Chen JA et al. (2005), Identification of novel genes affecting mesoder...

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Mech Dev 2005 Mar 01;1223:307-31. doi: 10.1016/j.mod.2004.11.008.

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Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus.

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

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The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.

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Species referenced: Xenopus tropicalis
Genes referenced: astl2c bix1.1 calu camp ccndx cdx2 cdx4 chrd crx dcun1d3 dlx5 dnajc19 etv1 eya1 foxa1 foxa4 foxd4l1.1 foxh1 foxi2 gal.2 gata4 gna11 gnaq gtf2h5 hand1 hes4 hes5.10 hey1 hmbs hmbs.2 hnrnpu hoxd1 inka1 itgb1bp2 laptm4a mef2d mespb msantd4 msx1 myb myf5 nodal nucks1 osr1 ovol2 pax3 pdlim5 pes1 ppp1r14b prkaca rhou serbp1 serpine1 sfrp1 shh sox21 spry2 srsf12 tbx6 tbxt tcf3 tcf7l1 tra2a unc13b vangl2 vegt wnt11b ywhae znf326 znf84

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	Fig. 3. Mesoderm related phenotypes. (A) Classification based on phenotype can be subdivided into two major groups: esoderm related phenotypes(gastrulation defect, anterior defect, dorsalanteriorized, extra axis, shortened A/P axis, and posterior defect), and on-mesoderm related phenotypes Values are given as percentages of numbers of identified active clones in the total (nZ82) and within the subgroup of mesoderm defect clones (nZ64). (B) Uninjected control at stage 34. (C) Control embryo injected with LacZ mRNA only and stained with X-gal. Lateral (D) and dorsal (E) view of gastrulation defect caused by injecting TGas002g05 (hypothetical protein LOC233805). (F) Embryo injected with clone TGas131g15 (Msx-1) displays anterior defect. (G) Injection with TGas059j09 (hairy2a), showing different degrees of a dorso-anteriorized phenotype. (H and I) Induced extra axis at injected site by TNeu118f24 (Lyn tyrosine kinase) and TGas127o03 (Otx5), respectively. (J) Embryos displaying shortened anteriorosterior axis following injection of TGas088n14 (similar to rat PKC-binding enigma). (K) Clone TNeu008i12 (Tbx6) gives rise to a posterior defect.
	Fig. 4. Example of clones giving rise to alteration in Xbra and Xmyf-5 expression. Vegetal and lateral views of embryos at the mid-gastrula stage (stage 11) stained for the expression of Xbra (A) and Xmyf-5 (B) (purple) by whole mount in situ hybridization. Embryos were injected at two cell stage into one side of marginal zone with 400 pg of clone mRNA along with 130 pg Lac-Z mRNA. Embryos were stained with X-gal (blue). Alterations in Xbra expression included inhibition, TGas131g15 (Msx1); shift, TNeu008i12 (Tbx6); and expansion to animal pole, TNeu118f24 (Lyn tyrosine kinase). Alterations of Xmyf-5 expression included inhibition, TGas131g15 (Msx1); expansion to dorsal lip, TNeu047o15 (Ga-11 protein); shift, TNeu008i12 (Tbx6), and expansion to ventral side, TNeu118f24 (Lyn tyrosine kinase).
	Fig. 6. Regionalized expression pattern of clones giving rise to gastrulation defects with effects on the expression of the early mesoderm markers. Ten clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 21); d, dorsal view (stage 11, 13, 15, 17 and 21); l, lateral view (stage 10, 30, and 32); v, vegetal view (stage 11).
	Fig. 7. Regionalized expression pattern of clones giving rise to gastrulation defects without effecting the expression of the early mesoderm markers. Eight clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 15 and 17); d, dorsal view (stage 17 and 19); l, lateral view (stage 28 and 30); v, vegetal view (stage 11).
	Fig. 8. Regionalized expression pattern of clones giving rise to anterior defects. Nine clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 17); d, dorsal view (stage 13, 15, 17 and 20); l, lateral view (stage 11, 28, and 30); v, vegetal view (stage 11).
	Fig. 10. Regionalized expression pattern of clones giving rise to extra axis. Six clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 17); d, dorsal view (stage 13 and 17); l, lateral view (stage 11, 28 and 30); v, vegetal view (stage 11).
	Fig. 11. Regionalized expression pattern of clones giving rise to shortened anteriorosterior axis. Seven clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 17); d, dorsal view (stage 17); l, lateral view (stage 11, 28 and 30); v, vegetal view (stage 11).
	Fig. 12. Regionalized expression pattern of clones giving rise to posterior defects. Three clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: d, dorsal view (stage 15 and 17); l, lateral view (stage 28 and 30); v, vegetal view (stage 11).
	Fig. 13. Regionalized expression pattern of clones giving rise to non-mesoderm related defects. Eleven clones from non-mesoderm related defect group gave rise to restricted expression patterns. These images are sub-grouped by synphenotypes and ordered by clone name. The upper two genes (TGas058e12, TNeu023n04) gave rise to a blister phenotype; the next five genes (TGas005m21, TGas007h22, TGas108b01, TNeu058o20, TNeu104c17) gave rise to ectopic pigmentation; the next gene (TGas111k06) gave rise to apopotisis; the next two genes (TGas139k18, TNeu101f16) gave rise to a bending phenotype and the last gene (TGas100b22) gave rise to a mixed phenotype. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: a, anterior view (stage 20); d, dorsal view (stage 15, 17 and 30); l, lateral view (stage 11, 21, 28 and 30); v, vegetal view (stage 11).
	Fig. 5. Non-mesoderm related phenotypes. (A) Classification based on phenotype can be subdivided into two major groups: mesoderm related phenotypes and non-mesodermal related phenotypes(blister, ectopic pigmentation, apotosis, bending and mix). Values are given as percentages of identified active clones in the total (nZ82) and within the subgroup of non-mesoderm defect clones (nZ18). (B) Blister phenotype caused by TNeu140g18 (connexin 43.4). (C) Embryo injected with TNeu058o20 (Sox21) shows ectopic pigmentation. (D) Stage 10 uninjected embryos. (E and F) Stage 10 and 35 embryos, respectively, injected with TNeu026a07 (hypothetical protein BC014729) show a phenotype characteristic of apoptosis. (G) Embryo injected with TGas139k18 (hydroxymethylbilane synthase) displaying a bent phenotype.
	Fig. 9. Regionalized expression pattern of clones giving rise to dorso-anteriorized phenotypes. Six clones from this synphenotype group gave rise to restricted expression patterns. The images are ordered by clone name. For each clone the species and annotation of the closest matching blastx hit is shown. Abbreviations are: d, dorsal view (stage 15 and 17); l, lateral view (stage 28 and 30); v, vegetal view (stage 11).
	dcun1d3 (DCN1, defective in cullin neddylation 1, domain containing 3) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	prkaca (protein kinase, cAMP-dependent, catalytic, alpha) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Vegetal View.
	prkaca (protein kinase, cAMP-dependent, catalytic, alpha) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view
	prkaca (protein kinase, cAMP-dependent, catalytic, alpha) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	myb (v-myb myeloblastosis viral oncogene homolog) gene expression in Xenopus tropicalis embryos, NF stage 21, as assayed by in situ hybridization. Anterior view: dorsal up.
	myb (v-myb myeloblastosis viral oncogene homolog) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	nodal (nodal growth differentiation factor) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Vegetal View.
	gnaq (guanine nucleotide binding protein (G protein), q polypeptide) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Vegetal view.
	gnaq (guanine nucleotide binding protein (G protein), q polypeptide) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	gnaq (guanine nucleotide binding protein (G protein), q polypeptide) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	pes1 (pescadillo ribosomal biogenesis factor 1) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization. anterior view, dorsal up.
	pes1 (pescadillo ribosomal biogenesis factor 1) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	ovol2 (ovo-like 2) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Lateral view.
	ovol2 (ovo-like 2) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	ovol2 (ovo-like 2) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	laptm4a (lysosomal-associated protein transmembrane 4 alpha) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Vegetal view.
	laptm4a (lysosomal-associated protein transmembrane 4 alpha) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal View, anterior left.
	laptm4a (lysosomal-associated protein transmembrane 4 alpha) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	fam212a (family with sequence similarity 212, member A) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view: anterior left.
	fam212a (family with sequence similarity 212, member A) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	fam212a (family with sequence similarity 212, member A) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	dnajc19(DnaJ (Hsp40) homolog, subfamily C, member 19) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	pdlim5 (PDZ and LIM domain 5) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	hnrnpu (heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A)) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Lateral view.
	hnrnpu (heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A)) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	hnrnpu (heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A)) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	ppp1r14b (protein phosphatase 1, regulatory (inhibitor) subunit 14B) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	ppp1r14b (protein phosphatase 1, regulatory (inhibitor) subunit 14B) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	nucks1 ( nuclear casein kinase and cyclin-dependent kinase substrate 1 ) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Lateral view.
	nucks1 ( nuclear casein kinase and cyclin-dependent kinase substrate 1 ) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	nucks1 ( nuclear casein kinase and cyclin-dependent kinase substrate 1 ) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	serbp1 (SERPINE1 mRNA binding protein 1) gene expression in Xenopus tropicalis embryos, NF stage 17, as assayed by in situ hybridization. Dorsal view: anterior left.
	serbp1 (SERPINE1 mRNA binding protein 1) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	unc13b (unc-13 homolog B) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Lateral view.
	unc13b (unc-13 homolog B) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view: anterior left.
	unc13b (unc-13 homolog B) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	tra2a (transformer 2 alpha homolog) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	itgb1bp2 (integrin beta 1 binding protein (melusin) 2) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view: anterior left.
	itgb1bp2 (integrin beta 1 binding protein (melusin) 2) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	ccndx (cyclin Dx) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Dorsal view: Anterior up.
	ccndx (cyclin Dx) gene expression in Xenopus tropicalis embryos, NF stage 11, as assayed by in situ hybridization. Anterior view: dorsal up.
	ccndx (cyclin Dx) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: Anterior left, dorsal up.
	hmbs.2 (hydroxymethylbilane synthase) gene expression in Xenopus tropicalis embryos, NF stage 21, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	hmbs.2 (hydroxymethylbilane synthase) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	znf84 (zinc finger protein 84) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization. Dorsal view: anterior left.
	znf84 (zinc finger protein 84) gene expression in Xenopus tropicalis embryos, NF stage 30, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up.
	ywhae (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior left.
	ywhae (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.