Kyuno J et al. (2008), A functional screen for genes involved in Xenop...

XB-ART-37709

Mech Dev 2008 Jul 01;1257:571-86. doi: 10.1016/j.mod.2008.03.001.

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A functional screen for genes involved in Xenopus pronephros development.

Kyuno J , Massé K , Jones EA .

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In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events.

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Species referenced: Xenopus laevis
Genes referenced: ahctf1 alg10 cops2 cpeb3 csnk1g1 csnk2b deaf1 dnaja2 dnajc15 dusp11 dusp4 ebag9 elavl2 fubp1 gadd45g gal.2 hsf3 KIAA0586 lrr1 mecr pax8 pcmtd1 phf20l1 snrpd1 sox13 sox7 spaca5 spaca5 srd5a1 srsf3 srsf5 supt6h tp53 triap1 vwa8 wdr73 wt1 xkr9
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	Fig. 2. Typical phenotypes caused by over-expression of the clones identified in the screen. The mRNA injection of the candidate putative individual clones identified from pool injection revealed 31 positive clones responsible for perturbation of pronephros development. Similar abnormalities displayed in more than 20% of injected embryos were adapted as phenotypes. Three classes of major phenotypes were identified as “reduced”, “absent” and “enlarged/ectopic”. (A) The percentage of clones displaying each major phenotype (open bar; Lim-1 staining at early tailbud stage, closed bar; Na+, K+-ATPase staining or 3G8/4A6 antibody staining at late tadpole stage). Because some of clones displayed more than one scorable phenotype, the sum of percentages is greater than 100%. (B–I) Typical embryos categorized into each of the major phenotypes. As a lineage tracer, LacZ mRNA was injected into embryos along with sample mRNA and detected by Red-gal staining, indicating the injected side of the embryo. Pronephric marker gene expressions were examined by whole mount in situ hybridization and recognized as purple staining. Lower panels indicate higher magnification of pronephros regions. Arrow heads indicate reduced staining. Open arrow heads indicate enlarged/ectopic staining. In all cases, lateral views of embryos are shown, orientated with anterior to the left. (B–E) Examples of embryos examined by Lim-1 staining at early tailbud stage. (F–I), Examples of embryos examined by Na+, K+-ATPase staining at late tadpole stage. (B and F) Embryos injected with mRNA from the clone identified as Supt6h are presented as examples of “reduced” pronephros phenotypes. (C and G) Embryos injected with Fubp1 mRNA are shown as examples of “absent” phenotypes. (D and H) Embryos injected with Wdr73 mRNA are used as examples of “enlarged/ectopic” phenotypes. Note that Wdr73 was not considered to have “enlarged/ectopic” phenotypes in Lim-1 staining, because the number of embryos scored with this phenotype was less than 20%. (E and I) Images of control embryos following LacZ mRNA injection displaying normal pronephros are also presented.
	Fig. 4. The expression patterns of clones identified by the functional screen, examined by whole mount in situ hybridization using X. tropicalis embryos. Twenty-nine out of the 31 positive clones identified in the screen displayed identifiable in situ patterns of expression. The clones were divided into each of seven functional groups on the basis of their predicted/established function (A, transcription; B, cell signaling; C, post-transcription/translation; D, cell-cycle/apoptosis; E, metabolism; F, other functions; G, functionally unknown). The images are ordered by gene names (black). The main expression domains of each clone at all stages analyzed are indicated in red, although not all of these domains are represented in the photographs presented (cc, ciliated cells; cns, central nervous system; dt, distal tubules; e, eye; it, intermediate tubules; ms, migrating somite; n, primary neuron; ns, nephrostomes; op, olfactory pit; ov, otic vesicle; pa, pharyngeal arches; pcv, posterior cardinal veins; pn, pronephros; s, somite; vbi, ventral blood island). The numbers in each photograph indicate the developmental stage of embryos shown. Other abbreviations are: A, anterior view (dorsal is up); D, dorsal view (anterior is left); L, lateral view (anterior is left, dorsal is up).
	Fig. 4. The expression patterns of clones identified by the functional screen, examined by whole mount in situ hybridization using X. tropicalis embryos. Twenty-nine out of the 31 positive clones identified in the screen displayed identifiable in situ patterns of expression. The clones were divided into each of seven functional groups on the basis of their predicted/established function (A, transcription; B, cell signaling; C, post-transcription/translation; D, cell-cycle/apoptosis; E, metabolism; F, other functions; G, functionally unknown). The images are ordered by gene names (black). The main expression domains of each clone at all stages analyzed are indicated in red, although not all of these domains are represented in the photographs presented (cc, ciliated cells; cns, central nervous system; dt, distal tubules; e, eye; it, intermediate tubules; ms, migrating somite; n, primary neuron; ns, nephrostomes; op, olfactory pit; ov, otic vesicle; pa, pharyngeal arches; pcv, posterior cardinal veins; pn, pronephros; s, somite; vbi, ventral blood island). The numbers in each photograph indicate the developmental stage of embryos shown. Other abbreviations are: A, anterior view (dorsal is up); D, dorsal view (anterior is left); L, lateral view (anterior is left, dorsal is up).
	Fig. 5. Further analysis of the five selected clones. Among the 31 clones identified in the screen, we selected five clones that had highly specific expression patterns in pronephros region and which had predicted/established function to analyze further. In addition to Lim-1 and Na+, K+-ATPase expression, effects by over-expression in Pax8 and Wt1 expression were examined by whole mount in situ hybridization. Embryos were injected at the 2-cell stage into the lateral marginal zone of 1 cell with 300 pg mRNA of SRY (sex determining region Y)-box 7 (Sox7), cytoplasmic polyadenylation element binding protein 3 (Cpeb3), and P53-inducible cell-survival factor (P53csv). 900 pg of mRNA was injected for mitochondrial trans-2-enoyl-CoA reductase (Mecr) and DnaJ (Hsp40)homolog, subfamily C, member 15 (Dnajc15). The injected embryos were fixed at early tadpole stage to examine Lim-1 and Pax8 staining, and at late tadpole stage to assay Wt1 and Na+, K+-ATPase staining. In each case, embryos were injected with 200 pg of LacZ mRNA along with sample mRNA as a lineage tracer and stained with Red-gal after fixation (red staining). The embryos only injected with LacZ mRNA were also prepared as control embryos. (A) The major types of over-expression phenotype. The phenotypes of each embryo were indicated by abbreviations in the panels (E, enlarged/ectopic; N, normal; R, reduced). First column; Lim-1 staining, second column; Pax8 staining, third column; Wt1 staining, fourth column; Na+, K+-ATPase staining. Arrow heads indicate reduced staining. Open arrow heads indicate enlarged/ectopic staining. In all cases, lateral views of embryos are shown, orientated with anterior to the left. (B) Bar chart to show the percentage of embryos displaying each phenotype. The numbers of embryos classified in each phenotype are shown in parentheses. The total number of embryos examined for each pronephric marker gene is indicated at right side of each bar (n). Yellow bar, normal staining; green bar, reduced staining; blue bar, absent staining; red bar, enlarged/ectopic staining.

	cpeb3 (cytoplasmic polyadenylation element binding protein 3) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
	dusp4 (dual specificity phosphatase 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.

	spaca5 ( sperm acrosome associated 5)gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.

	xkr9 (XK, Kell blood group complex subunit-related family, member 9 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	elavl2 (ELAV (embryonic lethal, abnormal vision)-like 2 (Hu antigen B) ) gene expression in Xenopus laevis embryos, NF stage 34, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	phf20l1 (PHD finger protein 20-like 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	phf20l1 (PHD finger protein 20-like 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.


	gadd45g (growth arrest and DNA-damage-inducible, gamma) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anterior left.




	wdr73 (WD repeat domain 73) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	vwa8 (von Willebrand factor A domain containing 8 )gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.
	triap (TP53 regulated inhibitor of apoptosis 1 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
	srd5a1 (steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1) ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	spaca5 (sperm acrosome associated 5) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.

	hsf3 (heat shock transcription factor 3) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.


	supt6h (suppressor of Ty 6 homolog ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
	supt6h (suppressor of Ty 6 homolog ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	csnk1g1 (casein kinase 1, gamma 1 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
	csnk1g1 (casein kinase 1, gamma 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	dusp11 (dual specificity phosphatase 11 (RNA/RNP complex 1-interacting)) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	csnk2b ( casein kinase 2, beta polypeptide) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	csnk2b (casein kinase 2, beta polypeptide) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.
	dusp4 (dual specificity phosphatase 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anterior left.
	srsf3 (serine/arginine-rich splicing factor 3) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.

	srsf5 (serine/arginine-rich splicing factor 5) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
	srsf5 (serine/arginine-rich splicing factor 5) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.
	snrpd1 (mall nuclear ribonucleoprotein D1 polypeptide 16kDa) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.