Voigt J et al. (2005), Expression cloning screening of a unique and fu...

XB-ART-2182

Mech Dev 2005 Mar 01;1223:289-306. doi: 10.1016/j.mod.2004.11.002.

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Expression cloning screening of a unique and full-length set of cDNA clones is an efficient method for identifying genes involved in Xenopus neurogenesis.

Voigt J , Chen JA , Gilchrist M , Amaya E , Papalopulu N .

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Functional screens, where a large numbers of cDNA clones are assayed for certain biological activity, are a useful tool in elucidating gene function. In Xenopus, gain of function screens are performed by pool screening, whereby RNA transcribed in vitro from groups of cDNA clones, ranging from thousands to a hundred, are injected into early embryos. Once an activity is detected in a pool, the active clone is identified by sib-selection. Such screens are intrinsically biased towards potent genes, whose RNA is active at low quantities. To improve the sensitivity and efficiency of a gain of function screen we have bioinformatically processed an arrayed and EST sequenced set of 100,000 gastrula and neurula cDNA clones, to create a unique and full-length set of approximately 2500 clones. Reducing the redundancy and excluding truncated clones from the starting clone set reduced the total number of clones to be screened, in turn allowing us to reduce the pool size to just eight clones per pool. We report that the efficiency of screening this clone set is five-fold higher compared to a redundant set derived from the same libraries. We have screened 960 cDNA clones from this set, for genes that are involved in neurogenesis. We describe the overexpression phenotypes of 18 single clones, the majority of which show a previously uncharacterised phenotype and some of which are completely novel. In situ hybridisation analysis shows that a large number of these genes are specifically expressed in neural tissue. These results demonstrate the effectiveness of a unique full-length set of cDNA clones for uncovering players in a developmental pathway.

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Species referenced: Xenopus
Genes referenced: csnk1d cul1 dlx5 esr1 etv1 fzd10 gtf2f1 mef2d prkacb rab32 sox2 sox3 srsf5 srsf7 stk17a triqk tubb2b vangl2 zbtb2 zgpat zic3

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	Fig. 1. Comparison of the phenotype penetrance between the redundant set and the unique full-length set screen. One hundred and six pools of a redundant neurula stage cDNA library (pool size 96 clones=10.176 cDNAs) and 120 pools of the unique full-length (FL) gastrula/neurula cDNA library (pool size eight clones=960 unique and full-length cDNAs) were screened by overexpressing ≈5 ng of in vitro transcribed RNA in one cell of two-cell stage embryos. Embryos were analysed at neurula and tadpole stages for changes in morphology and in the neural differentiation marker N-tubulin. Only phenotypes with a penetrance (percentage of affected embryos in a batch) of 40% or more were scored as ‘real’ but analysis for lower penetrance is also shown. In the redundant set screen ≈10% (n=11) of the screened pools showed a phenotype (≥40% penetrance) whereas this was raised to 50% (n=60) in the FL set screen (green star). At a higher penetrance (≥80%) there is also a big difference in phenotype recovery between the redundant and FL set screen (3 vs. 28%). Thus the FL set screen was five-fold more sensitive in detecting gain-of-function phenotypes compared to an unmodified cDNA library.
	Fig. 2. Phenotypic categories found when screening the unique FL set pools. Sixty pools of the unique full-length gastrula/neurula stage cDNA library gave rise to an overexpression phenotype (at least 40% penetrance). Nine major categories (x-axis) of phenotypes, for which representative images are shown below each bar, were observed and scored. Phenotypes were classified as secondary axes (rather than ectopic tissue outgrowth) if the ectopic tissue shows axial organisation, like secondary heads (at tadpole stage) or complete secondary neural tubes (at neurula stage). Ectopic tissue outgrowth comprises tumour-like growths, protrusions (which lack clear axial structures) and other abnormal thickenings observed. Blisters are oedema-like appearances, which do not contain tissue. Phenotypes such as loss/shedding of cells and or limited cell death were grouped into tissue loss. In contrast, embryos that showed global death are classified in death. Since most of the phenotypes observed were complex, many of the pools were included in more than one category (thus the sum of the percentages is greater than 100%). Of particular interest were phenotypes that affected the neural differentiation marker N-tubulin (stars), since they might contain an activity involved in the neurogenesis pathway. (Purple staining in images: N-tubulin, light blue staining: lineage tracer LacZ).
	Fig. 5. Additional detail on the overexpression of 4 selected genes and Rab32 expression. (a) XER 81 RNA injections expand X-Sox3 (purple) on the injected side. (b+c) Frizzled10B RNA injections enhance the medial (motorneuron) stripe of N-tubulin expression (white arrowhead, in wholemount (B) and transverse (C) view), in addition to the ectopic expression of N-tubulin observed in the floorplate (shown in Fig. 4b). (dashed line-midline, l-lateral, i-intermediate, m-medial). (d+e) 1 μm sections of Rab32 (1 ng) injected (E) and control Xenopus laevis (D) embryos. (D) In wild type embryos the melanosomes (darkly pigmented vesicles, arrow) are localised predominantly under the apical surface, but some can be found deeper within the cells (arrowhead). In contrast, when Rab32 was overexpressed the melanosomes were clustered tightly together and localised under the apical surface (arrows). (f) Transverse section (30 μm) of a bleached Xenopus tropicalis tailbud embryo in situ hybridised with an antisense probe to Rab32. Rab 32 is expressed in the retinal pigment epithelium (purple).
	Fig. 6. Creation of a unique full-length set. (a) The determination of the full-length representative in an EST cluster. 5′ sequenced ESTs from the X. tropicalis neurula and gastrula stage cDNA libraries that are 99% similar over a length of 100 nucleotides were aligned and clustered. Open reading frames of at least 210 base pair were identified. In order to ascertain that clusters contain full length EST clones in at least 2 and at least 50% of the EST clones at the same position a start codon (green) must be preceded by an in frame stop codon (red). To avoid 5′ UTR dependent negative translational control an EST with the shortest 5′UTR (arrow) was chosen as a full-length representative of the cluster/gene and re-arrayed into the unique and full-length set. (b) Criteria for selecting singletons as full-length. EST clones that did not align with any other clones were classified as singletons. Only singletons that contained a long ORF with a start codon preceded by a stop codon were considered for the full-length set. However, since the 50% rule (see above) cannot be applied in this case, Blast information was also used to determine if a singleton is full length. Only if the translational start of a matching protein in the database matched the start codon of the singleton, the latter was included in the full-length set.
	Fig. 3. Overexpression phenotypes of the single clones identified in the screen. (aCb) Approximately 400 pg in vitro transcribed RNA together with 200 pg beta-galactosidase RNA as a lineage tracer (light blue staining) were injected animally into one cell of two cell stage Xenopus laevis embryo. Embryos were analysed at neurula and tadpole stages for changes in the neural differentiation marker N-tubulin (purple) and morphology. Unless indicated otherwise, neurula stage embryos are viewed dorsally with anterior to the top and tadpoles are viewed laterally with anterior to the left. A transverse section is shown for clone 1.2B at tadpole stage.
	Fig. 3. Overexpression phenotypes of the single clones identified in the screen. (aCb) Approximately 400 pg in vitro transcribed RNA together with 200 pg beta-galactosidase RNA as a lineage tracer (light blue staining) were injected animally into one cell of two cell stage Xenopus laevis embryo. Embryos were analysed at neurula and tadpole stages for changes in the neural differentiation marker N-tubulin (purple) and morphology. Unless indicated otherwise, neurula stage embryos are viewed dorsally with anterior to the top and tadpoles are viewed laterally with anterior to the left. A transverse section is shown for clone 1.2B at tadpole stage.
	Fig. 4. (a,b) Expression patterns of the single clones identified in the unique full-length set screen. Xenopus tropicalis gastrula, neurula and tailbud stage embryos were in situ hybridised with antisense probes to the single clones identified in the unique full-length set screen. Gastrula stages embryos are shown vegetally with dorsal to the top (unless stated otherwise). At neurula stages an anterior and dorsal view is presented. Tailbud embryos are oriented with anterior to the left and dorsal to the top unless indicated otherwise. In come cases the wholemount images are supplemented by neurula and tailbud stage dissected embryos. If no specific staining was detected no image is shown.
	Fig. 4. (a,b) Expression patterns of the single clones identified in the unique full-length set screen. Xenopus tropicalis gastrula, neurula and tailbud stage embryos were in situ hybridised with antisense probes to the single clones identified in the unique full-length set screen. Gastrula stages embryos are shown vegetally with dorsal to the top (unless stated otherwise). At neurula stages an anterior and dorsal view is presented. Tailbud embryos are oriented with anterior to the left and dorsal to the top unless indicated otherwise. In come cases the wholemount images are supplemented by neurula and tailbud stage dissected embryos. If no specific staining was detected no image is shown.
	rab32 (RAB32, member RAS oncogene family ) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Dorsal view: anterior left.
	stk17a (serine/threonine kinase 17a) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	csnk1d (casein kinase 1, delta) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Anterior view: dorsal up.
	prkacb (protein kinase, cAMP-dependent, catalytic, beta) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	srsf5 (serine/arginine-rich splicing factor 5) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Anterior view: dorsal up.
	srsf5 (serine/arginine-rich splicing factor 5) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	gtf2f1 (general transcription factor IIF subunit 1) gene expression in Xenopus tropicalis embryos, NF stage 17, assayed by in situ hybridization, anterior view, dorsal up.
	gtf2f1 (general transcription factor IIF, polypeptide 1, 74kDa) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	zgpat (zinc finger, CCCH-type with G patch domain) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Vegetal view: dorsal up.
	zgpat (zinc finger, CCCH-type with G patch domain) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	zgpat (zinc finger, CCCH-type with G patch domain) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	triqk (triple QxxK/R motif containing) gene expression in Xenopus tropicalis embryos, NF stage 26, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	zbtb2 (zinc finger and BTB domain containing 2)gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Anterior view: dorsal up.
	zbtb2 (zinc finger and BTB domain containing 2) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.