Butler AM et al. (2018), A novel role for sox7 in Xenopus early primordi...

XB-ART-54309

Development 2018 Jan 08;1451:. doi: 10.1242/dev.155978.

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A novel role for sox7 in Xenopus early primordial germ cell development: mining the PGC transcriptome.

Butler AM , Owens DA , Wang L , King ML .

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Xenopus primordial germ cells (PGCs) are determined by the presence of maternally derived germ plasm. Germ plasm components both protect PGCs from somatic differentiation and begin a unique gene expression program. Segregation of the germline from the endodermal lineage occurs during gastrulation, and PGCs subsequently initiate zygotic transcription. However, the gene network(s) that operate to both preserve and promote germline differentiation are poorly understood. Here, we utilized RNA-sequencing analysis to comprehensively interrogate PGC and neighboring endoderm cell mRNAs after lineage segregation. We identified 1865 transcripts enriched in PGCs compared with endoderm cells. We next compared the PGC-enriched transcripts with previously identified maternal, vegetally enriched transcripts and found that ∼38% of maternal transcripts were enriched in PGCs, including sox7 PGC-directed sox7 knockdown and overexpression studies revealed an early requirement for sox7 in germ plasm localization, zygotic transcription and PGC number. We identified pou5f3.3 as the most highly expressed and enriched POU5F1 homolog in PGCs. We compared the Xenopus PGC transcriptome with human PGC transcripts and showed that 80% of genes are conserved, underscoring the potential usefulness of Xenopus for understanding human germline specification.

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Species referenced: Xenopus laevis
Genes referenced: bpnt2 cdc20 ctrl dazl ddx4 dnd1 fam168b fer germes grip2 h1-8 lmnb3 nasp odc1 pcna pgam1 pgat pgc piwil1 pou5f3.3 pphln1 ppp1r2 prpsap2 rtn3 sox7 tspan1 zfyve26
GO keywords: germ cell development
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???displayArticle.morpholinos??? pou5f3.3 MO7 sox7 MO2

???displayArticle.gses??? GSE102047: Xenbase, NCBI

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	Fig. 1. RNA-seq analysis of PGC versus endoderm cell transcripts in X. laevis embryos. (A) Two-dimensional principal component analysis (PCA) of PGC and Endo transcripts from st. 12.5-14 embryos. Variance was 73.5% and 11.3% in PC1 and PC2, respectively. (B) Differential expression analysis of PGC versus Endo transcripts; red dots indicate differentially expressed transcripts. (C) Heat map of the top 50 PGC-enriched transcripts; novel germline transcripts are underlined. Endo, endoderm cell sample. Asterisks indicate transcripts that express more than one copy. ‘.L’ and ‘.S’ indicate long and short chromosomes, respectively. Note that only the more highly expressed copy in PGCs is shown.
	Fig. 2. Analysis of PGC-enriched transcripts. (A) GeneGo direct interaction network connecting 53 of the 150 most highly expressed PGC-enriched transcripts (network key: https://portal.genego.com/help/MC_legend.pdf ). (B) Top 300 PGC-enriched transcripts categorized by molecular function. (C) Distribution of zygotically expressed maternal, vegetally enriched transcripts. DE, differentially expressed.
	Fig. 3. WISH analysis of PGC transcripts. Expression of selected PGC-enriched mRNAs at post-MBT st. 11.5 (gastrula), 16 (neurula) and 33/34 (tailbud). pgat expression marks germ plasm in PGCs. Transcripts detected in PGCs at neurula are shown at higher magnification in insets. WISH analysis was performed on ≥21 total embryos from at least three adult female frogs. Insets show magnifications of the boxed areas. A, anterior; b, brain; ba, brachial arches; cg, cranial ganglia; D, dorsal; gl, cement gland; isr, intersegmental region; L, left; n, notochord; np, nasal placodes; nt, neural tube; ov, otic vesicle; P, posterior; pn, pronephros; R, right; s, somites; V, ventral; vbi, ventral blood islands; Vg, vegetal view. Scale bars: 200 μm.
	Fig. 4. PGC number increased after PGC-directed sox7 knockdown or overexpression in early development. (A-D) The number of PGCs per embryo was determined in control, sox7-MO, sox7-FL, and sox7-MO rescue embryos at gastrula (st. 11.5) (A,B) and tailbud (st. 33-34) (C,D) stages. Representative images are shown (B,D). In box and whisker plots, middle bar is the median, top of the box is the third quartile, bottom of the box is the first quartile, and the whiskers represent the minimum and maximum number of PGCs observed in one embryo of the set analyzed (i.e. the range). ctrl n=23, sox7-MO n=24, sox7-FL n=23, sox7-MO rescue n=25. P<0.05, *P<0.005 (compared with uninjected control); # P<0.05 (compared with sox7-MO). pgat expression by WISH was used to identify PGCs. Analysis shown for one of at least three independent experiments. ctrl, control; FL, full-length, flagtagged; MO, morpholino. Scale bars: 500 μm (B); 1 mm (D).
	Fig. 5. PGC-directed sox7 knockdown or overexpression altered germ plasm localization in early PGC development. Control, sox7-MO, sox7-FL, and sox7-MO rescue embryos were analyzed for Piwi (germ plasm; pink) and PCNA (nuclear; green) expression by immunofluorescence at the indicated stages. (A) Germ plasm localization in each PGC was classified as membrane, nuclear or dispersed and quantified; n numbers of PGCs analyzed are indicated. (B) Representative images of each classification are shown for all stages analyzed. Representative PGC in each panel is outlined in white. P<0.05, *P<0.005 (compared with ctrl); # P<0.05 (compared with sox7-MO). ctrl, control; FL, full length, flag-tagged; MO, morpholino. All images were taken at 20×. Scale bars: 100 μm.
	Fig. 6. PGC-directed sox7 knockdown or overexpression increased the number of transcriptionally active PGCs in early development. Control, sox7-MO, sox7-FL, and sox7-MO rescue embryos were analyzed for Piwi (germ plasm; pink) and pSer2 (transcriptionally active nuclei; green) expression by immunofluorescence at the indicated stages. (A) The percentage of pSer2-positive PGCs was determined; n numbers of PGCs analyzed are indicated. (B) Representative images of PGCs from control and injected embryos are shown for each stage. Representative PGC in each panel is outlined in white; arrowheads indicate pSer2- positive PGCs; arrows indicate pSer2-positive somatic cell nuclei. P<0.05, *P<0.005 (compared with ctrl); # P<0.005 (compared with sox7-MO). ctrl, control; MO, morpholino; FL, full length, flag-tagged; pSer2, RNA polymerase II carboxy-terminal-domain phosphorylated at Ser2. All images were taken at 20×. Scale bars: 50 μm.
	Fig. 7. PGC-directed sox7 knockdown, but not overexpression, alters expression of germline genes. (A,B) RT-qPCR of expression of germline genes in PGC-directed scramble-MO control and sox7 knockdown embryos (sox7-MO) relative to uninjected control embryos (dashed line) (A) and uninjected control (ctrl) and PGC-directed sox7 overexpression (sox7-FL) embryos (B) at st. 8, 9, 10 and 11. *P<0.05 (compared with relative control). odc1 expression was used as the reference gene. Error bars represent s.d. of at least two experiments.
	Figure S1. Methods flow chart for RNA-seq analysis of PGC versus endoderm cell transcripts in X. laevis embryos. 1,2,3 denote PGC and Endo sample sets.

	Figure S3. sox7-MO does not inhibit sox7-FL rescue protein expression. Expression of full length, flag-tagged sox7 rescue construct (sox7-FL rescue) in the presence of sox7-MO (concentrations are indicated). Western blot (top); histogram of the western (bottom).
	Figure S4. pou5f3.3-MO inhibits pou5f3.3-FL and not pou5f3.3-FL rescue protein expression. Schematic of the pou5f3.3 (oct60) morpholino (pou5f3.3-MO) targeted region (A). Expression of full length, flag-tagged pou5f3.3 (pou5f3.3-FL) (B) and pou5f3.3-FL rescue (C) in the presence of pou5f3.3-MO (concentrations are indicated). Respective histograms are shown.

	Figure S6. Maternal vegt is expressed in PGCs. Raw reads from PGC and Endo samples were aligned to A) Sea urchin wnt8 (negative alignment control), Xenopus laevis wnt8 (positive alignment control), vegt region conserved between maternal and zygotic vegt isoforms (vegt Conserved Region; positive vegt control), maternal vegt (5’ 131bp region unique to maternal vegt, black, plus 70bp of the conserved region, green), and zygotic vegt (5’ 32bp region unique to zygotic vegt, black, plus 70bp of the conserved region, green), B) then counted. Transcription start site is indicated for maternal and zygotic vegt.
	Figure S6. Maternal vegt is expressed in PGCs. Raw reads from PGC and Endo samples were aligned to A) Sea urchin wnt8 (negative alignment control), Xenopus laevis wnt8 (positive alignment control), vegt region conserved between maternal and zygotic vegt isoforms (vegt Conserved Region; positive vegt control), maternal vegt (5’ 131bp region unique to maternal vegt, black, plus 70bp of the conserved region, green), and zygotic vegt (5’ 32bp region unique to zygotic vegt, black, plus 70bp of the conserved region, green), B) then counted. Transcription start site is indicated for maternal and zygotic vegt.
	cdc20 (cell division cycle 20 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	fam168b (family with sequence similarity 168 member B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	fer (fer (fps/fes related) tyrosine kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	lmnb3 (lamin B3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	pgam1 (phosphoglycerate mutase 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	rtn3 (reticulon 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	pphln1 (periphilin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	tspan1 (tetraspanin 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	ppp1r2 (protein phosphatase 1 regulatory inhibitor subunit 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.
	prpsap2 (phosphoribosyl pyrophosphate synthetase-associated protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33/34, lateral view, anterior right, dorsal up.

References [+] :

Aguero, Mechanisms of Vertebrate Germ Cell Determination. 2017, Pubmed, Xenbase