenotype. Instead, c-Jun appears selleck chemicals to have a specific function in adult cells, where it is required for activation of the repair program and timely suppression of the myelin program. The Schwann cell response to injury is commonly referred to as dedifferentiation, implying that adult denervated cells revert

to an earlier stage resembling the immature Schwann cells of perinatal nerves (Harrisingh et al., 2004; Jessen and Mirsky, 2005; Chen et al., 2007; Woodhoo et al., 2009). It is becoming clear, however, that this view is incomplete. These cells have a different structure, molecular profile, and function. Therefore, the immature cells, generated BMS-354825 molecular weight from Schwann cell precursors

during development, and Bungner cells generated in response to adult nerve injury, represent two distinct differentiation states. In injured nerves, myelinating Schwann cells, that are specialized to support fast conduction of action potentials, transform to Bungner cells that are specialized for the unrelated task of organizing nerve repair. This represents an unambiguous change of function, brought about by the combination of dedifferentiation and activation of an alternative differentiation program, the c-Jun Metalloexopeptidase dependent Schwann cell repair program. Transitions that share this set of features have been described in other systems, where they are generally referred to as transdifferentiation (Jopling et al., 2011). The regeneration defects in the c-Jun mutant are substantially more severe than those reported for other mouse mutants, in spite of the fact that the genetic defect is restricted to Schwann cells. The likely reason is the number and diversity of the molecules controlled by this single transcription factor. Among the 172 molecules that are abnormally expressed in the mutant are growth factors, adhesion molecules, growth-associated proteins, and transcription factors. This allows c-Jun to integrate a broad collection of functions that support nerve regeneration, and therefore to act as a global regulator of the Schwann cell repair program.

In contrast, positive peaks in scalp EEG tightly corresponded to negative peaks of depth EEG and to ON periods with rigorous spiking, in accordance with a depolarized up state. We set out to examine

quantitatively the relationship between sleep slow waves and the underlying spiking activity across all brain regions where units were detected (Figure 3). Individual slow waves were detected automatically in the depth EEG of each brain region separately (e.g., cyan dots in Figure 2), and unit spiking activity surrounding slow waves was averaged. When focusing on the highest amplitude waves in each channel (top 20%), positive and negative peaks in depth EEG were associated with marked decreases and increases in unit discharges, respectively (Figures 3A and 3B; n = 600). This result should be viewed as a lower limit on the modulation strength, since timing variability across individual check details neurons introduced a temporal jitter, thereby smearing the average result. Therefore, the wave-triggered average of spiking activity was computed in each unit separately, searching for the minimal (maximal) rate while allowing for different time offsets around EEG peaks (n = 600, average of 10,595 waves per neuron). The minimal firing rate around EEG positivity was 39% ± 1% compared with the mean firing rate in NREM (N2+N3) sleep, and the mean latency of such OFF periods was 72 ± 9 ms before the positive

EEG peak. Around EEG negativity, a maximal firing rate of 198% ± 11% was found across individual units, at 46 ± 10 ms before the negative EEG peak. In each subject and in each brain region, individual neurons whose activity find more was highly modulated by slow waves were identified (Figure 3C). Such neurons were found not only in neocortex, but also in limbic structures such as hippocampus and amygdala. Given the variability across individual neurons, we examined the percentage of neurons showing significant phase locking to sleep slow waves separately in each brain structure (Figure S3;

see Experimental Procedures). The results revealed considerable variability (Figure 3D): the lowest percentages Florfenicol of phase locked neurons were found in anterior cingulate (12% ± 11%, n = 84 units in 11 regions, mean and SEM across electrodes). Neocortical regions (41% ± 11%, n = 109 units in 16 regions), hippocampus (49% ± 7%, n = 100 units in 17 regions), and parahippocampal gyrus (55% ± 10%, n = 97 units in 13 regions) showed intermediate effects, while the highest percentages of phase locked neurons were found in the amygdala (87% ± 11%, n = 61 units in 9 amygdala regions), entorhinal cortex (84% ± 13%, n = 67 units in 10 regions), and posterior cingulate cortex (100% ± 0%, n = 30 units in three regions). Since slow waves were detected in the depth EEG recorded ∼4 mm away from unit activity, the percentages of modulated neurons should be regarded as a lower bound.

Furthermore, transmembrane proteins known to cycle through endosomes, including synaptotagmin ( Takei et al., 1996) and APP ( Haass et al., 1992), also accumulate at these TBs and partially colocalize with anti-HRP ( Figure 3C). Together, these data

show that overexpression of p150G38S causes a marked accumulation of endosomal membranes and proteins at NMJ TBs. To determine whether the accumulation of endosomes at Glued mutant TBs is due to disruption of dynein/dynactin function, we asked whether similar phenotypes are present in mutant alleles of genes encoding components of the dynein/dynactin complex. Because most available alleles BMN 673 supplier are early larval or embryonic lethal, we knocked down dynein/dynactin subunits in motor neurons by using RNAi ( Figure S4A). As expected, knockdown of three dynactin subunits (Gl, cpa, and p62) and three dynein subunits (dhc, dic, and dlic) phenocopies the

TB accumulation of anti-HRP immunoreactivity and Syt:GFP that we observed in D42 > p150G38S animals ( Figures 3C, 3E, and S4B). These data demonstrate that disruption of the dynein/dynactin complex causes an accumulation of endosomes within TBs of the NMJ. In filamentus fungi, the dynactin complex is required for MT plus-end localization of dynein GS 1101 and for the interaction between dynein and endosomes (Xiang et al., second 2000 and Zhang et al., 2010). To determine whether dynein is mislocalized in Glued animals, we analyzed the expression of the cytoplasmic dynein heavy chain (cDhc64C, referred to here as Dhc). Surprisingly, GlG38S larvae reveal a striking accumulation of Dhc at NMJ TBs in all segments in 100% of GlG38S and GlG38S/GlΔ22 animals; this phenotype is never observed in wild-type animals ( Figures 4A–4C and Figures S5A and S5B). At wild-type synapses, Dhc is localized

to small puncta at the periphery of all boutons ( Figure 4A), and occasionally small Dhc(+) puncta are observed near the center of the TB ( Figure 4E, arrow). In GlG38S animals, however, the mean Dhc signal intensity is increased ∼10-fold within TBs, with no significant differences between proximal and distal segments ( Figure 4B). Interestingly, in GlG38S larvae, Dhc predominantly accumulates at TBs of the longest branch in synapses with multiple branches ( Figures S5A and S5B). These accumulations are not seen in axons or motor neuron cell bodies ( Figure S5B and data not shown). Microtubules do not appear to be altered at GlG38S NMJs; however, we did note that mutant TBs with observable microtubule bundles did not accumulate dynein ( Figure S5C, arrow), in contrast to those TBs with no significant tubulin staining. These data suggest that dynein accumulates in GlG38S TBs lacking stable microtubules.

, 2011); and mouse anti-V5 (Invitrogen, 1:500). For immunofluorescence analyses, the following secondary antibodies were used: goat anti-mouse, rabbit, and rat F(ab′)2 fragments coupled to FITC (1:200), Cy3/DyLight549 (1:400) or Cy5/DyLight649 (1:200) (Jackson ImmunoResearch Bleomycin research buy Laboratories), as well as goat anti-mouse Alexa Fluor 568 (1:400; Invitrogen). As the V5 epitope was detectable using

anti-V5 antibody in western blots but not in cells or tissues, NetB was visualized using anti-NetB antibody. Images were collected using Zeiss/Bio-Rad Radiance2100, Leica TCS SP5II, and Zeiss LSM710 laser-scanning confocal microscopes. Immunofluorescence levels were determined using ImageJ; neurons were traced using Fiji Simple Neurite Tracer. For stainings shown in supplemental figures, see Supplemental Experimental Procedures. Detailed staining protocols are available upon request. We thank B. Altenhein, M. Brankatschk, B.J. Dickson, T. Hummel, C.H. Lee, R. Ueda, J.P. Vincent, the Bloomington Drosophila Stock Center, the Drosophila Genomics Resource Center, the Vienna Drosophila RNAi Center, the Kyoto Drosophila Genetic Resource Center, the National Institute of Genetics Fly Stock Center, and the Developmental Studies Hybridoma Bank for fly strains, antibodies, and plasmids. We thank C. Desplan for sharing lGMR-Gal80 transgenic flies and H. Apitz, C. Chotard, L. Ferreira, and Z. Ludlow for contributions

to the MH-Gal4 screen. We are grateful to F. Guillemot, E. Ober, J.P. Vincent, as well as H. Apitz, D. Brierley, E. Richardson, B. Richier, and N. Shimosako for critical reading of the manuscript.

This work is supported by a Marie Everolimus ic50 Curie Intra-European Fellowship (to W.J.) and the Medical Research Council (U117581332). “
“Brain functions are made possible by synapses, contacts formed between neurons or between a neuron and a target cell. The neuromuscular junction (NMJ) is a cholinergic synapse between motoneurons and skeletal muscle fibers that has most, if not all, features characteristic of a chemical synapse in the brain. Because of its simplicity, high spatial resolution, and accessibility, the NMJ has served as an informative model of synaptogenesis (Sanes and Lichtman, 1999, Sanes and Lichtman, 2001 and Wu et al., 2010). Its development requires the precise coordination between presynaptic Resminostat motoneurons and postsynaptic muscle fibers. Mechanisms by which motoneurons instruct postsynaptic differentiation are better characterized, whereas relatively little is known about retrograde signals from the muscle fibers. Agrin is a nerve-derived organizer of postsynaptic differentiation during NMJ formation (McMahan, 1990). It stimulates AChR cluster formation in myotubes in culture (Ferns et al., 1993 and Nitkin et al., 1987) and mice lacking agrin do not form the NMJ (Gautam et al., 1996). MuSK is a receptor tyrosine kinase that is essential for agrin-induced clustering and for NMJ formation in vivo (DeChiara et al., 1996, Glass et al.

Death of astrocytes shortly after their generation and the elevated expression of hbegf mRNA in endothelial cells compared to astrocytes ( Cahoy et al., 2008 and Daneman et al., 2010) support the hypothesis that astrocytes may require vascular cell-derived trophic support. MD-astrocytes show remarkable proliferative ability and can be passaged repeatedly over many months.

In contrast, most astrocyte proliferation in vivo is largely complete by P14 (Skoff and Knapp, 1991). To directly compare the proliferative capacities of MD and IP-astrocytes P7, we plated dissociated single cells at low density in a defined, serum-free 3MA media containing HBEGF and counted clones at 1, 3, and 7DIV (Figures S1Q–S1S). MD-astrocytes displayed a much higher proliferative capacity, 75% of them dividing once every 1.4 days by 7DIV. In contrast, 71% of IP-astrocytes divided less than once every 3 days (Figure S1S). Thus IP-astrocytes have a more modest ability to divide compared with MD-astrocytes, this is more in line with what is expected in vivo (Skoff and Knapp 1991). Using gene profiling, we determined if gene expression of cultured IP-astrocytes was more similar to that of acutely purified astrocytes, compared to MD-astrocytes. Total RNA was isolated from acutely purified astrocytes from P1 and P7 rat brains (IP-astrocytes P1 and

P7) and from acutely isolated cells cultured for 7DIV with HBEGF (IP-astrocytes P1 and P7 7DIV, respectively) and from MD-astrocytes (McCarthy Adenylyl cyclase and de Vellis, 1980). RT-PCR with cell-type specific primers was used to assess the purity of the isolated learn more RNA. We used GFAP, brunol4,

MBP, occludin, CX3CR1 as mentioned above, as well as chondroitin proteoglycan sulfate 4 (CSPG4) for OPCs and pericytes. MD-astrocytes consistently had some neuron contamination because of the high percentage of contaminating neural stem cells ( Hildebrand et al., 1997; Figure 4A). This was not observed in IP-astrocyte cultures. IP-astrocytes P1 and P7 7DIV cells had an expression profile resembling their acutely isolated counterparts, where only 118 and 54 genes respectively differed significantly (p < 0.05). In contrast, MD-astrocyte expression profiles were significantly different from that of acutely purified cells (Table 1; Figure 4B). With a very stringent statistical test (moderated t test) and posttest (Bonferroni correction) to identify the most significant changes, we found that 547 and 729 genes were significantly different (p < 0.05) between acute IP-astrocytes P1 or P7 cells and MD-astrocytes, respectively. These results strongly suggest that by gene expression, cultured IP-astrocytes are more similar to cortical astrocytes in vivo. Only 54 genes out of over 31,000 genes differed significantly between acute IP-astrocytes P7 and IP-astrocytes P7 7DIV (p < 0.05).

Elimination of catalytic activity or a moderate reduction in DAG binding affinity of the C1 domain disrupted RGEF-1b function in vivo. Chemotaxis was unaffected by mutations that inactivated Ca2+-binding EF hands or a conserved PKC phosphorylation site. Thus, RGEF-1b links external stimuli (odorants) and internal DAG to the control of behavior (chemotaxis) by differentially activating the LET-60-MPK-1 cascade in AWC neurons. A single gene, named rgef-1 (Ras GTP/GDP exchange factor-1), was identified by searching C. elegans genome, EST, and protein databases for RasGRP homologs. Cosmid F25B3 (GenBank)

contains the rgef-1 gene (4893 bp) and flanking DNA. RGEF-1 cDNA and protein DAPT molecular weight were not Sunitinib chemical structure previously characterized. Thus, RGEF-1 cDNAs were amplified by RT-PCR and cloned into a mammalian expression vector pCDNA3.1 (see Supplemental Experimental Procedures available online). Sequencing revealed that alternative splicing generated two cDNAs as diagrammed in Figure S1A (available online). RGEF-1a and RGEF-1b ( Figure S1E) cDNAs encode proteins composed of 654 and 620 amino acids, respectively ( Figure S1B).The isoforms are 98% identical and diverge only in a segment of unknown function that links a C1 domain to

the C-terminal region. Quantitative real time PCR (qR-PCR) analysis disclosed that RGEF-1b mRNA accounts for >95% of rgef-1 gene transcripts ( Figure S1C). Thus, studies were focused on RGEF-1b. The predicted RGEF-1b protein (Mr ∼70,000) contains structural (REM),

catalytic (GEF), and regulatory (two EF hands and C1) domains that share substantial amino acid sequence identity and similarity with corresponding domains in human RasGRPs (Figure S1D). By analogy, the RGEF-1b GEF domain will promote opening of the GTP/GDP binding site in small G-proteins (Bos et al., 2007). Guanine nucleotides will equilibrate between G protein and cytoplasm. Since the GTP:GDP ratio is ∼10, the net result is exchange of GTP for GDP. EF-hands, which contain five Asp or Glu residues, often regulate enzymatic activity by binding Ca2+ (Gifford et al., 2007). The C1 domain is predicted to mediate RGEF-1b translocation by binding membrane associated DAG (Hurley and Misra, 2000). ADP ribosylation factor Functions of RasGRP REM domains are unknown. Locations of domains along the RGEF-1b and RasGRP polypeptides are also preserved (Figure S1D). RGEF-1b translocates to membranes and catalyzes loading of GTP onto LET-60 in PMA-treated cells (see below). Together, these properties show that RGEF-1b is a new, but prototypical RasGRP. In C. elegans, unique genes encode a 21 kDa Ras homolog (LET-60) and a 21 kDa Rap1 polypeptide (RAP-1). LET-60 and RAP-1 cDNAs were inserted into a modified pCDNA3.1 plasmid that appends an N-terminal Flag epitope tag to encoded proteins. If RGEF-1b is a RasGRP, it will translocate to membranes and mediate loading of GTP onto colocalized LET-60 or RAP-1.

, 2003, Luk et al., 2009, Murray et al., 2003 and Serpell et al., 2000). Thus, we asked whether human α-syn pffs composed of α-syn-1-120, α-syn-1-89, α-syn-58-140, or α-syn-NAC could seed formation of LBs and LNs in neurons. We observed that α-syn-1-120 and α-syn-1-89 pffs induced robust accumulation of endogenous p-α-syn aggregates that were Tx-100-insoluble (Figure 2A; data not shown), and they are morphologically indistinguishable from those formed by α-syn-hWT pffs. α-syn-58-140 pffs also seeded formation of endogenous mouse α-syn aggregates that were hyperphosphorylated

Metformin mw (Figure 2A). Moreover, pffs composed of only the central hydrophobic α-syn-NAC domain also resulted in endogenous mouse α-syn fibrillar LB-like aggregates that were Tx-100-insoluble. Overall, our data demonstrate that α-syn pffs containing only the central, hydrophobic portion of α-syn-hWT are sufficient to seed conversion of endogenous α-syn into pathological aggregates. Mice typically do not develop LBs except in the case of transgenic lines overexpressing mutant human α-syn. We thus asked whether the formation of LB-like aggregates required human α-syn or whether they can be seeded by α-syn

pffs generated from recombinant mouse WT α-syn (α-syn-mWT) (Touchman et al., 2001). Immunoblots demonstrated that 14 days treatment of primary neurons with α-syn-mWT pffs induced appearance of p-α-syn in the Tx-100-insoluble fraction (Figure 2B). Immunofluorescence also showed that α-syn-mWT pffs induced formation of p-α-syn aggregates in neurites and somata. Thus, pathological PD-like α-syn aggregates can be induced by α-syn-mWT pffs and does not require the human protein. Examination PLX4032 of the α-syn aggregates using transmission and immuno-EM demonstrated abundant filaments in neurons treated with either α-syn-hWT or α-syn-1-120 pffs (Figure 3A) for 14 days, but not PBS-treated tuclazepam neurons (data

not shown). Remarkably, inclusions composed of 14- to 16-nm-thick filaments were seen throughout the cytoplasm, visualized by transmission EM. Two different immuno-EM detection systems, horse radish peroxidase (HRP) and immunogold amplification, were used to demonstrate that fibrils composed of p-α-syn are found throughout the neuron. P-α-syn-positive fibrils were seen in the soma (Figures 3B and 3C), adjacent to the active zone of presynaptic terminals (Figure 3D) and the postsynaptic terminal (Figure 3F) and throughout processes (Figure 3E). These data establish that the seeding and recruitment of endogenous mouse α-syn into hyperphosphorylated insoluble, filamentous aggregates recapitulate features of LBs and LNs in PD and other human synucleinopathies. To determine the temporal sequence of α-syn aggregate formation, α-syn-hWT pffs were added to the neurons at DIV5. P-α-syn immunostaining was not detectable until 4 days later when small aggregates began to appear, exclusively in the neurites, albeit at low levels (Figure 4A, upper series).

shRNA-mediated

silencing of MSH2 resulted in shorter repeat lengths suggesting that FRDA iPS cells could be a useful system to evaluate the mechanisms of repeat expansions and contractions in disease. It remains to be shown whether FRDA iPS cells will demonstrate cell-type-specific expansions of GAA repeats. GAA repeat mutations are unstable and progressive and postnatal instability occurs in various tissues throughout life. For example, large GAA repeat expansions are especially prominent in the dorsal root ganglia of FRDA patients, this website which harbor cell bodies of sensory neurons, a neuronal subtype especially affected in FRDA (De Biase et al., 2007). Given FRDA-iPS cells can be directed to differentiate into sensory neurons, as well as cardiomyocytes, the presence Temozolomide clinical trial and mechanisms of tissue-specific expansion should be testable (Liu et al., 2010). Disease modeling using human pluripotent stem cells might greatly benefit if the genome

of these cells could be readily modified. For instance, the generation of transgenic “reporter” cell lines using fluorescent reporter genes under the control of cell-type-specific promoters could enable the purification, tracking, and functional characterization of disease relevant cells after directed differentiation. It is our experience that use of such reporter genes is a significant consideration. Most in vitro differentiation strategies result in a heterogenous population of differentiated cells, which can include progenitors and a variety of cellular intermediates. Therefore, having the ability to prospectively identify, purify, and easily track the desired cell type by means of reporter-gene

expression can facilitate downstream disease-specific assays, which could be hindered by the presence of other cell types. The availability of stem cell lines harboring cell-type-specific reporters could also aid in the improvement of procotols for the directed differentiation of disease relevant cell types for nearly which efficient differentiation techniques are not yet available. In addition, the ability to overexpress or downregulate a particular gene of interest could be used in the future to recapitulate or rescue a disease-relevant phenotype. For instance, loss-of-function monogenic disorders could be mimicked using a wild-type cell line by downregulation of the particular disease-associated locus. Conversely, a loss-of-function disease-specific phenotype could be rescued by overexpression of the wild-type form of the gene. Finally, the use of gene-targeting strategies to correct or induce a particular genetic defect will allow for the generation of isogenic lines with and without a disease genotype.