Owens DA et al. (2017), High-throughput analysis reveals novel maternal...

XB-ART-53019

Development 2017 Jan 15;1442:292-304. doi: 10.1242/dev.139220.

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High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration.

Owens DA , Butler AM , Aguero TH , Newman KM , Van Booven D , King ML .

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During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA-sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.

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Species referenced: Xenopus laevis
Genes referenced: a2m acaca acsl1 acvr1 adpgkl ank1 arid5b atp1b1 atrx bicc1 casp3.2 cbx7 cbx8 cdk5r2 cdr2l chek1 ciita cnot4 cnppd1 crebbp ctrl ctsc cyp17a1 dand5 dazl ddx25 dnd1 e2f1 eeig1 efnb1 erbb2 esrra fam168b farp2 fgfr2 fndc3a fzd8 gdf1 germes gk grip2 hook2 ifrd2 irf8 kif13b ldlrap1 mapk8 midn mov10 nanos1 otx1 papola paqr6 parn pcsk6 perp pgam1 pgat pgc pld1 plin2 pou2f1 ppp1r2 prkag1 prr11 prrg2 raver1 rbpms2 rhobtb2 rmdn3 rnf38 rras2 rtn3 senp1 sf3a1 slain1 slc12a9 slc18a2 sox7 spata13 spire1 srgap1 ssr1 sulf1 sybu tesk1 tesk2 ticrr timm8b tmem192 tmem65 tob2 trank1 trappc14 trib1 trim36 ube2d1 vcl vegt wasl wnk2 wnt1 wnt11b wwtr1 xkrx zfp36l2
???displayArticle.morpholinos??? efnb1 MO1 otx1 MO1 sox7 MO2 wwtr1 MO1

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	Fig. 1. RNA-seq analysis of vegetal versus animal pole transcripts in stage VI X. laevis oocytes. (A) Total read counts for oocyte-paired vegetal and animal pole samples. Bars of the same color represent vegetal (n=3) and animal (n=3) pole samples extracted from the same oocytes. (B) Two-dimensional principal component analysis of vegetal and animal pole transcripts. Vectors V1, V2, V3 and A1, A2, A3 represent vegetal and animal pole samples, respectively. (C) Scatter plot comparing vegetal and animal pole transcripts. (D) Differential expression analysis of vegetal versus animal pole transcripts with FDR≤0.05 and FPKM≥5. GV, germinal vesicle.
	Fig. 2. Biological process and network analysis of vegetally enriched transcripts. (A) The 198 annotated transcripts upregulated in the vegetal pole categorized according to their biological function (GeneCards). (B) Sixty vegetally enriched transcripts with enzymatic activity grouped based on enzymatic function. (C) GeneGo pathway analysis using the direct interaction algorithm of vegetally enriched transcripts. (D) RT-qPCR verification of pathway hubs p300, irf8 and err1/esrra at animal (An) versus vegetal (Vg) poles. vg1, control for vegetal pole.
	Fig. 3. WISH of vegetal pole transcripts. The expression of a subset of vegetally enriched mRNAs was analyzed during oogenesis and embryo development by WISH. Expression patterns are grouped according to mRNA pathway: early (A), late (B) or both (C). xpat expression marks germ plasm. Probes, developmental stages and developmental structures are indicated in the illustrations at the top: germ plasm/PGCs (purple), pronephros (pink), ventral blood islands (lime green), eye (black), lens (white), otic vesicle (gray), cranial ganglia (yellow), brachial arches (green), nasal placodes (teal), intersegmental region (brown), notochord (orange), brain and neural tube (blue). Transcripts detected in PGCs at the tailbud stage are shown at higher magnification in insets. WISH analysis was performed on ≥40 total embryos from at least two adult female frogs. Dorsal (D), ventral (V), left (L), right (R), posterior (P), anterior (A), vegetal view (v); MC, mitochondrial cloud; N, nucleus; Oo, oocyte. Scale bars: 100 μm for stage I oocyte; 200 μm in remaining panels.
	Fig. 4. Overexpression of five out of six selected vegetally enriched mRNAs reduces PGC number. One-cell embryos were injected in the vegetal region with GFP (control) or the indicated transcripts (0.5 ng). Tailbud embryos (stage 32-35) were analyzed for xpat expression by WISH, and representative images are shown (A-G). The number of PGCs per embryo was quantified (H). GFP, n=16; spire1, n=39; e2f1, n=64; otx1, n=27; parn, n=35; rras2, n=48; wwtr1, n=35. *P<0.05, compared with GFP control. Analysis based on at least two independent experiments and shown as a box and whisker plot.
	Fig. 5. MO-mediated knockdown of a subset of vegetally enriched mRNAs increases PGC number. One-cell embryos were injected in the vegetal region with MOs (15 ng) targeting otx1 or wwtr1. Tailbud embryos (stage 32-35) were analyzed for xpat. Representative images are shown (A-C). The number of PGCs per embryo was quantified (D). Uninjected control (ctrl), n=32; otx1-MO, n=31; wwtr1-MO, n=25. *P<0.05 compared with control. Analysis based on at least two independent experiments.
	Fig. 6. Altering sox7 expression reduces PGC number. One-cell embryos were injected in the vegetal region with sox7dCEnR RNA (200 pg) and Xtsox7 RNA (200 pg), alone and in combination. Tailbud embryos were analyzed for xpat expression by WISH. Representative images are shown (A-D). The number of PGCs per embryo was quantified (E). Uninjected control (ctrl), n=28; sox7dCEnR, n=24; Xtsox7, n=27; sox7dCEnR and Xtsox7, n=28. *P<0.05 compared with uninjected control. #P<0.05 compared with sox7dCEnR. Analysis based on at least two independent experiments.
	Fig. 7. efnb1 is required for normal PGC number and migration. One-cell embryos were injected in the vegetal region with (A) efnb1-FL alone (200 pg), or efnb1-FL (200 pg) and efnb1-MO (16 ng); (B) scrambled-MO (16 ng), efnb1-MO alone (16 ng), or efnb1-MO (16 ng) and efnb1-FL-rescue (200 pg). Tailbud embryos were analyzed for xpat expression by WISH. Representative images are shown on the left. (A) The number of PGCs per embryo was quantified. (B) Percentage of embryos with mislocalized PGCs was calculated. Black lines indicate the boundaries of normal PGC location, between somites 5 and 11. Uninjected control (Ctrl), n=28; efnb1-FL, n=28; efnb1-FL and efnb1-MO, n=28; scrambled-MO, n=25; efnb1-MO, n=27; efnb1-MO and efnb1-FL-rescue, n=28. *P<0.05 compared with uninjected control. #P<0.05 compared with efnb1-FL (A) or efnb1-MO (B). Analysis based on at least two independent experiments.
	Fig. 8. Inhibition of p300 reduces PGC number. (A) Schematic of GeneGo-predicted p300 regulatory interconnections from our data set. (B-G) One-cell embryos were incubated with ≤0.04% DMSO (ctrl, n=31) or increasing concentrations of the p300 inhibitor C646: 0.2 μM, n=38; 0.5 μM, n=34; 1 μM, n=34; 2 μM, n=34. Tailbud embryos were analyzed for xpat expression by WISH. The average number of PGCs per embryo was quantified (B). *P<0.05 compared with uninjected control.
	Figure S1. Whole mount in situ hybridization (WISH) of select vegetal and animal pole transcripts. Expression of a subset of RNAs enriched in the vegetal (A-B) and animal (C) poles were analyzed during oogenesis and development by WISH. Probes, developmental stages, and developmental structures are indicated: germplasm/PGCs (purple), pronephros (pink), ventral blood islands (lime green), eye (black), lens (white), otic vesicle (gray), cranial ganglia (yellow), brachial arches (green), nasal placodes (teal), inter-segmental region (brown), notochord (orange), brain and neural tube (blue). Transcripts detected in primordial germ cells at the tailbud stage are magnified and embedded in their respective images. Black bars represent 100um in st. I oocyte panels, and 200um in all other panels. Please refer to Fig. 3 for use of xpat probe as a positive control for germ plasm localization.
	Figure S2. Morpholinos (MOs) inhibit expression of their target RNAs. A) Schematic of the efnb1, otx1, and wwtr1 morpholino targeted regions are indicated in red. B) Wheat germ extracts were incubated with either efnb1-FL (1ug), otx1-FL (250ng), or wwtr1-FL (250ng) transcripts in the presence of increasing concentrations of their respective MOs and subject to anti-flag western blot analysis. Quantification of respective flag expression is shown.
	Figure S3. Morpholino-mediated sox7 inhibition reduces PGC number in tailbud embryos. A) Schematic of the sox7 morpholino target region (top). Wheat germ extract was incubated with sox7-FL (500ng) in the presence of increasing concentrations of sox7-MO and subject to anti-flag western blot analysis. Quantification of respective flag expression is shown (bottom). B-D) Onecell embryos were injected in the vegetal region with sox7-MO (16ng). Tailbud embryos were analyzed for xpat expression by WISH. The number of PGCs per embryo was quantified (B). Representative images are shown (C-D). Uninjected control (ctrl) n=36, sox7-MO n=43. * statistically significant compared to ctrl (p<0.05). Analysis based on at least two independent experiments.
	e2f1 (E2F transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, vegetal view.
	e2f1 (E2F transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, vegetal view.
	e2f1 (E2F transcription factor 1) gene expression in bissectted Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	e2f1 (E2F transcription factor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	rras2 (related RAS viral (r-ras) oncogene homolog 2) gene expression in Xenopus laevis oocyte stage I, vegetal view.
	rras2 (related RAS viral (r-ras) oncogene homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, vegetal view
	rras2 (related RAS viral (r-ras) oncogene homolog 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	rras2 (related RAS viral (r-ras) oncogene homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	mov10 (Mov10 RISC complex RNA helicase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8, vegetal view.
	mov10 (Mov10 RISC complex RNA helicase) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	mov10 (Mov10 RISC complex RNA helicase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	spire1 ( spire-type actin nucleation factor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view.
	spire1 ( spire-type actin nucleation factor 1 ) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	spire1 ( spire-type actin nucleation factor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	wnk2 (WNK lysine deficient protein kinase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6 vegetal view.
	wnk2 (WNK lysine deficient protein kinase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	wnk2 (WNK lysine deficient protein kinase 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	dand5 (DAN domain family member 5, BMP antagonist) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view.
	hook2 (hook microtubule-tethering protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view
	hook2 (hook microtubule-tethering protein 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	hook2 (hook microtubule-tethering protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	parn (poly(A)-specific ribonuclease) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	parn (poly(A)-specific ribonuclease) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	rnf38 ( ring finger protein 38 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view
	rnf38 ( ring finger protein 38 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	rnf38 ( ring finger protein 38 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	trank1 (tetratricopeptide repeat and ankyrin repeat containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, oocyte stage i, vegetal view.
	trank1 (tetratricopeptide repeat and ankyrin repeat containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view.
	trank1 (tetratricopeptide repeat and ankyrin repeat containing 1) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	wwtr1 (WW domain containing transcription regulator 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	tob2 (transducer of ERBB2, 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, vegetal view
	tob2 (transducer of ERBB2, 2) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, lateral view, anterior right, dorsal up.
	tob2 (transducer of ERBB2, 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.

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