Top PDF Quantitative Analysis of Gene Function in the Drosophila Embryo

Quantitative Analysis of Gene Function in the Drosophila Embryo

Quantitative Analysis of Gene Function in the Drosophila Embryo

Tracey, W. D., Jr., M. E. Pepling, M. E. Horb, G. H. Thomsen hemorrhaging in the central nervous system and blocks definitive and J. P. Gergen, 1998 A Xenopus homologue of aml-1 reveals hematopoiesis. Proc. Natl. Acad. Sci. USA 93: 3444–3449. unexpected patterning mechanisms leading to the formation of Yang, M. Y., J. D. Armstrong, I. Vilinsky, N. J. Strausfeld and K. embryonic blood. Development 125: 1371–1380. Kaiser, 1995 Subdivision of the Drosophila mushroom bodies Tsai, C., and J. P. Gergen, 1994 Gap gene properties of the pair- by enhancer-trap expression patterns [see comments]. Neuron
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A Green Fluorescent Protein Reporter Genetic Screen That Identifies Modifiers of Hox Gene Function in the Drosophila Embryo

A Green Fluorescent Protein Reporter Genetic Screen That Identifies Modifiers of Hox Gene Function in the Drosophila Embryo

led to the genetic and molecular identification of Hox Studies of the Drosophila Extradenticle (Exd) HD- genes in a wide range of organisms covering the meta- containing protein and of the vertebrate related Pbx zoan phyla, including hydra, nematodes, arthropods, proteins have provided substantial support for the idea and humans. Hox genes are clustered within complexes, that protein cofactors contribute in multiple ways to are differentially expressed along the antero/posterior specify the function of Hox proteins (Mann and Chan (A/P) axis, and specify the diversified morphogenesis 1996). First, Exd can improve the affinity of a Hox of animal body parts (McGinnis and Krumlauf 1992; protein to DNA. This is best illustrated in the case of Botas 1993; Gellon and McGinnis 1998). They en- Labial (Lab), a Hox protein that binds DNA very poorly code homeodomain (HD)-containing transcription fac- on its own. When engaged in a complex with Exd, Lab tors thought to control different sets of subordinate adopts a conformation that overcomes an inhibitory targets required for morphogenetic processes (Garcia- activity of the hexapeptide motif (a short evolutionarily Bellido 1975; Graba et al. 1997). conserved motif lying upstream of the HD) on DNA The HD, a 60-amino-acid DNA binding domain, also binding and results in an increased affinity to target found in other classes of transcription factors (Duboule
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Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

ABSTRACT During embryonic development of Drosophila melanogaster, the maternal-to-zygotic transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. We utilized the NanoString nCounter instrument, which directly counts messenger RNA transcripts without reverse transcription or amplification, to study .70 genes expressed along the dorsal–ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first dataset of absolute numbers of transcripts during Drosophila development. We found that gene expression changes quickly during the MZT, with early nuclear cycle 14 (NC14) the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and we use mutants to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. We also uncovered elements within the gene regulatory network that maintain precise transcript levels for sets of genes that are spatiotemporally cotranscribed within the presumptive mesoderm or dorsal ectoderm. Using these new data, we show that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks, including coregulation of target genes and specific temporal input by transcription factors.
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Glial and Neuronal Functions of the Drosophila Homolog of the Human SWI/SNF Gene ATR-X (DATR-X) and the jing Zinc-Finger Gene Specify the Lateral Positioning of Longitudinal Glia and Axons

Glial and Neuronal Functions of the Drosophila Homolog of the Human SWI/SNF Gene ATR-X (DATR-X) and the jing Zinc-Finger Gene Specify the Lateral Positioning of Longitudinal Glia and Axons

Neuronal-specific functions of jing and DATR-X are required for repulsion of longitudinal axons and glia from the CNS midline: We targeted DATR-X and jing mutations to discern the neuronal and glial contribu- tions of each gene product to axon scaffold formation. DATR-X and jing expression was knocked down using conditional RNAi (H ammond et al. 2001; L ee and C arthew 2003). A total of 697- and 567-bp sequences from nonconserved regions of DATR-X and jing were separately subcloned into a P[UAST] derivative plasmid to produce intron-spliced hairpin RNA corresponding to the DATR-X and jing genes, respectively. The UAS/ Gal4 system (B rand and P errimon 1993) was then used to allow hairpin RNA to conditionally downregulate DATR-X and jing expression in specific cell lineages, which was confirmed by in situ hybridization. Pene- trance values of CNS axon phenotypes associated with neuronal and glial knockdown of jing and DATR-X ranged from 24 to 30% (see materials and methods ). Longitudinal connective formation was analyzed in homozygous jing mutant embryos and those with neuronal-specific knockdown of jing and DATR-X stained with an antibody to Fasciclin II (1D4). Neuronal specificity was directed by the ELAV-Gal4 driver. In these mutant embryos, FasII-positive longitudinal axons ab- errantly cross the CNS midline in stage 16 embryos (Figure 6, B–D, arrowheads; Figure 6M). In addition, there are breaks in the longitudinal connectives sug- gesting axon outgrowth defects (Figure 6, C and D, arrows). In embryos with simultaneous neuronal knock- down of jing and DATR-X all FasII-positive lateral fascicles fuse into a single tract at the CNS midline (Figure 6E). These results establish an autonomous neuronal requirement for jing and DATR-X function in the outgrowth and lateral positioning of longitudinal axons and a genetic synergy during this process.
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Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Additional evidence also suggests that TGF-ß signaling may also regulate the ind expression domains, but whether or not this signaling pathway functions through the A- box element was not known. Decapentaplegic (Dpp) is a TGFß/BMP homolog that is limited in its expression to dorsal regions of the embryo and functions as a morphogen to support patterning of the amnioserosa, at higher levels in dorsal-most regions of the embryo, and the non-neurogenic ectoderm, at lower levels in dorsal-lateral regions of the embryo (Ferguson and Anderson, 1992). A previous study found that in mutants in which Dpp signaling is expanded into lateral regions of the embryo, ind expression is lost (Von Ohlen and Doe, 2000). Likewise, ectopic expression of dpp in lateralized embryos that exhibit expanded ind expression throughout the embryo was able to repress ind in the domain where Dpp signaling was presented (Mizutani et al., 2006). Also, the ind CRM contains a 15 bp DNA sequence implicated in TGF- β signaling-mediated repression (Stathopoulos and Levine, 2005). Similar sites have been shown to mediate repression by recruiting a Dpp-dependent Schnurri/Mad/Medea (SMM) protein complex, but SMM dependent repression of ind has never been shown and in fact this mechanism of repression has only been shown to act at later stages of development (Dai et al., 2000; Pyrowolakis et al., 2004).
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The Drosophila retained/dead ringer gene and ARID gene family function during development

The Drosophila retained/dead ringer gene and ARID gene family function during development

The development of multicellular organisms requires tight spa- tial and temporal control of switches in the activity of key regulatory genes. Tissues are specified by transcriptional regulatory cas- cades that respond to a variety of extracellular and intracellular cues. It has become clear in recent years that the regulators of tissue-specific gene expression, the transcription factors, are assembled into multi-protein complexes. These complexes in- clude DNA-binding proteins, which recruit other components that are not able to bind DNA directly. The composition of complexes assembled on a specific promoter can be transient, as seen in the cascade of gene regulation in the early Drosophila embryo. Curi- ously, many transcription factors fall into a relatively small number of phylogenetically conserved families, although different family members can assume very different developmental roles in differ- ent organisms (see, for example, Lall and Patel, 2001). As a result, studies of members of conserved protein families very often provide new approaches to understanding mechanisms that regu- late a variety of developmental events.
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Analysis of slmo, a gene required for normal motor function in Drosophila melanogaster

Analysis of slmo, a gene required for normal motor function in Drosophila melanogaster

Reported in this thesis is an analysis of slowmo slmo, a gene previously shown to be expressed in the developing embryonic central nervous system of Drosophila.. Null mutants of slmo are[r]

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Gene Expression Analysis of the Function of the Male-Specific Lethal Complex in Drosophila

Gene Expression Analysis of the Function of the Male-Specific Lethal Complex in Drosophila

Probe preparation: One to three micrograms of linearized Immunostaining of chromosomes: Third instar salivary glands plasmid DNA of a selected gene was incubated with 20 ␮l of were dissected and fixed temporarily. The polytene chromo- digoxygenin labeling mixture (containing 10 mm ATP, 10 mm somes were immunostained with antibodies as described by Pal GTP, 10 mm CTP, 6.5 mm UTP, and 3.5 mm digoxygenin-conju- Bhadra et al. (1997). Chromosomes were probed with anti- gated UTP), including10⫻ transcription buffer and 2 ␮l of pro- JIL-1 antibodies at a dilution of 1:50. Cy5-conjugated goat anti- moter-specific enzyme (T7, SP6, or T3 RNA polymerase; Roche, chicken secondary antibodies were used for JIL-1 at a standard Indianapolis) at 37⬚ for at least 4 hr (Bhadra et al. 1999). After 1:200 dilution. In other experiments, the chromosomes were incubation, the reaction was terminated with 0.2 m EDTA, pH probed with anti-MSL1 antibodies at a dilution of 1:100 and 8.0. The labeled RNA was precipitated with 2.5 ␮l 4 m LiCl and subsequently with Cy5-conjugated anti-rabbit antibodies at a 75 ␮l prechilled ethanol at ⫺20⬚. The RNA was pelleted via 1:200 dilution. The chromosomes were mounted in a mixture centrifugation at 12,000 rpm, washed in 70% ethanol, and dis- of Vectashield mounting media with propidium iodide and solved in 25 ␮l double-distilled water. Probes prepared in this examined with a Bio-Rad 600 confocal microscope using a way could be stored for several months before use. In the case ⫻60 water immersion lens.
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Molecular analysis of the determination of developmental fate in the small intestinal epithelium in the chicken embryo

Molecular analysis of the determination of developmental fate in the small intestinal epithelium in the chicken embryo

Molecular mechanism of regionalization in gut development Our present results and many reports lead to the conclusion that developmental fate of the posterior gut endoderm is deter- mined early in the development while the anterior gut endoderm seems to retain the reactivity to the heterologous mesenchymes up to day 6 of incubation. The molecular mechanisms underlying the regionalization in gut development has not been clearly understood. We suppose that CdxA is involved as one of determi- nation factors. CdxA gene is specifically expressed in the endo- derm which becomes midgut and hindgut before 1.5 day (Ishii et al., 1997). Moreover, it was reported that mouse Cdx1 and Cdx2 are important in anterior-posterior patterning in the intestinal epithelium (Silberg et al., 2000). Cdx2 defines the stomach- intestine boundary and when the function of Cdx2 was down- regulated, the proximal part of the intestine formed the gastric- type tissue (Beck et al., 2003). From these reports, CdxA is thought to be an important factor involved in the determination of the developmental fate of the small intestinal epithelium in the chicken embryo. We are currently investigating the effects of ectopic expression of CdxA gene in the presumptive stomach endoderm of young embryo.
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Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo

Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo

gradient remains intact (Liu and Ma, 2013b). It has been suggested that specific pathways can become activated at the MBT to cause rapid degradation of maternal proteins such as Twine (Di Talia et al., 2013). If hb shutdown were to merely reflect a decaying Bcd gradient at nc14, we would have expected a shutdown process that initiates near mid-embryo (where the Bcd concentration is low) and ‘ spreads ’ toward the anterior (with increasing Bcd concentrations). But our results do not support this prediction (Fig. S8A,B; see also Liu and Ma, 2013a). In addition, neither the length constant nor the amplitude of the Bcd gradient profile is affected by the Bcd mutation (Fig. 3). Thus, our results show that the timing of Bcd-activated transcription during nc14 requires neither a physical disappearance of this maternal activator nor the accumulating activities of sequence-specific zygotic repressors. Instead, it is the functional potency of the maternal activator Bcd that is part of the mechanism of timing the molecular events in accordance with MZT morphological progression. Importantly, the potency of the Bcd activator can be either strengthened (this study) or weakened (Liu and Ma, 2013a) to tune – in opposite directions – the hb shutdown timing and hb expression level. We note that our bcd6-lacZ reporter results do not formally exclude the possibility that the shutdown of Bcd-activated transcription at nc14 involves a zygotic repressor(s) that operates by competing with Bcd binding to its DNA sites. But we currently do not favor this possibility because the position independence and rapidity of the shutdown event would likely require any such zygotic repressor not only to have the same/ overlapping Bcd binding specificity but also to accumulate in a spatially non-restricted (i.e. covering the entire hb expression domain) and temporally abrupt way at nc14.
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In Vivo Structure/Function Analysis of the Drosophila fat facets Deubiquitinating Enzyme Gene

In Vivo Structure/Function Analysis of the Drosophila fat facets Deubiquitinating Enzyme Gene

taining a faf cDNA with a Myc epitope tag between amino degradation. Genetic experiments have identified a sin- acids 53 and 54 cloned into the AscI site. For each of the faf⌬ gle gene, called liquid facets, that is likely to encode the constructs, the DNA sequence at the deletion breakpoint was determined to check that the reading frame was restored. critical substrate for Faf in the eye (Cadavid et al. 2000).

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Hox gene function and cell identity in Drosophila

Hox gene function and cell identity in Drosophila

(Mastick et al., 1995). Although both approaches have the advantage of isolating potential direct targets of UBX, immunopurification has been the most successful. This strategy benefited from more stringent conditions, as targets were isolated from wild type D ro s o p h ila embryos. Therefore unlike the yeast assay, immunopurification combined the presence of physiological UBX levels with the proteins involved in co-operative DNA binding with UBX (see Section 1.8). Gould and colleagues isolated several targets, including connectin {con), a gene involved in hemophilic cell-cell adhesion (Gould and White, 1992; Nose et al., 1992). Using a similar immunopurification approach, Heuer et al. (1995) isolated the ANTP target centrosomin (cnn), which encodes an essential centrosomal protein. As discussed in the next section, cnn has been implicated in the cell shape changes that occur during development of the second constriction of the midgut. In a third, closely related screen, this time incorporating DNA-protein cross-linking, scabrous (sea), a gene encoding a secreted protein involved in cellular communication during neurogenesis, was identified as a putative direct target of UBX (Graba et al., 1992).
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Dosage-Sensitive Maternal Modifiers of the Drosophila Segmentation Gene runt

Dosage-Sensitive Maternal Modifiers of the Drosophila Segmentation Gene runt

The protein encoded by the pair-rule gene runt functions as a transcriptional regulator during anterior- posterior patterning of the Drosophila embryo.. Results of over-express[r]

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A piggyBac Transposon Gene Trap for the Analysis of Gene Expression and Function in Drosophila

A piggyBac Transposon Gene Trap for the Analysis of Gene Expression and Function in Drosophila

This work describes the design and implementation of a new gene trap system for Drosophila. This system uses a piggyBac transposon to provide a different and, hopefully, broad spectrum of chromosomal insertion sites for functional genomic studies compared to that provided by previous P-element-based systems. We have demonstrated the ability of the PBss element to insert into new genes, splice with endogenous transcripts, and report the expression patterns of trapped genes. Fur- ther, a preliminary genetic analysis indicates that many of the insertions are lethal or mutant as homozygotes. Thus, the implementation of this system should signifi- cantly aid the analysis of gene function and expression in D. melanogaster. Below we focus this discussion on the
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Identification of downstream targets of Mirror, a transcription factor in Drosophila melanogaster

Identification of downstream targets of Mirror, a transcription factor in Drosophila melanogaster

using the heat shock subtraction model, confirmed that ectopic Mirror expression indeed inhibits the expression of most of the analysed genes. Thus, the bioinformatic analysis seems to reflect the situation in the embryo. This demonstrates that the microarray technique is sensitive enough to detect the different levels o f gene expression under the experimental conditions. However, the effect o f ectopic Mirror could only be confirmed for negatively regulated candidate genes in the embryo. Genes which were positively regulated according to the microarray analysis showed the normal wild type expression pattern in Mirror over-expressing embryos. It might be that Mirror acts primarily as a repressor in the embryo and that the upregulated genes are a result of the variability in the microarray analysis. Nevertheless, it cannot be ignored that the majority of analysed potential Mirror downstream targets seemed completely normally expressed in mirror loss-of-function embryos. The only exception from this observation was the gene glial cells missing, which encodes a transcription factor. However, it was expected that expression of gem is enhanced in an embryo which lacks Mirror function, which is not the case. The in situ hybridisation using a gem riboprobe and the antibody staining for its known positively regulated target Repo, showed reduced levels of expression. These results are inconsistent and might suggest that either the observed decrease of gem expression is an indirect effect of a negative regulatory feedback loop, activated in the absence of Mirror function, or that the way the microarray experiment was designed didn’t lead to the identification of direct Mirror targets.
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A regulatory code for neurogenic gene expression in the
Drosophila embryo

A regulatory code for neurogenic gene expression in the Drosophila embryo

This study provides direct evidence that Twist and Su(H) are essential for the specification of the neurogenic ectoderm in early embryos. The Twist protein is transiently expressed at low levels in ventral regions of the neurogenic ectoderm (Kosman et al., 1991). SELEX assays indicate that Twist binds the CACATGT motif quite well (K. Senger, unpublished). The presence of this motif in the vnd, brk and sim enhancers, and the fact that it functions as an essential element in the vnd and brk enhancers, strongly suggests that Twist is not a dedicated mesoderm determinant, but that it is also required for the differentiation of the neurogenic ectoderm. However, it is currently unclear whether the CACATGT motif binds Twist- Twist homodimers, Twist-Da heterodimers or additional bHLH complexes in vivo. Su(H) is the sequence-specific transcriptional effector of Notch signaling (Schweisguth and Posakony, 1992). The restricted activation of sim expression within the mesectoderm depends on Notch signaling (Morel and Schweisguth, 2000; Cowden and Levine, 2002); however, the rho, vnd and brk enhancers direct expression in more lateral regions where Notch signaling has not been demonstrated. Nonetheless, mutations in the two Su(H) sites contained in the brk enhancer cause a severe impairment in its activity. This observation raises the possibility that Su(H) can function as an activator, at least in certain contexts, in the absence of an obvious Notch signal.
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Survival Analysis of Life Span Quantitative Trait Loci in Drosophila melanogaster

Survival Analysis of Life Span Quantitative Trait Loci in Drosophila melanogaster

We used quantitative trait loci (QTL) mapping to evaluate the age specificity of naturally segregating alleles affecting life span. Estimates of age-specific mortality rates were obtained from observing 51,778 mated males and females from a panel of 144 recombinant inbred lines (RILs). Twenty-five QTL were found, having 80 significant effects on life span and weekly mortality rates. Generation of RILs from heterozygous parents enabled us to contrast effects of QTL alleles with the means of RIL populations. Most of the low-frequency alleles increased mortality, especially at younger ages. Two QTL had negatively correlated effects on mortality at different ages, while the remainder were positively correlated. Chromo- somal positions of QTL were roughly concordant with estimates from other mapping populations. Our findings are broadly consistent with a mix of transient deleterious mutations and a few polymorphisms maintained by balancing selection, which together contribute to standing genetic variation in life span.
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Dorsoventral Patterning of the Drosophila melanogaster Embryo by the Dorsal/NF-κB Signaling Pathway

Dorsoventral Patterning of the Drosophila melanogaster Embryo by the Dorsal/NF-κB Signaling Pathway

7 future gametes of developing embryos (25–27). φ31-integrase and specialized fly lines allow researchers to precisely control the genomic location of the transgene (27). Additionally, scientists have engineered an elegant system of Gal4-linked drivers and UAS-linked genes (28). The yeast protein Gal4 recognizes and binds to UAS sites in the promoter region of genes, turning on expression. This system allows researchers to express genes in specific tissues or at a specific time in development, essentially providing a system with much greater variety than could be generated through single injections. Since UAS-linked genes are not expressed except when Gal4 is present, researchers can maintain stocks containing deleterious mutations that would otherwise render flies non-viable. However, the Gal4/UAS system has its drawbacks. Some drivers are weaker than others, preventing full penetration of the desired phenotype. Furthermore, due to the limited number of chromosomes (four), it is difficult to manipulate multiple genes at the same time. Finally, these methods only allow researchers to add genes to the fly. In order to disrupt a gene, the DNA insertion must occur within that gene’s locus, a process that is difficult both to control and screen.
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Cell lineage analysis of pattern formation in the Tubifex embryo  II  Segmentation in the ectoderm

Cell lineage analysis of pattern formation in the Tubifex embryo II Segmentation in the ectoderm

cell (A), left NOPQ (B) or individual teloblasts (C, D) were injected with DiI and allowed to develop for 3 days before fixation. Wholemount preparations were viewed from the ventral side (A) or left side (B-D). In all panels, anterior is to the left; in B-D, dorsal is to the top. Bar, 100 µ m (A, B); 80 µ m (C, D). (A) Both the left and right germ bands (EGBl and EGBr, respectively) are labeled with DiI. Both GBs have coalesced with each other along the ventral midline in the anterior and mid regions of the embryo. Only the mid region of the embryo is in focus here. Note that GBs are divided into 50- µ m-wide blocks of labeled cells by intersegmental furrows, which are recognized as non-fluorescent transverse stripes. (B) The posterior portion of the left GB is shown. P and Q teloblasts are seen, but N and O teloblasts are out of the field. The arrow indicates the site where a fissure becomes evident in the ventralmost bandlet (i.e., n bandlet). The arrowhead indicates fissures at the dorsal side of the GB. (C) Fluorescent n and p bandlets in the left GB. These bandlets were de-
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Association analysis of the <em>Cadherin13</em> gene with schizophrenia in the Japanese population

Association analysis of the <em>Cadherin13</em> gene with schizophrenia in the Japanese population

haplotype may have a protective role. None of the SNPs in the promoter region of CDH13 evaluated in this study revealed a statistically significant association of the CDH13 locus with schizophrenia. One reason is that the sample size was too small to detect an association of CDH13 SNPs with schizophrenia. Based on the observed allele frequen- cies of rs12925602, rs7193788, rs736719, rs6565051, and rs7204454, the current combined samples provide powers of 0.058, 0.098, 0.050, 0.072, and 0.056, respectively, to detect nominally significant results. A recent mega analysis by the Psychiatric Genomics Consortium did not identify any association between CDH13 SNPs and schizophrenia. 35
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